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BMC Proceedings 2011, Volume 5 Suppl 8 

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MEETING ABSTRACTS Open Access 

22nd European Society for Animal Cell Technology 

(ESACT) Meeting on Cell Based Technologies 

Vienna, Austria. 15-18 May 2011 

Edited by Hansjörg Hauser 

Published: 22 November 2011 

These abstracts are available online at http://www.biomedcentral.com/1753-6561/5?issue=S8 

INTRODUCTION 

I1 

Cell Based Technologies: Abstracts of the 22 nd ESACT Meeting 2011 

in Vienna 

Hansjörg Hauser 

Helmholtz-Zentrum für Infektionsforschung GmbH, Department of Gene 

Regulation and Differentiation, 38124 Braunschweig, Germany 

E-mail: hansjoerg.hauser@helmholtz-hzi.de 

BMC Proceedings 2011, 5(Suppl 8):I1 

The European Society of Animal Cell Technology (ESACT) is a society that 

brings together scientists, engineers and other specialists working with 

animal cells in order to promote communication of experiences between 

European and international investigators and progress development of cell 

systems in productions derived from them. 

Animal cells are being used as substrates in basic research and also for the 

production of proteins. Tissue engineering, gene and cell therapies, organ 

replacement technologies and cell-based biosensors contribute to a 

considerable widening of interest and research activity based on animal cell 

technology. 

The abstracts of this supplement are from the 22 nd ESACT meeting that was 

held in Vienna, Austria, May 15-18, 2011. The abstracts review the 

presentations from this meeting and should be a useful resource for the 

state-of-the-art in animal cell technology. 

ORAL PRESENTATIONS 

O1 

Highly efficient, chemically defined and fully scalable biphasic 

production of vaccine viruses 

Ingo Jordan * , Volker Sandig 

ProBioGen AG, 13086 Berlin, Germany 

E-mail: ingo.jordan@probiogen.de 

BMC Proceedings 2011, 5(Suppl 8):O1 

Vectorial vaccines are predicted to yield novel therapeutic and protective 

approaches. They consist of recombinant live carriers that express antigen 

from an unrelated pathogen in the recipient. Promising viral carriers are hostrestricted 

pox viruses that trigger a strong immune response without ability 

to replicate in the human organism. A block in replication is an important 

safety feature that allows application even in immunocompromized 

recipients. However, with this type of attenuation there is not even limited 

amplification of the vector at the site of infection and therefore very high 

numbers of infectious units have to be given per dose for full efficacy. Hence, 

if such vectors are to be used in global vaccine programs highly efficient 

production processes will be required. Furthermore, to combat diseases such 

as AIDS, hepatitis C, tuberculosis, or malaria with these vectors, millions of 

the concentrated vaccine units will have to be provided annually. Any 

production process therefore should also be scalable and preferrably 

transferrable to newly industrialized countries. Latter requirement demands a 

robust process independent of the complex logistics and uncertainties 

associated with primary chicken cells, the current industrial substrate for 

these pox viruses and certain other vectors. 

Webelievethatwehavesolvedmostoftheupstreamchallengeswitha 

chemically defined suspension culture production process for three 

disparate members of the highly attenuated poxviruses [1]: modified 

vaccinia Ankara (MVA), fowlpoxvirus (FPV) and canarypoxvirus ALVAC. The 

process is independent of primary material and based on the continuous 

duck suspension cell line AGE1.CR specifically created as a vaccine substrate 

[2]. 

In contrast to production of influenza virus that readily replicates in the cell 

proliferation medium [3], development of a production process for 

poxviruses was surprisingly complicated and involves media formulations 

matched to two distinct phases, cell proliferation and virus production. Our 

process was adjusted to the different kinetics and requirements of the 

three examined viruses, and was studied in Wave and disposable 

bioreactors up to 50 L scale. For MVA, titers in the crude lysate without any 

processing reliably exceed the critical threshold of 10 8 pfu/mL and often 

are in the range of 5 × 10 8 to 2 × 10 9 pfu/mL. 

One hallmark of the biphasic process described here is controlled formation 

of suspension cell aggregates at the transition from cell proliferation to virus 

production. Such aggregate formation was achieved with distinct chemically 

defined media harmonized such that a highly efficient, robust and industrial 

biphasic processes was obtained that does not require perfusion, medium 

replacement or microcarriers. 

In addition to poxviruses, this approach was successful in production of an 

unrelated RNA vector and also for this reason we believe that we have 

developed a more general principle for scalable production of viruses that 

may benefit from cell-to-cell contacts: In the background of the AGE1.CR cell 

lines, packaging cells were created for SIN/VEE-chimeric alphavirus replicons 

[4]. The packaging cells were transfected for stable trans-complementation 

of envelope and capsid proteins from separate expression cassettes. 

Production of replicons was efficient using adherent (serum-dependent) 

cultures but only moderately efficient with suspension cultures of the 

packaging cells. After transfer and optimization of the pox virus production 

process to replicon-induced packaging cell cultures (shown in figure 1) we 

obtained yields beyond 10 8 pfu/mL. 

Appearance of suspension packaging cells just prior to infection and at 

various time points during virus production is shown in (A). Note induction of 

aggregates in presence of virus production medium and cytopathic effect 

24 h post induction. Yields of replicon is shown in (B) with peak titers 24 h to 

48 h post induction. 

© 2011 various authors, licensee BioMed Central Ltd. All articles published in this supplement are distributed under the terms of the 

Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and 

reproduction in any medium, provided the original work is properly cited.



Figure 1(abstract O1) Biphasic process adapted to alphavirus replicons. 

In summary, superior yields were obtained for unrelated viral vectors 

using separate chemically defined media for proliferation and virus 

production. The media are matched to allow highly efficient, robust and 

industrial biphasic processes that do not require perfusion or medium 

replacement. 

References 

1. Jordan I, Northoff S, Thiele M, Hartmann S, Horn D, Höwing K, Bernhardt H, 

Oehmke S, von Horsten H, Rebeski D, Hinrichsen L, Zelnik V, Mueller W, 

Sandig V: A chemically defined production process for highly attenuated 

poxviruses. Biologicals 2011, 39:50-58. 

2. Jordan I, Vos A, Beilfuss S, Neubert A, Breul S, Sandig V: An avian cell line 

designed for production of highly attenuated viruses. Vaccine 2009, 

27:748-56. 

3. Lohr V, Rath A, Genzel Y, Jordan I, Sandig V, Reichl U: New avian 

suspension cell lines provide production of influenza virus and MVA in 

serum-free media: studies on growth, metabolism and virus 

propagation. Vaccine 2009, 27:4975-82. 

4. Perri S, Greer CE, Thudium K, Doe B, Legg H, Liu H, Romero RE, Tang Z, 

Bin Q, Dubensky TW Jr, Vajdy M, Otten GR, Polo JM: An alphavirus replicon 

particle chimera derived from venezuelan equine encephalitis and 

sindbis viruses is a potent gene-based vaccine delivery vector. J Virol 

2003, 77:10394-403. 

O2 

Recombinant antibody mixtures; optimization of cell line generation 

and single-batch manufacturing processes 

Søren K Rasmussen * , Lars S Nielsen, Christian Müller, Thomas Bouquin, 

Henrik Næsted, Nina T Mønster, Frank Nygaard, Dietmar Weilguny, 

Torben P Frandsen, Anne B Tolstrup 

Symphogen A/S, Elektrovej 375, 2800 Lyngby, Denmark 

E-mail: skr@symphogen.com 


Background: Recombinant antibody mixtures represent an important new 

class of antibody therapeutics as demonstrated by the increasing amount 

of literature showing that combinations of two or more antibodies show 

superiority compared to monoclonal antibodies (mAbs) for treatment of 

cancer and infectious diseases [1-5]. Sym004, composed of two antibodies 

targeting non-overlapping epitopes of the epidermal growth factor 

receptor (EGFR) act in a synergistic manner to induce an efficient 

internalization of EGFR leading to subsequent degradation and exhibit 

superior anticancer efficacy as demonstrated in several preclinical in vivo 

models [5]. 

At Symphogen A/S, we have developed an expression platform, Sympress, 

for cost-efficient production of antibody mixtures. The antibody mixtures are 

produced using a single-batch manufacturing approach where a polyclonal 

working cell bank (pWCB) prepared by mixing the individual stable cell lines 

producing all the desired antibodies is used as seed material for a bioreactor 

process [6]. By using a single-batch approach the CMC development costs of 

Page 2 of 181 

antibody mixtures are comparable to costs for monoclonal antibodies. 

However, the single-batch manufacturing approach raises questions with 

regard to control of composition ratios, compositional stability and 

robustness of the cell banking procedure. 

Here, we present experimental data addressing these key questions and 

demonstrate that mixtures of recombinant antibodies can be produced 

under predictable, reproducible and stable conditions using the 

Sympress technology. 

Material and methods: The second generation Sympress technology is 

based on expression in the ECHO cell line, a genetically modified version 

of the dihydrofolate reductase (DHFR) negative Chinese Hamster Ovary 

(CHO) cell line DG44 [7]. 

The expression plasmid used for stable transfection contained a 

bidirectional CMV promoter construct enabling co-expression of the IgG 

light and heavy chains from one plasmid. A DHFR selection marker was 

coupled directly to the heavy chain via an internal ribosome entry site 

(IRES). 

ECHO parental cells were transfected separately with each of the individual 

antibody expression vectors using standard transfection technology, 

whereafter cells were subjected to a methotrexate (MTX) selection schedule. 

The selected stable pools were single-cell cloned by FACS and highexpressing 

clones were adapted to chemically defined cell culture medium, 

expanded and frozen, still as individual monoclonal cell lines. 

The preparation of polyclonal cell banks followed a two-tiered cell 

banking approach. First, the relevant monoclonal cell lines were thawed 

and expanded. They were then mixed in predefined ratios and frozen as 

polyclonal master cell banks (pMCB). The pMCB was subsequently 

thawed, expanded and frozen as polyclonal working cell bank (pWCB). 

Antibody purification was performed by capture on a MabSelect SuRe 

column and cation exchange chromatography (CIEX) was used to 

separate the different antibodies based on their charge differences. The 

characteristics of the chromatograms (peak area/peak height) were used 

to determine the relative distribution of the different antibodies in the 

mixtures. 

Results: Two ECHO cell lines producing two distinct antibodies were 

selected based on their growth and production characteristics for a study to 

examine how mixing of the cells at different ratios affected the antibody 

composition. Briefly, the two cell lines were thawed, expanded and mixed in 

five different rations (5:5; 4:6; 3:7; 2:8 and 1:9). The resulting mixtures were 

frozen as pMCBs. To examine the obtained ratios and the compositional 

stability over prolonged periods of cultivation the pMCBs were revived and 

subjected to six weeks cultivation in shakers, followed by a 14 days fed 

batch process in shakers. Supernatant samples were harvested once a week 

and at the end of the fed batch process. IgG was captured by protein A and 

the relative antibody ratio was determined by CIEX. The results clearly 

showed that all the compositions were very stable over time and, 

importantly, that the relative amount of each antibody could be controlled 

by mixing the cell lines in an appropriate ratio (Figure 1A). Furthermore, the 

was a very strong correlation (R2 = 0.997) between expected and measured 

percentage of Ab 1 as shown in figure 1B.



Figure 1(abstract O2) A: Relative antibody ratios in pMCBs generated by mixing two ECHO cell lines at five different ratios. The antibody ratios were 

determined once a week by CIEX (from week 1 to 6 of cultivation) and in the harvest of a 14-day fed batch cultivation initiated after 6 weeks of 

cultivation. Red lines represent antibody 1 (Ab 1) and blue lines represent antibody 2 (Ab 2). B: Expected percentages plotted against measured 

percentages of Ab 1 in the five different mixes after 6 weeks seed train. C: Antibody compositions in pWCB in fed batch shaker experiments after 14, 21 

and 28 days seed train, respectively. D:Antibody distributions in harvest from seven different 5 L bioreactor processes. Four pWCB-1 ampoules (blue 

columns) and three pWCB-2 ampoules (green columns) were used for seed train. The lighter colored columns represent mean ± sdev. 

We then examined the compositional stability in a more complex model 

composed of a mixture of six different antibodies. A seed train of 

approximately 3 weeks would be required to generate cells for 

inoculation of production reactors in a 10.000 L scaled-up manufacturing 

Page 3 of 181 

process. To examine the robustness of the relative cell compositions 

during this timeframe +/- one week the cells from pWCB were inoculated 

into a 14 days fed batch shaker after 14, 21 or 28 days of expansion, 

respectively. The antibody distributions at the three different time points



were very constant as illustrated in Figure 1C. Additionally, the growth 

and production properties were also very constant over time (data not 

shown). 

The reproducibility and robustness of the cell banking system was 

evaluated by comparing the antibody product composition produced from 

two different pWCBs generated from the same pMCB. A pMCB producing a 

mixture containing 6 antibodies was used for the study. We generated two 

polyclonal working cell banks (pWCB-1 and pWCB-2) from the same pMCB. 

Production of pWCB-2 was separated in time from production of pWCB-1 

by several months. Four ampoules from pWCB-1 were expanded and used 

for inoculation of four 5 L bioreactors for fed batch production. Several 

months later, three ampoules from pWCB-2 were expanded and used to 

perform three new 5 L bioreactor runs. The antibodies were purified and 

antibody distribution determined by CIEX. The observed variation, both 

within the same pWCB and between different pWCB in regard to the 

antibody distributing was very limited (Figure 1D). This strongly indication 

that the pMCB/pWCB concept provides a reliable cell banking strategy. The 

reproducibility was also confirmed with respect to cell growth and 

productivity (data not shown). 

Conclusion: Symphogen A/S has developed an expression platform, 

Sympress, that can be used for predictable, reproducible, and stable 

production of antibody mixtures in a cost effective setting. 

Here, we have shown that the relative antibody ratio in antibody mixtures 

can be effective controlled using appropriate mixing of the individual 

monoclonal cell lines before generation of the pMCB. Further, we have 

presented consistent data from fed batch productions with pWCB where the 

seed train lengths varied from 14 to 28 days. This strongly supports that the 

single-batch manufacturing concept is sufficiently robust for manufacturing, 

also at scales required for commercial manufacturing. The two-tiered cell 

bank approach composed of pMCB and pWCB is robust and reproducible, 

and showed very high consistency both within a given pWCB and between 

different polyclonal working cell banks generated from the same pMCB. 

References 

1. Ben-Kasus T, Schechter B, Lavi S, Yarden Y, Sela M: Persistent elimination 

of ErbB-2/HER2-overexpressing tumors using combinations of 

monoclonal antibodies: Relevance of receptor endocytosis. Proc Natl 

Acad Sci U S A 2009, 106:3294-3299. 

2. van der Horst EH, Chinn L, Wang M, Velilla T, Tran H, Madrona Y, Lam A, 

Ji M, Hoey TC, Sato AK: Discovery of fully human anti-MET monoclonal 

antibodies with antitumor activity against colon cancer tumor models in 

vivo. Neoplasia 2009, 11:355-364. 

3. Dong J, Demarest SJ, Sereno A, Tamraz S, Langley E, Doern A, Snipas T, 

Perron K, Joseph I, Glaser SM, Ho SN, Reff ME, Hariharan K: Combination of 

two insulin-like growth factor-I receptor inhibitory antibodies targeting 

distinct epitopes leads to an enhanced antitumor response. Mol Cancer 

Ther 2010, 9:2593-2604. 

4. Fuentes G, Scaltriti M, Baselga J, Verma CS: Synergy between trastuzumab 

and pertuzumab for human epidermal growth factor 2 (Her2) from 

colocalization: an in silico based mechanism. Breast Cancer Res 2011 in 

press. 

5. Pedersen MW, Jacobsen HJ, Koefoed K, Hey A, Pyke C, Haurum JS, Kragh M: 

Sym004: Novel synergistic anti-epidermal growth factor receptor 

antibody mixture with superior anticancer efficacy. Cancer Res 2010, 

15:588-597. 

6. Frandsen TP, Naested H, Rasmussen SK, Hauptig P, Wiberg FC, 

Rasmussen LK, Jensen AM, Persson P, Wikén M, Engström A, Jiang Y, 

Thorpe SJ, Förberg C, Tolstrup AB: Consistent manufacturing and quality 

control of a highly complex recombinant polyclonal product for human 

therapeutic use. Biotech and Bioeng 2011 in press. 

7. Nielsen LS, Baer A, Müller C, Gregersen K, Mønster NT, Rasmussen SK, 

Weilguny D, Tolstrup AB: Single-batch production of recombinant human 

polyclonal antibodies. Mol Biotechnol 2010, 45:257-266. 

O3 

Cell-based medicinal products and the development of GMP-compliant 

processes and manufacturing 

Luca Romagnoli * , Ilaria Giuntini, Marta Galgano, Chiara Crosta, 

Luigi Cavenaghi, Maria Luisa Nolli 

Areta International s.r.l., Gerenzano (VA), 21040, Italy 

E-mail: lromagnoli@aretaint.com 


Page 4 of 181 

When performing the GMP process development and scale up of cellular 

therapies, a critical review of the manufacturing process and all the 

materials and reagents involved in the production steps is the mandatory 

starting point to avoid potential issues related to the quality and safety of 

the product. The choice of the raw materials, plastics and all the additional 

equipmentthatcomesintodirectcontactwiththeproductmustbe 

performed always keeping in mind that cells, as drug products, cannot be 

terminally sterilized. The quality of the materials and reagents utilized is 

therefore directly related to the quality and degree of purity of the final 

product. Information about the available certification must be gathered for 

every component and, for critical materials, audits must be performed to 

the manufacturing sites to qualify the supplier. 

The protocols used for the cell expansion and processing (if necessary) 

must be designed trying to reduce at a minimum the dependence on 

growth factors and medium supplements. Each additional component that 

is added to the culture medium must be justified and its absence from the 

final product must be verified with validated methods. Residues that are 

not removed during the production steps must be accurately measured 

and limits must be set after performing a risk evaluation analysis, to ensure 

that these process-related impurities have no adverse effects on the 

patients. 

Supplements such as Fetal Bovine Serum (FBS) are permitted for the 

manufacturing of cellular therapies [1], as long as the serum is sourced 

from a TSE-free area. Anyway, the use of a medium containing FBS should 

be limited to the cases for which a valid alternative could not be found. 

However, continuous research and development is strongly advised in 

order to keep up to date with the latest advances in the field of medium 

formulations, in order to be ready to switch to an animal-free medium as 

soon as it is feasible. The reduction of growth factors and supplements is 

also effective in controlling the manufacturing costs of a cell therapy 

product. An evaluation of the economical aspects and market sustainability 

should be performed at an early stage to increase the chances for an 

industrial development of the cellular product. 

The manipulation steps performed during the manufacturing stage should 

be kept to a minimum, in order to reduce human intervention and 

decrease the risk of contamination. Media fill simulations must be 

performed in purposely stressed conditions to ensure that the process and 

the facility are able to support the production of a sterile product [2]. 

When manufacturing patient-specific therapies, extensive efforts should be 

directed towards the reduction in the variability of the starting material, that 

is usually a tissue sampled from the patient during hospitalization. Working 

with well-defined starting material allows for the set-up of a more robust 

process with comparable characteristics between batches dedicated to 

different patients. The specifications of the final product for parameters such 

as cell number, purity and potency must be wide enough to tolerate the 

normal biological variability of living organisms, but sufficiently narrowed 

down to generate comparable batches of drug. This uniformity is mandatory 

for the set up of clinical trials aiming at gathering a reliable analysis of the 

safety, tolerability and efficacy data obtained from treated patients, in order 

to speed up the clinical development of innovative medicinal products such 

as cellular therapies. 

References 

1. Note for guidance on the use of bovine serum in the manufacture of 

human biological medicinal products. , CPMP/BWP/1793/02. 

2. Annex 1 - Manufacture of Sterile medicinal Products. EU Guidelines to 

Good Manufacturing Practice EudraLex4. 

O4 

Biosynthetic pathway deflection – a new cell line engineering approach 

Hans Henning von Horsten, Thomas Rose, Volker Sandig * 

ProBioGen AG, Berlin, Germany 


With increasing information on genome, transcriptome and metabolome 

of commonly used production cell lines, engineering becomes an 

increasingly popular approach to achieve desired product attributes, 

growth behavior and nutrient consumption. Tools range from feeding 

intermediate metabolites, overexpression or deregulation of key enzymes 

of a pathway to knock-out and RNA silencing. While conceptionally 

simple, the latter approaches are either labor intensive or costly to apply 

at large scale.



Figure 1(abstract O4) Overview of GDP-L-Fucose Biosynthesis showing the point of substrate deflection within the fucose de-novo synthesis pathway. 

Fucose targeted glycoengineering: Aiming at glycan modulation we 

added another principle to this toolbox: enzymatic deflection of a 

biochemical pathway. Fucose is synthesized inside the cell from GDPmannose 

via short lived intermediates before it is transported to the 

Golgi apparatus for attachment to the nascent glycan (Figure 1). 

A bacterial enzyme is used to redirect synthesis towards a heterologous 

activated hexose that cannot be utilized by the cell resulting in depletion 

of the natural pathway (deflecting enzyme, Figure 1). To our surprise, 

even lowest level expression of the enzyme completely abolishes fucose 

synthesis in stably modified cells. 

The approach allows producing antibodies that are devoid of core fucose at 

Fc glycans of the CH2 domain [1]. This modification provides higher 

flexibility to the Fc-region of IgG1 antibodies and enhances their binding to 

the FcgRIIIa receptor of NK cells - the dominating effector cells in antibody 

dependent cytotoxicity (ADCC). Consequently, the potency of antibodies 

directed against tumor or infected cells is substantially increased. 

In contrast to other strategies the approach is easily applied to the starter 

cell line of choice and, moreover, allows modification of fully developed 

producer cell lines within weeks. 

Simultaneous regulation of multiple cellular pathways: Another concept 

for clone engineering is based on simultaneous modulation of multiple 

cellular processes. We found that the Rho GTPase cdc42 is a highly suitable 

effector molecule for this purpose. This pleiotropic modulator dramatically 

boosts antibody titers when overexpressed in the cytosol of pharmaceutical 

producer clones (Table 1). 

Table 1(abstract O4) CDC42-mediated relative mAb-titer 

increase over native clone titers. The clones represent 

five different products 

Titers of Naïve 

mAb producing 

clones [g/l] 

Titers of cdc42engineered 

mAb 

producing clones [g/l] 

Relative Fold 

Increase per 

modified clone 

0,8 1,65 2,06 

0,9 2,2 2,4 

2,3 3,0 1,3 

2,6 4,5 1,73 

0,8 1,65 2,06 

Page 5 of 181 

Reference 

1. von Horsten HH, Ogorek C, Blanchard V, Demmler C, Giese C, Winkler K, 

Kaup M, Berger M, Jordan I, Sandig V: Production of non-fucosylated 

antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4hexulose 

reductase. Glycobiology 2010, 20(12):1607-18. 

O5 

Fluorescence-based tools to improve biopharmaceutical process 

development 

Tiago M Duarte 1 , Manuel JT Carrondo 1,2 , Paula M Alves 1,2 , Ana P Teixeira 1,2* 

1 Instituto de Biologia Experimental e Tecnológica (IBET), Apartado 12, 2781-901 

Oeiras, Portugal; 2 Instituto de Tecnologia Química e Biológica – Universidade 

Nova de Lisboa (ITQB-UNL), Apartado 127, 2781-901 Oeiras, Portugal 

E-mail: anat@itqb.unl.pt 


Background: Process optimization and control are essential to match 

biopharmaceutical market demands. Early-steps in the development of new 

processes require screening hundreds of transfected clones in order to 

select those with the best expression characteristics, and identifying optimal 

environmental conditions for these clones to grow and express the protein 

of interest. While some culture systems include in situ analysis of cell 

density, most often by optical density or capacitance measurements, the 

majority of them still rely on off-line, laborious and time-consuming 

protocols for recombinant protein analysis. Spectroscopic methods have 

been proposed in literature to support bioprocesses development, because 

they are non-invasive and provide data on multiple components present in 

the culture bulks, thereby increasing information on process performance 

[1]. Here, we developed a fluorescence-based method for real-time 

monitoring of viable cells and antibody titers in bioreactor cultures, and 

extended this strategy for high throughput analysis of cellular productivity 

in 96-well plates. 

Materials and methods: For high-throughput cellular productivity analysis, 

three CHO-K1 cell clones with different IgG 4 monoclonal antibody specific 

productivities were grown in 125mL shake flasks. Cultures were sampled 

twice a day to assess cellular growth; cell supernatants were stored at -20°C 

for further antibody quantification and fluorescence analysis. The 

supernatant samples were distributed into black 96-well plates and the 

fluorescence maps were collected in a spectrofluorometer (Horiba Jobin 

Yvon) connected to a microwell plate reader via optical fibers, placed above



the microtiter plate [2]. For real-time monitoring of bioreactor cultures, the 

same three clones were grown in 650mL working volume bioreactors. 

Fluorescence analysis was performed in situ, with the optical fibres placed 

inside a submerged stainless steel probe, incorporating a quartz lens at the 

bottom. 

Both excitation and emission slit widths were set equal to 5 nm and 

integration time was set at 0.5 sec. Fluorescence excitation-emission matrix, 

also called fluorescence map, was recorded in the excitation range of 285 - 

485 nm, with a step of 20 nm, and in the emission range of 305 - 565 nm, 

with steps of 10 nm, giving a total of 176 excitation/emission wavelength 

pairs (lex/lem). The acquisition time of each map is approximately 4.5 min. 

Further details are described in the literature [2,3]. 

Results: The applicability of two dimensional (2D) fluorometry was studied 

using three CHO cell clones expressing an IgG 4 to monitor viable cell 

density and antibody titer in bioreactor cultures and also in 96-well plates, 

targeting high throughput screening of cellular productivity. For the 

bioreactor application, the fluorescence analysis of the culture bulk was 

performed in situ, whereas for the 96-well plate application the cultures 

were performed in shake flasks and only the cell supernatant (without 

cells) was analyzed in terms of fluorescence. The three cell clones grow at 

similar rates and reach similar maximum cell densities but produce 

significantly different antibody quantities. Comparing bioreactor to shake 

flask cultures, all cell clones reached higher cell densities and lower 

antibody titers in the former system (Figure 1A-D). 

As a result of cell growth and metabolism, significant changes were 

observed in all fluorescent regions. The global relative changes can be seen 

in Figure 1E-F, where it is plotted the ratios between fluorescence maps 

collected at late culture stages and maps collected after inoculation, in both 

Page 6 of 181 

culture systems under study. The larger changes occurred in the region of 

the cellular cofactors NAD(H)P (maximum at l ex/l em: 365/455nm), which 

increased significantly over culture time. In contrast, the fluorescence in the 

regions of tryptophan (maximum at lex/lem: 285/365nm) and flavins 

(maximum at lex/lem: 465/520) decreased along culture. 

As the excitation light or the light emitted by fluorophors can be absorbed 

or dispersed by cells or other medium components, the signal that reaches 

the detector does not correlate linearly with the fluorophor concentration. 

Related to this, no linear correlations could be obtained between individual 

l ex/l em pairs and our target bioprocess variables. Therefore, we adopted a 

multivariate statistical method, partial least squares, to construct the 

regression models linking the variation found in the spectra with the off-line 

measurements of the target variables. The dataset from both culture 

systems was split in two parts: one for calibration and the other for model 

validation. Both variables were predicted with good accuracies in the two 

configurations under study (Figure 1A-D). Importantly, the antibody profiles 

in the validation data sets could be described with an average error below 

7%. This is a very good result, particularly because the validation data sets 

include concentrations above the ranges used to calibrate the models, 

revealing the extrapolation potential of the developed models. 

Conclusions: Both cell density and secreted antibody could be predicted 

with good accuracies in culture supernatant samples, demonstrating the 

potential of the method to effectively analyze cellular productivity in 96-well 

plate format. This methodology allows to by-pass the time-consuming 

ELISA assays and thus contributing to shorten early-phases of process 

development. The same approach was successfully implemented for in-situ, 

real-time monitoring of viable cells and antibody titer in bioreactor cultures, 

allowing continuous evaluation of bioprocess performance. 

Figure 1(abstract O5) Correlations between predicted and measured cell density (A, B) or antibody titer (C, D), in 96-well plates or bioreactor cultures, as 

indicated. Open circles correspond to calibration data points and full circles correspond to validation data points. The optimum number of latent variables 

was selected such that the root mean squared error observed in the validation data set was minimum. For cell density, the best model structures are 

composed by 6 or 7 latent variables (LVs), corresponding to average errors of 10% or 9%, in the microtiter plates or bioreactor cultures, respectively. 

Larger models of 9 LVs allowed the best description for antibody titer, with average errors below 7%. Contour plots show the relative fluorescence 

changes in the map during a culture, analyzed in (E) 96-well plates and (F) in bioreactors. Colors reflect the magnitude of the ratio between maps from 

the end of a CHO cell culture and the beginning of the same culture.



Acknowledgments 

The authors gratefully acknowledge the financial support from Fundação 

para a Ciência e a Tecnologia, Portugal (PTDC/EBB-EBI/102750/2008) and 

MIT-Portugal Program. 

References 

1. Teixeira AP, Oliveira R, Alves PM, Carrondo MJT: Advances in on-line 

monitoring and control of mammalian cell cultures: Supporting the PAT 

initiative. Biotechnol Adv 2009, 27(6):726-732. 

2. Teixeira AP, Duarte TM, Oliveira R, Carrondo MJT, Alves PM: Highthroughput 

analysis of animal cell cultures using two-dimensional 

fluorometry. J. Biotechnol 2011, 151:255-260. 

3. Teixeira AP, Duarte TM, Carrondo MJT, Alves PM: Synchronous 

fluorescence spectroscopy as a novel tool to enable PAT applications in 

bioprocesses. Biotechnol & Bioeng 2011, 108(8):1852-1861. 

O6 

Towards rational engineering of cells: Recombinant gene expression in 

defined chromosomal loci 

Kristina Nehlsen 1 , Leonor da Gama-Norton 1,2 , Roland Schucht 1,3 , 

Hansjörg Hauser 1 , Dagmar Wirth 1* 

1 Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany; 

2 Instituto de Tecnologia Química e Biológica-Universidade Nova de Lisboa/ 

Instituto de Biológica Experimental e Tecnológica (ITQB-UNL/IBET), P-2781- 

901 Oeiras, Portugal; 3 InSCREENeX GmbH, 38124 Braunschweig, Germany 

E-mail: dagmar.wirth@helmholtz-hzi.de 


The strength of recombinant gene expression is a key property of cell lines 

for biopharmaceutical protein production. In most stable cell lines the 

expression vector is stably introduced into the host chromosomal DNA. 

Apart from the copy number and the used expression control elements the 

performance of recombinant expression vectors is modulated by genetic 

and epigenetic features provided by flanking host elements. Since targeted 

integration is very difficult cell clones with high expression of a recombinant 

Page 7 of 181 

vector are created by random integration and large scale screening for gene 

expression. This allows the isolation of those rare recombinant cells in which 

gene expression is optimal. This is usually due to locus-specific influences of 

the chromosomal surroundings. 

We have developed an efficient methodology for targeting expression 

cassettes to specific chromosomal sites [1,2]]. The method (Flp recombinase 

mediated cassette exchange - RMCE) allows the repeated use of defined loci 

by targeting constructs for expression of proteins and viruses, thereby 

allowing to exploit the positive features of a given integration site [3,4]]. 

Thereby, a systematic evaluation of the performance of a set of expression 

vectors in various chromosomal sites becomes feasible. 

In this study we screened for high performance integration sites in HEK293 

and CHO-K1 cells supporting expression cassettes driven by a potent 

promoter. As a read out, production of antibodies and recombinant retroviral 

vectors were used. Thereby, we could show that high level expression of a 

given promoter is restricted to defined integration sites, while other sites 

show only moderate expression (data not shown). An important new finding 

was that a given chromosomal site is not flexible with respect to the 

integrated cassette but requires the integration of specific promoters. As 

illustrated in figure 1, an integration site, identified for supporting high level 

expression of an SV40 promoter driven cassette fails to adequately support 

expression of MPSV and adenoviral major late promoter (AdmlP). Vice versa, 

another site, initially screened for supporting MSCV promoter expression, 

could restore expression of the highly homologous MPSV promoter while the 

SV40 promoter and the AdmlP promoter give only moderate expression. 

Finally, we tested the impact of the orientation of the cassette in a specific 

chromosomal site. We found that some integration sites are flexible with 

respect to the orientation of the expression cassettes while others support 

expression only in one direction (data not shown). 

While classical enhancer elements are known to activate promoters largely 

independent from the relative position this finding suggests that other cis 

acting elements affect the incoming cassettes in an orientation dependent 

manner. Together, this shows that not the nature of integration site and the 

design of the vector as such define the performance of a producer cell 

clone. Rather, the interplay between these components defines the level 

Figure 1(abstract O6) Mode of tagging defines the optimal targeting cassette. Two highly potent chromosomal integration sites were screened 

upon random integration of an expression cassette driven by the SV40 promoter and the MSCV promoter, respectively. By means of Flp recombinase 

mediated cassette exchange, the screening cassette was exchanged for various expression cassettes, thereby integrating expression cassettes driven by 

the SV40 promoter, the adenoviral major late promoter (AdmLP) or the MPSV promoter into the same chromosomal site. The expression level upon 

targeting the various cassettes was determined and related to the expression level of the tagged cell.



and stability of expression. Since these interactions cannot be predicted, the 

performance of a vector in a given site has to be evaluated empirically. 

In order to exploit favourable sets of chromosomal sites and vectors we made 

use of bacterial artificial chromosome (BAC) vectors. By recombineering, 

expression cassettes were integrated into pre-selected chromosomal sites as 

encoded by BAC vectors. These vectors were randomly integrated into cells 

by standard transfection protocols. Clones were isolated and evaluated for 

expression. As expected, highly reproducible expression characteristics were 

found in individual clones (data not shown). In conclusion, the definition of 

favorable combinations of specific integration sites and vector design allow 

the rational exploitation of given chromosomal sites. For this purpose, 

technologies for site specific genetic manipulation of mammalian cells are 

essential. This concerns both targeted integration of expression cassettes into 

defined loci (such as RMCE or site specific nuclease induced homologous 

recombination) or by transduction of large chromosomal domains (as 

provided by BAC vectors). These technologies pave the way for predictable 

and high expression of biotechnologically relevant products such as 

antibodies and recombinant viral vectors. 

References 

1. Wirth D, Gama-Norton L, Riemer P, Sandhu U, Schucht R, Hauser H: Road 

to precision: recombinase-based targeting technologies for genome 

engineering. Current opinion in biotechnology 2007, 18(5):411-419. 

2. Schucht R, Coroadinha AS, Zanta-Boussif MA, Verhoeyen E, Carrondo MJ, 

Hauser H, Wirth D: A new generation of retroviral producer cells: 

predictable and stable virus production by Flp-mediated site-specific 

integration of retroviral vectors. Mol Ther 2006, 14(2):285-292. 

3. Nehlsen K, Schucht R, da Gama-Norton L, Kromer W, Baer A, Cayli A, 

Hauser H, Wirth D: Recombinant protein expression by targeting preselected 

chromosomal loci. BMC Biotechnol 2009, 9:100. 

4. Gama-Norton L, Herrmann S, Schucht R, Coroadinha AS, Low R, Alves PM, 

Bartholomae CC, Schmidt M, Baum C, Schambach A, et al: Retroviral vector 

performance in defined chromosomal Loci of modular packaging cell 

lines. Hum Gene Ther 2010, 21(8):979-991. 

O7 

Human hair follicle equivalents in vitro for transplantation and 

chip-based substance testing 

R Horland 1* , G Lindner 1 , I Wagner 1 , B Atac 1 , S Hoffmann 1 , M Gruchow 1 , 

F Sonntag 2 , U Klotzbach 2 , R Lauster 1 , U Marx 1,3 

1 TU Berlin, Institute for Biotechnology, Faculty of Process Science and 

Engineering, 13355 Berlin, Germany; 2 Fraunhofer IWS Dresden, 01277 

Dresden, Germany; 3 TissUse GmbH, 15528 Spreenhagen, Germany 

E-mail: reyk.horland@tu-berlin.de 


Introduction: The ability to create an organoid, the smallest functional unit 

of an organ, in vitro across many human tissues and organs is the key to 

both efficient transplant generation and predictive preclinical testing 

regimes. The hair follicle is an organoid that has been much studied based 

on its ability to grow quickly and to regenerate after trauma. Replacing hair 

Page 8 of 181 

lost due to pattern baldness or more severe alopecia, including that induced 

by chemotherapy, remains a significant unmet medical need. By carefully 

analyzing and recapitulating the growth and differentiation mechanisms of 

hair follicle formation, we recreated human hair follicles in tissue culture that 

were capable of producing a hair shaft and revealed a striking similarity to 

their in vivo counterparts. Extensive molecular and electron microscopy 

analysis were used to track the assembly of follicular keratinocytes, 

melanocytes and fibroblasts into the final hair shaft producing microfollicle 

architecture. The hair follicle generation process was optimized in terms of 

efficiency, reproducibility and compliance with regulatory requirements for 

later transplantation. In addition, we developed a procedure to integrate the 

de novo created human microfollicles into our existing chip-based human 

skin equivalents for substance testing. This would allow the evaluation of 

the role of hair follicles in dermal substance transport mechanisms for 

cosmetic products. Finally, we describe the challenges and opportunities we 

are facing for first-in-man transplantation trials. 

Material and methods: Mesenchymal, ectodermal and neuro-ectodermal 

originated cells from dissected human hair follicles were isolated and 

expanded into multiple passages. Dermal papilla fibroblasts have been kept 

under low adherent culture conditions resulting in the formation of dermal 

papilla-like aggregates. These spheroids underwent extra cellular matrix 

protein coating which mimics basal membrane compositions and thereby 

retained their inductive properties. In subsequent co-culture procedures 

keratinocyte and melanocyte attachment to the spheroids was forced 

allowing further follicular development. Ultra-structural examinations by 

scanning electron microscopy and immunofluorescence staining of cryosectioned 

microfollicles were performed to reveal spatiotemporal 

development and to characterize hair follicle like structures. Microfollicles 

were further integrated into full skin equivalents by dermal surface 

application or by integration through micromanipulation. 

Results: The formation of functional neopapillae (dermal papilla condensates) 

needs more than 48 hours. At day 7 of the condensation process 

theaggregationofcellsismuchmoredenseandtheformationofan 

extracellular matrix becomes visible. After the addition of keratinocytes and 

melanocytes, the self-organizing microorganoids follow a stringent pattern 

of follicular-like formation by generating polarized segments, sheath 

formations and the production of a hair shaft – like fiber (Figure 1). Specific 

extracellular matrix proteins (e.g. Collagen IV, Chondroitin-4-sulfate, Versican) 

and defined mesenchymal and epithelial markers (e.g. Vimentin, CK15) were 

expressed. Selected proliferating (Ki67-positive) and apoptotic (TUNELpositive) 

keratinocytes were detected in the outer microorganoid layers but 

also in the innermost dermal papilla-like aggregate. Microfollicles in skin 

equivalents self-organize in specific distance. Stainings show the integration 

of microfollicles below the epidermis. 

Conclusion: We show that the de novo formation of human microfollicles 

in vitro is accompanied by basic hair follicle like characteristics. The 

microfollicles can be used to study mesenchymal-epithelial-neuroectodermal 

interactions and for the in vitro testing of hair growth-modulating and 

pigmentary effects of substances. Hair follicles represent a long-term 

reservoir of topically applied substances. As the hair follicle is highly 

vascularized, it supports penetration of substances into the skin and further 

Figure 1(abstract O7) Microfollicle formation. SEM images taken from developing microfollicles. After adding keratinocytes and melanocytes to the 

culture medium a loosen attachment to the neopapilla is seen (A). Polarization of the early aggregate (B). Assembly, orientation and sheath formation (C); 

microfollicle with fiber production (D).



into the bloodstream. Since 2009 all cutaneous resorption experiments of 

the cosmetic industry in the EU have to be performed in vitro. In vitro 

testing of topically applied substances might therefore be performed with 

significantly enhanced validity by the incorporation of a microfollicle into a 

dynamic chip-based bioreactor containing a skin equivalent which mimics a 

physiological penetration route. With further improvements, the generated 

microfollicles embedded in an artificial skin model might also in future be 

used as improved implants for treating wound conditions. Eventually 

GMP-compliant manufactured human neopapillae might be used as 

implants for treating reduced hair conditions in humans. 

O8 

Chromosome identification and its application in Chinese hamster 

ovary cells 

Yihua Cao 1 , Shuichi Kimura 2 , Joon-young Park 1 , Miyuki Yamatani 1 , 

Kohsuke Honda 1 , Hisao Ohtake 1 , Takeshi Omasa 1,2* 

1 Department of Biotechnology, Graduate School of Engineering, Osaka 

University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; 2 Institute of 

Technology and Science, The University of Tokushima, 2-1 Minamijosanjimacho, 

Tokushima 770-8506, Japan 

E-mail: omasa@bio.tokushima-u.ac.jp 


Introduction: Chinese hamster ovary (CHO) cells [1] are today a very 

important host for the commercial-scale production of protein 

pharmaceuticals. Two sub clones of CHO cells, proline-requiring CHO K1 [2] 

and the dihydrofolate reductase (DHFR) gene-deficient CHO DG44 [3], 

are the most widely used for both scientific research and industrial 

applications [4][5,6]. Previously, we constructed a genomic bacterial artificial 

chromosome (BAC) library from mouse Dhfr-amplified CHO DR1000L-4N cell 

genome, which was provided 5-fold coverage of the CHO cell genome and 

analyzed the structure of amplicons of exogenous Dhfr amplification [7]. The 

BAC clones of this library could be landmarks for a physical map for CHO 

cell genome that are essential to the basic research and industrial 

application of CHO cell genome. In this study, we constructed the detail 

chromosomal physical map of CHO DG44 cell and investigated the 

chromosome rearrangements among CHO K1, DG44, and primary Chinese 

hamster cells. Moreover, to determine the effect of the palindrome structure 

on Dhfr amplification in CHO cells, we constructed three types of expression 

vectors with or without the junction region of the proposed amplicon and 

investigated the gene amplification and expression levels in transfected 

CHO DG44 cells. 

Materials and methods: Cell lines, culture conditions, construction of 

vectors and transfection: CHO DG44, CHO K1 and primary Chinese 

hamster cells were used in this study. The primary Chinese hamster cells 

were isolated from lung tissue of 4 weeks old female Chinese hamster 

[7,8]. The structure of the Dhfr amplicon derived from CHO DR1000L-4N 

cells constructed from CHO DG44 cells was determined previously [7]. The 

structure has a large palindrome structure containing a small inverted 

repeat in the junction region. This small inverted repeat originates from 

the integrated vector. On the basis of this junction region, three expression 

vectors were constructed [8]. The pSV2-dhfr/GFP vector (vector A) was 

constructed from original vector [9] and GFP. The pcD-core region (vector 

B) was constructed from the core region (junction region containing two 

Dhfr copies and one GFP). The pcD-repeat free core region vector (vector 

C) was constructed from the repeat free core region (part of the junction 

region containing one Dhfr and one GFP). Three constructed plasmids were 

transfected into the CHO DG44 cells. In the Dhfr-amplification step, the 

transfected cells were cultivated with MTX at various concentrations of 50, 

100, 250 and 500 nM. 

Fluorescence in situ hybridization using BAC clones as hybridization 

probes (BAC-FISH) and construction of CHO physical map: Chromosome 

spreads were prepared from exponential-phase cultures and BAC-FISH to 

chromosome spreads was carried out as described previously [7]. In brief, 

the BAC probes were detected using fluorescein isothiocyanate (FITC)labeled 

streptavidin or an anti-DIG-rhodamine antibody. Chromosomes were 

counterstained with 4,6,-diamidino-2-phenylindole (DAPI) and observed 

under an Axioskop 2 fluorescence microscope. Photographs were taken 

with a CCD camera. After image processing was performed, the ImageJ 

software (http://rsbweb.nih.gov/ij/) was used to analyze the chromosomal 

Page 9 of 181 

loci of the BAC clone probes and the positions of the centromere on the 

chromosomes, and expressed as FLpter values [10]. 

Results and discussion: Construction of BAC-based physical map for 

Chinese hamster ovary cells: A CHO genomic BAC library consisted of 

122,281 clones was constructed in a previous study [7]. Three hundred BAC 

clones were randomly selected from this library and mapped onto each 

chromosome of CHO DG44 cells by BAC-FISH. The FISH signal location of 

BAC clone probes on each chromosome were determined by digital image 

analysis and expressed as FLpter values. The 185 BAC clones were also 

mapped on the chromosomes of CHO K1 cell line and 94 clones on 

primary Chinese hamster chromosomes to investigate the chromosome 

rearrangements. The karyotypic comparison between CHO DG44 and 

primary Chinese hamster cells was diagrammatically summarized in Fig. 1. 

The 20 chromosomes in CHO DG44 cell line were aligned in order of 

decreasing size and assigned letters from A to T. The normal Chinese 

hamster chromosome number were estimated using BAC-FISH, endsequencing 

and previous comparative study between mouse and Chinese 

hamster [11]. The chromosomes A, B, C, F, L, N, R and S were derived from 

normal Chinese hamster chromosomes without large rearrangements, and 

then these chromosomes were conserved between CHO DG44 and K1 cells 

(data not shown). This result suggested that these chromosomes were very 

stable and essential in CHO cells and supposedly conserved in other CHO 

cell lines. 

FISH analysis of gene-amplified chromosomal region of transfected 

cells [8]: The chromosomal site of integration of a transgene affects its 

transcription rate. This phenomenon is called a positive effect and is often 

observed in transgenic organisms in which the transcription of an inserted 

transgene is affected by the proximity of the transgene to heterochromatin 

[12]. To understand the effect of structure of Dhfr amplicon on the 

chromosomal site of integration, we performed two-color BAC-FISH analysis 

for transgene. In our previous study, we determined that the amplified gene 

is integrated in one specific chromosome (chromosome O). The constructed 

vectors B and C contain the palindrome structure obtained from the BAC 

clone Cg0031N14 located on chromosome O. The summarized results of 

integrated chromosomal sites under various MTX concentrations are shown 

in Table 1. Interestingly, the transfected cells whose transgene is located at 

the same chromosomal position of the BAC clone Cg0160E04 were 

abundantly observed in the vector B and C integrations. It is likely that the 

chromosomal position of the BAC clone Cg0160E04 on chromosome O is a 

hot spot for Dhfr amplification in CHO cells. 

In summary, we constructed a BAC-based physical map for CHO DG44 cells 

and analyzed genome-wide rearrangements of chromosome among CHO 

cells. This BAC-based physical map will greatly facilitate the studies of CHO 

cell genome. The BAC clones comprising this physical map could also 

provide a genome-wide resource for analysis of chromosome rearrangements, 

chromosome structure, comparative genome hybridization, gene 

targeting, and functional genomics. 

Acknowledgements 

The present work is partially supported by grants from the NEDO of Japan, 

the Program for the Promotion of Fundamental Studies in Health Sciences 

of the NIBIO, and the Grant-in-Aid for Scientific Research of the JSPS. CHO 

BAC library was constructed under the collaboration with Professor 

S. Asakawa at the University of Tokyo and Professor N. Shimizu at Keio 

University. 

References 

1. Puck TT: Development of the Chinese hamster ovary (CHO) cell for use 

in somatic cell genetics. New York: John Wiley 1985. 

2. Kao FT, Puck TT: Genetics of somatic mammalian cells, VII. Induction and 

isolation of nutritional mutants in Chinese hamster cells. Proc Natl Acad 

Sci U S A 1968, 60(4):1275-1281. 

3. Urlaub G, Chasin LA: Isolation of Chinese hamster cell mutants deficient 

in dihydrofolate reductase activity. Proc Natl Acad Sci U S A 1980, 

77(7):4216-4220. 

4. Griffin TJ, Seth G, Xie HW, Bandhakavi S, Hu WS: Advancing mammalian 

cell culture engineering using genome-scale technologies. Trends 

Biotechnol 2007, 25(9):401-408. 

5. Wurm FM: Production of recombinant protein therapeutics in cultivated 

mammalian cells. Nat Biotechnol 2004, 22(11):1393-1398. 

6. Omasa T, Onitsuka M, Kim WD: Cell engineering and cultivation of Chinese 

hamster ovary (CHO) cells. Curr Pharm Biotechnol 2010, 11:233-240. 

7. Omasa T, Cao Y, Park JY, Takagi Y, Kimura S, Yano H, Honda K, Asakawa S, 

Shimizu N, Ohtake H: Bacterial artificial chromosome library for



Page 10 of 181 

Figure 1(abstract O8) The physical map of CHO DG44 cells and karyotypic comparison to primary Chinese hamster cells on the basis of BAC-FISH results. 

Homologous region of CHO DG44 to Chinese hamster were colored according to the estimated Chinese hamster chromosomes. 

Table 1(abstract O8) Ratios of amplified genes located at same position of BAC clone Cg0160E04 on chromosome O 

MTX concentration (nM) Vector A (%) Vector B (%) Vector C (%) 

50 0 (0/14) a 

0 (0/11) a 

0 (0/15) a 

100 0 (0/10) a 

33.3 (4/12) a 

18.2 (2/11) a 

250 0 (0/10) a 

61.5 (8/13) a 

57.1 (8/14) a 

500 0 (0/10) a 

a, b 

70.8 (17/24) 66.7 (6/9) a 

a b 

(Number of chromosomes detected at same position of BAC clone Cg0160E04 on chromosome O/total number of analyzed chromosomes). Further results 

were obtained from the previous study [8].



genome-wide analysis of Chinese hamster ovary cells. Biotechnol Bioeng 

2009, 104(5):986-994. 

8. Park JY, Yamatani M, Wadano S, Takagi Y, Honda K, Omasa T, Ohtake H: 

Effects of palindrome structure on Dhfr amplification in Chinese hamster 

ovary cells. Process Biochem 2010, 45(12):1845-1851. 

9. Yoshikawa T, Nakanishi F, Itami S, Kameoka D, Omasa T, Katakura Y, 

Kishimoto M, Suga K: Evaluation of stable and highly productive gene 

amplified CHO cell line based on the location of amplified genes. 

Cytotechnology 2000, 33(1-3):37-46. 

10. Lichter P, Tang CJ, Call K, Hermanson G, Evans GA, Housman D, 

Ward DC: High-resolution mapping of human chromosome 

11 by in situ hybridization with cosmid clones. Science 1990, 

247(4938):64-69. 

11. Yang F, O’Brien PC, Ferguson-Smith MA: Comparative chromosome map 

of the laboratory mouse and Chinese hamster defined by reciprocal 

chromosome painting. Chromosome Res 2000, 8(3):219-227. 

12. Wilson C, Bellen HJ, Gehring WJ: Position effects on eukaryotic gene 

expression. Annu Rev Cell Biol 1990, 6:679-714. 

O9 

Development of an automated, multiwell plate based screening system 

for suspension cell culture 

Sven Markert * , Klaus Joeris 

Roche Diagnostics GmbH, Pharma Biotech Production and Development, 

Penzberg, Germany 

E-mail: Sven.Markert@roche.com 


Introduction: The automation of cell culture processes becomes more 

important in the pharmaceutical industry due to compressed timelines 

and the need to develop more products more efficiently. This drive to 

develop new processes faster and more efficient requires a streamlined 

workflow. 

Resource intensive approaches like the use of shake flasks limit the 

accessible design space for the development of highly productive 

processes or the characterization of established processes. Process 


automation provides the appropriate tools to address the following key 

points: 

Increasing experimental throughput ⇨ “Design of Experiments” 

using full factorial designs 

Increasing process information ⇨ improve process understanding 

(“Quality by Design”) 

Automate repetitive manual work ⇨ gain efficiency, focus on 

high value tasks 

Improve reproducibility ⇨ ensure robust processes 

A robotic plate handler based system was selected to meet the demands 

of a flexible, fast and modular screening system as presented in figure 1. 

Results: Scale-up prediction: The comparability of results obtained with 

this new multiwell plate based culture system for suspension adapted cell 

lines plates and bioreactors had to be verified. It could be shown that 6well 

plates were predictive for a scale-up to a 1000 L stirred tank reactor. 

The parameter profiles of viable cell density, lactate and antibody 

concentration were comparable in multiwell plates and bioreactors (2 L, 10 

L and 1000 L). The plates can be used for process scale-up prediction. 

Media screening: An automated media blend screening was carried out in 

a second experiment highlighting another main area of application. Two 

seed trains of a CHO cell line, media blends of two growth media and two 

feeding strategies were screened in 120 wells on 6 well plates. A success 

rate of 100% enabled the evaluation of all wells in terms of cell growth and 

productivity. An increase in viable cell density and product titer of about 

20% in comparison to the reference process was achieved. 

2 L bioreactor runs were performed to confirm these optimized parameters. 

A total of 6 bioreactor runs using the identified best combination of media 

blends, seed train and feeding strategy verified the predicted results from 

the multiwell plates and showed an increase in productivity of about 15%. 

Conclusion and outlook: The developed robotic screening system is 

capable of performing a fully automated workflow consisting of incubation, 

sampling, feeding and near real-time analytics. The scalability from the mLscale 

up to 1000 L scale could be shown. Furthermore, a successful screening 

application was carried out and an increase in product concentration could 

be achieved. This potential for a process improvement was confirmed in a 

bioreactor study. The robotic screening system has therefore been proven to 

be a suitable screening tool for process optimization. 

Figure 1(abstract O9) Schematic illustration of the developed robotic screening system prototype. Only the core system is shown with a robotic plate 

handler as key device connecting shaken cultivation, processing and analytical components.



Ongoing work is focusing on extending the analytical portfolio by 

including additional analytical methods and instrumentation. A second 

focus area is in the field of process characterization and process 

robustness studies. 

Acknowledgements: The authors would like to thank Prof. Dr. Thomas 

Scheper (Institute for Technical Chemistry, Leibniz University, Hannover, 

Germany) for being the doctoral advisor as well as Katrin Möser and 

Pawel Linke for their work on the system during their diploma theses. 

O10 

Utilization of multifrequency permittivity measurements in addition to 

biomass monitoring 

Christoph Heinrich * , Tim Beckmann * , Heino Büntemeyer, Thomas Noll 

Institute of Cell Culture Technology, Bielefeld University, 33615 Bielefeld, 

Germany 

E-mail: che@zellkult.techfak.uni-bielefeld.de 


Introduction: In recent years measurement of permittivity signal has been 

increasingly used for online biomass monitoring of cell cultures. Breweries 

use it as an established method for fermenter inoculation and bioprocess 

control for instance [1]. In the case of animal cell cultures the correlation 

between permittivity and viable cell densities determined offline varies 

along cultivation time. Hence, several authors have used the permittivity 

signal as an indirect method for measuring oxygen and glutamine 

consumption as well as intracellular nucleotide phosphate concentrations 

[2,3]. The latest generation of biomass monitoring devices allows parallel 

measurement of permittivity at a range of frequencies leading to an 

improvement in the correlation between biomass and permittivity and 

providing a tool to further explore other aspects related to the 

physiological state of the cells. 

Material and methods: In this study 10 different cell lines, among them 

industrial cell lines as well as cell lines distributed by ATCC, were cultivated. 

Cell type specific chemically defined and animal component-free media 

(TeutoCell AG) were used. Batch, fed-batch and perfusion bioreactor 

cultivations were carried out in controlled benchtop vessels (Sartorius AG or 

Applikon Biotechnology). The i-Biomass 465 sensor (FOGALE nanotech) was 

used for online multifrequency permittivity monitoring. In addition the 

permittivity signal was used to implement a fully automated cell bleed to 

maintain a constant viable cell density in a perfusion process. Furthermore, a 

fed-batch feed strategy was introduced to keep the substrate concentration 

at a certain level. Cell density and viability were determined using a CEDEX 

system (Innovatis-Roche AG). Glucose and lactate were measured with an 

YSI 2700 Biochemistry Analyzer (YSI Life Sciences). Amino acids were 

quantified using an in-house developed HPLC method. 


Results: The FOGALE i-Biomass 465 sensor was used to monitor the viable 

cell density of different human, CHO and hybridoma cell cultures online. 

A good correlation of the permittivity signal and the offline measured viable 

cell density for the growth phase was verified (R > 0.99), but pH-shifts and 

increased cell aggregation had a negative impact on the correlation. The 

linear factor to calculate the viable cell density from the online permittivity 

signal varied between 4.5·10 5 cells/(pF/cm) and 12.0 cells/(pF/cm). A clear 

relation between cell type (CHO, human or hybridoma) and the linear factor 

could not be established from the available data. 

Subsequently, the online biomass monitoring system was used to carry out 

a 1 L spin-filter perfusion process with constant viable cell density at a 

predefined setpoint. The application of a permittivity closed-loop controlled 

cell bleed resulted in a steady concentration of 10 7 viable cells/mL during 

perfusion, at a dilution rate of 1.0 d -1 . As soon as this threshold was reached, 

the cell bleed was automatically started and controlled based on the online 

signal of the i-Biomass 465 sensor. 

In addition to the correlation with viable cell density, a linear relationship 

(R 2 > 0.96) between the online i-Biomass 465 signal and the concentrations 

of numerous components, e.g. glucose, lactate, asparagine, glutamine, 

tyrosine, threonine, methionine, lysine, phenylalanine, serine, leucine and 

isoleucine, was found during the exponential growth phase of CHO-K1 and 

CHO DP-12 cultivations. The results indicated that the number of correlating 

substrates depended on the used cell line (CHO, human or hybridoma) and 

the process strategy (constant pH or pH-shift). Since, the established 

substrate correlations were more robust against process variations, they 

were investigated as a basis for a closed-loop feeding strategy in fed-batch 

cultivations. Compared to a pre-defined feeding schedule or to intermitted 

feeding this would have the advantage of avoiding nutrient limitations and 

substrate accumulation that might occur due to unexpected high or low cell 

growth. Also, feeding would be independent of human surveillance. The 

successful application of a completely automated permittivity-controlled 

feeding strategy was proved in two fed-batch runs with CHO DP-12 (ATCC 

CRL-12445) cells, as shown in Figure 1. 

The feeding of both runs was controlled only through the permittivity signal 

in order to maintain the asparagine concentration at a certain level. 

Asparagine was chosen due to its central role in cell metabolism and to the 

fact that it is usually a limiting substrate in CHO DP-12 cultures. These proofof-concept 

runs demonstrated that permittivity-based automated feeding 

can be a valuable tool for the optimization of fed-batch process parameters, 

such as feeding start, flow rate and composition. 

Conclusions: For suspension cultures with single cells and high viability a 

linear correlation (R 2 ≥ 0.98) of the permittivity signal with the viable cell 

density measured offline was obtained, at least for the exponential growth 

phase. Based on this correlation, a closed-loop controlled cell bleed was 

implemented in a perfusion process in which the permittivity signal was 

used to keep the viable cell density at a constant level of 10 7 viable cells/mL. 

Figure 1(abstract O10) Total viable cell count, viable cell density and asparagine concentration of the two proof-of-concept CHO DP-12 fed-batch 

processes (initial working volume: 1 L; 37°C; 40% DO; pH 7.1).



Furthermore, a linear correlation with the i-Biomass 465 signal was observed 

for several substrates independent of the correlation between viable cell 

density and permittivity. Based on these results, a closed-loop controlled 

feeding was successfully established resulting in a fully automated fed-batch 

process. 

Acknowledgements: We would like to thank FOGALE nanotech for 

providing the i-Biomass 465 system and TeutoCell AG for supplying the 

cell culture media and feed solutions. 

References 

1. Boulton CA, Maryan PS, Loveridge D: The application of a novel biomass 

sensor to the control of yeast pitching rate. Proc 22nd Eur Brew Conv 

Zurich European Brewing Convention Oxford: Oxford University Press 1989, 

653-661. 

2. Noll T, Biselli M: Dielectric spectroscopy in the cultivation of suspended 

and immobilized hybridoma cells. J Biotechnol 1998, 63:187-198. 

3. Ansorge S, Esteban G, Schmid G: Multifrequency permittivity 

measurements enable on-line monitoring of changes in intracellular 

conductivity due to nutrient limitations during batch cultivations of 

CHO cells. Biotechnol Prog 2010, 26:272-283. 

O11 

ATF4 over-expression increased IgG 1 productivity in Chinese hamster 

ovary cells 

Ahmad M Haredy 1 , Akitoshi Nishizawa 1 , Kohsuke Honda 1 , Tomoshi Ohya 2 , 

Hisao Ohtake 1 , Takeshi Omasa 1,3* 

1 Department of Biotechnology, Graduate School of Engineering, Osaka 

University, Suita, Osaka, 565-0871, Japan; 2 Tanabe Mitsubishi Pharma, 

Hirakata, Osaka, Japan, 573-1153; 3 Institute of Technology and Science, The 

University of Tokushima, Tokushima, 770-8506, Japan 

E-mail: omasa@bio.tokushima-u.ac.jp 


Introduction: The endoplasmic reticulum (ER) is the major organelle of 

synthesis of protein and forms a membranous network throughout the cell. 

According to Shimizu and Hendershot [1], about one third of the total 

proteins produced are synthesized in the ER. The ER lumen possesses a 

unique environment for high quality control for protein folding and 

assembly. It contains high concentrations of molecular chaperones, folding 

enzymes, and ATP, which aid in proper maturation of proteins [2]. However, 

if the amount of proteins to be folded exceeds the capacity of the folding 

machineries, unfolded proteins will accumulate in the ER and Unfolded 

protein response (UPR) will start. UPR aims at regaining the homeostasis 

inside the ER by attenuating the protein translation, activating the folding 

machineries, degradation of the unfolded proteins, and finally apoptosis. 

Activating transcription factor 4 (ATF4) is a central factor in the UPR 

pathways which is involved in folding and processing of secretory proteins. 

Our previous research showed that ATF4 over-expression is efficient for 

increasing the productivity of antithrombin-III [3,4]. In this study, we 

investigated if this approach is product-specific or not. 

Materials and methods: Cell line and medium: Serum-free adapted 

Chinese hamster ovary DP-12-SF (CHO DP-12-SF) cell line (producing human 

anti-IL-8 IgG 1) and Dulbecco Modified MEM medium supplemented with 

10% dialyzed fetal bovine serum (FBS) and 200nM methotrexate were used 

in this study. 

Vector construction: The ATF4 expression plasmid was constructed using 

CHO ATF4cDNA into KpnI/XbaI site of the pcDNA3.1/Hygro(+) expression 

vector (Invitrogen, Carlsbad, CA, USA), designated as pcDNA3.1/Hygro 

(+)-ATF4. 

Transfection and selection: The pcDNA3.1/Hygro(+)-ATF4 vector was 

transfected into CHO DP-12-SF cell line using a TransIT-LT1 transfection 

reagent (Mirus bio Madison, WI USA). Single cell clones were obtained 

using the limiting dilution method. The transfected cell lines were selected 

using 200μg/ml hygromycin. 

Chromosomal integration of ATF4: Genomic DNA was isolated after 72 h 

of cultivation using DNeasy blood and tissue extraction kit (Qiagen, 

Chatsworth, CA, USA). The primers 5`-TAATACGACTCACTATAGGG-3` and 5`- 

TAGAAGGCACAGTCGAGG-3` were employed for the amplification of noncoding 

region of pcDNA3.1/Hygro(+)-ATF4 for detection of the integration 

of the designated plasmid into the CHO chromosome. PCR was performed 

using Ex Taq polymerase (Takara Bio, Otsu, Shiga JAPAN) with 100 ng of the 

genomic DNA. 


Confirmation of ATF4 expression: RNA was isolated after 72 h of 

cultivation using RNeasy mini kit (Qiagen). cDNA equivalent to 1μg of the 

previously prepared RNA was prepared using omniscript RT kit (Qiagen) 

and PCR was performed using the same primers for chromosomal 

detection. 

Cell and antibody concentrations: Cell concentration was determined 

using Coulter Vi-Cell Automated Cell Viability Analyzer (Beckman Coulter, 

Inc., Fullerton, CA, USA). Antibody concentration was determined by 

sandwich enzyme linked immunosorbent assay (ELISA) [5]. Kinetic 

parameters were calculated as described previously [3]. 

Real time PCR: The quantification of mRNA of heavy and light chains of IgG 

was performed by the SYBR Green real-time RT-PCR method (Applied 

Biosystems, Foster City, CA, USA) using the StepOnePlus Real-Time PCR 

System. 

Results and discussion: The gene encoding ATF4 was cloned from CHO- 

K1 inserted into the multiple cloning site of pcDNA3.1 vector with 

hygromycin cassette as a selection marker. The ATF4 expression vector was 

then transfected into CHO DP-12-SF cell line [5], producing humanized anti 

IL-8 Immunoglobulin-1 (IgG 1). Twenty six single cell clones were then 

established using the limiting dilution method. To examine if pcDNA3.1/ 

Hygro(+)-ATF4 was integrated into the CHO chromosome or not, PCR 

analysis were performed using the primers designed for non-coding region 

of the expression vector. Only 5 clones were confirmed with the insert at 

1.2 Kb; CHO DP-12-ATF4-3, -9, -10, -12, and-16. Reverse Transcriptase- 

Polymerase Chain Reaction analyses revealed that only 3 clones, CHO DP-12- 

ATF4-10, -12, and -16, showed positive ATF4 expression. After 144 hours of 

cultivation, only clones with confirmed ATF4 expression showed significant 

increase in specific IgG 1 production rate ranging from 1.8 to 2.5 times the 

parental CHO DP-12-SF cell. Clone DP-12-ATF4-16 that showed the highest 

increase in specific productivity with about no change in the specific growth 

rate was subjected to further analysis for quantification of mRNA of heavy 

and light chains to determine if over-expression affects the transcription of 

mRNA or not. The result was found in agreement with our previous research 

that over-expression of ATF4 did not significantly change the level of the 

product mRNA [4]. It suggested that ATF4 over-expression might improve 

the translation and the secretion without affecting the transcription. 

References 

1. Shimizu Y, Hendershot LM: Organization of the functions and components 

of the endoplasmic reticulum. Adv Exp Med Biol 2007, 594:37-46. 

2. Gomez E, Powell ML, Bevington A, Herbert TP: A decrease in cellular 

energy status stimulates PERK-dependent eIF2alpha phosphorylation 

and regulates protein synthesis in pancreatic beta-cells. Biochem J 2008, 

410:485-493. 

3. Ohya T, Hayashi T, Kiyama E, Nishii H, Miki H, Kobayashi K, Honda K, 

Omasa T, Ohtake H: Improved production of recombinant human 

antithrombin III in Chinese hamster ovary cells by ATF4 overexpression. 

Biotechnol Bioeng 2008, 100:317-324. 

4. Omasa T, Takami T, Ohya T, Kiyama E, Hayashi T, Nishii H, Miki H, 

Kobayashi K, Honda K, Ohtake H: Overexpression of GADD34 enhances 

production of recombinant human antithrombin III in Chinese hamster 

ovary cells. J Biosci Bioeng 2008, 106:568-573. 

5. Kim WD, Tokunaga M, Ozaki H, Ishibashi T, Honda K, Kajiura H, Fujiyama K, 

Asano R, Kumagai I, Omasa T, Ohtake H: Glycosylation pattern of 

humanized IgG-like bispecific antibody produced by recombinant CHO 

cells. Appl Microbiol Biotechnol 2010, 85:535-542. 

O12 

Characterization of novel pneumatic mixing for single-use bioreactor 

application 

Brian Lee 1* , David Fang 2 , Matthew Croughan 3 , Manuel Carrondo 4 , 

Sang-Hoon Paik 5 

1 PBS Biotech Inc., Camarillo, California, 93012, USA; 2 Systems QbD, Newbury 

Park, California, 91320, USA; 3 Keck Graduate Institute (KGI), Claremont, 

California, 91711, USA; 4 Instituto de Biologia Experimental e Tecnologica 

(IBET), Lisbon, 2780-157, Portugal; 5 Green Cross Corp., Yongin-Si, KyungGi-Do, 

449-070, South Korea 

E-mail: blee@pbsbiotech.com 


Background: The novel bioreactor system from PBS Biotech® is the first 

with a pneumatic mixing device powered solely by gas buoyancy,



Table 1(abstract O12) 95% mixing time from 2L to 

5,000L working volumes in PBS prototype units 

2L 10L 50L 250L 1,000L 5,000L 

Gas Flow Rate (VVM) 0.07 0.06 0.05 0.04 0.03 0.02 

Mixing Time (sec) 20 25 30 38 53 62 

eliminating the need for an external mechanical agitator. The patented 

design of the Air-Wheel mixing device promotes not only high 

tangential fluid flow around the wheel but also efficient radial and axial 

flows. The leverage effect of the Air-Wheel mixing device also allows for 

lower gassing requirement (v/v) with increasing working volume, 

making the power utilization from the gas buoyancy more efficient with 

scale. 

A series of physical tests were performed to demonstrate that PBS systems 

can promote uniform, homogenous mixing over a wide range of working 

volumes and can offer a low-shear environment for cell culture. A series of 

biological tests were then performed at various evaluation sites to confirm 

the physical test results. 

Results: Mixing time was calculated in PBS systems ranging from 2L to 

5,000L working volume by measuring the change in conductivity readings 

from bolus additions of concentrated salt solution. 95% mixing times were 

found to be between 20 sec and 62 sec over this range of working 

volumes in the PBS systems (Table 1), significantly shorter than reported 

values in conventional stirred tank systems. 

Shear stress and turbulent kinetic energy dissipation rate (ε, inm 2 /s 3 ) were 

calculated from computational fluid dynamics (CFD) modeling on Star 

CCM+ software. Lower average shear stress (τ avg, in Pa) on the impeller 

and lower average energy dissipation rate (ε, inm 2 /s 3 )intheimpeller 

region were found in the PBS system compared to stirred-tank bioreactors 

at typical impeller speeds at the respective working volumes. Comparably 

low level of shear stress (



Background: There is a steadily increasing demand for producing higher 

yields of biopharmaceuticals as recombinant proteins, and it is 

anticipated that this will further expand during the next decades. Among 

the most frequent proteins produced are growth factors, monoclonal 

antibodies, hormones and blood coagulation factors. 

The production of recombinant proteins can be performed in expression 

systems derived from bacteria, yeast, plants, insects or mammals. 

Prokaryotic cells divide rapidly, making it possible to produce high yields 

of the protein at low costs. They are, however, normally not able to 

perform post-translational modification of proteins, which, for those of 

mammalian origin, is essential to ensure stability, proper folding and 

assembly and thus biological activity. Mammalian cells, on the other 

hand, confer post-translational modification. Unfortunately their 

cultivation is cost expensive and time consuming, they grow slower, are 

more sensitive to contamination and produce lower protein yields than 

their prokaryotic counterparts. Despite this, 60-70% of all recombinant 

proteins used in therapeutics are produced in mammalian cells. Yield 

improvement in mammalian systems is currently an area of major 

industrial importance [1]. 

To achieve this, two main strategies have been used: (1) optimising 

the components of the vector containing the gene of interest, and 

(2) optimising cell growth and selection. Optimisation of the vector by 

genetic engineering has mainly focused on increasing the efficiency by 

which the gene is transcribed. The concept being that a high level of 

transcription would ultimately lead to a higher protein yield due to 

increased availability of mRNA for translation. Vector design, the 

chromosomal environment of the plasmid integrated in the host genome 

and plasmid copy number, are among the parameters that can contribute 

to transcription efficiency. An increased level of mRNA coding for any 

secreted protein of interest, however, will only be beneficial if the 

transcript is correctly transported to the endoplasmic reticulum and then 

effectively translated. This area has hitherto been largely neglected. 


Efficient mRNA processing: UniTargetingResearch AS is now developing 

and commercialising tools to optimise protein synthesis and secretion, by 

ensuring that the mRNA encoding the protein of interest is efficiently 

processed. This has proved to be heavily dependent on the presence of 

specific genetic targeting elements, namely a selected signal sequence 

(SS) in combination with appropriate 5' and 3' untranslated regions (5' 

and 3'UTRs). Our focus is currently on the SS, which is translated into the 

signal peptide (SP). 

Earlier we observed a competition between a selected SS and the 3'UTR 

in mediating mRNA targeting to distinct classes of polysomes [2]. We 

therefore investigated the effect of different SPs derived from 

mammalian secretory proteins on the synthesis/secretion of Gaussia 

princeps (a marine copepod) luciferase (Gluc) used as a reporter protein 

[3]. The results showed that the choice of SP had a major impact on 

synthesis/secretion of Gluc in CHO cells. Contrary to what was expected 

the SP of albumin was extremely inefficient (



dependent on the AA sequence and even a single mutation at a specific 

position has a strong effect on protein yield. In order to test whether or 

not the total hydrophobicity of the hydrophobic core region (h-region) in 

the SP was of importance, hydropathy scores according to Eisenberg 

et al. [4] were calculated. From Table 1 it can be seen that there appears 

to be no direct correlation between yield and degree of hydrophobicity 

of the h-region and thus the latter is not a reliable measure for the 

prediction of the efficiency of an individual SP. 

The library concept: We thus adopted an alternative approach where 

we exploit the plethora of biological data we have accumulated in a 

bioinformatics context. A comparison of the success of individual SPs and 

an analysis of their AA composition has allowed us make predictions with 

respect to which AAs in which positions are likely to have a decisive 

influence on protein synthesis/secretion. Based on this we have 

developed a tool, termed UTR ® Tailortech, that provides us with the 

opportunity of generating rational SP libraries randomised at chosen 

positions. In contrast to a traditional non-rational approach which results 

in libraries of astronomic proportions not being manageable, with 

UTR ® Tailortech the libraries are considerably reduced in size while 

simultaneously being enriched for good performers. This increases the 

probability of finding “the needle in the haystack”. The tool also 

comprises the concept of generating libraries that are re-usable by 

constructing so-called “pre-made” libraries. These high-quality libraries 

can be linked in a seamless manner to any protein-coding region 

contained in any expression vector as outlined in Fig. 1. When combined 

with high-throughput screening technology, a tailored SP for any specific 

protein (including difficult-to-express proteins) can readily be defined. 

Conclusion: From our studies it is evident that there is considerable room 

for improvement of production of a recombinant protein by genetically 

modifying the SP utilised in a vector. To our knowledge UTR ® Tailortech is 

the first attempt to harness this potential in a rational way. 

References 

1. De Jesus M, Wurm FM: Manufacturing recombinant proteins in kg-ton 

quantities using animal cells in bioreactors. Eur J Pharm Biopharm 2011, 

78:184-188. 

2. Partridge K, Johannessen AJ, Tauler A, Pryme IF, Hesketh JE: Competition 

between the signal sequence and a 3'UTR localisation signal during 

redirection of beta-globin mRNA to endoplasmic reticulum: implications 

for biotechnology. Cytotechnology 1999, 30:37-47. 

3. Knappskog S, Ravneberg H, Gjerdrum C, Tröβe C, Stern B, Pryme IF: The 

level of synthesis and secretion of Gaussia princeps luciferase in 

transfected CHO cells is heavily dependent on the choice of signal 

peptide. J Biotechnol 2007, 128:705-715. 


4. Eisenberg D, Weiss RM, Terwilliger TC, Wilcox W: Hydrophobic moments in 

protein structure. Faraday Symp Chem Soc 1982, 17:109-120. 

O14 

High-capacity assay to quantify the clonal heterogeneity in potency of 

mesenchymal stem cells 

Kim C O’Connor 1* , Katie C Russell 1 , Donald G Phinney 2 , Michelle R Lacey 1 , 

Bonnie L Barrilleaux 1 , Kristin E Meyertholen 1 

1 Department of Chemical and Biomolecular Engineering, Tulane University, 

New Orleans, LA 70118, USA; 2 Scripps Research Institute, Jupiter, FL, 33458, 

USA 

E-mail: koc@tulane.edu 


Background: The regenerative capacity of mesenchymal stem cells (MSCs) 

is contingent on their content of multipotent progenitors [1]. Despite its 

importance to the efficacy of MSC therapies, the clonal heterogeneity of 

MSCs remains poorly defined. To address this deficiency, the current study 

presents a novel high-capacity assay to quantify the clonal heterogeneity 

in MSC potency and demonstrates its utility to resolve regenerative 

properties as a function of potency. Human bone marrow was the source 

of MSCs in this study. The versatility and accessibility of marrow-derived 

MSCs make them a standard for many therapeutic applications. 

Materials and methods: Primary MSCs were harvested from the iliac crest 

bone marrow of healthy adult volunteers and cultured as previously 

described [2]. The in vitro assay developed for this study utilizes a 96-well 

format to (1) clone fluorescent MSCs stained with CellTracker Green by 

limiting dilution, (2) generate matched clonal colonies, (3) differentiate 3 

matched colonies per clone to quantify trilineage potential to exhibit adipo-, 

chondro- and osteogenesis as a measure of potency, and (4) cryopreserve 

the 4th matched colony of each clone in situ in an undifferentiated state for 

future use. Clones of known potency were evaluated for their colonyforming 

efficiency as a measure of proliferation potential [3]. Expression of 

the heterotypic cell adhesion molecule CD146 on the surface of MSC clones 

was measured with flow cytometry. 

Results: All eight categories of trilineage potential were detected in 

human marrow MSCs. Multipotent MSCs had a higher proliferation 

potential than lineage-committed MSCs. Tripotent clones formed colonies 

with a median efficiency of 50%, as compared with 14% and 1% for biand 

unipotent clones, respectively (p < 0.01). Likewise, colonies that 

formed from tripotent clones had the largest median diameter. CD146 may 

be a biomarker of MSC potency. Histograms of fluorescence intensity from 

Figure 1(abstract O13) Seamless insertion of a randomised SS (SS*) from a pre-made library into a recipient vector. Grey boxes indicate special 

restriction sites used in the cloning process.



pooled tripotent clones labeled with anti-CD146 antibody shifted to higher 

CD146 expression relative to the parent MSC preparation from which the 

clones were generated; whereas, the histograms for parent MSCs and their 

unipotent progeny were similar. In particular, the mean fluorescence 

intensity of tripotent clones was nearly twice the value for the parent and 

unipotent MSCs (p < 0.05). 

Conclusions: The research presented here addresses a basic deficiency in 

stem cell technology by developing a quantitative and high-capacity assay 

to characterize the clonal heterogeneity of MSC potency. The data suggest a 

complex hierarchy of lineage commitment in which proliferation potential 

and CD146 expression diminish with loss of potency. The capacity of 

multipotent MSCs for ex vivo expansion and their differential expression of a 

potential potency marker will facilitate rapid production of efficacious MSC 

therapies with consistent progenitor content. The assay has numerous basic 

research and clinical applications given the importance of heterogeneity to 

the therapeutic potential of MSCs. 

Acknowledgements: We thank Alan Tucker for his assistance with flow 

cytometry, Dina Gaupp for preparing samples for histology, and Prof. 

Darwin Prockop for his helpful conversations and suggestions about this 

project. This research was sponsored by grants to Prof. O’Connor from the 

National Institutes of Health (R03EB007281) and National Science 

Foundation (BES0514242). 

References 

1. Barrilleaux BL, Phinney DG, Prockop DJ, O’Connor KC: Ex vivo engineering 

of living tissue with adult stem cells. Tissue Eng 2006, 12:3007-3019. 

Figure 1(abstract P1) NovaSeptum 100mL bag and NovaSeptum case configurations. 

2. Barrilleaux BL, Phinney DG, Fischer-Valuck BW, Russell KC, Wang G, 

Prockop DJ, O’Connor KC: Small-molecule antagonist of macrophage 

migration inhibitory factor enhances migratory response of 

mesenchymal stem cells to bronchial epithelial cells. Tissue Eng Part A 

2009, 15:2335-2346. 

3. Russell KC, Lacey MR, Gilliam JK, Tucker HA, Phinney DG, O’Connor KC: 

Clonal analysis of proliferation potential of human bone marrow 

mesenchymal stem cells as a function of potency. Biotechnol Bioeng 2011, 

108:2716-2726, doi:10.1002/bit.23193. 

POSTER PRESENTATIONS 


P1 

Improvement of cell freezing technologies: towards a fully closed 

process 

Aurore Polès-Lahille * , Brigitte Lafuente, Virginie Perrier, David Balbuena, 

Didier Peyret 

Merck Serono Biodevelopment, Martillac, France, 33650 

E-mail: Aurore.Lahille@merckgroup.com 

BMC Proceedings 2011, 5(Suppl 8):P1 

Cell culture from vials usually includes an open phase performed under 

laminar flow hoods. Even if disposable and small closed systems are 

available, at least the first cell culture step after vial thawing, which is a



centrifugation, is performed in disposable but open containers. This step is 

critical as the risk of contamination is high and it also has a direct impact 

on process timelines. 

In order to reduce the risk of contamination during thawing, Merck Serono 

Biodevelopment studied the possibility of freezing cell banks in bags and 

to directly thawing them directly in disposable and closed containers. 

The first step of this study was to evaluate if the DMSO present in 

freezing media had to be removed just after vial thawing or not. In 

general, this cryoprotective organic solvent is removed by centrifugation 

to avoid any toxic effect on cells. 

So vials from two different mammalian cell lines were thawed, each with 

and without a centrifugation step to remove DMSO, or not. Afterwards the 

cell amplification was performed in the same way for the two different 

thawing procedures. The population doubling level and the viability were 

monitored for more than 15 days and there was no significant impact 

neither on cell growth nor on viability whether DMSO was removed or not 

just after thawing, or not. The important parameter to take into account was 

the percentage of DMSO after dilution which had to be less than 0,5%. 

In order to reduce the risk of preparing cell banks, Merck Serono 

Biodevelopment looked at different disposable closed systems which allow 

cell banks to be prepared without laminar flow. It is possible to expand cells 

in closed containers such as spinners, shake flasks or rollers with deep tubes 

in addition to bags or disposable bioreactors. In order to concentrate cells or 

to change the medium from cell amplification to a cell freezing medium a 

centrifugation step may be necessary. Single use 500mL centrifuge tubes 

with deep tube and air vent are also available. In order to freeze cells, Merck 

Serono Biodevelopment evaluated several bags from several suppliers and 

chose the NovaSeptum® 100mL bag and the NovaSeptum® Case. The 

combination of these 2 elements ensured the integrity of the bag during 

freezing at -80°C (Figure 1-2). 

Thus 50mL of two different concentrated mammalian cell suspensions 

were frozen at -80°C and thawed in disposable and closed containers. 

The cells were not centrifuged just after thawing but expanded in order 

to seed a production bioreactor. The cells were also maintained in order 

to see if there was a long term impact of non-DMSO removal on viability 

and cell growth. The population doubling level, the cell viability obtained 

in disposable closed cell culture containers in addition to viable cell 

density, metabolism, molecule quantity and quality obtained in 

bioreactors were monitored. These parameters were compared to the 

same process performed with cells coming from vials. 

There was no process performance difference obtained between cells frozen 

in bags and cells frozen in vials. Cell freezing in bags without DMSO removal 

allows a fully closed cell culture and production process to be performed. It 

also allows process timelines to be reduced by least 2 weeks. 

The study will continue with a focus on bags which are compatible with 

nitrogen containers. The quality aspect will also be studied in order to be 

able to prepare a full or part of manufacturing of cell banks in bags. 

Figure 2(abstract P1) NovaSeptum 100mL bag and NovaSeptum case configurations. 


P2 

Disposable bioreactors: from process development to production 

Aurore Polès-Lahille 1* , Celine Richard 1 , Sandrine Fisch 1 , David Pedelaborde 1 , 

Sandy Gerby 2 , Nora Kadi 1 , Virginie Perrier 1 , Robert Trieau 1 , David Balbuena 1 , 

Laure Valognes 1 , Didier Peyret 1 

1 Merck Serono Biodevelopment, Martillac, France, 33650; 2 Ecole Nationale 

Supérieure de Technologie des Biomolécules de Bordeaux, Bordeaux, France, 

33000 

E-mail: Aurore.Lahille@merckgroup.com 


Single-use bioreactors are commonly used for seeding stainless steel 

bioreactors or for producing material. The profitability of these 

equipments has been well demonstrated on more that decade. But few 

data on its scalability have been published. 

In 2010-2011, Merck Serono Biodevelopment performed a study in order to 

evaluate the performance of several disposable bioreactors. As different 

technologies and scales were available, this study compared the 

performance of several types of mixing in single-use bioreactors for 

process development and pilot scale production. In addition, the 

evaluation was performed for both seeding applications and for clinical 

material production. 

Thus the feature of this study is the comparison of 3 to 50L disposable 

bioreactors with internal mixing system or external agitation (Table 1). In 

order to compare their performance with traditional bioreactors, gas flow 

rate applied in disposable bioreactors was scaled-down from seeding and 

production stainless steel bioreactors. Furthermore the ratio working volume 

on total volume and the power input applied to the 3L disposable 

bioreactor were similar to those in glass bioreactors. 

In order to compare the 5 different types of disposable bioreactors, a fedbatch 

process producing a highly glycosylated molecule was performed. 

The cell growth during the amplification phase was similar reflected by the 

doubling time of the cells. It was similar between traditional bioreactors 

(21h in 250L stainless steel seeding bioreactor) and disposable bioreactors 

(from 17h in the Nucleo and the Orbital to 19h in the STR). 

The quality of the molecule together with the molecule titer and the cell 

growth were compared in the 5 single use technologies during a production 

process. The process performance was also compared in 250L and 1.25kL 

bioreactors and to a 3.6L glass development bioreactor. The overall 

production process performance was similar in traditional and disposable 

bioreactors (Figure 1). 

This study was completed by a characterization of liquid/liquid and gas/ 

liquid transfers inside each disposable bioreactor in order to estimate their 

potential in terms of cell culture. 

These cells did not require a lot of oxygen. A low k La (0.44h –1 in the 

Nucleo) was sufficient to run this process. The air-flow rates applied in the



Table 1(abstract P2) Description of the different disposable bioreactors assessed 

Name Mobius® 

Cellready3L 

Supplier Millipore 

Nucleo TM 

Pierre Guerin 

ATMI 

orbital shaking bioreactor during the process were defined according to 

Merck Serono Biodevelopment and supplier experience. 

These flow rates should have been decreased as k La was higher (3.6h- 1 ) 

than in traditional bioreactors (2.6 h -1 ). 

Mixing time was measured in phosphate saline buffer at the maximum 

working volume and the agitation speed applied during production 

process. Mixing time was measured by injecting concentrated sodium 

hydroxide from the top of the bag and from the bottom of the bag. 

The Nucleo and the CellReady 3L system have classical probes, while the 

Orbital, the RM and the STR bioreactors have optical probes. Optical 

probes were more stable because they took a measure every five seconds. 

Classical probes were less stable and showed variations of around 0.03 pH 

unit even a few minutes after injection in the Nucleo. Nevertheless, mixing 

times on all disposable bioreactors (from 15s in CellReady 3L to 100s in 

Orbital) were higher than in traditional bioreactors (12s in 3.6L glass 

bioreactor to 28s in 1250L stainless steel bioreactor). For the STR, the 

mixing time was close to one minute, which is the recommended 

maximum for mammalian cell culture. The Orbital and the Nucleo had a 

mixing time below two minutes. Howerver, after sodium hydroxide 

injection, pH measured below or close to the final pH value. The 

disposable bioreactor which has a mixing time closest to traditional 

bioreactors is the CellReady 3L. 

Cultibag STR TM 

Agitation type Marine impeller Paddle 2 x 3-blade segment 

impeller 

Cultibag RM TM 

Sartorius 

Move back and 

forth 

Cultibag Orbital TM 

b-test 

Orbital shaking 

Minimum working volume 1L 8L 10L 5.5L 25L 

Maximum working volume 2,5L 20L 50L 20L 50L 

Oxygen regulation Micro or marcro Overlay + Overlay + Sparger Overlay only Overlay only 

sparger 

Sparger 

pH regulation with carbon 

dioxide 

Headspace or sparger Sparger Sparger Headspace Headspace 

Temperature regulation Blanket Jacket Jacket Blanket Blanket 

Figure 1(abstract P2) Quantity of highly glycosylated molecule produced in different bioreactors. 


Finally, an evaluation grid was applied to choose the best disposable 

bioreactor. All these comparisons allowed Merck Serono Biodevelopment 

to come to a conclusion about disposable bioreactor use and on the 

scalability (up and down) of these disposable systems. It also enabled us 

to highlight critical points for disposable bioreactor implementation such 

as tubing size and length, use of disposable or reusable probes or factory. 

P3 

Neuroprotective activity of a new erythropoietin formulation with 

increased penetration in the central nervous system 

Marina Etcheverrigaray 1* , Natalia Ceaglio 1 , Mónica Mattio 1 , Marcos Oggero 1 , 

Ignacio Amadeo 1,2 , Guillermina Forno 1,2 , Norma Perotti 1 , Ricardo Kratje 1 

1 Cell Culture Laboratory, School of Biochemistry and Biological Sciences, 

Universidad Nacional del Litoral. Ciudad Universitaria – C.C.242 – (S3000ZAA) 

Santa Fe, Provincia de Santa Fe, Argentina; 2 Zelltek S.A. Ruta Nacional 168 – 

PTLC – (3000) Santa Fe, Provincia de Santa Fe, Argentina 

E-mail: marina@fbcb.unl.edu.ar 


Background: Apart from its hematopoietic effect, erythropoietin (EPO) is a 

molecule with high neuroprotective potential. However, its prolonged



application may cause serious adverse effects due to the erythropoiesis 

stimulation. Therefore, an EPO derivative with neuroprotective properties 

but low hematopoietic activity, designated as neuropoietin (rhNEPO), was 

developed in our lab using an alternative purification process of the 

recombinant human erythropoietic counterpart (rhEPO) produced in CHO 

cells [1]. The in vitro cytoprotective activity of rhNEPO on neural phenotype 

cells and its brain uptake from blood are herein analyzed. 

Results: In vitro citoprotective activity of rhNEPO was analyzed on rat 

pheochromocytoma cells (PC-12) differentiated to neural phenotype with 

neural growth factor (NGF). Apoptosis was triggered by NGF and serum 

withdrawal from cell cultures. Thus, nuclear DNA fragmentation was 

analyzed by colorimetric TUNEL detection. One-way analysis of variance 

was carried out followed by Dunnett´s multiple comparison test. 

Probabilities lower than 0.05 were considered significant (p



rhNEPO distribution into the nervous system, causing a faster appearance 

into the action site. Moreover, the rapid clearance of rhNEPO from plasma 

represents an advantage in the treatment of neurological diseases, 

because the continuous presence of EPO in blood is the stimulus that 

triggers the production of sanguineous cells with the resulting appearance 

of adverse side-effects. 

Conclusions: The in vitro anti-apoptotic effect of rhNEPO becomes a 

remarkable fact to predict its neuroprotective action in the nervous system. 

Furthermore, despite its short plasma half-life, rhNEPO appears promptly 

within the CSF, being also a further and a significant fact that encourage the 

study of neuroepoetin as a potential drug for the treatment of neurological 

diseases, in which, a highly neuroprotective activity with low side effects 

and a fast blood-to-brain influx are desirables. 

References 

1. Mattio M, Ceaglio N, Oggero M, Perotti N, Amadeo I, Orozco G, Forno G, 

Kratje R, Etcheverrigaray M: Isolation and characterization of a subset of 

erythropoietin glycoforms with cytoprotective but minimal 

erythropoietic activity. Biotechnol Progr 2011, doi: 10.1002/btpr.633. 

2. Egrie JC, Browne JK: Development and characterization of novel 

erythropoiesis stimulating protein (NESP). Nephrol Dial Transplant 2001, 

16(Suppl 3):3-13. 

3. Fukuda MN, Sasaki H, Lopez L, Fukuda M: Survival of recombinant 

erythropoietin in the circulation: the role of carbohydrates. Blood 1989, 

73:84-89. 

P4 

New reporter cell clones to determine the biological activity of human 

type I interferons 

Milagros Bürgi 1 , Claudio Prieto 1 , Marcos Oggero 1 , Mariela Bollati-Fogolín 2 , 

Marina Etcheverrigaray 1 , Ricardo Kratje 1* 

1 Cell Culture Laboratory, School of Biochemistry and Biological Sciences, 

Universidad Nacional del Litoral. Ciudad Universitaria – C.C. 242 – 

(S3000ZAA) Santa Fe, Provincia de Santa Fe, Argentina; 2 Cell Biology Unit, 

Institut Pasteur de Montevideo. Mataojo 2020 (11400) Montevideo, Uruguay 

E-mail: rkratje@fbcb.unl.edu.ar 


Background: Interferons (IFNs) are potent biologically active proteins 

synthesized and secreted by somatic cells of all mammalian species. They 

play an important role in the immune response and defence against viruses 

because they have antiproliferative, antiviral and immunomodulatory 

activities [1]. They are widely used as biopharmaceuticals, so their potency 

must be correctly identified. Usually, the biological activity is quantified by a 

bioassay based on its capacity to induce an antiviral state in target cells. 

Antiviral assays may be subject to high inter- and intra-assay variations 

having the drawbacks of using viruses [2]. Therefore, a set a new human cell 

lines derived from different tissues were developed using the enhanced 

green fluorescent protein (eGFP) gene under the control of type I IFNinducible 

Mx2 promoter. In addition, having reporter gene assays derived 

from cells of different tissue origins would allow us to design studies aiming 

to evaluate how IFNs induce their actions through the human body. 

Results: Three human tissue-derived cell lines: A549 (lung cancer cells), 

HEp-2 (epidermoid larynx carcinoma cells) and HeLa (cervical adenocarcinoma 

cells) were used to develop new reporter gene assays based on 

the expression of the eGFP gene under the control of type I IFN-inducible 

Mx2 promoter. 

Six stably transfected Mx2/eGFP lines (two of each wild type cell line) 

were obtained and selected during 10 days with the antibiotic neomycin. 

The Mx promoter sequence, cloned upstream to the eGFP, responded 

specifically and quantitatively to type I IFNs (IFN-a and IFN-b) usingthe 

new reporter cell lines (Figure 1). Therefore, the three cell lines showed 

that eGFP percentage (measured by flow cytometry) rose as IFN increased, 

reaching maximum values of 25-85% activated cells, depending on the cell 

lines and the type of IFNs. No eGFP expression was observed for negative 

controls, indicating that there was no basal expression of the reporter 

protein. 

Two subtypes of IFN-a and IFN-b were assayed: the E. coli-derived IFNs-a 

having the amino acid position 23 occupied by the residue Lys (IFN-a2a) 

or by the residue Arg (IFN-a2b) and the E.coli-derived IFN-b with the 

residue Cys 17 replaced by Ser (IFN-b1b) or the glycosylated CHO cellderived 

IFN-b (IFN-b1a). The dose-response relationships corresponding to 

both IFN-b subtypes showed higher slopes (43 ± 5 IU/ml) than those of 

IFN-a subtypes (23 ± 12 IU/ml). Considering that type-I IFNs act through a 

common receptor complex (ifnar) and that mutational analysis revealed 

distinct binding sites for these IFNs on ifnar [3], the above mentioned 

results give more evidences that differences in IFN a/b recognition may be 

associated with cytoplasmic signaling. 

Analyzing in more detail the linear dose-response relationships for each 

cell line (Table 1), A549 cells showed the lowest detection limit for all IFN 

subtypes (0.24 – 0.48 IU/ml). Comparing IFN-a subtypes, no differences in 

detection limit were observed using any of the cell lines. Contrarily, 

differences in responses were observed when comparing the activity of 

IFN-b subtypes in HEp cell lines. Therefore, the standardization of several 

assays to measure the potency of IFNs might be carried out using the cell 

lines herein documented. 

Conclusions: These systems have several advantages when compared to 

antiviral activity assays and other reporter gene systems: they can 

Figure 1(abstract P4) Reporter gene assays based on the eGFP gene expression after the induction of Mx2 promoter by type I IFNs. 


Table 1(abstract P4) Linear dose-response relationships from reporter gene assays employing different human cell 

lines 

IFN HEp L1D7 HEp L1G5 HeLa C5G6 HeLa C6C3 A549L2A6 A549L2G9 

IFN-a2a 1.52 – 390 12.20 – 25,000 7.8 – 1,000 1.95 – 500 0.24 – 16 0.24 – 125 

IFN-a2b 0.76 – 390 12.20 – 50,000 7.8 – 1,000 1.95 – 500 0.24 – 4 0.24 – 8 

IFN-b1a 0.05 – 200 3.05 – 6,250 3.9 – 250 1.95 – 31.25 0.24 – 32 0.48 – 62 

IFN-b1b 3.05 – 200 12.20 – 50,000 3.9 – 1,000 3.90 – 500 0.24 – 32 0.48 – 62



determine the potency of type I IFNs using only one cell line; they are very 

fast having the specificity of the Mx promoter. Also, they are sensitive and 

safe, showing reproducible responses in a dose-dependent manner. 

Outstandingly, the main contribution of this work was the development of 

alternative reporter systems as suitable candidates to evaluate the way 

that IFNs induce their activity in different human tissues. 

References 

1. Billiau A: Interferon: the pathways of discovery I. Molecular and cellular 

aspects. Cytokine Growth Factor Rev 2006, 17(5):381-409. 

2. Girad DJ, Fleischaker RJ: A study showing a high degree of 

interlaboratory variation in the assay of human interferon. J Biol Stand 

1984, 12(3):265-269. 

3. Piehler J, Schreiber G: Mutational and structural analysis of the binding 

interface between type I interferons and their receptor ifnar2. J Mol Biol 

1999, 294(1):223-237. 

P5 

Analytical techniques for characterization of raw materials in cell 

culture media 

Chandana Sharma * , Barry Drew, Kevin Head, Rani Pusuluri, Matthew V Caple 

Cell Sciences and Development, SAFC, 13804 W 107 th St, Lenexa, KS 66215, 

USA 


Figure 1(abstract P5) Analytical RMC process flow. 


Background: A prioritized list of 100 “high-risk” raw materials was 

developed based on a risk assessment performed within SAFC. This poster 

will focus on the analytical screening of certain “high-risk” raw materials 

within the prioritized list to identify any variability and critical contaminants 

present. In order to achieve this, orthogonal methods were used that 

include ultra-high performance liquid chromatography-mass spectrometry 

(U-HPLC/MS) for non-volatile polar components and gas chromatographymass 

spectrometry (GC/MS) for volatile non-polar materials. Inductively 

coupled plasma-optical emission spectrometry (ICP-OES) was also used to 

identify any trace metal contamination present. In addition, the solubility of 

the raw materials is also tested to identify any variability within a vendor or 

between different vendors. 

Results: Figure 1 shows the process flow followed within the analytical 

RMC initiative. It describes how each raw material is screened and the 

strategy for analyzing any possible contaminant. 

Conclusions: The orthogonal methods cited to characterize raw materials 

have proven to be robust and reliable for the intended purpose. The 

solubility experiment described for amino acids illustrates a significant 

difference in solubility limits of amino acids and establishes intra- and 

inter-vendor variability. This program has helped SAFC get better insight 

into their suppliers’ manufacturing processes. This is a long term initiative 

within the organization and the most important goal through the 

program is to develop a better understanding of raw materials to deliver 

superior products of the finest quality.



Table 1(abstract P5) Summary of solubility findings on multiple lots of three amino acids 

Solubility in 100 ml of a neutral buffer 

Vendor Lot No. Lysine.HCl (g) Cystine.2HCl (mg) Tyrosine.2Na (mg) 

A 1 5.0 12.4 175.1 

2 12.5 35.0 185.8 

3 10.0 35.2 185.5 

B 1 15.0 30.3 185.8 

2 12.5 50.8 195.2 

3 12.0 48.2 200.7 

C 1 60.0 32.0 N/A 

2 60.0 35.2 N/A 

3 60.0 32.8 N/A 

D 1 60.0 N/A N/A 

2 60.0 N/A N/A 

Acknowledgements: Our sincere thanks to Mr. T. Bower and Mr. S. Jones 

of Sigma-Aldrich for running samples on ICP and on LC/MS respectively. 

P6 

Next-generation sequencing of the CHO cell transcriptome 

Jennifer Becker 1* , Christina Timmermann 1 , Tobias Jakobi 2 , Oliver Rupp 2 , 

Rafael Szczepanowski 3 , Matthias Hackl 4 , Alexander Goesmann 2 , Andreas Tauch 3 , 

Nicole Borth 4 , Johannes Grillari 4 , Alfred Pühler 3 , Thomas Noll 1 , Karina Brinkrolf 3 

1 Department of Cell Culture Technology, Center of Biotechnology (CeBiTec), 

Bielefeld University, 33615 Bielefeld, Germany; 2 Bioinformatics Resource 

Facility, Center of Biotechnology (CeBiTec), Bielefeld University, 33615 

Bielefeld, Germany; 3 Department of Genome Research and Systems Biology, 

Center of Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, 


Germany; 4 Department of Biotechnology, University of Natural Resources 

and Applied Life Sciences Vienna, 1180 Vienna, Austria 

E-mail: jbecker@uni-bielefeld.de 


Since 1957 Chinese hamster ovary (CHO) cells are used for in vitro 

cultivation as they require assimilable low sustenance [1]. Today, CHO cell 

lines represent the most commonly used mammalian expression system 

for the production of therapeutic proteins and are considered as the 

mammalian equivalent of E. coli in research and biotechnology [2]. The 

production of biopharmaceuticals in CHO cells is superior to protein 

production in bacteria, because mammalian cell lines procure complex 

folding and post-translational modifications like glycosylation. However, 

contrary to the increasing importance in biotechnology and industry, 

Figure 1(abstract P6) Self-Self Hybridization experiment of the customized CHO microarray. The dye ratio (M-value) is plotted against the adjusted 

p-value (student´s t-test, FDR controlled, a=0.05). The dye ratio was calculated using the mean intensity values (A-value) of all probes belonging to one 

transcript from four microarray replicates. Different thresholds were used as evidence for quality: p > 0.05, M= - 1< M < 1 (pink); p ≤ 0.05, -1 < M < 1 

(dark blue); p ≤ 0.05, M ≤ -1 || M ≥ 1, A < 6 (red); p ≤ 0.05, M ≤ -1 || M ≥ 1, A ≥ 6 (green); p > 0.05, M ≤ -1 || M ≥ 1 (light blue).



comprehensive genome and transcriptome information of CHO cell lines is 

still rare. 

In this study, the pyrosequencing technology from 454 Life Sciences and a 

novel assembly approach for cDNA sequences were used to achieve a 

major step forward towards unraveling the transcriptome of CHO cells. 

CHO cDNA samples derived from different CHO cell lines and growth 

conditions were used for the generation of 1.84 mill. high quality 

sequencing reads with an average read length of 373 nt summing up to 

603 Mb data. Assembly of the sequencing data resulted in 41,039 

contiguous sequences. These contigs were grouped by the Newbler 

software into 36,383 isotigs and 28,039 isogroups. 

Taxonomical classification and comparison to the Mus musculus 

transcriptome demonstrated the actual quality of the CHO cell line 

sequences. 

Metabolic pathways of the central carbohydrate metabolism and 

biosynthesis routes of sugars used for protein N-glycosylation were 

reconstructed from the transcriptome data. All relevant genes representing 

major steps in the N-glycosylation pathway and the central metabolism of 

CHO cells were detected. Only fructose-1,6-bisphosphatase (3.1.3.11) and 

6-phosphogluconolactonase (3.1.1.31) were not identified within the 

pentose phosphate pathway. 

The newly sequenced CHO cell line transcriptome was the basis for the 

design of a customized CHO microarray. Contig sequences were used for 

the design of 94.580 probes. The designed probes cover 31,905 splice 

variants of CHO transcripts. With a Self-Self Hybridization experiment 

(Figure 1) the functionality of the probes was demonstrated. This 

experiment was performed with the same RNA as which was used for 

sequencing. Half of this RNA was labeled with Cy3, the other half with Cy5. 

For this study the dye intensity, the dye ratio and the adjusted p-value 

(student´s t-test, FDR controlled, a=0.05) of four microarray replicates were 

analyzed. It is expected, that the two labeled RNA samples bind equally to 

the probe, if a transcript is expressed (Figure 1: pink, dark blue, red, light 

blue). Only the probes for one transcript could be rejected, because of 

their dysfunctionality (Figure 1, green). 

This CHO microarray is now available for further experiments and will 

support transcriptional analysis of CHO cells under process conditions for 

cell line and process optimization. It was used already used successfully 

for a gene expression study of CHO DP-12 cells cultivated under sodium 

butyrate treatment [3]. 

Acknowledgements: JB, TJ, and JS acknowledge the receipt of a 

scholarship from the CLIB Graduate Cluster Industrial Biotechnology. CT is 

supported by BIO.NRW of the Cluster Biotechnology of North Rhine 

Westphalia. MH is supported by a BOKU DOC grant. NB and JG are 

supported by FWF Biotop, JG is supported by GENAU. 

References 

1. Puck TT, Cieciura SJ, Robinson A: Genetics of somatic mammalian cells. III. 

Long-term cultivation of euploid cells from human and animal subjects. 

J Exp Med 1958, 108:945-956. 

2. Puck TT: Development of the Chinese Hamster Ovary (CHO) Cell. 

Molecular Cell Genetics 1985, 1:37-64. 

3. Klausing S, et al: Bioreactor cultivation of CHO DP-12 cells under sodium 

butyrate treatment and comparative transcriptome analysis with CHO 

cDNA microarrays. in press. 

P7 

A strategy to obtain recombinant cell lines with high expression levels. 

Lentiviral vector-mediated transgenesis 

Claudio Prieto * , Diego Fontana, Marina Etcheverrigaray, Ricardo Kratje 

Cell Culture Laboratory, School of Biochemistry and Biological Sciences, 

Universidad Nacional del Litoral. Ciudad Universitaria – C.C.242 – (S3000ZAA) 

Santa Fe, Provincia de Santa Fe, Argentina 

E-mail: cprieto@fbcb.unl.edu.ar 


Background: The primary goal of any recombinant protein production is 

to achieve successful gene transfer and expression in a target cell. There 

are two general categories of delivery vehicles/vectors employed in 

protein expression protocols. The first category includes the non-viral 

vectors, ranging from direct injection of DNA to complexing DNA with 

cationc lipds, polylysine, etc. The second category comprises DNA and 

RNA viral vectors. 

Viruses have evolved specific mechanism to deliver their genetic material 

to target cell nuclei. Virus members of family Retroviridae, e.g. retroviruses 

and lentiviruses, are among the most widely used viral vectors. The use 

of lentiviral vectors has been increasing because the vector system has 

attractive features. Lentiviruses have an advantage over retroviruses in 

that they can infect both dividing and non-dividing cells and therefore 

have attracted much attention regarding the potential as vectors for 

gene delivery/therapy. Once integrated into the genome, recombinant 

cell lines are selected using different selection mechanisms. 

Results: Lentivirus particles were produced by simultaneous cotransfection 

of HEK 293T cells with four plasmids. The packaging construct 

(pMDLg/pRRE) [1], the VSV-G-expressing construct (pMD.G) [2], the Revexpressing 

construct (pRSV-Rev) [1], and the self-inactivating (SIN) lentiviral 

vector construct containing the green fluorescent protein (GFP) reporter 

gene (pLV-PLK-eGFP). The medium containing lentiviral particles was 

collected 48 h after transfection, clarified by centrifugation 10 min at 2000 

rpm and then stored at -80°C. To determine viral titers, HEK 293T cells 

were seeded at 3 x 10 4 cell/ml in 6-well plates and mantained for 18 h. The 

supernatant was replaced with 1 ml of diluted lentiviral particles 

supernatant containing pLV-PLK-GFP, followed by incubation overnight. 

Then, the supernatants were replaced with fresh medium. The cells were 

analized by flow cytometry and the percentage of GFP positive cells were 

counted 96 h post transduction. Titer was calculated from the dilutions at 

which the percentage of eGFP-positive cells fall within the range of 1-30% 

using the following formula [3,4]: Titer (TU/ml) = [F x C/V] x D; where TU/ 

ml: transduction units/ml, F: frequency of GFP-positive cells, C: total 

number of cells in the well at the time of transduction, V: volume of 

inoculum in ml, and D: lentivirus dilution. 

The viral titer was as high as 4.4 x 10 8 TU/ml. 

After that, HEK 293T cells were seeded at a density of 6 x 10 4 cell/ml in 6well 

plates and after 24 h the supernatant was replaced by 1 ml medium 

containing lentiviral vectors. 96 h post-transduction the cells were 

analyzed by flow cytometry (t=0); then, the cells were incubated with the 

puromycin selection agent to obtain stable recombinant cell lines. Two 

protocols were employed: A-) Single-step selection protocol: the cells were 

Table 1(abstract P7) eGFP expression level of different recombinant cell lines according to puromycin concentration 

Recombinant cell line Puromycyn (µg/ml) Single-step selection protocol Multistep gradual selection protocol 

CL1 1 1.0 

Fold increase eGFP expression (X-mean) 

1.0 

CL5 5 2.0 2.0 

CL10 10 2.0 2.0 

CL50 50 2.0 2.0 

CL100 100 nv 4.5 

CL150 150 nv 4.5 

CL200 200 nv 6.0 

CL250 250 nv nv 

nv: non viable cells. 

Page 24 of 181



Figure 1(abstract P7) Flow cytometry of different eGFP recombinant cell lines. 

incubated with 1, 5, 10, 50, 100, 150, 200 and 250 µg/ml of puromycin in 

different plates, and B-) Multistep gradual selection protocol: the cells were 

incubated from 1 up to 250 µg/ml of puromycin, but the selection agent 

was gradually changed each 7 days on the same plates (Table 1). 

Once the cells were resistant to different concentrations of puromycin, 

recombinant cell lines were cryopreserved and analyzed by flow 

cytometry to compare the expression of eGFP (x-mean). Recombinant cell 

lines showed different eGFP expression levels according to puromycin 

concentration. The cell line CL200 showed the highest eGFP expression 

level (Figure 1). 

Conclusions: Employing the gradual selection protocol, it was possible to 

maintain the cells in culture condition up to 200 µg/ml puromycin and 

achieve higher expression levels of the reporter gene, between 2 and 6 

times depending on puromycin concentration. Contrarily, in the singlestep 

selection protocol cells cultures were resistant only up to 50 µg/ml 

and expression levels of eGFP were lower. Simultaneously, resistant cell 

lines were cloned by limit dilution methods and the resulting cell clones 

were also analyzed by flow cytometry. The eGFP expression of each clone 

was consistent with the ones observed in the respective resistant cell 

lines (data not shown). Therefore, with this strategy of recombinant cell 

line selection, it was possible to obtain high eGFP producing stable 

cell clones without the use of any gene amplification system. 

References 

1. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L: A 

third-generation lentivirus vector with a conditional packaging system. 

J Virol 1998, 72:8463-8471. 

2. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, 

Trono D: In vivo gene delivery and stable transduction of nondividing 

cells by a lentiviral vector. Science 1996, 272:263-267. 

3. White SM, Renda M, Nam NY, Klimatcheva E, Zhu Y, Fisk J, Halterman M, 

Rimel BJ, Federoff H, Pandya S, et al: Lentivirus vectors using human and 

simian immunodeficiency virus elements. J Virol 1999, 73:2832-2840. 

4. Sastry L, Johnson T, Hobson MJ, Smucker B, Cornetta K: Titering lentiviral 

vectors: comparison of DNA, RNA and marker expression methods. Gene 

Ther 2002, 9:1155-1162. 

P8 

In-situ cell density monitoring and apoptosis detection in adherent 

Vero cell bioreactor cultures 

Emma Petiot 1* , Amal El-Wajgali 1 , Geoffrey Esteban 2 , Cécile Gény 3 , 

Hervé Pinton 3 , Annie Marc 1 

1 

Laboratoire Réactions et Génie des Procédés, UPR-CNRS 3349, Nancy- 

Université, Vandœuvre-lès-Nancy, France; 2 FOGALE nanotech, Nîmes, France; 

3 

Sanofi pasteur, Marcy L’Etoile, France 

E-mail: emma.petiot@nrc-cnrc.ca 


Background: In cell-based processes, and particularly in viral vaccine 

production, cell growth and death are strategic informations to obtain for 

process monitoring (ie. scale-up, determination of MOI, TOI, and harvest 

time). Dielectric spectroscopy is a tool which was increasingly 

implemented on cell-culture bioreactors as it presents great potentials, 

compared to other methods, for the in-line monitoring of these two 

crucial parameters. Considering viral vaccine production, Vero cells are 


one of the most employed cell platform. But, due to its adherent 

characteristics, few in-line techniques were developed for cell density 

monitoring, on the contrary to the ones available for suspension cells. In 

addition, it should be underline that no in-line technique exists for 

quantification or detection of the mammalian cell death, despite the 

importance of this parameter for cell culture processes. 

Materials and methods: The Vero cell line employed in this study was 

provided by Sanofi Pasteur. Cells were cultivated in serum-free conditions; 

either in a reference medium or in a modified medium with alanineglutamine 

peptide (Glutamax®) substituted to glutamine. The adhered cell 

population was numbered on haemacytometer after crystal violet treatment. 

The apoptotic cells, labelled with annexin V, were quantified by flow 

cytometry (Guava). The in-line recording of permittivities at different 

frequencies and of the characteristic frequency of the cell population, fc, 

were performed by a Fogale Biomass system®. 

Theoretical background: The application of the permittivity theory to 

mammalian cell density quantification was well described in previous 

studies [1]. For a basic comprehension it has to be precised that, in this 

method, viable cells are considered as miniature condensators, and 

permittivity measurement corresponds to the amplitude of the decharge 

curve of cell culture suspension. This curve modelling allowed to relate 

permittivity to different physical parameters, such as cell size, cell 

membrane capacitance or cell intracellular conductivity as described by the 

following equations [2]. From these mathematical relations, a parameter that 

will be called specific permittivity of the cells (Δε fogale / C) was calculated. 

Most of these physical parameters could be potentially impacted by 

changes in the cell physiology and especially by modifications induced by 

cell death. 

Δε =9.r.B.CM /4=3.ð.r 4 .C.CM 

fc = si /(2.B.r.C M) 

Δε: permittivity, pF.cm -1 

fc: characteristic frequency, Hz 

B : biovolume (percentage of viable cell volume in the culture suspension) 

C : cell concentration, cell.mL -1 

r : cell radius, m 

C M : membrane capacitance, F. m -2 

si : intracellular conductivity, mS.cm -1 

Results: 

Dielectric properties of adherent Vero cells: A valid correlation between 

biovolume and cell concentration was observed for batch cultures 

performed in both media, confirming that the Schwan model [3], originally 

developed for spherical cells, was also implementable to cells attached on 

microcarrier surface. The slope of the correlation between permittivity and 

cell concentration highlighted the impact of the medium composition on 

the Vero cell dielectric properties. Indeed, in the modified cell culture 

medium, the slope was lower suggesting a reduction of specific permittivity, 

and so potential changes in cell dielectric properties (C M and si) (Figure 1). 

The Fogale Biomass system® also allowed to observe the impact of cell 

density on cell morphology. Indeed, for culture in the reference medium, a 

linear correlation was observed under 10 x 10 5 cell.mL -1 . Beyond this cell 

density, the specific permittivity decreased indicating a decrease of the 

biovolume per cells. This was confirmed by microscopic observation at 

different time points of the culture, demonstrating a reduction of cell size 

due to carrier surface saturation. So, an accurate monitoring of Vero cells 

adhered on microcarriers could be realized with Fogale biomass system®,



in different culture media and during the different exponential or decline 

culture phases. 

In-line detection of Vero cell death: The Fogale biomass system® was 

also applied to in-line detect Vero cell death. We only focused on apoptosis, 

while no necrotic cells were detected in these Vero cell cultures. Dielectric 

spectroscopy parameters were plotted and correlated to apoptotic cell 

death occurrence. Thus, a drastic increase of cell apoptosis was observed to 

be concomitant with fc parameter increase, whatever the culture medium or 

the feeding process used (batch / fed-batch). Plotting the characteristic 

frequency derivative, dfc/dt, with the apoptotic cell concentration 

highlighted that this derivative always became equal to zero when the 

apoptosis occurred. As a proof-of-concept, an apoptotic inducer, the 

actinomycin D, was added during the exponential cell growth phase of a 

batch culture. In that case the fc parameter presented the same behaviour 

confirming the relationship between apoptosis physiological modifications 

and the physical parameters impacting fc (r, C M, si). 

Conclusion: The first major impact of this work was to demonstrate that 

no model adaptation was needed to monitor adherent cell concentration 

by using permittivity measurements. An accurate monitoring of adhered 

Vero cell concentration was obtained with Fogale Biomass system® until 

1x10 6 cell.mL -1 . A further development of the method should be to 

monitor higher densities of adherent cells. This could be easily achieved by 

monitoring the cell size evolution and correcting the changes induced in 

the correlations. The second major impact of this work was to demonstrate 

the potentials of this method for the risk-mitigation strategies. Indeed, it 

was possible to detect a high increase of cell apoptosis in cell culture 

performed with reference operating conditions, but also, an abnormal 

increase of apoptotic cell concentration artificially induced during the 

culture process. This observation validates the fact that detection of 

apoptosis occurrence, due to viral infection for example, could be 

monitored with this system. 

References 




CHO cells. Biotechnol Progr 2010, 26:272-283. 


Figure 1(abstract P8) Evolution of the relative permittivity, Δε fogale, with the adhered Vero cell concentration and microscopic observations of 

microcarriers at different time points of the culture. 

2. Markx GH, Davey CL: The dielectric properties of biological cells at 

radiofrequencies: Applications in biotechnology. Enzyme Microb Technol 

1999, 25:161-171. 

3. Schwan HP: Electrical properties of tissue and cell suspensions. Adv Biol 

Med Phys 1957, 5:147-208. 

P9 

Does earlier use of productivity enhancers during cell line selection 

lead to the identification of more productive cell lines? 

Alison J Porter * , Atul Mohindra, Juana Maria Porter, Andrew J Racher 

Lonza Biologics plc, 228 Bath Road, Slough, Berkshire, SL1 4DX, UK 

E-mail: alison.porter@lonza.com 


Background: After selection of a recombinant cell line for the production 

of a therapeutic protein, attempts are often made to increase volumetric 

productivity. Various techniques have been employed during the 

production process when trying to increase product concentration. Some 

aim to increase the time integral of viable cell concentration (IVC; the 

number of hours viable cells are available to produce the product) by 

increasing the maximum viable cell concentration and maintaining high 

viabilities. Techniques include (i) optimization of media and feeds, (ii) 

optimization of feeding strategies and (iii) genetic manipulation. Others 

aim to increase specific production rate (Q P) by the deliberate inhibition of 

cell growth (controlled proliferation). Three methods commonly used to 

control proliferation are use of (i) chemical agents, (ii) hypothermic 

conditions and (iii) genetic manipulation. In this study, we focused on 

increasing volumetric productivity by manipulating Q P; in particular, by the 

use of chemical agents. 

As a cell line has typically already been selected as a manufacturing cell 

line prior to assessing such methods to increase Q P, the likelihood of 

success is not predictable: resulting in the frequently heard comment that 

results are ‘cell line specific’. 

But what if we were to look at these methods with many cell lines, at a 

much earlier stage of development (before the final cell line has been



Figure 1(abstract P9) Full distribution of product concentration values for the control, NaBu and NaAc cultures. 

selected)? It could be that the cell line with the highest product 

concentration is typically a non-responder. 

The work described looks to answer the questions: (1) To what extent 

does the response to such methods vary in a large panel of cell lines 

producing the same antibody? and (2) Would their use in an earlier stage 

of cell line development enable the selection of a ‘better’ manufacturing 

cell line? 

Materials and methods: Cell lines: A panel of 148 GS-CHO cell lines was 

generated by transfecting the host cell line CHOK1SV with the GS vector 

pEE12.4 containing the gene for the model antibody cB72.3 (Porter et al, 

2010, Biotechnol Prog 26: 1455-1464). 

Cell culture: The cell lines were assessed in a scale-down model (fedbatch 

shake-flask cultures) of Lonza Biologics’ final production bioreactor 

process, using CDACF medium and feeds. Three cultures were initiated 

for each cell line. The first was a control culture, in the second the culture 

medium was supplemented with 1 mM Sodium Butyrate (NaBu), and in 

the third the culture medium was supplemented with 7.5 mM Sodium 

Acetate (NaAc). The cell concentration was determined using a Vi-CELL 

automated cell counter. Product concentration was determined using 

Protein A HPLC. 

Results: The distribution of the values for the parameters IVC, Q P and 

product concentration were investigated for each condition (control, NaBu 

and NaAc). For IVC, the distribution of the values for both the NaBu and 

NaAc conditions are lower than that of the control. For Q P, the distribution 

of values for the NaBu condition are higher that that of the control. The 

NaAc condition shows no improvement. For product concentration, the 

distribution of values for the NaBu condition are lower than that of 

the control. Little difference is observed between the control and the NaAc 

condition. Data were analyzed by one-way ANOVA and Tukey’s multiple 

comparison test at a 5% significance test. The analysis reveals that there is 

a significant difference between the control and NaBu conditions for IVC, 

QP and product concentration. In addition, there is a significant difference 

between the control and NaAc conditions for IVC but not for Q P and 

product concentration. 

Review of individual cell lines (Figure 1) reveals that 

■ The majority of cell lines did not achieve a higher product 

concentration compared to the control, when either NaBu or NaAc was 

added 

■ The use of NaBu resulted in an increase in productivity, compared to 

the control, for 27% of the cell lines 

■ The use of NaAc resulted in an increase in productivity, compared to 

the control, for 41% of the cell lines 

■ An increase in productivity was approximately twice as likely to be 

observed with the use of NaAc compared to the use of NaBu 


■ For both NaBu and NaAC, an increase in product concentration 

compared to the control was more likely if the cell line was a low 

producer 

■ For those cell lines exhibiting an increase in product concentration 

compared to the control when NaBu or NaAc was added, 80% and 62% 

respectively were in the lower 50% of producers when ranked by control 

product concentation 

■ The highest product concentration was achieved using control 

conditions 

■ No advantage of NaAc or NaBu if looking to identify a more productive 

cell line from the population 

Conclusions: The data generated in the rigorous testing support the 

anecdote that the ability of productivity enhancers to increase Q P is ‘cell line 

specific’. On average, no increase in mean product concentration was seen 

and only ~25-50% cell lines exhibited a benefit. The question ‘Would the 

use of productivity enhancers in an earlier stage of cell line development 

enable the selection of a ‘better’ manufacturing cell line?’ has been 

answered: There is no advantage in their use. If the selection strategy has 

identified a high producing cell line, the productivity enhancers are unlikely 

to be effective. Also, the highest product concentration was achieved with 

control conditions. 

Acknowledgements: Lonza Biologics plc, Slough, GB: Cell Culture 

Development Groups; Analytical Development Groups. 

P10 

Quantification of intracellular nucleotide sugars and formulation of a 

mathematical model for prediction of their metabolism 

Ioscani Jiménez del Val 1* , Judit M Nagy 2 , Cleo Kontoravdi 1 

1 Department of Chemical Engineering, Imperial College London, London, 

SW7 2AZ, UK; 2 Institute of Biomedical Engineering, Imperial College London, 

London, SW7 2AZ, UK 


The US FDA and the European Medicines Agency have recently proposed 

the implementation of the Quality by Design (QbD) paradigm to the 

manufacture of biopharmaceuticals. Its implementation requires the use of 

all available knowledge of a given product, including the parameters that 

affect its quality, for the design, optimization and control of the 

manufacturing process. The goal is to ensure that quality is built into the 

product at every stage of the manufacturing process. Most licensed 

monoclonal antibodies (mAbs) are based on the immunoglobulin G isotype 

and contain a consensus N-linked glycosylation site on the Cg2 domains of 

their heavy chains. Studies have found that the oligosaccharides attached to



this site dramatically influence the efficacy of mAbs as therapeutics either by 

reducing their serum half-life or by directly affecting the mechanisms they 

trigger in vivo[1,2], thus defining glycosylation as a critical quality attribute of 

mAbs under the QbD scope. It has been recently proposed that detailed 


mathematical models will play a critical role in the design, control and 

optimization of biopharmaceutical manufacturing processes under the QbD 

scope [3]. To our knowledge, there are currently no mathematical models 

that relate mAb glycosylation with cell culture conditions. 

Figure 1(abstract P10) Model reproduction of the experimental data. A, B, C and D show reproduction of viable cell counts (Xv), dead cell counts 

(Xd), glucose, glutamine, ammonia and product concentrations in conventional culture media (SF-RPMI) and optimized chemically defined media (PF- 

BDM). E, F, G and H show reproduction of intracellular NSD concentrations.



Several reports have shown that glycosylation is directly affected by the 

intracellular availability of nucleotide sugar donors (NSDs) [4] which are 

the co-substrates for the glycosylation reactions that occur in the Golgi 

apparatus. During culture, cells synthesize all the relevant NSDs from 

glucose through the nucleotide sugar metabolic pathway. In an effort to 

relate process conditions with mAb glycosylation, we have generated a 

dynamic mathematical model for this metabolic pathway. The NSD 

pathway described in KEGG [5] was used as the starting point. In the 

full pathway, four potential carbon sources are converted into the eight 

main NSDs (UDP-GlcNAc, UDP-Glucose, UDP-Galactose, UDP-GalNAc, 

UDP-GlcA, GDP-Man, GDP-Fuc and CMP-Neu5Ac) through 31 enzymatic 

reactions. However, many of the intermediary species are difficult to 

measure throughout the course of cell culture. For this reason, the 

kinetic model was reduced based on the methodology described by 

Nolan and Lee [6] whereby sequential reactions along different 

branches of the pathway were lumped into single reactions. As an 

additional simplification, glucose was considered as the only carbon 

source for the pathway. In order to relate NSD metabolism with 

macroscopic cell culture variables, a model for cell growth, nutrient 

depletion, metabolite accumulation and product secretion was 

formulated based on conventional Monod kinetics. Both models were 

linked by defining the intracellular glucose accumulation needed for the 

NSD model as a function of the glucose maintenance energy term (m s, 

glc) from the cell culture model; the outlet of NSDs from the cells was 

associated to the product secretion rate. 

In order to estimate the unknown parameters of the combined model, 

experimental data from Kochanowski and collaborators [7] was used. 

First, the parameters from the macroscopic model were estimated from 

the data, including the maintenance energy term for glucose. The results 

are shown in panels A, B, C and D of Figure 1. Once the cell culture data 

was reproduced accurately with the estimated parameters, the unknown 

kinetic parameters from the NSD component of the model were 

estimated with the intracellular NSD data from Kochanowski et al. [7]. 

These results are shown in panels E, F, G and H of Figure 1. 

Figure 1 shows that, overall, the model reproduces the experimental data 

accurately. The only exceptions are the UDP-GlcNAc concentration 

profiles for both culture media and the UDP-GalNAc profiles for the SF- 

RPMImedium.InthecaseofUDP-GlcNAc,themodelpredictshigher 

accumulation of this NSD towards the end of the data set, whereas the 

experimental data suggests that the profile flattens out. It is likely that 

the model overestimates UDP-GlcNAc accumulation because experimental 

data for CMP-Neu5Ac was unavailable and therefore, this NSD was not 

considered within the model. From the reduced metabolic network for 

NSDs, it is known that CMP-Neu5Ac is produced from UDP-GlcNAc. If 

CMP-Neu5Ac is not considered within the model, it is natural that the 

model will predict additional accumulation of UDP-GlcNAc. In the case of 

UDP-GalNAc for the SF-RPMI medium, overaccumulation for this NSD is 

also predicted by the model. It is possible that this is due to the excess 

accumulation of UDP-GlcNAc. From the metabolic network, it is known 

that UDP-GalNAc is directly synthesized from UDP-GlcNAc. If the model 

predicts higher accumulation of the latter, it will certainly predict higher 

UDP-GalNAc accumulation as well. 

To our knowledge, the model presented in this work is the first to link cell 

culture variables with intracellular metabolic processes through the glucose 

maintenance energy term (m s,glc). Furthermore, it is the first model to relate 

cell culture variables with intracellular NSD concentrations and the results 

show that it is capable of reproducing experimental data accurately. 

However, in order to achieve better reproduction of experimental data and 

obtain higher confidence in the estimated parameters, additional 

experimental data is needed. Specifically, the concentration profiles of GDP- 

Fuc and CMP-Neu5Ac throughout cell culture would lead to improved 

reproduction of experimental data and predictive capabilities of the model. 

Furthermore, fed-batch cultures are also necessary to validate the model 

and its parameters. 

Once validated with additional data, our mathematical model for NSD 

metabolism, can be coupled to a model for Golgi N-linked glycosylation. 

This combined model would generate a direct link between extracellular 

glucose concentration, which is a readily measurable process variable, 

and protein glycosylation. Such a combined model has great potential for 

the design, control and optimization of manufacturing processes that 

produce mAbs with built in glycosylation-associated quality as proposed 

under the QbD paradigm. 

Acknowledgements: Ioscani Jiménez would like to thank CONACYT and 

The Mario Molina Foundation for their financial support. Cleo Kontoravdi 

would like to acknowledge the support of Lonza Biologics for her 

Fellowship. 

In memoriam Dr. Judit M. Nagy. 

References 

1. Chen X, Liu YD, Flynn GC: The effect of Fc glycan forms on human IgG2 

antibody clearance in humans. Glycobiology 2009, 19:240-249. 

2. Raju TS: Terminal sugars of Fc glycans influence antibody effector 

functions of IgGs. Curr Opin Immunol 2008, 20:471-478. 

3. Jimenez del Val I, Kontoravdi C, Nagy JM: Towards the implementation of 

quality by design to the production of therapeutic monoclonal 

antibodies with desired glycosylation patterns. Biotechnol Prog 2010, 

26:1505-1527. 

4. Wong DCF, Wong KTK, Goh LT, Heng CK, Yap MGS: Impact of dynamic 

online fed-batch strategies on metabolism, productivity and Nglycosylation 

quality in CHO cell cultures. Biotechnol Bioeng 2004, 

89:164-177. 

5. Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, 

Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y: KEGG for linking 

genomes to life and the environment. Nucleic Acids Res 2008, 36: 

D480-D484. 

6. Nolan R, Lee K: Modeling the dynamics of cellular networks. Methods in 

Bioengineering: Systems Analysis of Biological Networks Artech House 

Publishers: Jayaraman A, Hahn J 2009. 

7. Kochanowski N, Blanchard F, Cacan R, Chirat F, Guedon E, Marc A, 

Goergen JL: Influence of intracellular nucleotide and nucleotide sugar 

contents on recombinant interferon-g glycosylation during batch and 

fed-batch cultures of CHO cells. Biotech Bioeng 2008, 100:721-733. 

P11 

Design and simulation of a controller system for metabolic shift 

regulation in mammalian cells 

Damián Baeza 1,3* , Ziomara P Gerdtzen 2,3,4 , Cristian J Salgado 1,2,3 

1 Laboratory for Process Modeling and Distributed Computing, Department 

of Chemical Engineering and Biotechnology, Faculty of Physical and 

Mathematical Sciences, University of Chile, Santiago, Chile; 2 Millennium 

Institute for Cell Dynamics and Biotechnology, Department of Chemical 

Engineering and Biotechnology, Faculty of Physical and Mathematical 

Sciences University of Chile, Santiago, Chile; 3 Department of Chemical 

Engineering and Biotechnology, Faculty of Physical and Mathematical 

Sciences, University of Chile, Santiago, Chile; 4 Centre for Biochemical 

Engineering and Biotechnology, Department of Chemical Engineering and 

Biotechnology, Faculty of Physical and Mathematical Sciences University of 

Chile, Santiago, Chile 


Background: Experimental evidence shows the existence of multiple 

steady states in mammalian cell culture with distinct cellular metabolism 

[1]. Different metabolic states are represented by different lactate to 

glucose stoichiometric ratios (ΔL/ΔG).AsitisshowninTable1,the 

existence of multiple steady states involves the interaction between 

metabolic and gene [2]. 

Changes on the cell culture’s metabolic state have been found to be 

related to the amount of residual glucose in a reactor [3]. Experimental 

results indicate that cells in a low metabolic state respond immediately to 

pulse additions of glucose. 

The problem of providing an optimized strategy for glucose feeding in 

order to achieve a specific metabolic state is yet to be studied. We 

propose a model based strategy for designing a control system for 

Table 1(abstract P11) Gene level fold changes 

corresponding to low over high ΔL/ΔG states 


Enzyme Real Time PCR Microarray cDNA 

Lactate Dehydrogenase ↓ 2.4 ↓ 1.9 

Pyruvate Kinase ↓ 2.9 ↓ 2.0 

Phosphofructokinase ↓ 2.0 ↓ 1.3



metabolic state regulation that considers the biological complexity of the 

regulation of the cellular system. 

Materials and methods: A detailed metabolic model for mammalian cell 

metabolism was formulated (complete model); a system of ordinary 

differential equations for the main metabolic variables of the following 

form: 

� 

dC � � � 

=S⋅r −t(C) 

dt 

wherein is the metabolite concentration� vector, � S is the stoichiometric 

matrix, is the reaction rate vector, and t(C) the transport/consumption 

rate vector. The reaction rates can be described by: 

r i = ri f (C) 

max 

� 

⋅ i 

� 

where max 

r is the reaction rate constant i and 

i f(C) , is a non-linear 

i 

function that describes the reaction-reactant dependency. Furthermore, 

cell growth rate was modeled as the following first order monod function: 

m = m 

max 

e 

C glc I 

K +C I +C 

X 

lac 

e 

X 

X 

⋅ ⋅ 

glc 

glc 

lac 

e 

lac 

The model’s parameters were obtained from literature and through a 

fitting process to experimental data; said fitting process was by the least 

squares method using the Nelder-Mead simplex method [4]. 

Once the experimental curves were obtained with the detailed model a 

simplified model was traced that includes the main metabolic reactions 

in CHO cells, and the same fitting process was carried out. Stability 

analysis was carried out on both the detailed and the simplified model in 

order to establish the number of feasible steady states for both models. 

Enzyme kinetic for lactate dehydrogenase was weighted with an enzyme 

factor F, which varies between 0 and 1: 

r’ LDH = F ⋅ rLDH 

The objective of the same was to associate low ΔL/ΔG with low values of 

F. Based on the estimated F values, a Hill activation function that 

depends on the residual glucose concentration was adjusted to obtain 

the experimental curves of the culture with metabolic shift. 

Figure 1(abstract P11) Simulation results for detailed and simplified model. 

Results: The results of the curve fitting of the detailed and simplified 

models are depicted in Figure 1a and 1b, respectively. However, the 

same parameters cannot be used to simulated a cell culture that presents 

metabolic shift; the results of the use of the same parameters to simulate 

a MAK cell culture induced to a lower metabolic state do not portray the 

metabolic response expected for said cell culture (see Figure 1c). 

Stability analysis confirmed that both the simplified and detailed 

metabolic models exhibit only one steady state. The existence of only 

one stable attractor supports the idea that metabolic regulation alone 

cannot explain the metabolic shift. Therefore, gene regulation is an 

element that should be considered in a model that correctly describes 

this phenomenon. 

(a) Simulation results for complete model without metabolic shift, 

(b) Simulation results for unregulated simplified model without metabolic 

shift, (c) Simulation results for unregulated simplified model with 

metabolic shift, (d) Simulation results for regulated simplified model with 

metabolic shift. - : Simulated Lactate, - : Simulated Glucose, - : 

Simulated Cell Conc., � : Lactate, ■ : Glucose, • : Cell Conc. 

The implemented regulation model consists of a Hill activation function 

that depends on the residual glucose concentration, which modifies the 

enzyme kinetic for lactate dehydrogenase in the following manner: 

r’ = b ⋅ 

LDH 

n 

e 

C glc 

K + C 

X 

( ) 

n 

n 

e 

( glc ) ( glc ) 

⋅ r 

LDH 


wherein b = 1 is the maximal expression level, K glc = 1.24 [mM] 

is the activation coeficient, n = 14.64 is the Hill coeficient, and C e glc is 

the glucose concentration within the bioreactor. The result of 

the implementation of the above function is presented in Figure 1d. The 

maximal expression level was fixed at 1 due to the upper limit of the 

named enzyme activity, the activation coefficient was retrieved from 

literature [3] and the Hill coefficient was adjusted through the previously 

used curve fitting process. Additionally, the sum of square residuals of 

glucose, lactate and cell concentration was 15.74 when comparing the 

simplified regulated model with another source of experimental data, 

further supporting the proposed metabolic model. 

Conclusions: Metabolic shift is caused by regulation at gene expression 

and metabolic levels. A reduction of the ΔL/ΔG ratio is accompanied by a 

variation in the expression levels of certain genes that participate in 

glycolisis, which justifies the use of a regulation function that varies from 0



to 1. Stability analysis concludes that the current metabolic models are 

capable of reproducing only one steady state, making explicit the need to 

implement a regulation model. Moreover, the immediate response 

capability to glucose feed pulses indicate that said regulation model must 

be continuous. A Hill activation function was for supplying the gene 

regulation due to its structure; glucose concentration has been noted 

experimentally to be the main factor that produces a metabolic shift in 

mammalian cells and the modification of enzyme kinetics leads to a 

different ΔL/ΔG ratio, thus to a different steady state. 

The final objective of said model is to design a model based controller 

capable of maintaining a low metabolic state (ΔL/ΔG ratio under 0.5) under 

continuous operation. The tentative input variables are the glucose and 

lactate concentrations as well as the ΔL/ΔG ratio. The controller will modify 

the response of the system by manipulating the dilution rate and the 

glucose feed concentration of the culture. 

Acknowledgments: Conicyt, This work has been supported by 

FONDECYT Initiation Grants 11080016 and 11090268 

References 

1. Europa A, Gambhir A, Fu P-F, Hu W-S: Large scale gene expression 

profiling of metabolic shift of mammalian cells in culture. Biotechnol 

bioeng 2000, 67:25-34. 

2. Korke R, Gatti M, Lau A, Lim J, Seow T, Chung M, Hu W-S: Multiple steady 

states with distinct cellular metabolism in continuous culture of 

mammalian cells. J Biotech 2004, 107:1-17. 

3. Cruz HJ, Moreira JL, Carrondo MJT: Metabolic shifts by nutrient 

manipulation in continuous cultures of BHK cells. Biotechnol bioeng 1999, 

66:104-113. 

4. Lagarias JC, Reeds JA, Wright MH, Wright PE: Convergence properties of 

the Nelder-Mead simplex method in low dimensions. J Optim 1998, 

9:112-147. 

P12 

The way to a design space for an animal cell culture process according 

to Quality by Design (QbD) 

Robert Puskeiler 1* , Jan Kreuzmann 1 , Caroline Schuster 1 , Katharina Didzus 2 , 

Nicole Bartsch 1 , Christian Hakemeyer 1 , Heike Schmidt 3 , Melanie Jacobs 3 , 

Stefan Wolf 3 

1 

Roche Diagnostics GmbH, Pharma Biotech, Development Fermentation, 

Penzberg, Germany, 82377; 2 Roche Diagnostics GmbH, Pharma Research and 

Early Development Cell Sciences, Penzberg, Germany, 82377; 

3 

Roche Diagnostics GmbH, Pharma Biotech, Manufacturing Fermentation, 

Penzberg, Germany, 82377 

E-mail: robert.puskeiler@roche.com 


Background: The strategy of implementation of the QbD (Quality by 

design) approach in upstream processing of therapeutic proteins consists 

of the identification of critical process parameters (CPPs) that have a 

statistically significant influence on the critical quality attributes (CQAs) of 

a specific process. By applying the acceptance criteria to the CQAs, 

proven acceptable ranges (PARs) for the CPPs can be deduced from 


experimental data. The multidimensional combination of these ranges 

form the design space and thus assures the quality of the product. 

The QbD approach according to the ICH guidelines Q8, Q9 and Q10 may 

be subdivided in the work packages scale down model qualification, risk 

analysis, process characterization and range studies. The foundation of 

the QbD approach is represented by the scale down model. Several 

different scale down criteria were applied and adapted until a satisfactory 

match of scale down to commercial scale data was achieved. The scale 

down model is then used to investigate cause effect relationships 

between process parameters and quality attributes of the production 

process. 

Since a standard cell culture process from thawing of the vial up to the 

final production fermenter can comprise up to 100 process parameters, a 

risk based approach is helpful to filter the most important ones. Those 

parameters are then experimentally investigated to verify their criticality 

for the quality attributes of the process. This approach relies on design of 

experiment (DoE) to reduce the number of required experiments to a 

manageable number while maintaining meaningful results. During the 

range studies, those critical parameters will be investigated with the help 

of a high resolution DoE matrix in order to be able to reveal possible 

interactions and higher order effects. 

Scale down model: Based on development data a scale down model at 

2 L scale was established. Predefined scale down criteria (power input, 

volumetric aeration rate, tip speed) were applied while taking the specific 

clone properties into account. The qualification of the scale down model 

was carried out by considering an acceptance criteria for several critical 

quality attributes and key performance indicators for at least three scale 

down fermentation runs. The acceptance criteria consisted of matching 

the 2-fold standard deviation range with the mean of the small scale 

data and the 3-fold standard deviation range with individual data points. 

Risk analysis: Being confronted with a large number of process 

parameters, risk assessment tools are used to focus the experimental 

efforts on the most relevant parameters. A Failure Mode and Effects 

Analysis (FMEA) based tool was applied to rate hypothetical deviations of a 

parameter from a previously defined observation range. The hypothetical 

deviation is rated by its severity, occurrence and detectability yielding a 

ranking of all parameters according to their risk priority number. 

Process characterization / range studies: The experimental investigation 

was started with a first round fractional factorial screening design with the 

parameters filtered by the risk analysis tool. The resulting models were 

optimized such that model quality was sufficient (p < 0.05) and no lack of 

fit was observed. The parameters were evaluated in terms of statistical 

significance by the t-ratio and p-value. Relevance of certain parameters 

wascheckedbycomparisonoftheestimatesizetothecenterpoint 

variation (Figure 1). 

The results of that work package allowed to eliminate some process 

parameters from further experimentation due to their lack of significance 

and/or relevance. The resulting parameters were investigated in a second 

round of multivariate experimentation with a central composite design 

aiming at the definition of the unit operation design space. The higher 

resolution models were then subsequently used to define the design space 

mathematically. To that aim, each quality attribute was evaluated with its 

own optimized model only covering significant terms. 

Figure 1(abstract P12) Statistical data evaluation from process characterization data. Models with satisfying ANOVA data and lack of fit were created to 

evaluate all significant and relevant process parameters. Plots created with JMP®, SAS, Cary, USA. DF = degrees of freedom, C. Total = total sum of 

squares, Prob> |t|= probability of getting a higher t-Ratio by chance (p-value).



Figure 2(abstract P12) Example of the visualization of a design space for 4 parameters by the combination of 2 internal and 2 external axes. 

Design space visualization: The mathematical definition of the design 

space can, for example, be transformed into the following graphical 

representation (Fig. 2). Herein, the combination of two external and two 

internal parameter axes allow the visualization of 4 parameters in 9 twodimensional 

plots. Every single plot depicts the proven acceptable range in 

white restricted by the mathematical models represented by the colored 

shaded areas. The models are derived from the experimental results and 

thus cut those areas that represent exceeded acceptance criteria. 

A fully representative view of the 4-dimensional design space can thus be 

achieved if all possible permutations of the external and internal axes is 

shown. 

P13 

Improvement of stem cell performance by supplementation with 

metabolic enhancers 

Abi M Abitorabi 1* , Christopher Wilcox 2 

1 2 

GIRUS Life Sciences, Inc., Sunnyvale, CA, 94085, USA; Sheffield BioScience, 

Beloit, WI, 53511, USA 

E-mail: abi@girusinc.com 


Practical culture methods for expansion of human stem cells are needed 

for research and industrial applications. In general, ingredients of known 


and unknown composition have been used in stem cell cultures based on 

past studies and practices. To harness the substantial potential of stem 

cells in treating human diseases, products with improved characteristics 

are needed to support the manufacturing of stem cells for cell therapy use. 

Safety, definition, cost and consistency are key considerations for all new 

stem cell products. Based on data mining and cell-based screens, we have 

designed the RS Novo defined small molecule metabolic enhancers for 

expansion of human embryonic (ESC), mesenchymal (MSC) and 

hematopoietic stem cells with limited differentiation. Cell growth, viability, 

phenotype and stem cell potential were endpoints of interest in our 

studies. Our aim was to minimize media components that would “trigger” 

stem cells into differentiation, apoptosis, and necrosis. We also aimed to 

minimize undefined components like serum that could introduce 

inconsistencies. Here, we cover some RS Novo results with human MSC 

and ESC and introduce the GEM Novo serumfreemediumforamore 

complete culture system for stem cell expansion. Cells grown in this 

culture system maintained their stem cell phenotype and potencies. For 

example, at different passages, human ESC H7 clone maintained the Oct-4 

marker of undifferentiated cells more consistently in this system versus a 

culture system optimized by others (Table 1), and human MSC expanded 

in GEM Novo containing RS Novo and then transferred to differentiation 

media could differentiate into adipocytes (Figure 1). Altogether, this new 

culture system provides a consistent, high performance condition for 

human stem cell expansion.



Table 1(abstract P13) Consistent marker expression at different passages of human ESC culture in GEM Novo medium 

containing RS Novo, determined by mean fluorescence intensity (MFI) of Oct-4 staining by flow cytometry 

Marker Control Optimized Conditions MFI GEM Novo & RS Novo MFI 

Passage 3 Oct-4 75.8 81.5 

Passage 8 Oct-4 66.1 81 

Figure 1(abstract P13) Human MSC expanded in GEM Novo containing RS Novo can later differentiate into adipocytes as demonstrated here by Oil Red 

O staining. 

P14 

Use of Micro Bioreactor systems to streamline cell line evaluation and 

upstream process development for monoclonal antibody production 

Steve R C Warr * , Jai Patel, Rongzan Ho, Katy V Newell 

GlaxoSmithKline, Stevenage, Hertfordshire, SG1 2NY, UK 


Introduction: The development of monoclonal antibody (mAb) processes 

conventionally involves the generation of multiple cell lines in multiwell 

plates, cell line screening in shake flasks followed by final cell line selection 

and process optimisation in bioreactors. This process can typically take up 

to 18 months to generate a robust process suitable for early phase 

manufacturing and therefore any opportunity to streamline this process 

impacts directly on drug development timelines. 

This work describes the potential integration of the Micro-24 Bioreactor 

system (Pall Life Sciences) and the Duetz Microflask system (Applikon 

Biotechnology) into cell line development and early process development 

and demonstrates how these systems can be used for cell line evaluation 

and process optimisation. The Micro-24 Bioreactor system comprises 24 

bioreactors (7ml working volume) each capable of independent 

temperature, dissolved oxygen and pH control. Cell cultures are carried 


out in a presterilised polycarbonate mammalian cell culture cassette with 

a central vent and are inoculated manually in a laminar flow cabinet 

before sealing with Type A single use closures and incubation under 

experimental conditions. 

Methods: The performance of a number of cell lines in the Micro-24 

Bioreactor and the Duetz Microflask system was compared to that in shake 

flasks and bioreactors using cell numbers, viability and product titre. This 

data was then used to rank cell lines according to specific parameters. 

Unless otherwise stated a standard hydrolysate containing complex 

medium and standard experimental conditions were used throughout this 

work. For shake flasks these were: 35°C, 5% CO 2, 140 rpm; for Duetz 

Microflasks: 35°C, 5% CO2, 200 rpm, 80% humidity and for Micro-24 

Bioreactors: 35°C, 650rpm, 6.95 pH, 30% DO. Viable cell numbers and 

viability were determined using a ViCell Cell Viability Analyser (Beckman 

Coulter) and antibody titres were determined using an Immunochemistry 

System (Beckman Coulter). 

Reproducibility: A hydrolysate containing complex media fed batch 

process was run in each of the 24 bioreactors using the same model CHO 

cell line. Viable cell numbers (VCC), viability and titre were measured in 

each bioreactor and the coefficients of variation (CV) were calculated for 

each time point. This data was also used to calculate the specific 

productivity (SPR) for each bioreactor. At each time point CVs were



Figure 1(abstract P14) Comparison of performance rank order of clones in different bioreactor systems in a hydrolysate containing medium batch 

process (Figure 1A and B) and fed batch process (Figure 1C, 1D, 1E and 1F). 

generally less than 10% for each parameter measured and there was no 

significant difference between different rows of the cassette. 

Cell Line Selection: To demonstrate the potential of this system to 

identify candidate mAb producing cell lines a series of experiments was 

carried out using the Micro-24 Bioreactor system and the results 

compared to those obtained in our standard cell line selection process. 

After initial screening in static multiwell plates during scale up a further 

screen in the Duetz Microflask system indicated significant differences 

in the performance of the remaining cell lines. In the standard 

hydrolysate containing batch process peak titres varied by up to 60% 

and there was a 2 fold difference in the overall SPR across the different 

cell lines. 

Based on titre and SPR data from the Duetz Microflask screen 12 of 

these cell lines were selected for further evaluation. Using the standard 

batch process conditions these cell lines were grown in parallel in the 

Micro-24 Bioreactors, Duetz Microflasks and conventional shake flasks. 

Although there were some differences in absolute titres the rank order 

of cell lines was similar in each of the systems tested here (Figure 1a 

and b) with R 2 =0.66 (Micro-24 v Duetz) and R 2 =0.72 (Micro-24 v shake 

flasks).The rank order of peak VCC was also similar in the Micro-24 and 

shake flasks (R 2 = 0.7) although there was less similarity in overall SPR 

(R 2 =0.4). 

This data from the batch process was used to select 6 cell lines for 

evaluation in the standard fed batch process which was run in parallel in 

the Micro-24 Bioreactor and shake flasks. 

For each cell line the effect of feeding was similar in Micro-24 Bioreactors 

to shake flasks (Figure 1C) and the rank orders of titre, VCC and SPR 

achieved in the Micro-24 Bioreactors were similar to those achieved in 

shake flasks (Figure 1D, E and F). 

Bioreactor validation: Clones ranked 1, 2, 5 and 12 in the Micro-24 

Bioreactors were tested in conventional 2 litre bioreactors. In bioreactors 

the titre produced by the top ranked clone was approximately 20% higher 

than the clone ranked 12. The remaining 2 clones (ranked 2 and 5 in the 

Micro-24) produced intermediate titres. 

Product quality: SEC, CE-IEF and NGHC data for the mAb produced in 

the Micro-24 showed no significant differences to that produced in 

control shake flasks. 

Process optimization: The Micro-24 Bioreactor also enables early stage 

process optimisation to be carried out at a small scale. The potential for 

media development is shown by the data in Table 1A which demonstrates 

that the same trend in peak titres is observed when cells are grown in 4 

different media in shake flasks, conventional bioreactors and the Micro-24. 

Similarly a further experiment in the Micro-24 Bioreactor to investigate the 

effect of pH and temperature stepdown on performance demonstrated 

Table 1(abstract P14) Process optimization 

Table 1A – Medium Development 

Medium Shake flasks (120mL) Bioreactor (2000mL) Micro-24 (7mL) 

Medium 1 100% 100% 100% 

Medium 2 129% 125% 124% 

Medium 3 121% 131% 140% 

Medium 4 

Table 1B – Condition Optimisation 

153% 175% 159% 

pH Temperature Relative titre 

Condition 1 High Low 58% 

Condition 2 High High 100% 

Condition 3 Low Low 96% 

Condition 4 Low High 141% 

Page 34 of 181



that condition dependent titre improvements could be identified using this 

instrument (Table 1B). 

Discussion: This data demonstrates that the Micro-24 Bioreactor can be 

used successfully to select high producing cell lines and to carry out 

initial process optimisation experiments. Although there were some 

differences in absolute data the rank orders and process trends identified 

in the Micro-24 Bioreactors were similar to those in conventional systems. 

Therefore this work has shown that the Micro-24 Bioreactor system could 

be used to replace shake flasks and bioreactors in cell line evaluation and 

early process improvement work. 

P15 

Cell line selection using the Duetz Microflask system 

Steve R C Warr * , Sharon L White, Yuen-Ting Chim, Jai Patel, Hella Bosteels 

GlaxoSmithKline, Stevenage, Hertfordshire, SG1 2NY, UK 


Introduction: The identification of a small number of monoclonal 

antibody (mAb) producing candidate cell lines from the large number of 

clones generated post transfection is one of the bottlenecks of cell line 

development. Clone numbers are reduced significantly during initial 

medium exchange and static scale up stages but significant numbers can 

still progress to evaluation in shaking cultures. This is often carried out in 

shake flasks where the number of clones that can be evaluated may be 

restricted due to resource limitations. 

The Duetz Microflask system is a microtitre plate based system which uses 

‘Sandwich Covers’ to convert the individual wells of 24 well plates into 

individual ‘mini reactors’. The‘Sandwich Covers’ consist of a stainless steel 


cover, a 0.2µm filter, microfibre inlays and a flexible silicone sealing layer 

to ensure adequate oxygen transfer rates to individual wells. 

This work describes the use of the Duetz Microflask system to evaluate cell 

lines in culture prior to scale up to production shake flasks. We have 

compared the growth and rank order of performance for a number of cell 

lines in the Duetz Microflask system with data obtained from shake flasks 

and demonstrated this system can be used successfully to identify 

candidate cell lines for further progression. 

A series of preliminary experiments was completed to determine suitable 

volumes and operating conditions to minimise evaporation. Unless 

otherwise stated these were: Fill volume – 1.5mL, Shaker speed – 200rpm, 

Humidity – 80%. 

Cell line evaluation: After initial transfection a typical cell line development 

process would involve plating cell lines to multiwell plates followed by 

several static media exchange, scale up and selection steps. These are used 

to reduce the number of cell lines carried forward to larger scale static 

T-flasks and eventually to shake flasks for further evaluation and selection. 

Although the multiwell plate steps are amenable to automation the manual 

handling aspects of culturing multiple cell lines in shake flasks can be 

resource limiting and therefore the ability to improve cell line selection using 

shaking multiwell plates offers significant time line and resource advantages. 

Complex medium process: The performance of a series of 28 mAb 

producing clones in hydrolysate containing media in Duetz Microflasks 

was compared with standard shake flask evaluation data. 

The rank order of titre performance of these clones in ‘Early’ Duetz 

Microflasks (ie inoculated before repeated shake flask subculture) was 

compared to that in shake flasks inoculated after the cultures had been 

subcultured for 6 passages. There were some differences in the rank orders 

resulting in a correlation R 2 = 0.46. However this improved significantly 

Figure 1(abstract P15) Titre rank order of clones in batch shake flasks compared with top clones in batch ‘Early’ ‘Late’ Duetz Microflasks (Figure 1A). 

Correlation of the % feed response (Figure 1B) and titre rank order (Figure 1C) of clones in a fed batch process in shake flasks and ‘Late’ Duetz 

Microflasks is also shown.



(R 2 = 0.92) when a further set of Duetz Microflasks (‘Late’ Duetz) was run in 

parallel to the flasks. 

This data indicates that although Duetz Microflasks can be used at an 

early screening stage to identify high producing clones the correlation 

with subsequent performance is improved after the cell lines are adapted 

to shaking growth conditions (ie after repeated subculture in shake 

flasks). 

Figure 1A shows that the top 8 cell lines identified in a ‘Late’ Duetz 

screen were the same as the top 8 lines identified in shake flasks. In 

contrast only 5 of the top 8 lines in the ‘Early’ Duetz screen were in the 

top 8 lines in shake flasks. Therefore the likelihood of discarding a high 

producing line is increased the earlier the Duetz screening is carried 

out. 

Similar results were obtained using a hydrolysate feed fed batch process 

in which the titre rank order and the response to feed in Duetz 

Microflasks was similar to that in shake flasks. Thus Figure 1B shows good 

correlation between the effect of the feed (% increase in titre) on cell 

lines in shake flasks and in ‘Late’ Duetz Microflasks and Figure 1C shows 

the correlation between the (titre) rank order in the two systems. 

Chemically defined medium process: A similar experiment was carried 

out with a different mAb producing CHO cell line in a chemically defined 

fed batch process. The standard (bioreactor and SF model) process 

involves a series of feeds during the fermentation. This multiple feed 

strategy was found to be impractical at the Microflask scale and so a 

compromise ‘single feed’ model was used in Duetz Microflasks. The 

correlation of rank order between ‘Early’ Duetz Microflasks and shake 

flasks was poor (R 2 = 0.34) but was significantly improved after Duetz 

Microflasks were inoculated with cells previously subcultured in shake 

flasks (R 2 = 0.73). 

Discussion: This data has demonstrated that, although there are some 

limitations around the adaptation of cell lines to shaking culture, Duetz 

Microflask screening can be used successfully to reduce the number of 

cell lines carried forward in the cell line selection process to shake flask 

or bioreactor screening. 

Although correlation with subsequent shake flasks is improved after 

repeated subculture (to allow complete adaptation to shaking) this data 

has also demonstrated that screening in Duetz Microflasks inoculated 

directly from static cultures or after minimal shake flask subculturing can 

be used to differentiate between cell lines that are high and low 

performers in conventional screening systems. We have used this system 

to reduce the number of cell lines carried forward to shake flask 

screening by approximately 70%. 


The simplicity of this system combined with the standard multiwell plate 

footprint facilitates automation and potentially enables large numbers of cell 

lines to be screened simultaneously and we are currently incorporating this 

system into our automated cell line generation process. 

P16 

Evaluation of an online biomass probe to monitor cell growth and cell 

death 

Angelo Perani 1* , Benjamin Gloria 1 , Dongmao Wang 1 , Anne-Sophie Buffier 2 , 

Olivier Berteau 2 , Geoffrey Esteban 2 , Fiona E Smyth 1 , Andrew M Scott 1 

1 Ludwig Institute for Cancer Research, Melbourne-Austin Branch, Heidelberg, 

Victoria, 3084, Australia; 2 Fogale Nanotech, Nimes, 30900, France 

E-mail: angelo.perani@ludwig.edu.au 


Background: The estimation of cell density and cell viability of mammalian 

cell lines in cell culture has traditionally been performed using the exclusion 

dye trypan blue that stains “dead” cells when their cell membrane is 

damaged. In large scale cell cultures using bioreactors this estimation is 

performed off-line. The online biomass probe is based on the principle that 

under the influence of an electric field between two electrodes, ions in 

suspension migrate toward the electrodes. The cell plasma membrane is 

non-conductive so that the cells with intact plasma membranes are 

polarized and act as tiny capacitors and it has been shown that capacitance 

increases as the cell concentration does. The measurement is based on the 

linear relationship between the permittivity difference ε1- ε2 and the viable 

biomass concentration as it has been described by Ansorge et al. [1]. This 

study compares the data obtained using the biomass probe against the cell 

counts and viability determined by trypan blue exclusion with two GS-CHO 

cell line productions in bioreactors. Apoptosis determination measurements 

using rhodamine-123 and pan-caspase activation by flow cytometry will be 

compared to permittivity values. 

Material and methods: Cell Culture – Production: CHO-K1 SV cells were 

transfected with the vector coding for LICR C and LICR D antibodies using a 

Glutamine Synthetase expression vector (Lonza) and maintained in CD-CHO 

(Invitrogen) + 25mM of methionine sulfoximine (MSX) (Sigma). LICR C S1: 3L 

stirred-tank bioreactor (STR) (Applikon) in batch mode + temperature shift 

in CD-CHO + 32mM MSX (base 1); LICR C S2: 3L STR (Applikon) in batch 

mode + temperature shift in CD-CHO + 32mM MSX (base 2). LICR D S1: 3L 

STR (Applikon) in batch mode + 32mM MSX with addition of 4, 10 and 

20 mM NH4+. Biomass: Online monitoring was performed with an iBiomass 

Figure 1(abstract P16) Detection of apoptosis using a pan- caspase inhibitor labeled with FITC and rhodamine 123 during early stages of cell culture 

using flow cytometry in parallel with the online measurement of Δε with the online biomass probe.



465 system (Fogale Nanotech). Viable Cell Concentration – Cell size: Viable 

Cell Concentration and cell size were estimated by off-line measurements 

using an automated trypan blue cell counter (Cedex, Roche). Apoptosis: 

Apoptosis related measurements were performed with a FACS Aria III 

(Becton Dickinson) using rhodamine-123 (Sigma) at 1mg/mL in buffered 

saline (PBS) for 10 6 cells or with 10 mM of CaspACE FITC-VAD-FMK 

(Promega) for 10 6 cells. FCS data was analysed with Gatelogic v3.08 (Inivai). 

Off-line measurement vs. on-line measurement: Correlation off-line and online 

measurements obtained by comparing the values of Xv.Dm4 to those of 

Δε (Xv: viable cell concentration estimated by Cedex in cells/mL; Dm: 

average diameter of cells estimated by Cedex in mm and Δε: cell 

permittivity measured by Fogale Biomass probe in pF/cm). Fluorescent 

Microscopy: 40μL of rhodamine 123 and CaspACE FITC-VAD-FMK stained 

samples were observed under fluorescent microscopy (FITC filter) with a 20x 

objective. 

Results: Permittivity vs. Cell Concentration: The comparison of the viable 

cell concentrations (Xv) profiles obtained with the Cedex and the 

permittivity profiles obtained with the biomass probe for the production 

of LICR C in bioreactors with different process controls show a clear 

correlation between the two measurements. The fitting of a linear 

regression curve gives a value of R 2 = 0.8777. (Data not shown). 

Diameter vs. permittivity related parameter: There is a direct correlation 

between the cell size (diameter) and viable cell concentration (Xv) measured 

by the Cedex and the permittivity measured with the biomass probe for the 

measurements obtained from LICR C bioreactor productions. The fitting of a 

linear regression curve gives a value of R 2 = 0.8798. (Data not shown). 

Apoptosis and permittivity: FACS analysis of a bioreactor run of LICR D cell 

lines under metabolic stress shows a different rhodamine-123 subpopulation 

distribution (forward scatter (FSC) vs. rhodamine 123 channel) 

less than 2h after first stress signal. There is no change in the caspase 

distribution (FSC vs. caspase). These results were confirmed with visual 

observation of the cells by fluorescence microscopy (Figure 1) using the 

same samples that were maintained in a FACS-fixing buffer: 16g of 

D-glucose (Sigma), 40% formaldehyde solution (Sigma) in 500 ml of 0.01M 

(1X) PBS pH7.2, stored at +4 0 C. 

Conclusion: The measurements obtained with the biomass probe 

demonstrated correlation between viable cell concentration and permittivity. 

These observations suggest that it is possible to use such a probe in the 

routine monitoring strategy for production of biopharmaceuticals using GS- 

CHO cell lines in bioreactors. Early detection of apoptosis in bioreactor 

cultures seems possible by using measurements obtained with the biomass 

probe. This type of measurements can be of critical value when developing 

processes that aim to minimise apoptosis. Future experiments need to be 

performed to correlate these observations with process parameter changes 

and mitochondrial modifications. 

Acknowledgement: The authors thank FOGALE Nanotech for the use of 

the iBiomass 465 unit and technical support. 

Reference 

1. Ansorge S, Esteban G, Schmid G: On-line monitoring of infected Sf-9 

insect cell cultures by scanning permittivity measurements and 

comparison with off-line biovolume measurements. Cytotechnology 2007, 

55:115-124. 

P17 

Impact on product quality of high productive GS-CHO cell lines 

Angelo Perani * , Benjamin Gloria, Dongmao Wang, Roger Murphy, 

Harjit Khangura Singh, Fiona E Smyth, Andrew M Scott 

Ludwig Institute for Cancer Research, Melbourne-Austin Branch, Heidelberg, 

Victoria, 3084, Australia 

E-mail: angelo.perani@ludwig.edu.au 


Background: The Clinical Program of the Ludwig Institute for Cancer 

Research (LICR) aims to translate basic laboratory discoveries into early 

phase clinical trials in cancer patients. A key component of the LICR 

approach has been to focus on the identification of antibodies selectively 

targeting antigens preferentially expressed in tumour tissue, and the 

molecular engineering of chimeric or humanised antibodies to these 

targets. The development of robust and high producing cell lines is 

crucial in the development of each antibody construct. The Cell Biology 

Group at the LICR Melbourne-Austin Branchisresponsibleforthe 


production of recombinant proteins including monoclonal antibodies 

from hybridomas or from industrially relevant mammalian cell lines (CHO, 

NS0, etc). The Cell Line Development activities are driven by the 

successful combination of industrial relevant cell lines transfected with 

cancer-relevant DNA targets using the Glutamine-Synthetase system (GS) 

from Lonza. Here we present two case-studies of high-productive cell 

lines in terms of product quality and biological activity of the protein 

produced with these cell lines. 

Material and methods: Cell line development / Production: CHO-K1 SV 

cells were transfected with the vector coding for monoclonal antibody 

LICR A within a GS expression vector (Lonza) and maintained in CD-CHO 

(Invitrogen) + 25μM methionine sulfoximine (MSX) (Sigma). LICR B (mu) 

murine hybridomas were generated against LICR B antigen and 

maintained in DMEM + 15% FBS (Invitrogen). Shake flask productivity 

screens were performed in E250 Erlenmeyer flasks with CD-CHO + 25μM 

MSX in batch mode for LICR A and LICR B and fed-batch for LICR B. 

Bioreactor productions: LICR A : 15L stirred-tanks bioreactors (STR) 

(Applikon) in batch mode + temperature shift in CD-CHO 32μM MSX;LICR 

B (mu): 15L STR in batch mode + temperature shift in DMEM (Invitrogen) + 

15% FBS and LICR B : 7L STR (Applikon) in fed-batch mode + temperature 

shift in CD-CHO + 32μM MSX. Purification: Supernatants were filtered and 

purified with Protein A eluted with 100mM glycine (pH 2.5), then adjusted 

to pH 8.0 with 1M Tris buffer then dialysed into PBS. ELISA assay: An antihuman 

IgG (g-chain specific) antibody for coating plates and an antihuman 

IgG (g-chain specific) alkaline phosphatase conjugate as secondary 

antibody. Purified antibody was used to set up a standard curve for 

calculating antibody concentration. Plates were read at 405 nm. 

SDS PAGE: Antibody (5μg) in a final volume of 20μl wasloadedunder 

both reducing and non-reducing conditions on NuPage, 4-12% Bis-Tris 

gels (Invitrogen) along with 20μl of molecular weight standards. Protein 

bands were detected with Coomassie blue staining. Biosensor analyses 

were conducted on a BIAcore 2000 instrument (GE). The epitope for the 

recombinant antibody was immobilized in a Ni-NTA chip and a blank 

control channel was for correction of refractive index effects. Sample at 

increasing concentrations were flowed over the chip surface and then 

washed out. The chip was regenerated with 0.1M EGTA between each 

analysis. FACS analysis was performed with a FACS Canto II cytometer 

(Becton Dickinson), Cell line expressing antigen A at 5x10 5 cells/sample; 

Negative control: isotype IgG (10μg /mL); Positive control: Purified LICR A 

(10μg/mL); Secondary antibody PE conjugated (1:1000) (Sigma); Data 

analysis using Gatelogic v 3.08 (Inivai). 

Results – discussion: LICR A: Production of the LICR A antibody 

increased from 265 mg/L in shake-flask production to 721 mg/L in 15L 

bioreactor (Results not shown). The FACS analysis of LICR A antibody with 

cells expressing the A antigen shows an increased binding when using 

purified LICR A antibody from the bioreactor production at 50 μg/mL 

(Mean Fluorescent Intensity (MFI) for PE from LICR A shake flask: 4591 

and MFI PE from LICR A bioreactor: 9396). Physicochemical analysis by 

SDS-PAGE of the product shows that the heavy and light chains (under 

reduced conditions) and the banding pattern (under non-reduced 

conditions) are typical for IgG. On SE-HPLC the main peak (94.7% for 

shake flask and 91.1% for bioreactor) is eluting at the expected size of an 

intact IgG. 

LICR B: Production of the LICR B antibody showed a continued increase 

from the production in bioreactor of the parental LICR B (mu): 109 mg/L 

to537mg/Linshake-flaskbatchmodeandto1252mg/Linshake-flask 

fed-batch mode (Results not shown). The highest productivity observed 

by ELISA was observed with the supernatant from the 7L bioreactor in 

fed-batch mode: 2244 mg/L. The BIAcore comparative analysis of the 

material purified from the LICR B (mu) bioreactor and LICR B bioreactor 

fed-batch shows an increased binding (decreased K d):KdLICRB(mu) 

bioreactor: 0.41 nM; K d LICR B bioreactor fed-batch: 1.13 pM (Figure 1). 

Physicochemical analysis by SDS-PAGE of the product shows that the 

heavy and light chains (under reducedconditions)andthebanding 

pattern (under non-reduced conditions) are typical for IgG. On SE-HPLC 

the main peak [96.8% for LICR B (mu) and 95.9% for LICR B] is eluting as 

an intact IgG (Results not shown). 

Conclusion: We have successfully constructed two GS-CHO cell lines 

producing functional intact antibodies against cancer-related epitopes. 

The product concentration was substantially increased for both cell lines 

without having a negative effect of product quality. Analyses of complex 

glycans present on LICR A and LICR B antibodies are to be performed.



Figure 1(abstract P17) Binding to cells expressing the LICR A antigen analysed by FACS for purified LICR A antibody and Binding to LICR B antigencoated 

chips analysed by BIAcore for purified LICR B antibody. 

P18 

Anti-diabetes effect of water containing hydrogen molecule and Pt 

nanoparticles 

Sanetaka Shirahata 1,2* , Takeki Hamasaki 1 , Keisuke Haramaki 2 , 

Takuro Nakamura 1 , Masumi Abe 1 , Hanxu Yan 2 , Tomoya Kinjo 2 , 

Noboru Nakamichi 3 , Shigeru Kabayama 3 , Kiichiro Teruya 1,2 

1 Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu 

University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; 2 Division of 

Life Engineering, Graduate School of Systems Life Sciences, Kyushu 

University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; 3 Nihon 

Trim Co. Ltd., 1-8-34 Oyodonaka, Kita-ku, Osaka 531-0076, Japan 

E-mail: sirahata@grt.kyushu-u.ac.jp 


Background: Electrochemically reduced water (ERW) contains a lot of 

hydrogen molecule (H 2) and scavenges reactive oxygen species (ROS) to 

protect DNA from oxidative damage [1]. ERW also contains small amounts of 

Pt nanoparticles (NPs) and elongates the lifespan of C. elegans [2]. Pt NPs are 

newly recognized multi-functional ROS scavengers [3]. ERW exhibits antidiabetes 

effects in vitro and in vivo [4-6][7]. We proposed mineral 

nanoparticle active hydrogen reduced water hypothesis to explain the 


activation mechanism of H 2 to hydrogen atom (H)[4]. Recently, H 2 has been 

reported to scavenge ROS and suppress a variety of oxidative stress-related 

diseases [8], however, the action mechanism of H 2 has not been clarified 

thoroughly. Here, we examined anti-diabetes effects of H2 and Pt NPs. 

Materials and methods: Pt NPs of 2-3 nm sizes were synthesized from 

H 2PtCl 6 by the citrate reduction method. L6 rat myoblast cells (1.2 x 10 5 

cells) were inoculated into a 35 mm culture dish and a day later, the cells 

were treated with or without 25mM N-acetylcystein in the presence of BES- 

H2O2, a H 2O 2-specific detection reagent in DMEM for 2 h. After washing the 

cells, molecular hydrogen treatment was performed in a dark condition by 

cultivating cells in a fresh DMEM medium in a mixed gas incubator under an 

atmosphere of 75%N 2/20%O 2/5%CO 2 or 75%(H 2 and N 2 mixed gas)/20%O 2/ 

5%CO 2 for 1.5 h, followed by flowcytometric analysis. In this condition, 

culture medium contained maximum 0.4-0.5 ppm of dissolved hydrogen. 

Glucose uptake of differentiated myotube L6 cells was examined after 

treating the cells with 3 H-2-deoxyglucose for 10 min. Gene expression of 

catalase (CAT), glutathione peroxidase (GPx) and hemoxoigenase (HO-1) was 

examined using RT-PCR method. Three weeks old type 2 diabetes model 

mice (KK-A y )werefedH 2 and/or Pt Nps-containing water ad lib for 6 weeks. 

Results: H 2 stimulated glucose uptake into L6 cells. Pt NPs catalyzed the 

activation of H 2 to hydrogen atom (H) to scavenge DPPH radical in vitro. 

The combined use of molecular hydrogen and Pt NPs resulted in



extremely stimulated glucose uptake into L6 cells, suggesting that H 

produced from H 2 by catalyst action of Pt NPs regulated glucose uptake 

signal transduction. As oppose to the paper by Ohsawa et al.[8], H 2 of 25 

to 75% concentration in the mixed gas significantly scavenged 

intracellular H 2O 2 in rat fibroblast L6 cells (Figure 1) and induced the 

gene expression of antioxidative enzymes such as CAT, GPx and HO-1 via 

activation of Nrf2 (Figure 2). H 2, Pt NPs and their combination 

significantly suppressed the levels of fasting blood glucose and improved 

Figure 1(abstract P18) The scavenging effect of hydrogen molecule on intracellular hydrogen peroxide in rat myotube L6 cells. ***, p



the impaired sugar tolerance abilities of obese insulin-resistant type 2 

diabetic KK-A y mice. 

Conclusion: H 2, Pt NPs, and their combined use resulted in activation of 

glucose uptake signal transduction pathways and stimulation of glucose 

uptake into L6 myotubes. In the groups of H2, Pt NPs and their combined 

use groups, blood sugar levels and impaired sugar tolerance of type 2 

diabetes model mouse (KK-A y ) were significantly improved, suggesting that 

H 2, Pt NPs and H are redox regulation factors in animal cells. 

References 

1. Shirahata S, Kabayama S, Nakano M, Miura T, Kusumoto K, Gotoh M, 

Hayashi H, Otsubo K, Morisawa S, Katakura Y: Electrolyzed-reduced water 

scavenges active oxygen species and protects DNA from oxidative 

damage. Biophys Biochem Res Commun 1997, 234:269-274. 

2. Yan H, Tian H, Kinjo T, Hamasaki T, Tomimatsu K, Nakamichi N, Teruya K, 

Kabayama S, Shirahata S: Extension of the lifespan of Caenorhabditis 

elegans by the use of electrolyzed reduced water. Biosci Biotech Biochem 

2010, 74:2011-2015. 

3. Hamasaki T, Kashiwagi T, Imada T, Nakamichi N, Aramaki S, Toh K, 

Morisawa S, Shimakoshi H, Hisaeda Y, Shirahata S: Kinetic analysis of 

superoxide anion radical-scavenging and hydroxyl radical-scavenging 

activities of platinum nanoparticles. Langmuir 2008, 24:7354-7364. 

4. Shirahata S, Hamasaki H, Teruya K: Advanced research on the health 

benefit of reduced water. Trends Food Sci Tech 2011, DOI 10.1016/j. 

tifs.2011.10.009. 

5. Li Y–P, Nishimura T, Teruya K, Maki T, Komatsu T, Hamasaki T, Kashiwagi T, 

Kabayama S, Shim S–Y, Katakura Y, Osada K, Kawahara T, Otsubo K, 

Morisawa S, Ishii Y, Gadek Z, Shirahata S: Protective mechanism of 

reduced water against alloxan-induced pancreatic b-cell damage: 

Scavenging effect against reactive oxygen species. Cytotechnology 2002, 

40:139-149. 

6. Li Y–P, Hamasaki T, Nakamichi N, Kashiwagi T, Komatsu T, Ye J, Teruya K, 

Abe M, Yan H, Kinjo T, Kabayama S, Kawamura M, Shirahata S: Suppressive 

effects of electrolyzed reduced water on alloxan-induced apoptosis and 

type 1 diabetes mellitus. Cytotechnology 2010, DOI 10.1007/s10616-010- 

9317-6. 

7. Kim M–J, Kim H–K: Anti-diabetic effects of electrolyzed reduced water in 

streptozotocin-induced and genetic diabetic mice. Life Sciences 2006, 

79:2288-2292. 

8. Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, 

Katsura K, Katayama Y, Asoh S, Ohta S: Hydrogen acts as a therapeutic 

antioxidant by selectively reducing cytotoxic oxygen radials. Nature Med 

2007, 13:688-694. 

P19 

Metabolic enhancers: a new paradigm in cell culture media 

optimization 

Abi M Abitorabi 1* , Christopher Wilcox 2 

1 2 

GIRUS Life Sciences, Inc., Sunnyvale, CA, 94085, USA; Sheffield BioScience, 

Beloit, WI, 53511, USA 

E-mail: abi@girusinc.com 


Our primary focus is enabling and accelerating biological drug 

manufacture through the development of cost-effective technologies that 


facilitate rapid bioprocess development and improve manufacturing 

“bang-for-the-buck”. Using data mining and cell-based screens we have 

designed the Regocel small molecule yield enhancers for a number of 

mammalian cell platforms used in biomanufacturing such as CHO and NS0 

cells. Chemically defined molecules were screened against several 

parameters: growth, viability and most importantly protein production. 

Formulations were developed that are defined, animal-free, non-nutritional 

and compatible with different media, nutritional supplements, culture 

formats and scales. Because they target ubiquitous cellular pathways such 

as apoptosis, cell cycle and protein synthesis, these formulations provide 

consistent results with a variety of clones and can shorten bioprocess 

times and cost. Our results with Regocel supplements demonstrated 

increased protein production in a variety of commercial media (Figure 1), 

increased yields of different proteins such as antibodies and mammalian 

target of rapamycin (mTOR), immediate yield enhancements (from passage 

1), and persistence of yield enhancements over many passages with no 

permanent alterations to cells, as determined by return of productivity to 

control levels upon Regocel supplement removal from the medium. 

These new small molecule formulations provide a new means of 

improving cell culture outcome independently of mammalian cell clone, 

cell culture medium and process. 

P20 

Neuroprotective effects of 3,5-di-o-caffeoylquinic acid in vitro and 

in vivo 

Junkyu Han 1,2 , Hiroko Isoda 1,2* 

1 Graduate School of Life and Environmental Sciences, University of Tsukuba. 

Tsukuba, Ibaraki 305-8572, Japan; 2 Alliance for Research on North Africa 

(ARENA), University of Tsukuba. Tsukuba, Ibaraki 305-8572, Japan 

E-mail: isoda.hiroko.ga@u.tsukuba.ac.jp 


Background: Caffeoylquinic acid (CQA) derivatives are natural functional 

compounds isolated from a variety of plants and possess a broad range of 

pharmacological properties, including antioxidant, hepatoprotectant, 

antibacterial, antihistaminic, anticancer, and other biological effects [1]. 

Recently, it has been demonstrated that CQA derivatives possess 

neuroprotective effects in Ab-induced PC12 cell toxicity and in 

tetrahydropapaveroline (THP)-induced C6 glioma cell death [2]. One of the 

animal models that is used to study AD and aging is the senescenceaccelerated 

mouse (SAM). The SAM model was developed in 1981, which 

originally consisted of nine major senescence-accelerated-prone mice 

(SAMP) substrains and three major senescence-accelerated-resistant mice 

(SAMR) substrains, each of which exhibits the characteristic disorders. 

Methodology: As in vitro experiment, the human neuroblastoma clonal 

SH-SY5Y cell were maintained at 37°C under 5% CO 2 /95%air.Asin vivo 

experiment, the CQA-treated mice were orally administered with 3,5-di-O- 

CQA mixed with drinking water (6.7 mg/kg · day) for 1 month using oral 

administration tube and syringe. Proteomics analysis, real-time PCR, 

measurement of intracellular ATP content, Moris water maze were carried 

out to investigate the neuroprotective effect of CQA. 

Results: 3,5-di-O-CQA had neuroprotective effect on Ab 1–42 treated cells. 

The mRNA expression of glycolytic enzyme (phosphoglycerate kinase-1; 

PGK1) and intracellular ATP level were increased in CQA treated SH-SY5Y 

Figure 1(abstract P19) Antibody production by Chinese hamster ovary (CHO) cells increase with the Regocel small molecules (RS-CHO) in different 

commercial media (M1-M6).



Figure 1(abstract P20) Effect of CQA on the spatial learning and memory of SAMP8 mice in MWM. 

cells. We also found that CQA administration induced the improvement of 

spatial learning and memory on SAMP8 mice, and the overexpression of 

PGK1 mRNA. 

Conclusion: CQA has a neuroprotective effect on Ab1–42 treated SH-SY5Y 

cells. The mRNA expression of glycolytic enzyme (PGK1) and the intracellular 

ATP level were increased in CQA-treated SH-SY5Y cells. We also found that 

CQA administration induced the improvement of spatial learning and 

memory on SAMP8 mice, and the overexpression of PGK1 mRNA level. 

These findings suggest that CQA has a neuroprotective effect through the 

induction of PGK1 expression and ATP production activation. 

References 

1. Basnet P, Matsushige K, Hase K, Kadota S, Namba T: Four di-O-caffeoyl 

quinic acid derivatives from propolis. Potent hepatoprotective activity in 

experimental liver injury models. Biol Pharm Bull 1996, 19:1479-1484. 

2. Soh Y, Kim JA, Sohn NW, Lee KR, Kim SY: Protective effects of quinic acid 

derivatives on tetrahydropapaveroline induced cell death in C6 glioma 

cells. Biol Pharm Bull 2003, 26:803-807. 

P21 

Improvement of insulin resistance by Cyanidin 3-glucoside, anthocyanin 

from black beans through the up-regulation of GLUT4 gene expression 

Tetsuya Inaguma 1 , Junkyu Han 1,2 , Hiroko Isoda 1,2* 

1 Graduate School of Life and Environmental Sciences University of Tsukuba, 

Tsukuba, Japan; 2 Alliance for Research on North Africa (ARENA) University of 

Tsukuba, Tsukuba, Japan 



Introduction: Black beans have been suggested to have a protective effect 

against obesity. Cyanidin 3-glucoside (Cy-3-G) belongs to the flavonoid class 

of molecules and is a member of the anthocyanin family that is present in 

black beans. Some studies indicated that Cy-3-G from black beans may be 

beneficial for the improvement of obesity and type Ⅱ diabetes. It has been 

reported that Cyanidin 3-glucoside ameliorates insulin sensitivity due by 

down-regulating the retinol binding protein 4 expression in diabetic mice [1]. 

Accumulation of neutral lipids, triglyceride (TG) in particular, is highly related 

to the development of insulin resistance and its consequences, such as type 

Ⅱ diabetes. Lipid droplet size regulation is central in the regulation of 

metabolism and in adipocytokines secretion such as TNF-a and adiponectin 

[2,3]. However, little is currently known about how Cy-3-G influences 

adipocyte differentiation and insulin resistance in 3T3-L1 adipocytes. The 

purpose of the present study was to determine the effects of Cy-3-G on 

GLUT4 gene expression to improve insulin resistance in 3T3-L1 adipocytes. 

Materials and methods: 

Cell culture and treatment: Adipocytes, 3T3-L1 cells (Riken Cell Bank, 

Ibaraki, Japan), were maintained in Dulbecco’s modified Eagle’s medium 

(DMEM) (Sigma Chemical Co, St. Louis, MO, USA) supplemented with 10% 


fetal bovine serum (FBS) (Biowest, Miami, FL, USA, Japan) and 1% 5,000 

units/ml penicillin, 5,000 μg/mL streptomycin (PS) (Lonza Walkersville, 

MD,USA) and incubated in 37 °C in 5% CO 2. For adipocyte differentiation, 

3T3-L1 cells were cultured at 3.0×10 5 cells/well in 6-well plates. Cells were 

maintained until full confluence, which is often around 48h, prior to 

treatment, . And then, cells were treated with differentiation medium 

containing Dexamethazone (DEX) Solution, 3-Isobuthyl-1-Methylxanthine 

(IBMX) Solution, Insulin Solution, and Cy-3-G. DEX Solution, IBMX Solution, 

and Insulin Solution were purchased from Cayman Chemicals (Ann. Arbor, 

MI, USA). Cy-3-G derived from black soybean was purchased from Fujicco 

Co., Ltd. (Kobe, Japan). After 72 h of induction, medium was changed to 

DMEM containing insulin and Cy-3-G was added every 2 days. After 4 

days of incubation from the initiation of differentiation, bioassays were 

performed. 

RNA extraction and real-time PCR: Total RNA was isolated from the 3T3- 

L1 cells using ISOGEN (Nippon Gene, Tokyo, Japan) according to the 

manufacturer’s instructions. The integrity of the RNA extracted from all 

samples was verified and quantified using NANO DROP 2000 (Agilent 

Technologies, CA). Total RNA was used for the single strand cDNA 

synthesis with a cDNA synthesis kit, SuperScript® III Reverse Transcriptase. 

Gene expression levels were analyzed by quantitative real-time PCR, using 

the Applied Biosystems 7500 FAST INSTURMENT (Applied Biosystems, 

Foster City, CA, U.S.A.). The oligonucleotide primers of mouse were 

obtained from Applied Biosystems (Foster City, CA, U.S.A.). The cDNA was 

denatured at 95 °C for 10 min, followed by 40 cycles of PCR (95 °C, 15 sec; 

60 °C, 60 sec). 

Results and discussion: In Cy-3-G-treated cells, the number of droplets 

increased, while the lipid droplet size decreased. Cy-3-G significantly 

promoted the mRNA expressions of peroxisome proliferrator-activated 

receptor-g (PPARg) [4], CCAAT/enhancer binding protein a (C/EBPa) [4], 

and glucose transport 4 (GLUT4) [Table 1] in differentiated 3T3-L1 cells. 

PPARg and C/EBPa mainly control adipocyte differentiation. GLUT4 takes in 

glucose on cell membrane. According to the results, Cy-3-G promotes 

adipocyte differentiation and uptake of glucose in a dose-dependent 

manner. In the present study, the concentrations of Cy-3-G treated were 

20 μM, and 100 μM. Cy-3-G at 20 μM, and 100 μM significantly decreased 

TNF-a concentration and ROS production, and increased the adiponectin 

concentration in differentiated 3T3-L1 cell in a dose-dependent manner 

[4]. These effects suggest that Cy-3-G would contribute to the 

improvement of insulin resistance and its consequences. However, when 

we utilize black beans as food, the doses of Cy-3-G in black beans would 

be lower than the concentrations that we used in this study. Therefore, the 

study on the effects of Cy-3-G at lower concentrations, such as 1 μM, 5 μM 

and 10 μM, on adipocyte differentiation and adipocytokines secretion in 

differentiated 3T3-L1cells will be needed. Also, the anti-fat effects of Cy-3- 

G in vivo using diabetic model mice have not yet been widely reported. 

From our study, Cy-3-G derived from black bean as a food factor is 

expected to prevent life style-related disease.



Table 1(abstract P21) Effect of Cy-3-G on the gene 

expression of GLUT4 in 3T3-L1 adipocytes 

Sample Relative GLUT4 gene expression level 

(% of Control) 

Control 100 ± 1.5 

Cy-3-G 20 µM 208.2 ± 1.4 * 

Cy-3-G 100 

226.57 ± 15.2 

µM 

* 

Cy-3-G significantly induced the gene expression of GLUT4 in a dosedependent 

manner. *p< 0.05 (vs Control). 

Conclusion: Cy-3-G has the potential to improve insulin resistance and its 

consequences in 3T3-L1 adipocytes through the up-regulation of GLUT4 

gene expression. Additional study on insulin resistance using 3T3-L1and 

anti-fat effect using diabetic model mice will be needed to verify these 

results in vivo. 

Acknowledgement: This research was partially supported by JST-JICA’s 

Science and Technology Research Partnership for Sustainable 

Development (SATREPS). 

References 

1. Sasaki R, Nishimura N, Hoshino H, Isa Y, Kadowaki M, Ichi T, Tanaka A, 

Nishiumi S, Fukuda I, Ashida H, Horio F, Tsuda T: Cyanidin 3-glucoside 

ameliorates hyperglycemia and insulin sensitivity due to downregulation 

of retinol binding protein 4 expression in diabetic mice. Biochem Pharm 

2007, 74:1619-1627. 

2. Okuno A, Tamemoto H, Tobe K, Ueki K, Mori Y, Iwamoto K, Umesono K, 

Akanuma Y, Fujiwara T, Horikoshi H, Yazaki Y, Kadowaki T: Troglitazone 

increases the number of small adipocytes without the change of white 

adipose tissue mass in obese Zucker rats. J Clin Invest 1998, 

101:1354-1361. 

3. Kadowaki T: Insights into insulin resistance and type 2 diabetes from 

knockout mouse models. J Clin Invest 2000, 106:459-465. 

4. Han J, Inaguma T, Isoda H: Cyanidin 3-glucoside anthocyanin from black 

beans has potential to protect insulin resistance on 3T3-L1 adipocytes 

by inhibiting TNF-a release. Br J Nutr , (in press). 


P22 

Engineering CHO cell growth by stable manipulation of miRNA expression 

Noëlia Sanchez * , Nga Lao, Clair Gallagher, Martin Clynes, Niall Barron 

National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, 

Ireland 


Background: MiRNAs are small non-coding RNAs involved in many 

biological functions such as cell proliferation and apoptosis (1), cell cycle (2), 

homeostasis (3) and cell metabolism (4). They are highly conserved between 

species. They are capable of regulating hundreds of genes in a posttranscriptional 

manner by translation repression and/or mRNA degradation 

(5). These characteristics make miRNAs attractive tools for CHO cell 

engineering as multiple genes may be targeted simultaneously and possibly 

entire biological pathways can be manipulated. After temperature-shifting 

CHO cell culture from 37°C to 31°C, several miRNAs were found differentially 

regulated of which miR-7 was found to be down-regulated. Our laboratory 

has previously demonstrated that transient overexpression of miR-7 using 

mimics of mature endogenous miR-7, leads to a significant decrease in cell 

growth (6). On the other hand, transient inhibition using inhibitors of 

mature endogenous miR-7 provoked increased cell growth, though to a 

lesser extent. As this antisense technology is transient, another strategy was 

chosen to study the impact of stable miR-7 inhibition on CHO phenotypes. 

“Sponge” technology is an effective tool to stably sequester endogenous 

miRNAs (7) by introducing a decoy target reporter gene. This approach can 

be used to understand post-transcriptional regulation in CHO cells and to 

identify cellular pathways related to cell growth, cell viability and cell 

productivity - key phenotypes for cell bioprocess improvement. 

Materials and methods: Binding sites for either miR-7 (sponge miR-7) or a 

non-specific control sequence (sponge NC) were inserted into pCMV-deGFP 

(Professor.Sharp’s laboratory). SEAP-secreting CHO-K1 cells were transfected 

with 5µg of these constructs in a 6-well plate. Selection pressure was 

applied on day 1 after transfection by addition of hygromycin at 350µg/ml. 

Cells were transferred to a T-75 flask. From the mixed population, cells with 

high and medium GFP positivity were selected for single cell sorting using 

FACS flow cytometer in 96-well plates. Expanded clones were transferred to 

24-well plates in adherent and suspension culture. GFP positivity and cell 

growth were monitored by Guava EasyCyte flow cytometer. 

Figure 1(abstract P22) CHO-K1 SEAP cell growth in sponge NC and sponge miR-7 expressing cell clones at day 3 of cell culture.P-value=0.0024 

in student t-test.



Results: Design of sponge NC/miR-7: Four tandemly repeated miR-7 

binding sites were cloned downstream of a destabilised enhanced GFP to 

increase efficacy of endogenous miRNA sequestration. The negative 

control consisted of the same cassette with non-specific sequences to 

replace miR-7 binding sites. The deGFP, a modified enhanced GFP, 

consists of a fusion of the ornithine decarboxylase degradation domain 

from mouse (MODC) and the C-terminal of an enhanced variant of GFP 

(eGFP) conferring a shorter half-life than eGFP (2h). The change of deGFP 

fluorescence induced by the binding of endogenous miR-7 to the sponge 

can be correlated to the change in active miRNA levels. To avoid RNAitype 

cleavage by Argonaute 2 and degradation, the sponges were 

designed with a bulge region (position 9-11). 

Impact of sponge miR-7 on CHO phenotypes in a mixed population: 

After transfection of the sponge miR-7 and sponge NC in SEAP-secreting 

CHO-K1 cells, GFP positivity and cell growth were monitored in both mixed 

populations and single clones. In mixed populations, sponge miR-7 

transfected cells were found to have reduced GFP positivity compared to 

control pools (sponge miR-7: 34.9% and control 56.1%) and significantly 

improved cell density at day 3 (1.6x10 6 cells/ml versus 1.16x10 6 cells/ml 

respectively) and at day 4 of culture (3.4x10 6 cells/ml and 2.9x10 6 cells/ml, 

respectively). 

Impact of sponge miR-7 on CHO phenotypes in single clones: In single 

clones, the average of GFP positivity was lower in sponge miR-7 expressing 

clones than in control clones (49.6% compared to 30.9%). The average cell 

density for 23 clones was significantly higher in sponge miR-7 expressing 

clones than in the control expressing clones (1.3x10 6 cells/ml and 

1.01x10 6 cells/ml). Moreover, 12 clones from the sponge miR-7 expressing 

clones achieved more than 1.4x10 6 cells/ml whereas only one clone from 

the control expressing cells could reached this density. (Figure 1). 

Conclusions: In this study, we showed that miRNAs may be used as tools 

to improve CHO phenotypes, in this case cell density, and also as potential 

tools for understanding of CHO cellular pathway regulation. The application 

of miRNA expression manipulation in large-scale culture could be a novel 

technology to increase significantly the performance of biopharmaceutical 

production processes. 

Acknowledgements: This work was supported by NICB, DCU and 

Science Foundation Ireland. 

References 

1. Cheng AM, Byrom MW, Shelton J, Ford LP: Antisense inhibition of human 

miRNAs and indications for an involvement of miRNA in cell growth and 

apoptosis. Nucleic Acids Res 2005, 33(4):1290-7. 

2. Lal A, Navarro F, Maher CA, Maliszewski LE, Yan N, O’Day E, et al: miR-24 

inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle 

genes via binding to “seedless” 3 ‘ UTR MicroRNA recognition elements. 

Mol Cell 2009, 35(5):610-25. 

3. Li X, Cassidy JJ, Reinke CA, Fischboeck S, Carthew RW: A MicroRNA imparts 

robustness against environmental fluctuation during development. Cell 

2009, 137(2):273-82. 

4. Gao P, Tchernyshyov I, Chang T, Lee Y, Kita K, Ochi T, et al: c-myc 

suppression of miR-23a/b enhances mitochondrial glutaminase 

expression and glutamine metabolism. Nature 2009, 458(7239):762-U100. 

5. Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, et al: 

Microarray analysis shows that some microRNAs downregulate large 

numbers of target mRNAs. Nature 2005, 433(7027):769-73. 

6. Barron N, Kumar N, Sanchez N, Doolan P, Clarke C, Meleady P, et al: 

Engineering CHO cell growth and recombinant protein productivity by 

overexpression of miR-7. J Biotechnol 2011, 151(2):204-11. 

7. Ebert MS, Neilson JR, Sharp PA: MicroRNA sponges: Competitive 

inhibitors of small RNAs in mammalian cells. Nature Methods 2007, 

4(9):721-6. 

P23 

Expression of recombinant human coagulation factors VII (rFVII) and IX 

(rFIX) in various cell types, glycosylation analysis, and pharmacokinetic 

comparison 

Ernst Böhm 1* , Michael Dockal 1 , Michael Graninger 1 , Meinhard Hasslacher 1 , 

Martin Kaliwoda 1 , Christian Konetschny 1 , Artur Mitterer 1 , Eva-Maria Muchitsch 2 , 

Manfred Reiter 1 , Friedrich Scheiflinger 2 

1 2 

Baxter BioScience, Orth/Donau, A-2304, Austria; Baxter BioScience, Vienna, 

A-1220, Austria 



Introduction: Clearance mechanisms for rFVII (or the active enzyme rFVIIa) 

and rFIX are influenced by post-translational modifications, especially 

N-glycosylation. This should be considered when choosing a recombinant 

expression system in view of the varying ability of frequently used cell lines 

to perform modifications similar to human proteins. 

Differences in the pharmacokinetic properties of recombinant FVIIa versus 

plasma-derived (pd)FVII or desialylated rFVIIa are known for human FVII(a). 

Asialo rFVIIa clears quickest, whereas pdFVII, having a higher degree of 

sialylation, is cleared to a lesser extent [1]. In the case of FIX, the degrees 

ofserinephosphorylationandtyrosinesulfationintheactivationpeptide 

have been postulated to influence pharmacokinetic behavior, especially 

in vivo recovery [2]. 

We chose CHO, BHK and HEK293 cells for expression of rFVII to compare 

post-translational protein modifications, and HEK293-derived cell lines to 

generate highly phosphorylated and sulfated rFIX for in vivo studies. rFIX 

from the same clone and production run was purified using two different 

down-stream processes: The first to enrich high phosphorylated and 

sulfated protein, the second to purify total rFIX at high yield. These HEK293derived 

rFIX isoforms were compared with CHOrFIX and pdFIX in a 

pharmacokinetic study in FIX knock-out mice. 


Proteins: pdFVII and pdFIX were from HTI, recombinant, CHO-derived FIX 

was from Wyeth. 

Cell culture: BHK (BHK-21; ATCC#CCL-10) and HEK293 cells (ATCC#1531) 

were from American Type Culture Collection, CHO DXB11 from University 

of Columbia. All were cultivated in DMEM/Ham’s F12 medium containing 

fetal bovine serum (FBS). BHK and HEK293 cell-derived rFVII producer 

clones were selected by antibiotic resistance. CHO DXB11-derived 

producer clones were generated by methotrexate gene co-amplification. 

rFVII was produced in vitamin K-containing medium without FBS. 

HEK293 cells producing human FIX were selected by antibiotic resistance. 

Clones were adapted to serum-free suspension culture in Excell293 medium 

containing vitamin K, and cultivated in repeated batch mode fermentation 

runs. 

Purification: rFVII was purified using Q-Sepharose FF for capture, and 

a tandem step containing a cellufine sulfate column connected to 

Q-Sepharose FF. 

HEK293-derived rFIX from the same clone from one fermentation run was 

purified from culture supernatants with two different purification schemas: 

for high yield, rFIX was loaded in the presence of EDTA on Q-Sepharose FF, 

the column was washed with EDTA at high salt concentration, FIX was 

eluted at low salt CaCl2. For enrichment of the highly phosphorylated and 

sulfated protein fraction, rFIX was loaded in the presence of EDTA on 

fractogel TMAE, washed with EDTA at high salt concentration, and eluted 

with a salt gradient in the presence of CaCl 2. 

Analytics: Purified FVII forms were analyzed using reversed phase HPLC, 

LC-MS for intact protein or peptide mapping after trypsin digestion to 

confirm sequences and post-translational modifications, and anion 

exchange HPLC for monosaccharide composition, sialic acid quantification, 

and comparison of N-glycans. 

Degrees of FIX-phosphorylation/sulfation were monitored by LC-MS. Nglycosylation 

was characterized by oligosaccharide mapping (HPLC 

method). 

In vivo pharmacokinetic study: FIX preparations were administered to 

FIX-knock-out mice intravenpusly at a nominal dose of 75 U/kg. Citrate 

plasmas were obtained by heart puncture. FIX levels in plasmas were 

determined by antigen enzyme-linked immunoassay and by activated 

partial thromboplastine time (APTT) clotting assay. 

Results and conclusions: Identical protein structures and minor differences 

in post-translational modifications between rFVII from BHK, CHO, HEK293 

cells, and pdFVII were found, except for N-glycosylation. Protein activities of 

rFVII determined by antigen and activity assays were similar for each cell 

type. Most N-glycans of pdFVII are bi- and triantennary, non-fucosylated 

structures with almost complete sialylation [3]. In contrast, all rFVII forms 

were sialylated only in part. Approximately 50% N-glycans of BHKrFVII were 

composed of a core-fucosylated biantennary complex-type with two 

terminal sialic acids. Another 20% contained terminal GalNAc, a sugar 

moiety with a high affinity for the asialoglycoprotein receptor involved in 

the in vivo clearance of glycoproteins from circulation. GalNAc were not 

found on N-glycans on CHO-derived rFVII. Most oligosaccharides on 

CHOrFVII were core fucosylated, biantennary structures carrying two sialic 

acid residues. HEK293rFVII showed major differences from all other rFVII



Table 1(abstract P23) AUCs of pdFIX, CHOrFIX, HEK293rFIX, and high-phospho/sulfo HEK293rFIX in a FIX-knock-out 

mouse pharmacokinetic study. Only relative AUC values are shown. Statistically significant different ratios to CHOrFIX 

(at the 5 % level) are marked with an asterisk*. The ratio of HEK293rFIX high phospho/sulfo to HEK293- total rFIX was 

also statistically significantly different for both antigen and activity 

Relative value of AUC: antigen Relative value of AUC: clotting activity 

pdFIX 1.5* 1.8* 

CHOrFIX 1 1 

HEK293rFIX high phospho/sulfo 0.7* 0.7* 

HEK293rFIX 0.4* 0.4* 

forms, and, unexpectedly, from pdFVII. Most oligosaccharide structures were 

not comparable to those published for pdFVII [3], or found on rFVII from 

the other cell lines. A high content of fucosylation and terminal GalNAc, 

and a lower degree of terminal sialic acids were measured, and tri- or tetraantennary 

structures were not found. Some oligosaccharides were of a 

hybrid type with high-mannose structures. These N-glycan structures do not 

favor the use of HEK293 cells as a production platform for human 

biotherapeutics, when protein recovery and half-life in the circulation are of 

importance, and influenced by N-glycosylation. 

Pharmacokinetic differences between pdFIX and CHO-derived rFIX were 

as expected from the literature; HEK293rFIX materials (total rFIX and 

high phospho/sulfo rFIX) were both inferior to CHO-derived rFIX in terms of 

area under the curve (AUC) (Table 1) and in vivo recovery. Similar terminal 

half-lives and mean residence times, but different in vivo recoveries between 

rFIX and pdFIX preparations were confirmed by statistical analysis. High 

phospho/sulfo rFIX from HEK293 had a larger AUC and in vivo recovery than 

total rFIX from the same HEK293-derived clone and fermentation run, 

indicating an influence of these modifications on pharmacokinetics (Figure 1). 

Oligosaccharide mapping showed high glycosylation heterogeneity 

for HEK293 and pdFIX, and more defined peak-groups representing mono- to 

tetrasialylated glycans for CHO-derived material. Sialic acid content of Nglycans 

was 25% lower for HEK293- than for CHOrFIX. Glycosylation and 

phosphorylation/sulfation degrees contributed to pharmacokinetics of FIX 

preparations. If glycosylation was similar in case of the two HEK293rFIX 

preparations, the effects of phosphorylation and sulfation became evident. 

In conclusion, these data showed that HEK293 cells were not adequate for 

rFIX or rFVII production due to improper N-glycosylation compared with the 

material from other cell lines, or from human plasma. Consequently, an 


advantage of this human cell line for the production of “more-human-like” 

biotherapeutics could not be observed. 

References 

1. Appa RS, Theill C, Hansen L, Møss J, Behrens C, Nicolaisen EM, Klausen NK, 

Christensen MS: Investigating clearance mechanisms for recombinant 

activated factor VII in a perfused liver model. Thromb Haemost 2010, 

104(2):243-251. 

2. Ewenstein BM, Joist JH, Shapiro AD, Hofstra TC, Leissinger CA, Seremetis SV, 

Broder M, Mueller-Velten G, Schwartz BA, Mononine Comparison Study 

Group: Pharmacokinetic analysis of plasma-derived and recombinant F 

IX concentrates in previously treated patients with moderate or severe 

hemophilia B. Transfusion 2002, 42(2):190-197. 

3. Fenaille F, Groseil C, Ramon C, Riandé S, Siret L, Chtourou S, Bihoreau N: 

Mass spectrometric characterization of N- and O-glycans of plasmaderived 

coagulation factor VII. Glycoconj J 2008, 25(9):827-842. 

P24 

Complex medium supplements optimized for the reduction of 

animal-derived components in vaccine production media 

James F Babcock * , Karen A Benedict, Amanda L Perlman 

Sheffield Center for Cell Culture Technology, Sheffield Bio-Science, A Kerry 

Group Business, Ithaca, NY USA 

E-mail: james.babcock@kerry.com 


Introduction: A series of cell-line specific complex media supplements 

have been developed for enhancing performance of various vaccine 

Figure1(abstract P23) Pharmacokinetic comparison of pdFIX, CHOrFIX, HEK293rFIX, and high-phospho/sulfo HEK293rFIX in FIX-knock-out mice. Plasma 

concentrations of FIX after administration are shown as mean values ± standard deviations with n = 10 mice for each timepoint. Dose adjustment was 

done by normalizing to start material concentrations.



production systems. Created using in-house media optimization methods, 

these supplements are manufactured using innovative process technology 

which allows for the formulation of complex and/or chemically 

defined animal-component free media additives into a single homogenized 

functional supplement. These supplements have been optimized 

for individual cell lines, and have proven to be suitable for use in a range 

of basal media. Data are presented which demonstrate the effectiveness 

of these optimized supplements for application as performance 

enhancers, and as a vehicle for serum-reduction in CEF, MDCK and BHK 

culture media. While these particular supplements do contain some 

undefined components, preliminary research indicates that this same 

technology may be applied to chemically-defined, multi-component 

supplements. 

Materials and methods: CEF and MDCK cells were maintained in T-75 

flasks with a working volume of 15 ml. CEF cells were grown in DMEM + 

5% Tryptose Phosphate Broth (TPB) + 5% FBS. MDCK cells were grown in 

DMEM + 10% FBS. 

To generate growth curves for both CEF and MDCK, triplicate T-25 flasks 

were seeded at 2.0 x10 5 cells/ml with a working volume of 7.5 ml. Cells were 

incubated at 37°C in 5% CO 2 . One set of triplicates was set up for each day 

cells were to be counted. On days 3-6, one set of triplicates was trypsinized 

and re-suspended in fresh medium for counting. 

BHK cultures were grown in 125 ml shake-flasks containing a final medium 

volume of 35 ml. The basal medium consisted of DMEM plus 10% FBS. 

Triplicate cultures were seeded at 2.0 x10 5 cells/ml, and incubated at 37°C 

in 5% CO 2 at 130 rpm for 12 days. Hydrolysate supplementation was 

achieved via the use of filter-sterilized 100 g/l stock solutions prepared in 

the basal medium. 

On days 3-7, 1.0 ml of the culture supernatants were removed for assessing 

cell counts and viability. All cells were counted using a Nova BioProfile Flex 

automated cell counter. 

Results: In a medium for Chicken Embryo Fibroblast (CEF) cells, incorporation 

of non-animal components into a complex supplement allows for 

reduction in the final concentration of serum in the basal medium 

formulation. This results in a significant reduction in animal-derived products 

as compared to the traditional CEF medium formulation containing 5%TBP + 

5% FBS. The non-animal supplement out-performs the conventional 

medium with respect to cell growth when the serum level is reduced to 1% 

(Figure 1). 

As with the CEF cells, various hydrolysates were screened to determine 

the most compatible with MDCK cells. Sheff-VAX is a hydrolysate-based 

animal component free supplement which can be employed to reduce 

FBS concentration from 10% to 3% or less. When Sheff-VAX is used in 

conjunction with 3% FBS, higher cell densities are achieved than with the 

control medium containing 10% FBS. 

In BHK-21 culture, supplementation with Sheff-VAX or Sheff-VAX Plus ACF 

allows for significantly improved cell counts while simultaneously 


reducing the serum requirement. The control media for both cell lines 

contain 10% FBS, and both cell lines reached similar maximum cell 

densities. However, the magnitude of improvement achieved with the 

animal component free supplements was significantly greater with the 

BHK-21 cells as compared to the MDCK cells. 

Summary: The data presented illustrate how complex animal component 

free supplements may be employed to reduce or eliminate serum from 

many traditional vaccine production media, while providing equal or 

better performance with respect to cell growth. Partial or complete serum 

elimination was achieved in media used to cultivate CEF, MDCK and BHK- 

21 cell lines. These supplements have also proven to facilitate the 

transition from anchorage dependent to suspension culture. 

P25 

Application of complex and chemically-defined medium supplements 

toward cell line specific performance enhancement of 

biopharmaceutical production systems 






Introduction: A series of cell-line specific complex media supplements have 

been developed for enhancing performance of various biopharmaceutical 

production systems. Created using in-house media optimization methods, 

these supplements are manufactured using innovative, proprietary process 

technology which allows for the combination of complex and/or chemically 

defined animal-component free media additives into a single homogeneous 

functional supplement. These supplements have been optimized for 

individual cell lines, and have proven to be suitable for use in a range of 

basal media. Data are presented which demonstrate the effectiveness of 

these optimized supplements as performance enhancers for application in 

CHO and SP2/0 batch culture, and as feed supplements in fed-batch 

systems. Preliminary data will also be presented on a parallel series of strictly 

chemically-defined supplements, similarly optimized for specific cell lines. 

Materials and methods: CHO data were collected using a transfected 

CHO-K1 line (ATCC #CCL-61), adapted to serum-free suspension culture, and 

engineered to constitutively express secreted embryonic alkaline 

phosphatase (SEAP) by means of a modified human cytomegalovirus 

(hCMV) promoter. 

Hybridoma data were collected using a murine hybridoma suspension cell 

line (ATCC # CRL-1753). This line was derived from primary spleen cell 

cultures of animals immunized with purified human immunoglobulin. 

These spleen cells were then fused with Sp2/0-Ag14 myeloma cells to 

create the hybridoma. 

Figure 1(abstract P24) Growth performance of an animal component free medium supplement in Chicken Embryo Fibroblast (CEF) adherent cell culture.



Triplicate 125 ml shake-flasks contained a final medium volume of 35 ml. 

Duplicate spinner flasks had a working volume of 150 ml. The basal 

medium consisted of 100% chemically defined medium (CDM). Cultures 

were incubated at 37°C in 5% CO2. Medium supplement stock solutions 

were prepared at 100 g/l in the basal medium and sterilized through a 

2.0 µm filter. 

At appropriate points during each experiment, 1.0 ml of the culture 

supernatants were removed for assessing cell counts and viability. Cells 

were counted using a Nova BioProfile Flex automated analyzer. On the 

final day of each run, 500 µl of the culture supernatants were removed 

for SEAP or IgG analysis. Levels of SEAP in the supernatants were 

measured using anion-exchange HPLC on a Waters Model 2695 HPLC 

Figure 1(abstract P25) Performance comparison of defined and undefined medium supplements in CHO batch culture. 

Page 46 of 181



separations module equipped with a dual-wavelength absorbance 

detector. IgG was quantified using a Protein G affinity column. 

Results: Using in-house media optimization methods, both an undefined 

and a chemically-defined supplement for CHO cells were developed from 

a variety of individual components. Themostfavorabledosageforeach 

supplement was determined through a series of dose-response 

experiments. While both supplements improved cell culture performance, 

the undefined supplement achieved a significantly higher peak cell 

density than the chemically defined supplement (Figure 1). 

Both SEAP titer and specific productivity (Qp) were improved when the 

supplements were employed (Figure 1). As reflected in the growth data, 

the undefined provided a significantly greater benefit. While the titer 

increase for the chemically-defined supplement was substantial, there 

was little increase in the Qp. The significant Qp increase seen with the 

undefined supplement undoubtedly accounts for the higher SEAP titer 

obtained, and may be a function of un-characterized elements within the 

hydrolysate based supplement. 

Summary: Medium optimization is an integral part of biopharmaceutical 

process development. There is an on-going debate within the industry as 

to the various advantages and disadvantages of both defined and 

undefined media and media components. The choice of which type of 

system to employ is often motivated by risk mitigation with respect to 

consistency of performance, as weighed against the underlying goal of 

achieving the highest possible product titers for any given system. Defined 

and undefined supplementation solutions may not be mutually exclusive. 

P26 

A comparison of performance enhancing synergy among 

ultrafiltered yeast extracts and recombinant human serum 

albumininCHO-K1cells 






Introduction: We have previously demonstrated a synergistic reaction 

between a wheat hydrolysate and recombinant human serum albumin used 

to supplement a chemically defined growth medium for SP2/0 hybridoma 

cells. The data presented here illustrate the synergystic performance 

enhancing effect obtained when ultrafiltered yeast extract and recombinant 

human serum albumin are co-supplemented in CHO cell media. Each 

combination has its own distinctive effect on the growth and productivity of 

transfected cells. Cell viability, cell proliferation and target protein 

production all may be improved, yet these effects are not necessarily 

observed concurrently in a given system. 

Materials and methods: Data were collected using a transfected CHO-K1 

line, adapted to serum-free suspension culture, and engineered to 

constitutively express secreted embryonic alkaline phosphatase (SEAP) by 

means of a modified human cytomegalovirus (hCMV) promoter. 

Figure 1(abstract P26) Growth Curves and SEAP Titers for rHSA, HyPep YE and UltraPep YE Supplemented Batch Cell Cultures. 

Page 47 of 181



Cultures were grown in 125 ml shake-flasks containing a final medium 

volume of 35 ml. The basal medium consisted of 100% chemically defined 

medium (CDM) supplemented with 1 mg/ml G-418. Triplicate flasks were 

seeded at 4.0 x10 5 cells/ml, and incubated at 37°C in 5% CO2 at 130 rpm 

for 12 days. Medium supplement stock solutions were prepared at 100 g/l 

in the basal medium and sterilized through a 2.0 µm filter. 

At days 5, 7, 8, 9 and 12, 1.0 ml of the culture supernatants were 

removed for assessing cell counts and viability. Cells were counted using 

a Nova BioProfile Flex automated analyzer. At Day 12, 1.0 ml of the 

culture supernatants were removed for SEAP analysis. Levels of SEAP in 

the supernatants were measured using anion-exchange HPLC. 

Results: Maximum cell density increased with respect to the Medium 

Control when cultures were dosed with rHSA at 1 g/l, but not when 

supplemented with HyPep YE at 1 g/l. When both supplements were 

used together, an even greater increase in cell density was observed. The 

synergystic effect was also seen with rHSA and UltraPep YE. However, the 

UltraPep YE/rHSA combination out-performed the HyPep YE/rHSA with 

respect to maximum cell density (Figure 1). All cultures were assayed for 

total SEAP production on Day 12. When dosed at 1g/l, all of the 

supplements (HyPep YE, UltraPep YE and rHSA) yielded higher titers than 

the Medium Control. The greatest increases were seen when HyPep YE or 

UltraPep YE were used in conjunction with rHSA (Figure 1). 

Summary: The data presented here illustrate the performance-enhancing 

synergy that may be realized by supplementing various cell culture 

media with a combination of yeast extract and recombinant human 


serum albumin. When the two supplements are used together, cell 

culture performance results exceed those achieved when using each 

supplement individually. Overall performance was further improved by 

varying the individual dosages of yeast extract and recombinant human 

serum albumin. In four separate basal media, cell response to cosupplementation 

for each of the yeast extract/recombinant albumin 

combinations tested was shown to be both medium and dosage 

dependent. The optimized combination provided significant overall 

performance improvement in all media tested. 

P27 

Targeted metabolomics for bioprocessing 

Denise Sonntag * , Francesca M Scandurra, Torben Friedrich, Michael Urban, 

Klaus M Weinberger 

BIOCRATES Life Sciences AG, Innsbruck, Austria 

E-mail: denise.sonntag@biocrates.com 


Background: Bioprocesses like the cell-based production of biologicals, i.e. 

mainly recombinant proteins and monoclonal antibodies, require optimal 

culture conditions to obtain a high yield of quality products. The 

performance of a bioreactor highly depends on the cell characteristics as well 

as on the composition of the cell culture medium and the process 

conditions. As the metabolic activity of the cells is very high during 

Figure 1(abstract P27) Comparison of the metabolite spectrum of commercially available cell culture media supplements derived from biological sources.



fermentation, the external and internal metabolite compositions vary 

tremendously throughout the process. The quantification of a wide range of 

metabolic substrates and products is a prerequisite to understand and 

optimize the underlying cell-based activities. Furthermore, metabolite 

quantification reveals the composition of biologically derived cell culture 

supplements, thus serving as a tool to monitor supplement quality or 

providing the base for the formulation of a chemically defined medium 

supplement. 

Materials and methods: Targeted metabolomics was carried out using 

a mass spectrometry-based platform. Analyses were performed with 

commercially available KIT plates that allow the simultaneous quantification 

of 180 metabolites [1]. The fully automated assay is based on PITC 

(phenylisothiocyanate)-derivatization in the presence of internal standards 

followed by FIA-MS/MS (for acylcarnitines, lipids, hexoses) as well as LC-MS/ 

MS (amino acids and biogenic amines) using a AB SCIEX 4000 QTrap mass 

spectrometer in the multiple reaction monitoring detection mode with 

electrospray ionization. On the same instrument, a validated HPLC-MS/MS 

quantification method is used for the analysis of energy metabolism 

intermediates. The concentrations of individual fatty acids are determined as 

their corresponding methyl ester derivatives (FAME’s) using GC-MS on an 

Agilent 7890 GC/5795 MSD instrument after derivatization. Vitamins were 

determined using LC-ESI-MS/MS technique after their pre-separation into two 

fractions (water- and fat-soluble vitamins). 

Results: The metabolomics approach to bioprocess monitoring made it 

possible to determine the concentration of a multitude of analytes from 

several metabolite classes in a sample- and time-efficient way by using small 

sample volumes and a multi-assay strategy. The analyte portfolio went 

beyond routinely determined culture media components, as it covered 

proteinogenic and non-proteinogenic amino acids, e.g. ornithine and 

citrulline, and intermediates of the energy metabolism, e.g. lactate, hexoses, 

succinate, as well as acylcarnitines, biogenic amines, glycerophospholipids, 

sphingomyelins, fatty acids, and vitamins (Figure 1). 

Conclusions: Targeted metabolomics comprises a rapid and comprehensive 

method to determine the composition of cell culture supplements of 

biological origin. 

The method is also well suited to rapidly characterize fermentational 

processes metabolically by monitoring changes in medium composition and 

cellular metabolite pools. The received quantitative data can be directly 

related to growth, vitality, and productivity of the cell. 

Reference 

1. [http://www.freepatentsonline.com/20070004044.html]. 

P28 

Toolbox approach for fast generation of stable CHO production cell 

lines from different hosts 

Susanne Seitz * , Henning von Horsten, Thomas Rose, Volker Sandig, 

Karsten Winkler 

ProBioGen AG, Berlin, 13086, Germany 

E-mail: Susanne.Seitz@probiogen.de 


Figure 1(abstract P28) Overview of PBG Cell Line Development. 


Background: Protein production in CHO cell lines is a highly dynamic 

process, where the yield limiting step can shift among transcription, 

translation, and secretion under different cell growth stages and culture 

conditions. In addition, the choice of CHO cell line has been shown to affect 

target protein quality and quantity. 

Further advancements in titer and specific productivity require elimination 

of cellular/process bottlenecks along the generation of stable production 

cell lines including vector design, clone selection, genetic engineering, 

and media and feed optimization. 

To overcome these limitations, we developed a toolbox for all process 

phases from transfection to production. This toolbox is based on a 

chemically defined media platform and includes pre-adapted CHO-K1 or 

CHO-DG44 cell lines, optimized vectors for single and multi-chain proteins 

as well as fine-tuned protocols. In addition, it enables the production of 

antibodies with specialized glycan structures (GlymaxX® [1]) as well as clone 

engineering with regard to the expression and secretion machinery (CDC42). 

Materials and methods: ProBioGen‘s toolbox expression vectors 

encoding an Fc-protein or therapeutic glycoprotein were generated using 

gene-optimized sequences and optimized signal peptides. 

To create stable cell lines, expression vectors were transfected into CHO- 

DG44 and CHO-K1 cells by microporation. Two serum-free selection 

strategies were applied using both MTX and puromycin: selection in 

semi-solid medium followed by automated clone picking and deposition 

into 96 wells using the ClonePix FL (Genetix), and combined selection 

and cloning in 96-well plates. For a first expression analysis monoclonal 

primary clones were subjected to 96-well plate assay. Clones showing 

best performance in 96-well were expanded and taken into batch and 

fed-batch for productivity assessment. Best performing primary clones 

were subjected to a second cloning step by limiting dilution and 

analyzed in 96-well. Following clone scale-up highest ranking sub-clones 

clones were assessed in shake flask using fed-batch. Prior to master cell 

banking candidate lead clones were tested for fed-batch bioreactor 

performance in a Cellferm-pro® (DASGIP AG). Protein concentrations were 

determined using either an ELISA method, the Gyrolab SIA Ligand 

Binding Assay or an HPLC method. 

Protein secreted from stable cell lines was purified either by protein A 

affinity chromatography (Fc-protein) or Ion Exchange Chromatography 

using weak anion exchangers (glycoprotein) and evaluated by Size- 

Exclusion HPLC (SEC), Western blot and SDS-PAGE. 

To determine the stability of clones a continuous passage of roughly 80 

population doublings with analyzing protein batch titers as well as mRNA 

levels and gene copy numbers (StepOnePlus RT-PCR System) at defined 

time points was performed. Cells were cultivated without selection 

pressure. 

Results and conclusion: Here we describe an innovative parallel 

platform cell line development for CHO-K1 and CHO-DG44 (Figure 1) 

exemplified by two completely different recombinant proteins, an Fcprotein 

and a therapeutic glycoprotein, with the glycoprotein showing 

differences in glycosylation pattern in the two starter cell lines. 

Our toolbox approach enables a fast and highly reproducible generation 

of high producer clones stably expressing the transgene for more than 80



Table 1(abstract P28) Growth and protein production data of CHO producer clones in different assay formats 

Fc-protein Glycoprotein 

Assay format Cell line Max VCD [viable cells/mL] Max Titer [mg/L] Max VCD [viable cells/mL] Max Titer [mg/L] 

96-well plate assay K1 nd a 

50 nd a 

268 

(5 days) DG44 nd a 

176 nd a 

607 

Batch K1 2.3E+07 610 1.1E+07 1772 

(5 or 7 days) DG44 8.6E+06 980 6.4E+06 2200 

Fed batch K1 3.0E+07 2500 1.9E+07 8100 

(12-17 days) DG44 1.7E+07 4500 3.0E+07 13200 

a not determined. 

generations without selection pressure and resulting in upstream yields of 

4.5-13 g/L (Table 1). 

Acknowledgement: We would like to thank the PBG teams for their hard 

and excellent work. 

Reference 

1. von Horsten HH, Ogorek C, Blanchard V, Demmler C, Giese C, Winkler K, 

Kaup M, Berger M, Jordan I, Sandig V: Production of non-fucosylated 

antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4hexulose 

reductase. Glycobiology 2010, 20:1607-1618. 

P29 

Growth characterization of CHO DP-12 cell lines with different high 

passage histories 

Christoph Heinrich 1* , Timo Wolf 1 , Christina Kropp 1 , Stefan Northoff 2 , 

Thomas Noll 1 

1 

Institute of Cell Culture Technology, Bielefeld University, Bielefeld, Germany; 

2 

TeutoCell AG, Bielefeld, Germany 



Introduction: For industrial pharmaceutical protein production fast 

growing, high producing and robust cell lines are required. To select pHshift 

permissive and faster growing sub-populations, the CHO DP-12 cell line 

was serially subcultured for more than four hundred days in shaker flasks. 

Initial adaptation to growth in suspension was carried out in chemically 

defined medium without hypoxanthine and thymidine (HT), while the final 

medium used for long term cultivation contains HT. Cell samples were 

cryopreserved at four different time points after 21, 95, 165 and 420 days. 

Cultivations of these four sub-populations (SP) in shaker flasks and 

bioreactors revealed considerable differences in specific growth rates 

and product formation as well as in the metabolism of glucose, lactate and 

several amino acids. For the elucidation of the intracelluar mechanism 

behind these alteration in growth characteristics and metabolism additional 

probes were analyzed using proteomic and metabolomic approaches [1]. 

Material and methods: In this study the CHO DP-12 clone#1934 (ATCC 

CRL-12445) was used as reference organism. It co-expresses the variable 

light and heavy chains of the murine 6G4.2.5 monoclonal antibody (ATCC- 

HB-11722) which inhibits binding of interleukin 8 to human neutrophile. 

CHO DP-12 cells were cultivated in CD-ACF medium TC 42 (TeutoCell AG) 

and PowerCHO-2 (LONZA AG) for the first steps of suspension adaptation. 

200 nM methotrexate was present at any time. Precultures and parallel 

cultivations were carried out in 125 mL and 250 mL polycarbonate 

Erlenmeyer flasks (Corning Life Sciences). Incubator conditions were set to 

37°C, 5% CO 2 and relative humidity of 80%. A shaker revolution of 185 rpm 

or 125 rpm with an orbital movement of 2” was chosen. For bioreactor 

cultivations four parallel vessels (Applikon) controlled by CellfermPro 2.3 

software (DASGIP AG) were used. Cultivation parameters were set to 37°C, 

40% DO and pH 7.1. 

Cell concentration and viability were determined with a CEDEX system 

(Innovatis-Roche AG). The anti IL-8 antibody was quantified using Protein A 

HPLC. 

A MACSQuant® Analyzer (Miltenyi Biotec GmbH) was used for the 

measurements of intracellular IgG-product pools. Intracellular detection of 

the antibodies required permeabilization of the cell membrane with 

detergents. IgG light and heavy chains were stained with fluorochrome- 


conjugated antibodies that bind to Fc and kappa chains of IgGs within 

fixed CHO cells (all solutions from Miltenyi Biotec GmbH). 

Results: The initial adaptation to growth in suspension of the CHO DP-12 

cells led to considerable changes in average cell diameter and specific 

growth rate. Within the first 100 days of serial subculturing the cell 

diameter dropped from a maximum of about 17 µm to 12 µm as the 

lowest value. In the same period the cell specific growth rate increased 

from initially 0.2 d -1 up to values greater than 1.0 d -1 . After further 320 

days cells had an average diameter about 14 ± 0.74 µm and a mean 

specific growth rate of 0.82 ± 0.12 d -1 . 

The growth characterizations of the four sub-populations SP21, SP95, SP165 

and SP420 were carried out in several parallel controlled and uncontrolled 

batchcultivations.Inadditiontotheinfluenceoftheinsulinlikegrowth 

factor 1 (IGF) the presence of hypoxanthine and thymidine (HT) was 

investigated as well. Most important characteristics for growth and 

productivity determined in this experimental setup are presented in Figure 1. 

Though the sub-populations SP95, SP165 and SP420 seemed to possess 

comparable growth rates in shake flask cultivations, they differed 

remarkably in their maximum cell density when HT-mix was present. For 

SP21 and SP420, maximum cell densities of about 1.1·10 7 cells/mL and 

2.1·10 7 cells/mL, respectively, were determined. These results indicate an 

influence of the HT-mix on the maximum reachable cell densities. 

Furthermore, an increased passage number seemed to cause decreasing 

growth performance in the controlled bioreactor system compared to the 

results from the shake flask cultivations with HT containing medium. This 

fact could be associated to the adaptation of subcultivated cells to pHshifts 

that occur during cultivation under uncontrolled conditions. 

Additionally, the observed differences between the four sub-populations 

in terms of the metabolism of glucose, lactate and several amino acids 

might play a role in this context. In bioreactor cultivations a two-fold 

higher lactate formation was observed for SP420 as compared to SP21, 

for instance. 

The highest specific production rate of 10.5 pg/(cell·day) was obtained for 

the sub-population SP95 resulting in a final antibody titer of 334 mg/L. 

Considering long-term cultivation, cell specific productivity increased 

during first passages and was lost with ongoing subcultivation. Further 

cytometry analysis regarding intracellular productivity of examined CHO 

DP-12 cells revealed that serial subculturing resulted in accumulation of a 

subclone expressing only the light chain of the IL-8 antibody. 

Conclusions: Serial subculturing over an extended period led to the 

selection of faster growing and pH-shift permissive cells, thus resulting in 

higher viable cell densities, especially in uncontrolled shake flask 

cultivations. Furthermore, cells or rather sub-populations with distinct 

metabolic characteristics were enriched along the subculturing process. 

This indicated that a targeted experimental approach could be used e.g. 

to specifically select cells adapted to low glutamine concentrations and 

therefore, a reduced consumption rate. On the other hand, the observed 

loss of productivity shows that the selection pressure given by 200 nM 

methotrexate and deprivation of hypoxanthine and thymidine could not 

prevent an increase of sub-populations expressing no or only incomplete 

(non-native, deficient) product. This problem could be caused by the 

vector design maybe used for the CHO DP-12 cells, which resulted in a 

non associated integration of the dihydrofolate reductase (dhfr) and anti- 

IL 8 sequences. Hence, it might be interesting to monitor the intracellular 

expression during clone selection or even seed-train development by 

flow cytometry.



Figure 1(abstract P29) Overview of growth performance (A) and product formation (B) of the four sup-populations in controlled and uncontrolled 

culture systems with varying media supplementation. 

Acknowledgements: The authors would like to thank Miltenyi Biotec 

GmbH for providing the MACSQuant® analyzer and consumables. 

Reference 

1. Beckmann T, Thüte T, Heinrich C, Büntemeyer H, Noll T: Proteomic and 

metabolomic characterization of CHO DP-12 cells with different high 

passages histories. Proc 22nd ESACT Meeting Vienna 2011. 

P30 

Utilization of multifrequency permittivity measurements in addition to 

biomass monitoring 

Christoph Heinrich * , Tim Beckmann * , Heino Büntemeyer, Thomas Noll 


Germany 



Introduction: In recent years measurement of permittivity signal has been 

increasingly used for online biomass monitoring of cell cultures. Breweries 

use it as an established method for fermenter inoculation and bioprocess 

controlforinstance[1].Inthecaseofanimalcellculturesthecorrelation 

between permittivity and viable cell densities determined offline varies 

along cultivation time. Hence, several authors have used the permittivity 

signal as an indirect method for measuring oxygen and glutamine 

consumption as well as intracellular nucleotide phosphate concentrations 

[2,3]. The latest generation of biomass monitoring devices allows parallel 

measurement of permittivity at a range of frequencies leading to an 

improvement in the correlation between biomass and permittivity and 

providing a tool to further explore other aspects related to the physiological 

state of the cells. 

Material and methods: In this study 10 different cell lines, among them 

industrial cell lines as well as cell lines distributed by ATCC, were cultivated. 

Cell type specific chemically defined and animal component-free media 

(TeutoCell AG) were used. Batch, fed-batch and perfusion bioreactor 

cultivations were carried out in controlled benchtop vessels (Sartorius AG or 

Applikon Biotechnology). The i-Biomass 465 sensor (FOGALE nanotech) was 

used for online multifrequency permittivity monitoring. In addition the 

permittivity signal was used to implement a fully automated cell bleed to 

maintain a constant viable cell density in a perfusion process. Furthermore, a 

fed-batch feed strategy was introduced to keep the substrate concentration 

at a certain level. Cell density and viability were determined using a CEDEX 

system (Innovatis-Roche AG). Glucose and lactate were measured with an 

YSI 2700 Biochemistry Analyzer (YSI Life Sciences). Amino acids were 

quantified using an in-house developed HPLC method. 

Results: The FOGALE i-Biomass 465 sensor was used to monitor the 

viable cell density of different human, CHO and hybridoma cell cultures 


online. A good correlation of the permittivity signal and the offline 

measured viable cell density for the growth phase was verified (R > 0.99), 

but pH-shifts and increased cell aggregation had a negative impact on 

the correlation. The linear factor to calculate the viable cell density from 

the online permittivity signal varied between 4.5·10 5 cells/(pF/cm) and 

12.0 cells/(pF/cm). A clear relation between cell type (CHO, human or 

hybridoma) and the linear factor could not be established from the 

available data. 

Subsequently, the online biomass monitoring system was used to carry out 

a 1 L spin-filter perfusion process with constant viable cell density at a 

predefined setpoint. The application of a permittivity closed-loop controlled 

cell bleed resulted in a steady concentration of 10 7 viable cells/mL during 

perfusion, at a dilution rate of 1.0 d -1 . As soon as this threshold was reached, 

the cell bleed was automatically started and controlled based on the online 

signal of the i-Biomass 465 sensor. 

In addition to the correlation with viable cell density, a linear relationship 

(R 2 > 0.96) between the online i-Biomass 465 signal and the concentrations 

of numerous components, e.g. glucose, lactate, asparagine, glutamine, 

tyrosine, threonine, methionine, lysine, phenylalanine, serine, leucine and 

isoleucine, was found during the exponential growth phase of CHO-K1 and 

CHO DP-12 cultivations. The results indicated that the number of 

correlating substrates depended on the used cell line (CHO, human or 

hybridoma) and the process strategy (constant pH or pH-shift). Since, the 

established substrate correlations were more robust against process 

variations, they were investigated as a basis for a closed-loop feeding 

strategy in fed-batch cultivations. Compared to a pre-defined feeding 

schedule or to intermitted feeding this would have the advantage of 

avoiding nutrient limitations and substrate accumulation that might occur 

due to unexpected high or low cell growth. Also, feeding would be 

independent of human surveillance. The successful application of a 

completely automated permittivity-controlled feeding strategy was proved 

in two fed-batch runs with CHO DP-12 (ATCC CRL-12445) cells, as shown in 

Figure 1. 

The feeding of both runs was controlled only through the permittivity 

signal in order to maintain the asparagine concentration at a certain 

level. Asparagine was chosen due to its central role in cell metabolism 

and to the fact that it is usually a limiting substrate in CHO DP-12 

cultures. These proof-of-concept runs demonstrated that permittivitybased 

automated feeding can be a valuable tool for the optimization of 

fed-batch process parameters, such as feeding start, flow rate and 

composition. 

Conclusions: For suspension cultures with single cells and high viability a 

linear correlation (R 2 ≥ 0.98) of the permittivity signal with the viable cell 

density measured offline was obtained, at least for the exponential 

growth phase. Based on this correlation, a closed-loop controlled cell 

bleed was implemented in a perfusion process in which the permittivity



Figure 1(abstract P30) Totalviablecellcount,viablecelldensityandasparagineconcentration of the two proof-of-concept CHO DP-12 fed-batch 

processes (initial working volume: 1 L; 37°C; 40% DO; pH 7.1). 

signal was used to keep the viable cell density at a constant level of 10 7 

viable cells/mL. Furthermore, a linear correlation with the i-Biomass 465 

signal was observed for several substrates independent of the correlation 

between viable cell density and permittivity. Based on these results, a 

closed-loop controlled feeding was successfully established resulting in a 

fully automated fed-batch process. 

Acknowledgements: We would like to thank FOGALE nanotech for 

providing the i-Biomass 465 system and TeutoCell AG for supplying the 

cell culture media and feed solutions. 

References 

1. Boulton CA, Maryan PS, Loveridge D: The application of a novel biomass 

sensor to the control of yeast pitching rate. Proc 22nd Eur Brew Conv 

Zurich European Brewing Convention Oxford: Oxford University Press 1989, 

653-661. 

2. Noll T, Biselli M: Dielectric spectroscopy in the cultivation of suspended 

and immobilized hybridoma cells. J Biotechnol 1998, 63:187-198. 




CHO cells. Biotechnol Prog 2010, 26:272-283. 

P31 

CHO cell lines generated by PiggyBac transposition 

Mattia Matasci 1 , Virginie Bachmann 1 , Lucia Baldi 1 , David L Hacker 1 , 

Maria De Jesus 2 , Florian M Wurm 1,2* 

1 

Laboratory of Cellular Biotechnology, Faculty of Life Sciences, Ecole 

Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland; 

2 

ExcellGene SA, CH-1870 Monthey, Switzerland 

E-mail: florian.wurm@epfl.ch 


A major bottleneck in the manufacture of recombinant therapeutic proteins 

is the time and effort needed for the generation of stable, high-producing 

mammalian cell lines. Conventional gene transfer methods for stable cell 

line generation rely on random transgene integration, resulting in 

unpredictable and highly variable levels of expression of the transgene in 

individual clones [1,2]. As a consequence, a large number of stably 

transfected cells must be analyzed to recover a few high-producing clones. 

Recently, we described the use of a PiggyBac (PB) transposon for the 

generation of high-producing mammalian cell lines [3]. The PB dual vector 

system consists of 1) a donor vector carrying an artificial transposon with a 

mammalian expression cassette for the recombinant transgene and 

puromycin selection marker and 2) a helper vector driving transient 

expression of the PB transposase (PBase). PB transposition mediates stable 

transgene integration via a “cut and paste” mechanism in which the PBase 

excises the artificial transposon sequence from the plasmid and catalyzes its 


insertion into the host cell genome. A main advantages of the PB system 

over conventional passive integration are an improved efficiency of 

transgene integration resulting in a more integration events and more 

stable clones. Furthermore, PB favors transgene integration into actively 

transcribed regions of the host genome [4]. The PB transposon has a high 

cargo capacity of up to 14 Kb and transposition results in the stable 

genomic integration of well-defined sequences, thus reducing the 

probability of integration of truncated, non-functional transgenes [5]. Finally, 

recent reports have demonstrated the feasibility of using the PB system to 

obtain persistent expression of multiple genes carried either on a single or 

on distinct donor vectors [6]. 

We initially determined the efficiency of the PB system in the generation of 

stable lines using suspension adapted mammalian cells. CHO and HEK 293 

cells were co-transfected with an eGFP-bearing donor plasmid along with 

the PB helper plasmid. The cells were then grown in the absence of 

puromycin, and the percentage of GFP-expressing cells in each culture was 

determined by flow cytometry on a daily basis. By 21 days post-transfection, 

the remaining GFP-positive cells were assumed to be recombinant. 

Compared to conventional transfection of plasmid DNA, PB transposition 

resulted in an improvement in the efficiency of stable cell line generation up 

to 20-fold for both CHO and HEK 293 cells (Figure 1A). 

To further evaluate the PB system, CHO cells expressing a tumour necrosis 

factor receptor:Fc fusion protein (TNFR:Fc) were generated either by PBtransposition 

or by conventional transfection. Clonal cell lines were 

recovered following selection in 50 or 10 µg/mL puromycin for two weeks. 

Recovered lines were grown in suspension culture for 7 days in 24-well 

plates after which the medium was analyzed by ELISA to determine TNFR:Fc 

productivity. Transposition increased the frequency of high-producing 

clones in the transfected population (Figure 1B). To further characterized for 

the level and stability of transgene expression the original cell pools 

generated by PB transposition or conventional transfection, as well as the 

top 4 producers from each transfection were cultivated in the absence of 

selection in serum-free suspension culture, over a period of 16 or 14 weeks, 

respectively. When compared to clones and cell pools generated by 

conventional transfection, PB-derived cell lines and cell pools produced up 

to 4-fold more recombinant protein and had greater transgene expression 

stability (Figure 1C) 

In conclusion our results demonstrate that stable cell lines derived by PB 

transposition are efficiently generated and are more productive than cell 

lines generated by conventional transfection methods. Therefore, the PB 

system represents a valuable and practical alternative to standard 

plasmid transfection to efficiently generate cell clones with stable and 

enhanced transgene expression. 

Acknowledgements: This work was supported by the Ecole Polytechnique 

Fédérale de Lausanne and the CTI Innovation Promotion Agency of the 

Swiss Federal Department of Economic Affairs (n. 10203.1PFLS-LS) under a 

collaboration with ExcellGene SA (Switzerland).



Figure 1(abstract P31) A) Enhanced stable integration efficiency in CHO and HEK 293 cells by PB transposition. B) Productivity analysis of CHO clonal cell 

lines sorted from cell pools generated by PB transposition (PB) or conventional transfection (TX). C) Analysis of the stability of TNFR:Fc expression over 

time and transgene copy number, for cell pools [PB(50), PB(10), TX(50), and TX(10)] and clonal lines generated by PB transposition (PB50 and PB10) or 

conventional transfection (TX50 and TX10). 

References 

1. Matasci M, et al: Recombinant therapeutic protein production in 

cultivated mammalian cells: current status and future prospects. Drug 

Discovery Today: Technologies 2009, 5(2-3):e37-e42. 

2. Wurm FM: Production of recombinant protein therapeutics in cultivated 

mammalian cells. Nat Biotechnol 2004, 22(11):1393-8. 

3. Matasci M, Baldi L, Hacker DL, Wurm FM: The PiggyBac transposon 

enhances the frequency of CHO stable cell line generation and yields 

recombinant lines with superior productivity and stability. Biotechnol 

Bioeng 2011, doi: 10.1002/bit.23167. 

4. Galvan DL, et al: Genome-wide mapping of PiggyBac transposon 

integrations in primary human T cells. J Immunother 2009, 32(8):837-44. 

5. Ding S, et al: Efficient transposition of the piggyBac (PB) transposon in 

mammalian cells and mice. Cell 2005, 122(3):473-83. 

6. Kahlig KM, et al: Multiplexed transposon-mediated stable gene transfer in 

human cells. Proc Natl Acad Sci U S A 2010, 107(4):1343-8. 

P32 

Transposon mediated co-integration and co-expression of transgenes in 

CHO-DG44 cells 

Sowmya Balasubramanian, Mattia Matasci, Lucia Baldi, David L Hacker, 

Florian M Wurm * 

Laboratory for Cellular Biotechnology (LBTC), Faculty of Life Sciences, École 

Polytechnique Fédérale de Lausanne CH-1015 Lausanne, Switzerland 



Background: Transposon systems mediate stable integration of exogenous 

DNA elements into a host cell genome, and have been successfully used in 

mammalian cells for the generation of stable cell lines. The piggyBac (PB) 


transposon system has been shown to have several advantages over the 

other transposon system available [1-3]. It has also been shown to generate 

stable cell lines at significantly higher frequency than the conventional 

transfections [3]. Here, we investigated the efficiency of the piggyBac (PB) 

transposon to facilitate the co-expression of multiple artificial transposons, 

each bearing a single transgene and the puromycin resistance gene for 

selection. Green fluorescent protein (eGFP), red fluorescent protein (mKate), 

and a human IgG1 antibody were used as model proteins [4]. The effect of 

the stringency of selection on pool productivity was determined with 

increasing concentrations of puromycin. The duration of selection necessary 

for the generation of recombinant cell pools was also tested by selecting for 

a period of either 5 or 10 days. 


CHO-DG44 transfection: Cells were transfected using linear 25 kDa 

polyethylenimine (PEI) (Polysciences, Eppenheim, Germany). All the 

transfections are done in a final volume of 10 mL. Transfected cultures 

were incubated at 37°C in 5% CO 2 and 85% humidity with agitation at 

180 rpm. The ratio of plasmid coding for Gene of Interest (GOI) to the 

plasmid coding for the transposase was kept constant at 9:1. 

Generation of pools and clones: For the generation of stable pools, two 

days post transfection the cells were seeded at a density of 5 x 10 5 cells/mL 

in ProCHO5 and puromycin. The cells were placed under selection pressure 

for 5 or 10 days. In case of a 10 day selection period, the puromycin 

concentration in the cultures was replenished on day 7 post transfection by 

seeding at a density of 5 x10 5 cells/mL in fresh ProCHO5 with puromycin. 

For productivity analysis the cells were seeded at a density of 3 x10 5 cells/ 

mL and analyzed at day four. 

Stable clones were generated by limiting dilution of the pools. The 

productivity of the clones was analyzed after five days of culture. 

Analyses: A Guava EasyCyte microcapillary flow cytometer (Millipore) with 

excitation and emission wavelengths of 488 and 532 nm, respectively, was



Figure 1(abstract P32) Comparison of the A) %GFP positive cells and B) IgG1 productivity between pools generated using standard transfection (left) 

and PB transposition (right). 

used to measure EGFP-specific fluorescence. The IgG concentration in the 

culture medium was determined by sandwich ELISA as previously 

described [4]. 

Results: 

PB Transposition significantly enhances co-expression of multiple 

genes from stable pools compared to standard transfection: Cells were 

co-transfected with three artificial transposons namely, eGFP and the heavy 

and light chains of a human IgG1 antibody along with the plasmid coding 

for the transposase. Cell populations recovered after selection showed 

30 % of GFP positive cells for standard transfections, whereas for pools 

generated by PB transposition, the percentage of cells producing GFP was 

up to 80%, which represent a 2.5 fold improvement (Fig. 1A). IgG1 

productivity up to 120 mg/L was achieved using transposition, whereas in 

standard transfections the highest titers obtained were only 5 mg/L, 

corresponding to a 24 fold improvement (Fig. 1B). 

No significant differences were observed in the percentage of GFP positive 

cells and IgG productivity in cell pools generated by transposition with 5 or 

10 days of selection. However, increased IgG productivity and % GFPpositive 

cells were observed with 10 days of selection in case of standard 

transfection (data not shown). This shows that a longer duration of selection 

pressure is necessary to generate pools by standard transfections compared 

to transposition. Furthermore, increased antibody productivity was observed 

with increasing puromycin concentration (Fig. 1). 

PB transposition strongly improved clonal productivity: Four different 

plasmids coding for (1) EGFP, (2) mKATE, (3) the light and (4) heavy chain 

genes of a human IgG1 antibody. Clones for both the standard and the 

transposition-based transfections were generated by limiting dilution of the 

cell pools after selection for 10 days. The IgG productivities of 65 clones of 

each standard transfection and transposition were analyzed. About 96% of 

the clones generated by the standard transfection were found to be lowproducing. 

However, more than 40% of clones generated by transposition 

produced more than 20 mg/L and about 12% of the clones were highproducers 

(Table 1). 

Conclusions: Based on the above results we conclude that the piggyBac 

transposon system provides an efficient method for the co-integration of 

multiple genes. The use of the PB transposon system for the co- 

Table 1(abstract P32) Distribution of clones based on 

their IgG productivity 

Productivity Range Standard Transfection Transposition 

60 mg/L 0% 13% 


integration of multiple genes generates a higher frequency of highproducing 

clones than standard transfection. The GFP-expressing cell 

population was larger and the volumetric antibody productivity of the 

pools were higher with higher stringency of selection. A selection period 

with puromycin of only 5 days was sufficient for the generation of pools 

from transposon mediated transfection. 

Acknowledgments: This work has been supported in part by the CTI 

Innovation Promotion Agency of the Swiss Federal Department of Economic 

Affairs (n. 10203.1PFLS-LS), in collaboration with the company ExcellGene SA 

in Monthey, Switzerland. 

References 

1. Kahlig KM, Saridey AK, Kaja A, Daniels MA, George AL Jr, Wilson MH: 

Multiplexed transposon-mediated stable gene transfer in human cells. 

Proc Natl Acad of Sci USA 2010, 107(4):1343-8. 

2. Wilson MH, Coates CJ, George AL Jr: PiggyBac Transposon-mediated Gene 

Transfer in Human Cells. Molecular Therapy 2007, 15:139-145. 

3. Matasci M, Baldi L, Hacker DL, Wurm FM: The PiggyBac Transposon 

Enhances the Frequency of CHO Stable Cell Line Generation and Yields 

Recombinant Lines with Superior Productivity and Stability. Biotechnol 

Bioeng 2011, doi: 10.1002/bit.23167. 

4. Meissner P, Pick H, Kulangara A, Chatellard P, Friedrich K, Wurm FM: 

Transient gene expression: recombinant protein production with 

suspension-adapted HEK293-EBNA cells. Biotechnol Bioeng 2001, 

75:197-203. 

P33 

The use of filler DNA for improved transfection and reduced DNA 

needs in transient gene expression with CHO and HEK cells 

Divor Kiseljak, Yashas Rajendra, Sagar S Manoli, Lucia Baldi, David L Hacker, 


Laboratory for Cellular Biotechnology, Faculty of Life Sciences, École 

Polytechnique Fédéral de Lausanne, CH-1015 Lausanne, Switzerland 



Background: Transient gene expression (TGE) is a rapid method for the 

production of recombinant proteins. Protein productivity in TGE has 

improved significantly over the past decade, reaching 300 mg/L and 1 g/L in 

CHO DG44 (CHO) and HEK 293E (HEK) cells, respectively [1,2]. However, the 

amount of plasmid DNA needed for transfection remains relatively high, 

contributing significantly to the overall cost of the TGE process. In order to 

reduce the amount of plasmid DNA in TGE, we examined the possibility of 

partially replacing it with herring sperm DNA (non-coding “filler” DNA) in 

transfections of CHO and HEK cells.




Transfections: Suspension-adapted CHO were centrifuged and 

resuspended in ProCHO5 (Lonza, Verviers, Belgium) at a density of 4 x 

10 6 cells/mL. Transfections of 5 mL were performed in TubeSpin® 50 

bioreactors (TPP, Trasadingen, Switzerland) using 0.625 µg DNA/1x10 6 

cells and 2.5 µg/1x10 6 cells of linear 25 kDa polye-thyleneimine (PEI; 

Polysciences, Eppenheim, Germany). pA3 carrying the genes for a human 

IgG light and heavy chains was used for transfections [1]. The transfected 

cultures were incubated at 31 °C in 5% CO2 and 85% humidity with 

agitation at 180 rpm. HEK cells were centrifuged and resuspended at a 

density of 20 x 10 6 cells/mL in RPMI 1640 medium (Lonza). To each 

culture, 1.0 µg DNA/1x10 6 cells and 3.0 µg PEI/1x10 6 cells were added. At 

3 h post-transfection, cells were diluted with Ex-Cell293 TM medium 

(Sigma) medium to a density of 1 x 10 6 cells/mL, and valproic acid was 

added to a final concentration of 3.75 mM. The transfected cultures were 

incubated at 37 °C as above. 

Analyses: The IgG concentration was determined by sandwich ELISA [3]. 

To quantify plasmid DNA copy number, total cellular DNA was extracted 

using DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s 

protocol. To estimate the mRNA transgene levels, total RNA was extracted 

from cells using the GenElute mRNA kit (Sigma) according to the 

manufacturer’s protocol. DNA-free RNA was reverse transcribed using M- 

MLV reverse transcriptase (Sigma) and oligo dT as the primer. The RTqPCR 

was carried out in a LightCycler 480 Real-Time PCR System (Roche 

Applied Science, Basel, Switzerland) with the ABsolute QPCR SYBR Green 

ROX mix (Thermo Fisher Scientific) according to the manufacturer’s 

instructions. 

Results: 

Filler DNA allows considerable reduction in coding pDNA amounts: 

We tested the efficiency of herring sperm DNA as filler for TGE in CHO 

and HEK cells. We reduced the amount of pA3 to 17% or 33% of the 

optimum amount for each cell line and added filler DNA to 100%. The 

total amount of PEI was kept constant for all conditions. We observed 

that antibody titers increased when filler DNA was co-transfected with 

pA3 as compared to transfection with a reduced amount of pA3 alone 

(Fig. 1). These results showed that up to 83% of the coding pDNA could 

be replaced by filler DNA with only a minimal negative impact on yield. 

Filler DNA does not influence the delivery of coding pDNA: We 

investigated whether the use of filler DNA could improve pDNA delivery 

and/or intracellular pDNA stability by quantifying the plasmid copy 

number by qRT-PCR. The results showed that the plasmid copy number 

decreased proportionally with the amount of pA3 transfected in the 

presence or absence of filler DNA in both CHO and HEK cells (data not 

shown). Therefore, filler DNA did not influence the delivery of coding 

pDNA to transfected cells. 

Filler DNA enhances transgene mRNA levels and improves release 

of coding pDNA: We tested the hypothesis that the use of filler DNA 

could influence transcriptional competence of coding pDNA. By qPCR, we 

found that transgene mRNA levels were significantly higher in the 

presence of filler than in its absence (data not shown). This may explain 

the improved protein titers observed in the presence of filler DNA. We 

then hypothesized that filler DNA could influence the strength of PEI:DNA 

complexes and pDNA release from the complex upon delivery. With an in 

vitro dextran sulfate displacement assay we observed that with increasing 


PEI:DNA ratios, complex strength increased. However, when filler DNA 

was added to the complex, the release of pDNA from the complex was 

improved (data not shown). 

Conclusions: Our data show that in TGE the amount of the coding vector 

could be reduced considerably by replacement of a significant proportion 

of pDNA with filler DNA (herring sperm DNA) without a major negative 

impact on recombinant protein productivity. However, filler DNA did not 

influence the delivery or stability of pDNA. The addition of filler DNA to the 

DNA-PEI complex, however, relaxed the complex in vitro. Basedonthese 

results, we speculate that the presence of filler DNA results in a more 

efficient intracellular release of the pDNA from the DNA-PEI complex and 

thus to improved transgene transcription. 

Acknowledgments: This work has been supported in part by the CTI 

Innovation Promotion Agency of the Swiss Federal Department of 

Economic Affairs (n. 10563.1PFLS-LS) under a collaboration with 

ExcellGene SA (Monthey, Switzerland). TPP (Trasadingen, Switzerland) is 

acknowledged for providing TubeSpin® bioreactor 50 tubes. 

References 

1. Rajendra Y, Kiseljak D, Baldi L, Hacker D, Wurm FM: A simple high-yielding 

process for transient gene expression in CHO cells. J Biotechnol 2011, 

153:22-26. 

2. Backliwal G, Hildinger M, Chenuet S, Wulhfard S, De Jesus M, Wurm FM: 

Rational vector design and multi-pathway modulation of HEK 293E cells 

yield recombinant antibody titers exceeding 1 g/l by transient 

transfection under serum-free conditions. Nuc Acid Res 2008, 36(15). 

3. Meissner P, Pick H, Kulangara A, Chatellard P, Friedrich K, Wurm FM: Transient 

gene expression: recombinant protein production with suspensionadapted 

HEK293-EBNA cells. Biotechnol Bioeng 2001, 75:197-203. 

P34 

Rapid recombinant protein production from pools of transposongenerated 

CHO cells 

Mattia Matasci 1 , Virginie Bachmann 1 , Lucia Baldi 1 , David L Hacker 1 , 

Maria De Jesus 2 , Florian M Wurm 1,2* 

1 



2 

ExcellGene SA, CH-1870 Monthey, Switzerland 



Transient gene expression (TGE) is the most commonly used technology for 

the rapid production of moderate quantities of recombinant proteins for 

preclinical studies or for analytical assay development [1,2]. Whereas TGE can 

provide milligram to gram amounts of recombinant proteins within a time 

period as short as few days, technical limitations and high costs of plasmid 

DNA hamper the application of this technology at larger scales. Here we 

describe an alternative method for the fast production of recombinant 

proteins from pools of mammalian cells stably transfected with the PiggyBac 

(PB) transposon. Expression from stably integrated transgene is highly 

dependent upon the chromatin structure surrounding the site of integration 

[3]. As a consequence stable cell populations generated by conventional 

transfection techniques generally show low productivity and reduced 

expression stability. Several studies showed that the PB transposase mediated 

Figure 1(abstract P33) Effect of filler DNA on transient IgG production in A) CHO and B) HEK cells. IgG titers were measured on day 7 post-transfection 

by ELISA. The DNA amounts are presented in μg/10 6 cells.



Table 1(abstract P34) Analysis of the stability or recombinant protein expression in 5 independent pools of CHO cells 

CHO-Pool Days post transfection % eGFP positive cells (*) 

TNFR:Fc productivity (mg/L) (°) 

30 92.8 ± 2.1 430 ± 46 

1 60 93.3 ± 0.2 455 ±10 

90 92.5 ± 1.2 473 ± 10 

30 90.0 ± 0.8 348 ± 36 

2 60 91.4 ± 0.5 370 ± 8 

90 94.0 ± 1.1 325 ± 35 

30 94.0 ± 1.5 432 ± 25 

3 60 94.2 ± 1.1 451 ± 28 

90 91.9 ± 1.8 494 ± 16 

30 92.0 ± 0.2 432 ± 30 

4 60 90.1 ± 2.0 407 ± 23 

90 92.3 ± 1.3 406 ± 28 

30 96.1 ± 1.5 388 ± 52 

5 60 92.4 ± 1.4 466 ± 19 

90 93.6 ± 0.2 372 ± 12 

(*) determined by GUAVA flow cytometry; (°) determined by ELISA. 


Figure 1(abstract P34) (A) Schematic representation of the protocol for the rapid production of recombinant proteins from pools of transposed cells. This 

protocol was successfully used to produce a recombinant monoclonal antibody (B) and two variants of TNFR:Fc (C and D). For each bioprocess shown, the 

percentage of viable cells (dotted lines) the viable cell density (dashed lines), and the recombinant protein titer (solid lines) were measured at the timesindicated.



transgene insertion predominantly into actively transcribed regions of the 

mammalian genome [4,5], moreover we recently demon-strated that PB 

transposition generates high-producing CHO cell lines at a higher frequency 

than conventional plasmid transfection [6]. These results prompted us to 

develop a technology based on the use of stable pools generated by PB 

transposition for the rapid and scalable production of recombinant proteins. 

Initial experiments were aimed at determining the proportion of transgene 

expressing cells as well as the level and stability of recombinant protein 

production in PB transposed pools. First, we analyzed transgene expression in 

952 cell lines recovered from a pool of CHO cells generated by PBtransposition 

to express tumor necrosis factor receptor as an Fc fusion protein 

(TNFR:Fc). The cell lines were analyzed by ELISA for TNFR:Fc expression in 4day 

batch cultures. The clones showed levels of TNFR:Fc expression up to 360 

mg/L with an overall mean of 96 +/- 54 mg/L. PB-transposition resulted in a 

high percentage (more than 98%) of TNFR:Fc expressing clones (data not 

shown). Analyses on the stability of transgene expression over time were 

conducted using a bicistronic PB-donor plasmid allowing co-expression of 

TNFR:Fc and the enhanced green fluorescent protein (eGFP). Five 

independent pools of cells were generated by transposition and recovered 

after 10 days of selection in puromycin. The pools were cultivated for 3 

months in the absence of selection and eGFP expression was monitored 

periodically by flow cytometry. For each pool, the percentage of eGFPpositive 

cells remained stable over time (Table 1). The stability of transgene 

expression was further confirmed by TNFR:Fc productivity studies performed 

in 50-ml cultures at different time points post-transfection. At one month 

post-transfection, the five pools showed comparable growth and production 

characteristics, reaching TNFR:Fc titers in the range of 350-500 mg/L in 14-day 

batch cultures. Similar results were obtained when productivity was tested 

2 – 3 months post-transfection (Table 1). 

We developed a protocol for protein production from transposed cell pools 

at the 0.5-L scale (Figure 1A). Starting at 2 d post-transfection cells were 

subjected to 10 days of puromycin selection during which cells were 

expanded from TubeSpin® Bioreactor 50 tubes into orbitally shaken 250-mL 

cylindrical bottles. The batch bioprocess was finally started at 12 d posttransfection 

using orbitally shaken TubeSpin® Bioreactor 600 tubes. Using 

stable cell pools expressing either an IgG antibody (Fig. 1B) or two TNFR:Fc 

variants (Fig. 1C, D), we produced 500-750 mg of recombinant protein 

within a month after transfection. 

Our results demonstrated an improved level and stability of transgene 

expression in transposed pools, indicating usefulness of PB transposed 

cell pools as a valuable alternative to TGE for the rapid production of 

recombinant proteins. 

Acknowledgements: This work was supported by the Ecole Polytechnique 


Swiss Federal Department of Economic Affairs (n. 10203.1PFLS-LS) under 

collaboration with ExcellGene SA (Switzerland). 

References 

1. Baldi L, Hacker DL, Adam M, Wurm FM: Recombinant protein production 

by large-scale transient gene expression in mammalian cells: state of 

the art and future perspectives. Biotechnol Lett 2007, 29(5):677-84. 

2. Hacker DL, De Jesus M, Wurm FM: 25 years of recombinant proteins from 

reactor-grown cells - where do we go from here? Biotechnol Adv 2009, 

27(6):1023-7. 

3. Kwaks TH, Otte AP: Employing epigenetics to augment the expression of 

therapeutic proteins in mammalian cells. Trends Biotechnol 2006, 24(3):137-42. 

4. Ding S, et al: Efficient transposition of the piggyBac (PB) transposon in 

mammalian cells and mice. Cell 2005, 122(3):473-83. 

5. Galvan DL, et al: Genome-wide mapping of PiggyBac transposon 

integrations in primary human T cells. J Immunother 2009, 32(8):837-44. 




Bioeng 2011, doi: 10.1002/bit.23167. 

P35 

Influence of glutamine on transient and stable recombinant protein 

production in CHO and HEK-293 cells 

Yashas Rajendra, Divor Kiseljak, Lucia Baldi, David L Hacker, Florian M Wurm * 

Laboratory for Cellular Biotechnology (LBTC), École Polytechnique Fédéral de 

Lausanne (EPFL), CH-1015 Lausanne, Switzerland 




Background: Glutamine is an essential component in culture media for 

most of the mammalian cell lines. It is often used as an alternative source of 

energy by cells, along with glucose. Glutamine metabolism induces 

ammonia accumulation in cell culture. Elevated ammonia concentration 

above 2 mM has been shown to have negative impact on both cell growth 

and recombinant protein productivity [1-4]. In this study we investigated the 

effects of decreased glutamine concentration in the medium for CHO-DG44 

and HEK-293E cells during transient gene expression (TGE). The rationale 

was to reduce ammonia accumulation in the culture, and consequently, 

improve cell viability and recombinant protein productivity. 


CHO-DG44 transfection: The cells were centrifuged and resuspended in 

ProCHO5 medium (Lonza, Verviers, Belgium) at a density of 5x10 6 cells/mL 

in orbitally shaken 250 ml glass bottles. Each transfection was performed 

in 100 mL of culture using 0.6 µg of plasmid DNA and 3.0 µg of linear 

25 kDa polyethyleneimine (PEI, Polysciences, Eppelheim, Germany; pH 7) 

per million cells. The transfected cultures were incubated at 31 °C in 5% 

CO2 and 85% humidity with agitation at 120 rpm. 

HEK-293E transfection: The cells were centrifuged and resuspended at 

density of 20x10 6 cells/mL in RPMI 1640 medium. Each transfection was 

performed in 100 mL of culture using 1.5 µg of plasmid DNA and 3.0 µg of 

linear 25 kDa PEI per 10 6 cells. Three hours post-transfection, cells were 

diluted with Ex-Cell293 medium (Sigma, Saint-Louis, USA) to a density of 

2x10 6 cells/mL, and valproic acid was added to a final concentration of 

3.75 mM. The transfected cultures were incubated at 37 °C in 5% CO 2 and 

85% humidity with agitation at 120 rpm. 

Stable Clone and Pool: A cell line and cell pool expressing anti-Rhesus 

IgG was kindly provided by Tatiana Benavides from our lab. Cell line was 

established by transfection of plasmid containing both light and heavy 

chain into CHO-DG44 cells, followed by flow cytometer sorting and 

limiting dilution. Cell pool was established by transfection of plasmid 

containing both light and heavy chain into CHO-DG44 cells, followed by 

selection under puromycin for two weeks. 

Metabolic analytes and IgG levels: The levels of glucose, glutamine, 

ammonium, and lactate were determined with a BioProfile 200 Bioanalyzer 

(Nova Biomedical Corp., Waltham, MA). The IgG concentration in the 

culture medium was determined by sandwich ELISA as previously 


Results: 

Low glutamine concentration improved transient IgG production: 

Different concentrations of free glutamine in medium, ranging from 0 mM 

to 6 mM were tested. Both cell lines showed improved production of IgG 

with a reduced glutamine concentration (Fig. 1 panel A). The optimal 

concentration of glutamine in terms of IgG production was 2 mM for CHO- 

DG44 cells and 0 mM for HEK-293E cells. We observed a 50% improvement 

in IgG production for both CHO-DG44 and HEK293 cells. We did not 

observe a significant difference on cell density or viability between the 

tested concentrations of glutamine for both CHO-DG44 and HEK293 cells 

during the entire course of the culture (data not shown). 

Higher initial concentration of glutamine resulted in higher concentration 

of ammonia, up to 5 mM for the highest glutamine concentration tested 

(Fig 1 panel B). 

Lactate accumulation in both CHO-DG44 and HEK-293E cells was observed 

during the initial phase of the cultures, while the cells were actively 

growing. In CHO-DG44 cells, consumption of lactate started immediately 

after its accumulation. In HEK-293E cells, lactate levels remained either at a 

constant level or, in the absence of glutamine, continued to increase over 

the entire culture (data not shown). Glucose and glutamine consumption 

rate remained unaffected under all the conditions tested. 

Lower initial glutamine concentration improved stable IgG production 

in growth-arrested stable CHO-DG44 cells and pools: A stable cell clone 

and cell pool of CHO-DG44 cells expressing IgG were cultivated in the 

presence of different glutamine concentrations under mild hypothermia 

conditions (31 °C). Both the clone and the cell pool showed improved 

production of IgG with reduction in glutamine concentration. The IgG titers 

were approximately 80% higher for the clone and 60% higher for the pool 

at 0 mM glutamine, compared to titers obtained with 6 mM glutamine 

(data not shown). 

Conclusions: The effects of different concentrations of glutamine on IgG 

production in growth arrested cells were investigated. Recently published 

results show that CHO-DG44 cells are arrested in G1 phase of the cell cycle 

under mild hypothermia at 31 °C [6]. For HEK-293E cells growth arrest was 

induced with VPA [7]. We conclude that a lower glutamine concentration



Figure 1(abstract P35) Effect of glutamine concentration on relative A) IgG production and B) Ammonia accumulation in CHO-DG44 cells and HEK-293E 

cells under transient transfection conditions on day 7. 

results in improved transient antibody titers in CHO-DG44 and HEK-293E 

cells mainly due to lower accumulation of ammonia in the culture, which 

has previously been shown to have a negative impact on cellular 

productivity [1-4]. Glutamine reduction also had a positive impact on 

recombinant IgG production in a stable clone and a pool of recombinant 

CHO-DG44 cells at 31°C. These data suggest that this strategy may be 

successful in both transient and stable gene expression processes under 

conditions of growth arrest. 

Acknowledgments: We thank Tatiana Benavides and Mattia Matasci for 

providing the CHO clone and pool. Part of this work was supported by the 

Swiss Innovation Promotion Agency KTI/CTI of the Swiss Federal Department 

of Economic Affairs (n. 10203.1PFLS-LS) under a collaboration with 

ExcellGene SA (Switzerland). 

References 

1. Canning WM, Fields BN: Ammonium chloride prevents lytic growth of 

reovirus and helps to establish persistent infection in mouse L cells. 

Science 1983, 219:987-988. 

2. Hansen HA, Emborg C: Influence of ammonium on growth, metabolism, 

and productivity of a continuous suspension Chinese hamster ovary cell 

culture. Biotechnol Prog 1994, 10:121-124. 

3. Ito M, McLimans WF: Ammonia inhibition of interferon synthesis. Cell Biol 

Int Rep 1981, 5:661-666. 

4. Reuveny S, Velez D, Macmillan JD, Miller L: Factors affecting cell growth 

and monoclonal antibody production in stirred reactors. J Immunol 

Methods 1986, 86:53-59. 

5. Meissner P, Pick H, Kulangara A, Chatellard P, Friedrich K, Wurm FM: 

Transient gene expression: recombinant protein production with 

suspension-adapted HEK293-EBNA cells. Biotechnol Bioeng 2001, 

75:197-203. 

6. Wulhfard S, Tissot S, Bouchet S, Cevey J, De Jesus M, Hacker DL, Wurm FM: 

Mild hypothermia improves transient gene expression yields several fold 

in Chinese hamster ovary cells. Biotechnol Prog 2008, 24:458-465. 

7. Greenblatt DY, Vaccaro AM, Jaskula-Sztul R, Ning L, Haymart M, 

Kunnimalaiyaan M, Chen H: Valproic acid activates notch-1 signaling and 

regulates the neuroendocrine phenotype in carcinoid cancer cells. 

Oncologist 2007, 12:942-951. 

P36 

kLa as a predictor for probe-independent mammalian cell bioprocesses 

in orbitally shaken bioreactors 

Stéphanie Tissot, Dominique T Monteil, Lucia Baldi, David L Hacker, 



Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland 




Background: Orbitally shaken flasks are commonly used at an early stage 

of bioprocess development with mammalian cells. In contrast to large-scale 

stirred-tank bioreactors, shaken flasks are usually operated in probeindependent 

bioprocesses, i.e. without strictly controlling the pH or 

dissolved oxygen concentration (DO). As a consequence, gas transfer issues 

are thought to limit the effectiveness of orbitally shaken flasks and 

bioreactors (OSRs). To define optimal operating conditions for probeindependent 

bioprocesses in OSRs, we tested the effects of the mass 

transfer coefficient of oxygen (k La) on mammalian cell growth, recombinant 

protein production, and environmental conditions of the culture (pH, DO). 

Materials and methods: The k La was measured by the dynamic method 

describedin[1]usingnon-invasiveO 2 sensors (PreSens, Regensburg, 

Germany). A recombinant CHO DG44-derived cell line expressing a human 

IgG monoclonal antibody (CHO-IgG) [3] was cultivated in suspension as 

described [4]. To investigate the effects of the k La on cell growth, CHO-IgG 

cells were into 1-L cylindrical bottles with working volumes from 200 to 600 

mL. The bottles were equipped with vented caps and orbitally shaken at 

110 rpm in an incubator at 37°C with 5% CO 2. To test the k La as a scale-up 

factor, CHO-IgG cells were inoculated at 0.3 million cells/mL in a 200-L OSR 

(Kühner AG, Birsfelden, Switzerland) with a working volume of 100 L and 

agitated at 57 rpm. Air containing 5% CO 2 was flushed into the OSR at 1 L 

min -1 . After overnight cultivation, samples were withdrawn from the 100-L 

culture and used to inoculate satellite cultures in 1- and 5-L bottles with 

vented caps. The volume of the cultures in bottles was adjusted to obtain 

the same k La as the one in the 200-L bioreactor (7 h −1 ), and the bottles were 

agitated at 110 rpm. 

Results: In a 1-L OSR the k La decreased from 11 to 3 h-1 as the working 

volume increased from 200 to 600 mL (Fig. 1a). As the working volume of 

the cultures increased in the 1-L OSR, the DO decreased (Fig. 1b). In all the 

cultures, the pH decreased with time of cultivation (Fig. 1c) At working 

volumes greater than 400 mL (k La



CHO-IgG cells. Cell cultivation in a 200-L OSR without pH or DO controllers 

resulted in similar cell densities, recombinant protein titers and pH values 

as in 1- and 5-L OSRs when the three types of OSRs were operated at the 

same k La. These results suggest that large-scale bioprocesses can be 

operated without pH or DO controllers as long as a sufficient kLa is 

maintained through appropriate cultivation conditions (e.g. working 

volume, agitation rate, geometry of the vessel). 

Acknowledgments: We thank Dr. Mattia Matasci for providing the CHO- 

IgG cell line. We gratefully acknowledge: Kühner AG, and Sartorius-Stedim 

Biotech, for the considerable support of equipment and material. This work 

has been supported by the CTI Innovation Promotion Agency of the Swiss 

Federal Department of Economic Affairs and by the Swiss National Science 

Foundation (SNSF). 

References 

1. Gupta A, Rao G: A study of oxygen transfer in shake flasks using a noninvasive 

oxygen sensor. Biotechnol Bioeng 2003, 84:351-358. 

2. Oberbek A, Matasci M, Hacker DL, Wurm FM: Generation of stable, highproducing 

CHO cell lines by lentiviral vector-mediated gene transfer in 

serum-free suspension culture. Biotechnol Bioeng 2011, 108:600-610. 




Figure 1(abstract P36) Effects of the k La on CHO-IgG cell cultures. The k La was measured in 1-L OSR with working volumes from 200 to 600 mL (a). The 

CHO-IgG cells were cultivated in 1-L OSR in 200 (⃝), 300 (⃞), 400 (Δ), 500 (●) and 600 mL (■). The DO (b), pH (c) and viable cell density (d) were 

measured at the times indicated. The shaking diameter was 5 cm. 


Bioeng 2011, DOI: 10.1002/bit.23167. 

4. Muller N, Girard P, Hacker DL, Jordan M, Wurm FM: Orbital shaker 

technology for the cultivation of mammalian cells in suspension. 


P37 

Transient transfection of insect Sf-9 cells in TubeSpin® bioreactor 

50 tubes 

Xiao Shen 1† , Patrik O Michel 1† , Qiuling Xie 1,2 , David L Hacker 1 , 

Florian M Wurm 1* 

1 



2 

Institute of Bioengineering, Jinan University, Guangzhou 510632, 

Guangdong, P. R. China 



Background: Sf-9 cells, derived from Spodoptera frugiperda, arewidely 

used for recombinant protein production using the baculovirus expression



vector system (BEVS). However, this results in a productive viral infection 

and cell lysis. Therefore, a non-lytic, plasmid-based expression system for 

suspension Sf-9 cells would be a valuable alternative to the BEVS for rapid, 

scalable, and high-yielding recombinant protein production and for the 

generation of stable Sf-9 cell lines [4]. In this work, we present a simple, 

efficient and cost-effective plasmid-based method for transient expression 

of recombinant proteins in Sf-9 cells cultivated in serum-free suspension 

mode in a high-throughput culture system, TubeSpin® bioreactor 50 tubes 

(TubeSpins). 

Materials and methods: Sf-9 cells were maintained in suspension in 

TubeSpins (TPP, Trasadingen, Switzerland) at 28 °C [5]. The human tumor 

necrosis factor receptor-Fc fusion protein (TNFR-Fc) gene was cloned into 

pIEx10 (Novagen, Merck, Darmstadt, Germany) to generate pIEx-TNFR-Fc. 

The cells in exponential growth phase were inoculated in fresh Sf900 II 

medium (Invitrogen, Carlsbad, CA) one day prior to transfection. The next 

day, the cells were transfected with 1.5 µg pTNFR-Fc and 2.25 µg linear 

25 kDa polyethylenimine (PEI, Polysciences, Warrington, PA) per 10 6 cells. 

The DNA and PEI were first mixed in de-ionized water at room temperature 

for 10 min prior to addition to the culture. At the time of transfection the 

cell density was 20 x 10 6 cells per mL. Cells were subsequently diluted to 4 x 

10 6 cells per mL with fresh Sf900 II media to allow for growth. The culture 

was maintained at 28 °C in an incubator shaker for 7 d [5]. The TNFR-Fc 

concentration in the medium was determined by ELISA as described [6]. 

Results: Sf-9 cells were transfected in TubeSpins with pIEx-TNFR-Fc. By 7 d 

post-transfection, the TNFR-Fc concentration reached 42 mg/L (Figure 1). 

In a separate transfection with a plasmid expression the enhanced green 

fluorescent protein (GFP) gene, 58% of the cells were GFP-positive at 5 d 

post-transfection (data not shown). 


Conclusions: This study validates the use of PEI for transient expression in 

suspension Sf-9 cell cultures. This system was used to produce both 

intracellular (GFP) and secreted (TNFR-Fc) proteins. The results show that 

PEI-mediated transient transfection is a fast and efficient alternative to BEVS 

for high-yielding protein expression in Sf-9 cells. 

Acknowledgements: This work has been supported by the CTI Innovation 

Promotion Agency of the Swiss Federal Department of Economic Affairs, by 

grants from the Guangdong Provincial Department of Science and 

Technology (2008B030301349), the MOE of China (211 Grant), and the 

Academy of Finland (decision no. 135820). TPP (Trasadingen, Switzerland) is 

acknowledged for providing TubeSpin® bioreactor 50 tubes. 

References 

1. De Jesus M, Wurm FM: Manufacturing recombinant proteins in kg-ton 

quantities using animal cells in bioreactors. Eur J Pharm Biopharm 2011, 

78:184-188. 

2. Harrison RL, Jarvis DL: Transforming lepidopteran insect cells for continuous 

recombinant protein expression. Methods Mol Biol 2007, 388:299-316. 

3. Jarvis DL: Baculovirus-insect cell expression systems. Methods Enzymol 

2009, 463:191-222. 

4. Li E, Brown SL, Dolman CS, Brown GB, Nemerow GR: Production of 

functional antibodies generated in a nonlytic insect cell expression 

system. Prot Expr Purif 2001, 21:121-128. 

5. Xie Q, Michel PO, Baldi L, Hacker DL, Zhang X, Wurm FM: TubeSpin 

bioreactor 50 for the high-density cultivation of Sf-9 insect cells in 

suspension. Biotechnol Lett 2011, 33:897-902. 

6. Oberbek A, Matasci M, Hacker DL, Wurm FM: Generation of stable, highproducing 

CHO cell lines by lentiviral vector-mediated gene transfer in 

serum-free suspension culture. Biotechnol Bioeng 2011, 108:600-610. 

Figure 1(abstract P37) Sf-9 cells were transfected with pTNFR-Fc (■) plasmid DNA and PEI . The TNFR-Fc concentration in the medium was determined 

by ELISA at the times indicated.



P38 

Transient gene expression with CHO cells in conditioned medium: 

a study using TubeSpin® bioreactors 

João Pereira, Yashas Rajendra, Lucia Baldi, David L Hacker, Florian M Wurm * 


Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland 



Background: Transient gene expression (TGE) allows rapid protein 

production in mammalian cells and has become an important tool in the 


pharmaceutical product development pipeline [1]. Polyethylenimine (PEI)mediated, 

high-density transfection allowed to express recombinant 

proteins at yields exceeding 1 g/L in only a few weeks [2]. Although highly 

efficient protocols are available, volumetric scale-up of TGE is still a 

challenge. A major issue is the need to perform the transfection in fresh 

medium rather than in conditioned (spent) medium. This implies a 

medium exchange step just before transfection. In CHO-DG44 [3] cells we 

observed up to a 100-fold decrease in volumetric protein production if 

transfections were performed in conditioned medium, compared to fresh 

medium. The reasons for such a negative effect of conditioned medium on 

transfectability and/or protein production expression are not yet known. 

To study this problem we transfected CHO cells at small-scale in TubeSpin® 

Figure 1(abstract P38) Using the TubeSpin® bioreactors, 38 commercially available cell culture media were studied for their impact in cell growth and in 

transient protein production in a 7 day batch process at the 5 mL scale. Error < 5 % for growth rate. (n=2).



bioreactor 50 tubes using 41 different commercially available serum-free 

media formulations in combination with different transfection parameters 

and culture conditions. By comparing the transient production of a 

recombinant IgG antibody among the different media, we observed 

variation of up to 400-fold when transfecting in fresh media and up to 

20-fold when using conditioned medium. The optimization of the PEI:DNA 

ratio allowed a significant improvement in yields of transfection in 

conditioned medium. 

Methods: Suspension-adapted CHO-DG44 cells [3] were routinely 

cultivated in TubeSpin® bioreactor 50 tubes (TPP, Trasadingen, 

Switzerland) in ProCHO5 medium (Lonza, Vervier, Belgium) supplemented 

with 13.6 mg/L hypoxanthine, 3.9 mg/L thymidine, and 4 mM glutamine. 

The 38 media samples for transfection were provided by Excellgene SA. 

Conditioned medium is defined as a cell culture medium where cells 

have been growing for more than two days up to a density between 4-5 

million cells/mL. Cell growth was accessed with the Packed Cell Volume 

method. The dual expression vector pXLGCHO-A3, containing the cDNAs 

coding for human anti-Rhesus D IgG1 heavy and light chain cloned in 

separate expression cassettes in a head-to-head orientation, was kindly 

provided by Excellgene SA [4]. Transfections are performed at a cell 

density of 5.5 million cells/mL at a volume of 5 mL by the direct addition 

of 15 µg of pDNA and 76 µg of linear 25 kDa Polyethylenimine (PEI, 

Polysciences, Eppenheim, Germany). 

Results: Cells were inoculated in 38 different commercially available serum 

free media formulations tested, and cell growth was assessed daily by the 

packed cell volume method [5], over 3 days of culture. We identified 16 

media formulations that allowed for fast cellular growth (doubling time 

< 20h) up to the cell density of transfection (Figure 1). The same TGE 

protocol was applied in the 38 media formulations tested in a 7-day long 

batch process. Antibody productivity was analyzed by ELISA at day 7. 

Recombinant IgG production was observed only in 13 among the 38 

media formulations studied, and only in 5 media titers over 100 mg/L were 

achieved. Transfection in conditioned media yielded in the best cases only 

1/10 th of the production titers observed in fresh medium. Interestingly, the 

chemically defined media which sustained the fastest cellular growth (1-5, 

Figure 1) were not suitable for transient gene expression under the 

transfection conditions applied in this study. 

In a separate study, we tested different transfection conditions that could 

lead to an improvement of the process yields (data not shown). We 

observed that increasing the PEI and DNA concentrations could improve 

transient gene expression titers 3-fold when transfecting in conditioned 

medium. 

Conclusion: This work shows that the cell culture medium has a strong 

impact on the transient gene expression process. We have observed that 

even in media formulations that sustain a very good cell growth, 

polycation-mediated transfection is not very efficient. When transfecting in 

conditioned medium with a transfection procedure optimized for fresh 

medium, the transient gene expression process is not satisfactory in any of 

the media formulation tested. We were able to improve the transfection 

process by changing the PEI:DNA ratio (data not shown). In order to design 

a robust transfection protocol that would be effective in fresh medium as 

well as in conditioned medium, it is necessary to understand what factors 

affect the transfection negatively in the latter. This study suggests that a 

process without a medium exchange can be designed, facilitating easier 

scale-up of transient gene expression. 

Acknowledgments: This work was supported by the Ecole Polytechnique 


Swiss Federal Department of Economic Affairs (n. 10563.1PFLS-LS) under 

a collaboration with ExcellGene SA (Monthey, Switzerland). TPP 

(Trasadingen, Switzerland) is acknowledged for providing TubeSpin® 

bioreactor 50 tubes. 

References 

1. Baldi L, Hacker DL, Adam M, Wurm FM: Recombinant protein 

production by large-scale transient gene expression in mammalian 

cells: state of the art and future perspectives. Biotechnol Lett 2007, 

29(5):677-84. 

2. Geisse S: Reflections on more than 10 years of TGE approaches. Protein 

Expr Purif 2009, 64(2):99-107. 

3. Urlaub G, Chasin LA: Isolation of Chinese hamster cell mutants deficient 

in dihydrofolate reductase activity. Proc Natl Acad Sci U S A 1980, 

77(7):4216-20. 


4. Miescher S, Zahn-Zabal M, De Jesus M, et al: CHO expression of a novel 

human recombinant IgG1 anti-RhD antibody isolated by phage display. 

Br J Haematol 2000, 111(1):157-66. 

5. Stettler M, Jaccard N, Hacker D, et al: New disposable tubes for rapid and 

precise biomass assessment for suspension cultures of mammalian cells. 

Biotechnol Bioeng 2006, 95(6):1228-33. 

P39 

Hydrodynamic stress in orbitally shaken bioreactors 

Stéphanie Tissot 1 , Martino Reclari 2 , Samuel Quinodoz 3 , Matthieu Dreyer 2 , 

Dominique T Monteil 1 , Lucia Baldi 1 , David L Hacker 1 , Mohamed Farhat 2 , 

Marco Discacciati 3 , Alfio Quarteroni 3 , Florian M Wurm 1* 

1 Laboratory of Cellular Biotechnology, Faculty of Life Sciences, Ecole 

Polytechnique Fédérale de Lausanne; 2 Laboratory of Hydraulic Machines, 

School of Engineering, Ecole Polytechnique Fédérale de Lausanne; 3 Chair of 

Modeling and Scientific Computing, School of Basic Sciences, Ecole 

Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland 



Background: Orbitally shaken bioreactors (OSRs) of nominal volume from 

50 mL to 2’000 L have been developed for the cultivation of suspensionadapted 

mammalian cells. Here we study the hydrodynamics of OSRs for 

mammalian cells. The results are expected to allow the determination of 

key parameters for cell cultivation conditions and will facilitate the scaleup 

of OSRs. 

Materials and methods: CHO-DG44 cells were cultivated in suspension 

in 1-L bottles as described in [1]. To determine conditions under which 

the shear stress was harmful for the cells, the bottles were orbitally 

shaken on an ES-X platform (Kühner AG, Birsfelden, Switzerland) at 

agitation rates from 150 to 200 rpm for 24 h. Control cultures were run 

in parallel with agitation at 110 rpm. The velocity fields, shear stress, 

and free surface of a 1-L bottle at 110 rpm were simulated with 

Computational Fluid Dynamics (CFD). The Navier-Stokes equation was 

approximated with the finite element method. The simulations were all 

based on a mesh containing 50’000 tetrahedra and 10’000 vertices. The 

area of a finite element was 9.8 cm 2 . Because of the chosen 

discretization, each time step required the resolution of a linear system 

composed of 190’000 unknowns. 

Results: According to the CFD simulations of a 1-L bottle at 110 rpm, the 

maximal shear stress value was situated at the tip of the wave and was 

about 0.075 Pa (Figure 1a). The shear stress was higher at the walls than in 

the center of the liquid (Figure1b). Most of the zones in the liquid phase 

had a shear stress value equal to or lower than 0.02 Pa (Figure1b). The 

zones showing the maximal shear stress values represented less than 1% 

of the liquid phase (Figure1b). 

The cell growth rate was similar for the CHO culture agitated at 110 rpm 

and those at 150 or 160 rpm. According to CFD simulations, the maximal 

shear stress was ≤ 0.17 Pa at these higher agitation rates. At 170 rpm, 

the cell growth rate decreased and cell damage was observed. The 

maximal shear stress value at this agitation rate was about 0.19 Pa. The 

maximal values of shear stress were always situated at the tip of 

the wave independently of the agitation rate. The maximal value of shear 

stress increased with the agitation rate. However, only a small number of 

zones had these maximal values. 

Conclusions: The maximal shear stress value in a 1-L OSR agitated at 

110 rpm with a working volume of 300 mL was one to two orders of 

magnitude lower than values reported to be harmful for CHO cells [2]. This 

indicates that standard cultivation conditions in OSRs are safe for sensitive 

mammalian cells. The maximal shear stress was located at the tip of the 

wave of the free surface. However, as the wave of the free surface rotates 

with time, many zones of the liquid will be affected by the wave tip over 

time. Therefore, its potential to damage cells can not be neglected. Our 

study suggests that different wave patterns may lead to different maximal 

shear stress values. Further analysis of the correlation between the shape 

of the wave and the maximal shear stress level are required to determine a 

scale-up factor for hydrodynamic stress in OSRs. 


We thank Dr. Mattia Matasci and Dr. Agata Oberbek for providing cell lines. 

We gratefully acknowledge Kühner AG and Sartorius-Stedim Biotech for the



Figure 1(abstract P39) The shear stress in a 1-L bottle at 110 rpm was simulated by CFD at the free surface (a) and in the liquid phase (cut view) 

(b). The shaking diameter was 5 cm and the working volume was 100 mL. The arrow indicates the tip of the wave. 

considerable support of equipment and material. This work was supported 

by the CTI Innovation Promotion Agency of the Swiss Federal Department of 

Economic Affairs and the Swiss National Science Foundation (SNSF). 

References 

1. Muller N, Girard P, Hacker DL, Jordan M, Wurm FM: Orbital shaker 

technology for the cultivation of mammalian cells in suspension. 


2. Tanzeglock T, Soos M, Stephanopoulos G, Morbidelli M: Induction of 

mammalian cell death by simple shear and extensional flows. Biotechnol 

Bioeng 2009, 104:360-370. 

P40 

A platform fed-batch process for various CEMAX® producer cell lines 

Benedikt Greulich * , Marika Poppe, Henry Woischnig, Karlheinz Landauer, 

Andreas Herrmann 

Celonic AG, Basel, Switzerland 

E-mail: Benedikt.Greulich@celonic.ch 


The random nature of transgene integration harbours various pitfalls for 

development of production cell lines including clonal variation in 

expression level and growth characteristics. The CEMAX system is an 

expression system for targeted integration of expression cassettes via DNA 

double-strand break induced homologous recombination. Stable high 

producers are available within 4 weeks without the need of extensive 

clone screening and process development. 

Stable and high expression rates are ensured with the CEMAX system due 

to targeted integration of a single copy of the gene of interest at a 

transcriptionally highly active site in the host cell genome. Producer cell 

lines for various therapeutic product candidates were established. These 

cell lines produced antibodies and highly glycosylated antibody fusion 

proteins and showed high clonal similarity. This made a platform fed-batch 

process profitable. 

The CEMAX expression system is based on a genetically modified CHO cell 

line bearing a tag at a highly active genomic transcription site. The gene of 

interest (GOI) is integrated via site-directed DNA double-strand breakinduced 

homologous recombination. The target site comprises a screening 

and selection cassette, which was used for initial development of the high 

producer host cell. This exchangeable cassette is flanked by elements that 

facilitate site-directed integration by homologous recombination. These 

elements include regions of homology for recombination with the CEMAX 


vector, rudimentary selection markers that are activated during 

recombination, and cleavage sites for the homing endonuclease I-SceI 

(Meganuclease). 

Transfection of the CEMAX vector comprising the GOI along with transient 

Meganuclease activity triggers a chain of events after cotransfection: DNA 

gets cleaved by I-SceI and cellular DNA repair machinery is induced by free 

DNA double-strand breaks. The CEMAX vector comprising the GOI 

functions as a repair matrix using recombination between homologous 

elements flanking the DNA lesion. The GOI gets integrated and the 

rudimentary selection markers become activated upon homologous 

recombination. CEMAX producer cells were selected at multi-well plate 

scale followed by analysis for outgrowth of stable producer cells. The 

platform process for fed-batch cultivation of various CEMAX producer cells 

was applied after expansion to the scale needed for production of protein 

material. 

The idea of a platform process that is suitable for all CEMAX producer cells 

was based on theoretical consideration about similarity of cells due to sitedirected 

integration and the observation that cell growth and metabolism of 

CEMAX cell lines were comparable. This was verified by the results of this 

study. Applying the fed-batch process cell growth of CEMAX host cells and 

CEMAX producer cells was comparable (Figure 1). The development of the 

platform fed-batch process was based on three small scale development 

steps: (1) basal media screening, (2) feed medium optimization, and 

(3) improvement of feeding regime. Process development in 1 L bioreactors 

is currently ongoing. 

The fed-batch process comprises a chemically defined and commercially 

available basal medium and the chemically defined feed solution CeloFeed 

(Celonic) supplemented according to the improved feeding regime. The 

glucose level was maintained between 4.5 g/L and 7.5 g/L. Glutamine was 

kept at a concentration above 0.8 mM. By applying the platform fed-batch 

process product concentrations up to 690 mg/L were achieved with 

CEMAX cell lines producing a glycosylated Fc fusion protein after sitedirected 

integration of the GOI (Figure 1). Achievable productivity for 

different protein products varied from product to product. This was most 

probably due to different requirements in protein processing and 

secretion. 

The platform fed-batch process was developed by optimization based on 

commercial available and proprietary basal and feed media formulations. It 

was suitable for all producer cell lines derived of a particular CEMAX host 

cell due to highly similar growth behavior after site-directed integration of 

the gene of interest. The platform process made product concentrations of 

up to 0.7 g/L achievable. Animal derived component free chemically



Figure 1(abstract P40) Platform fed-batch process for CEMAX cell lines. Cells were cultivated in comparison to the CEMAX host cell line in shake flask 

scale. The basal medium was a commercially available chemically defined medium and was used in combination with the proprietary chemically defined 

feed solution CeloFeed. Product concentration was measured by Protein A HPLC. 

defined medium and feed solution ensure a robust and regulatory 

compliant process. By combining the platform process with site-directed 

integration timelines in cell line development and process development 

were shortened for faster entry in first-in-man studies. 

P41 

Development of a chemically defined CHO medium by engineering 

based on a feed solution 

Karlheinz Landauer * , Henry Woischnig, Natalie Hepp, Benedikt Greulich, 

Andreas Herrmann 

Celonic AG, Basel, Switzerland 

E-mail: Karlheinz.Landauer@celonic.ch 


The use of chemically defined culture media for production of 

biopharmaceuticals has become state of the art. In combination with a 

suitable feed solution chemically defined media are the most potent driving 

factor for yield improvement in fed-batch processes. Although various 

chemically defined media are available of-the-shelf and deliver good results, 

those media are developed with the aim to sustain growth for various host 

cell lines, and need to be optimized for specific projects. 

We describe here the development of CeloCHO, a chemically defined 

medium for culture of recombinant Chinese hamster ovary cells. The 

medium was developed by engineering based on an optimised proprietary 

feed medium, which was developed previously. The approach was straightforward 

and linked superior results with minimum investments in medium 

development. 

In a first step of development, the components of the proprietary feed 

medium were categorized into five component groups: bulk salts, trace 

elements, vitamins, amino acids, and other components. Necessary 

components not present in the feed medium were considered while 

establishing a basic medium formulation based on theoretic considerations 

of cellular needs in culture (buffer system, adaptation of amino acid 

concentrations, glucose, growth factors, et cetera). This basic formulation 

was combined with various x-fold concentrates of the component group 

stock solutions. 

Seven media formulations were developed this way and were used for 

adaptation in the second step of development. At this stage cell densities 

up to 4×10 6 cells per ml in batch experiments were achieved using a CHO- 

K1 derived recombinant cell line producing a humanized antibody (IgG A). 

Albeit maximum viable cell density and integral of viable cell density were 

significantly less than achieved with the chemically defined reference 


medium for this cell line, product concentration was improved by 7% with 

the newly designed medium. 

The most suitable formulation was selected for fine tuning in the third phase 

of medium development. Therefore, the concentrations of the component 

groups were varied and individual components as well as the buffer system 

were optimized. Vitamins, for example, had a profound effect on cell growth 

and cell specific productivity. The integral of viable cells was reduced with 

vitamin concentrations, while productivity increased. Vitamin concentrations 

above 35% were not suitable for cultivation as observed by diminished cell 

growth. 

The final medium, CeloCHO, was tested with different CHO clones producing 

either IgG or fusion proteins. Figure 1 shows a batch experiment after 

adaptation of a recombinant cell line producing IgG A compared to the 

commercial available reference medium. Maximum viable cell density was 

improved 3-fold compared to the initial medium design with 12×10 6 cells 

per ml and exceeded the performance of the chemically defined reference 

medium. The product concentration was improved by 14% in the batch 

experiment and 57% in the fed-batch experiment compared to the 

reference medium (Table 1). Product concentration was 1.4 g/L without 

further process optimization. The improvement in fed-batch indicated a 

good compatibility of basal and feed medium. The feed medium that was 

used for supplementation was also used for the development of the initial 

basal medium formulation, which was probably the reason for the good 

performance. Product concentration in fed-batch experiments with other 

CHO-K1 derived producer cell lines was improved between 6% and 25% 

demonstrating suitability for different CHO-K1 derived cell lines (Table 1). 

The strategy for the development of a basal medium formulation based on 

a previously optimized feed solution proved suitable for medium 

development approaches without the need of extensive analytical work. 

Components were classified into five stock solutions based on chemical 

properties and combined as x-fold concentrates for fast media development. 

After a round of modifications regarding vitamins, buffer and salt 

optimization, cell densities up to 12×10 6 cells per ml were achieved in 

batch experiments with the chemically defined, animal derived component 

free CHO medium. The product concentration in fed-batch experiments 

was improved up to 57% compared to the reference medium for the cell 

line producing IgG A. Data of the third phase of medium development 

delivered a starting point for medium optimization to clonal demands, 

since the effects of several key components were studied in detail. Cell 

growth and specific productivity, for example, could be modulated in a 

vitamin-dependent way. Six cell lines were studied for growth and 

recombinant protein production using CeloCHO and demonstrated 

feasibility for universal use.



Figure 1(abstract P41) Adaptation of cell line producing IgG A to CeloCHO and batch experiment. The experiment was performed in shake flask scale 

without process optimization. 

Table 1(abstract P41) Relative product concentration in batch and fed-batch mode using CeloCHO with 4 different cell 

lines 

Cell line, CHO-K1 derived IgG A, batch IgG A, fed-batch IgG B, fed-batch IgG C, fed-batch 

Reference medium a , Titer b 

100% 100% 100% 100% 

CeloCHO, Titer b 

114% 157% 125% 106% 

a b 

Chemically defined reference Medium optimized for each cell line. Normalized to product concentration in reference medium. 

P42 

Process development of ATROSAB, an anti TNFR1 Monoclonal 

Antibody: in three steps from research to GMP 

Karlheinz Landauer 1* , Cuneyt Unutmaz 1 , Simon Egli 1 , Verena Berger 2 , 

Simone Lais 1 , Timo Liebig 1 , Daniel Steiner 1 , Jo Maier 1 , Ina Rostalski 1 , 

Francois Forcellino 1 , Andreas Herrmann 1,2 

1 Celonic AG, Basel, Switzerland; 2 Celonic GmbH, Jülich, Deutschland 

E-mail: Karlheinz.Landauer@celonic.ch 


The humanized monoclonal antibody ATROSAB is targeting specifically 

the TNF receptor 1(TNFR1). TNF is a central mediator of inflammation and 

key target for intervention in inflammatory diseases such as rheumatoid 

arthritis, psoriasis and Crohn’s Diseases. Notably, blockade of the second 

TNF receptor, TNFR2, has been associated with increased sensitivity to 

viral infections or increased susceptibility to demyelinating disorders and 

lymphomas. In this context, a selective inhibition of TNF-induced TNFR1 

but not TNFR2 signaling activity holds great promises to overcome 

undesired effects observed with less specific TNF antagonists currently 

used in clinic. 

The recently humanized antibody was used to establish a rCHO-K1 cell line 

under serum-free, chemically defined conditions (1). The process 

development was based on two sets of shake flask experiments, three 10L 

bioreactor runs and finally 2 GMP production runs in 300 L scale. The scale 

up strategy was based on quality by design considerations specified in the 

design space for the scale-up parameters. The main parameter for the scaleup 

development was mixing time as a function of the bioreactor geometry, 

tip-speed, Reynolds number, and the power input of the systems. Those 

parameters are generally considered as important parameters during upstream 

process development. 

Protein characteristics were specified based on several considerations. 

A major part was the ICH-Q8 guideline as well as antibody and other 


drug guidelines. The other part is based on experience with antibody 

development and the specific protein in special. The specifications are 

described in Table 1. The analytical methods used to characterize the 

antibody were SDS-PAGE, western blot, HPLC-SEC, protein A HPLC, 

HPAEC-PAD, Isoelectric focusing and a cell based potency assay. As the 

antibody is declined to bind to the TNF-R1 on target cells, it was 

engineered not to elicit ADCC or CDC. Therefore those parameters were 

not checked during process development. 

The basal medium was fixed to a commercial available chemically defined 

medium during clone screening. In a batch process, performed in a fully 

equipped 1L bioreactor, a titer of approximately 180 mg/L was obtained. 

At this stage the antibody was characterized and specifications were 

partly narrowed and some were newly set. In the first step of process 

development different feeding solutions and concentrations thereof were 

tested in shake flask experiments. Metabolite data were monitored on a 

daily basis comprising glucose, lactate, ammonia, and amino acid levels. 

Additionally cell density and protein concentration were measured at the 

same time interval. For in-depth protein characterization, supernatant was 

purified after 6 days of fermentation and at harvest, in order to check the 

quality attributes already early at the development stage. This step led to 

an increase of about 2.5 fold in product concentration, while the protein 

characteristics stayed within specifications at both sampling time points. 

The second step was the optimization of the feeding strategy as well as 

the fortification of the solutions with certain chemically defined 

supplements. This led to another increase of about 2.5 fold regarding 

final product concentration. Product quality changed slightly, but stayed 

within specifications. Especially the glycosylation structure changed 

towards less glycosylated versions. However, no impact on the bioassay 

was observable. The process was finally scaled up to the 300L stirred tank 

bioreactor in the GMP facility. An engineering run was performed to test 

the process and product parameters. Some influence was measured, thus 

the process was slightly changed prior the first GMP run. The obtained 

product quality was well within specifications, the achieved titer was



Table 1(abstract P42) specified scale-up parameters and defined protein characteristics 

scale-up parameter protein characteristics 

Parameter Design Space 300L GMP specifications defined at the 

project start 

300L GMP 

Mixing Time [s] 10 - 70 max. 60 Isoforms (IEF) pH 7.6 - 8.8 

Tip-Speed [m/s] 0.6 - 1.2 max. 1.0 Glycosylation 

Mol% of different forms ± 20% from clone 

(HPAEC-PAD) 

screening 

Reynolds 

Number 

1 - 8 Mio max. 6 Mio Potency 40% - 160% from clone screening 

Power Input [W/ 25 - 1100 max. 650 Molecular weight 

Main Band at app. 150 kDa 

m3] 

(SDS-PAGE) 

Gassing Head Space / Ring Head Space & Ring Immunogenic glycostructures none 

Sparger 

Sparger 

Agitation orbital sh., Rushton I. Rushton I. Purity (HP-SEC) > 90% 

Figure 1(abstract P42) The left diagram shows the cell density of two GMP runs in the 300L bioreactor. The right diagram focuses on the achieved 

product concentrations during the different process development steps. The batch experiment was performed in 1L bioreactors, the feed screening was 

done in 250 mL shake flasks. The feed optimization was performed in 10L bioreactors and the GMP runs were done in a 300L stirred tank bioreactor in a 

GMP facility. 

more than 1.2 g/L, which was a more than 7 fold increase in comparison 

to the initial batch process. Although the cell density profile of the 

process changed between run 1 and 2, the volumetric product concentration 

was only slightly below 1.2 g/L and the product characteristics 

were identical to the first run. This indicates the robustness of the 

developed process and the validity to define and specify design spaces 

for process development especially during scale-up development. The 

analysis of protein characteristics employing SDS-PAGE, IEF, HP-SEC, 

potency assay and glycosylation pattern with HPAEC-PAD ensures the 

quality of the protein. 

Reference 

1. Zettlitz KA, Lorenz V, Landauer K, Münkel S, Herrmann A, Scheurich P, 

Pfizenmaier K, Kontermann R: ATROSAB, a humanized antagonistic antitumor 

necrosis factor receptor one-specific antibody. mAbs 2 2010, 

2(6):639-647. 

P43 

Cell line development using the SEFEX system 

Benedikt Greulich 1* , Verena Berger 2 , Marika Poppe 1 , Natalie Hepp 1 , 

Karlheinz Landauer 1 , Andreas Herrmann 1,2 

1 Celonic AG, Basel, Switzerland; 2 Celonic GmbH, Jülich, Deutschland 

E-mail: Benedikt.Greulich@celonic.ch 



Cell lines producing biopharmaceuticals with high yield and high quality 

in a regulatory compliant environment are a prerequisite for cost 

effective bioproductions. The development of these production cell lines 

often includes screening strategies combined with gene amplification and 

limited dilution experiments, a time consuming process. Especially gene 

amplification tends to interfere with clonal stability. Limited dilution,



especially using a serum-free culture environment is prone to failure and 

low clone yields. 

We present here the SEFEX platform technology for the development of 

non-amplified high yield production cell lines. The strategy is based on a 

regulatory compliant method for transfection and single cell cloning using 

a proprietary, fully tested, CHO-K1 host cell line adapted to chemically 

defined medium. Fast track cell lines were generated by selection of single 

cell derived clones within 2.5 months after transfection. 1.0 g/L product 

concentration were achieved after two rounds of process development 

using these cell lines. Optimized cell lines were developed based on fast 

track cell lines employing a second transfection. These cell lines were 

capable for production of 2.6 g/L during an early phase of process 

development. 

Cell line development with the SEFEX technology comprises steps carried 

out entirely under serum-free conditions including transfection, selection, 

and single cell cloning. Fast track cell lines were developed within 2.5 

months by transfection and selection of stably transfected cells followed by 

single cell cloning. During single cell cloning, semi-automated photo 

documentation at the single cell level is used to assure and document 

clonality in a regulatory compliant manner. Table 1 summarizes cell specific 

productivity and product concentration in fed-batch processes. More than 

1.0 g/L were achieved in a GMP process at 300 L scale after two steps of 

process development followed by scale-up development. A similar scale-up 

strategy including GMP production was summarized by Landauer et al. in 


this issue [1]. Nevertheless, production of proteins that are difficult to 

express like, IgG C in Table 1, suffered from low productivity and low yield. 

Optimized cell lines, which were obtained by a second serial transfection of 

the antibody expression vector including regulatory sequences, were 

suitable to deliver high rates of antibody production. The productivity of IgG 

C was improved 6-fold to18 pg/c/d. This allowed production of the difficult 

to express protein at a product concentration of 1.1 g/L in fed-batch mode 

(Table 1). Other examples of optimized cell lines provided product 

concentrations of 1.7 g/L without any optimization during cell line 

development (IgG A, Table 1), or 2.6 g/L after two steps of upstream process 

development at 1 L bioreactor scale (IgG B, Table 1, Figure 1). 

Figure 1 shows growth curves of the fast-track and optimized cell line 

producing IgG B. Optimization of maximum viable cell density was not 

addressed during upstream process development so far. This optimization 

is currently ongoing and leaves room for further improvements. Specific 

productivity of the optimized cell lines varied between 18 and 36 pg/c/d, 

which was equivalent to a two- to six-fold improvement compared to the 

fast track cell lines. Productivity was suitable for production of high volume 

products. Scale-up to 300 L stirred tank and 1000 L wave bioreactor for 

production of clinical phase II drug product was shown for production cell 

lines generated with the SEFEX platform technology. 

The SEFEX platform technology for the development of non-amplified high 

yield production cell lines is based on a regulatory compliant, proprietary 

CHO-K1 host cell line adapted to chemically defined medium. Fast track 

Table 1(abstract P43) Cell specific productivity and product concentration obtained with fast track cell lines and 

optimized cell lines 

specific productivity [pg/cell/day] product concentration fed-batch [g/L] 

Project fast track cell line optimised cell line fast track cell line optimised cell line 

1 (IgG A) 11 36 0.6 a 

1.7 a 

2 (IgG B) 12 25 1.0 b 

2.6 c 

3 (IgG C) 3 18 no fed-batch 1.1 d 

a b 

Fed-batch experiment during cell line development without any optimization using chemically defined basal medium and proprietary CeloFeed. Fed-batch 

experiment after two steps of process development and scale-up to 10 L using chemically defined basal and feed medium. c Fed-batch experiment at 1 L 

bioreactor scale after two steps of optimization using chemically defined basal medium and proprietary CeloFeed. d Fed-batch experiment at shake flask scale 

using chemically defined basal medium and proprietary CeloFeed. 

Figure 1(abstract P43) Fed-batch experiments for production of IgG B. The fast-track cell line (left hand graph) was cultivated at 10 L scale after two 

steps of process development and scale up using chemically defined basal and feed medium. The optimized cell line (right hand graph) was cultivated 

at 1 L scale after basal media and feed media screening. A chemically defined medium was used in combination with the proprietary feed solution 

CeloFeed.



cell lines, which were available within 2.5 months produced 1.0 g/L 

product. Optimized cell lines, which were developed based on fast track 

cell lines, were capable for production of 2.6 g/L in early phase of process 

development. Using the SEFEX platform technology, all development steps 

were carried out under entirely serum-free or chemically defined media 

conditions including transfection, selection, and single cell cloning. 

Clonality was assured and documented in a regulatory compliant manner 

using photo documentation of single cells in cell cloning experiments. The 

serial transfection cell line development strategy described here provides 

the possibility to develop production cell lines meeting industrial demands 

employing simplest process development procedures within minimized 

time frames. 

Reference 

1. Landauer K, Unutmaz C, Egli S, Berger V, Lais S, Liebig T, Steiner D, Maier J, 

Rostalski I, Forcellino F, Herrmann A: Process Development of ATROSAB, 

an anti TNFR1 Monoclonal Antibody: in Three Steps from Research to 

GMP. BMC Proceedings 2011 in press, this issue. 

P44 

Platform process for production of monoclonal antibodies for research 

purposes – improvement option 

Joern Meidahl Petersen 1* , Claus Kristensen 2 

1 Biopharm Manufacturing Development, API Support, Gentofte, Denmark, 

DK-2820; 2 Biopharmaceutical Research, Mammalian Cell Technology, 

Maaloev, Denmark, DK-2760 

E-mail: jmp@novonordisk.com 


Background: In 2006 Novo Nordisk decided to invest in building a pipeline 

within inflammatory disease management. To support this strategy the cell 

culture units had to establish technology for expression and production of 

Figure 1(abstract P44) Yields of 11 anitbodies in shake flask and bioreactors. 

monoclonal antibodies in CHO cells. A number of technology providers at 

that time offered proven platform processes for this purpose. It was decided 

to in-license one of these technology platforms, the one developed at Lonza 

Biologics, and focus research resources on product innovation rather than 

development of an in-house production system. The platform has now been 

fully implemented and the work flow optimised and standardised. 

Platform process review: The platform process comprises: 

Host cell line/expression system 

Medium/feeds (chemically defined – animal derived component free) 

Process parameters 

Scale-down model (shake flask, 100 ml working volume). 

The platform process has been applied for cell line development and 

antibody production for R&D purposes for five years. During this period 11 

monoclonal antibodies have been transferred from laboratory scale to pilot 

plant production. The performance of the process platform across projects 

has been reviewed. The scope of the review was to compare yields obtained 

in the scale down model and yields obtained in the bioreactor process for 

all 11 antibodies. Figure 1 shows the results of the review. 

In conclusion the review show that 

The cell lines are yielding 1.9 – 4.5 g/l mAb. 

The scale down model is predictive for the bioreactor process. 

Evaluation of an improvement option: An upgrade of the medium, 

feeds and process protocols was offered by Lonza Biologics. A b-version 

of the latest process from Lonza has been tested and compared to the 

previous version in a study including five cell lines. The experiments were 

carried out in 100 ml shaker flask and the scale down version of the 

process was applied. A comparison of the two fed batch procedures is 

shown below: 

Current version: Version 6 

Medium and feeds CD-ACF 

Two feed solution 

Feed rates based on 

Table 1(abstract P44) Result of the comparison of the two versions of the fed batch process 

mAb Current version: Version 6 New version: Version 8 

ICA* ) 

Lactate mAb ICA Lactate mAb 

10E6 viable cell days/ml mmol/l g/l 10E6 viable cell days/ml mmol/l g/l 

C 108 57 3.5 92 28 7.1 

D 103 61 2.2 90 29 5.2 

E 162 65 5.5 104 17 9.0 

H 100 55 3.2 120 25 7.0 

J 148 62 3.2 164 21 4.3 

* ) ICA: Integrated Cell Area. 

Page 68 of 181



○ Viable Cell Density 

○ Residual glucose 

New version: Version 8 

Medium and feeds CD-ACF 

Five feed solutions 

Feed rates based on 

○ Viable Cell Density 

○ Residual glucose 

○ Time 

The results of the study are compiled in table 1. 

The comparison of the b-test of improved platform process and the 

current process showed: 

mAb yields improved 1.3 – 2.4 fold 

No change in integrated cell area 

Improved lactate control may be the key to the yield improvement. 

Acknowledgements: The following people all contributed with cell lines, 

data and/or stimulating discussions 

Novo Nordisk:Birgitte Friedrichsen, Bjarne Rask Poulsen, Mark Chadfield, 

Ann Merete Mygind, Ida Mølgaard Kundsen, Max Wellerdiek, Gitte 

Johnsen. 

Lonza Biologics:Alison Porter, Jeetendra Vaghjiani. 

Figure 1(abstract P45) Cluster analysis of MALDI-TOF MS spectra from 92 different cell lines. 


P45 

Development and validation of a protocol for cell line identification by 

MALDI-TOF MS 

Guido Vogel 1* , André Strauss 1 , Bernard Jenni 1 , Dominik Ziegler 1 , 

Eric Dumermuth 2 , Sylvie Antz 2 , Claudia Bardouille 3 , Beat Wipf 3 , 

Christian Miscenic 3 , Georg Schmid 3 , Valentin Pflüger 1 

1 Mabritec AG, 4125 Riehen, Switzerland; 2 Novartis Pharma AG , 4056 Basel, 

Switzerland; 3 F. Hoffmann-La Roche AG, 4070 Basel, Switzerland 

E-mail: guido.vogel@mabritec.com 


Summary: Misidentification or cross-contamination of cell lines used in 

biotechnology or diagnostic settings are a challenge for laboratories and cell 

culture repositories. Masters et al. [1] among others reported the occurrence 

of large numbers of unrecognized and unreported misidentification or crosscontamination 

of cell lines. Current methods for the authentication of cell 

lines such as karyotyping, 2D-gel-electrophoresis, restriction fragment length 

polymorphism (RFLP) or short tandem repeats (STR) are expensive, labor 

intensive and not routinely applied. In the last decade MALDI-TOF MS 

became a powerful tool for the rapid and cost effective identification and



Table 1(abstract P45) Example of problematic samples 

sample name expected species MALDI-TOF MS results 

Sf9 Spodoptera frugiperda Trichoplusia ni 

HepG2 Homo sapiens Mus musculus 

SK-MEL-30 Homo sapiens Homo sapiens profile with strongly altered mass composition: contamination possible 

CHO-K1 Cricetulus griseus unknown mass profile 

J558L Mus musculus Mus musculus profile with strongly altered mass composition: contamination possible 

WS1 Homo sapiens unknown mass profile 

taxonomic classification of microorganisms directly from whole cell extracts. 

We adapted this protein fingerprinting approach for a fast species 

identification of eukaryotic cell lines and established as well as validated a 

reference database. 

In addition we demonstrate that this new approach has a potential for the 

rapid characterization of recombinant protein expression systems. Accurate 

protein expression and the determination of the molecular mass of the 

recombinant expressed proteins (20-120kDa) can directly be analyzed from 

stable transfected and virus infected cultures. 

Materials and methods: 1 ml of fresh culture was transferred into an 

Eppendorf tube and washed once with 1x PBS. The pellet was resuspended 

in 70% EtOH for transport and storage at RT. 1µl of the cell pellet was 

transferred with a plastic loop to a new Eppendorf tube and mixed with 

20µl formic acid (10%). The suspension was mixed with 40µl of saturated 

sinapinic acid matrix. 1µl of sample suspension was spotted on a steel target 

plate in duplicates. Spectra were acquired on Shimadzu Confidence MALDI- 

TOFMSinlinearmodeinamassrangefrom2-50kDa.Peaklistswere 

imported into SARAMIS software for Marker pattern definition and 

automated identification. 

Results: In a first phase we analyzed 146 different eukaryotic cell lines 

derived from 11 different taxa (Figure 1), including common lines from 

culture collections as well as customer specific in-house lines. Marker 

patterns were established for these 11 taxa and validated in a blinded 

study with 9 mammalian and 48 insect cell lines. All 57 test samples were 

correctly identified on the species level. Even two intentionally mixed 

samples were assigned to the right species. 

In a second phase we analyzed more than 178 mammalian and 240 

insect cell lines for identity authentication from different customers in 

order to evaluate the usefulness of this service. Out of 418 samples 

analyzed, we revealed 6 samples with either wrong identities, strongly 

altered or unknown mass profiles (Table 1). 

When necessary cell lines were also analyzed for their expression profile 

after infection or transfection with viral vectors. It was possible to 

determine precise masses of overexpressed proteins as compared to 

controlled samples. For these additional experiments, no further sample 

preparation was necessary. 

Conclusion: We developed a fast, simple and accurate method with high 

throughput capacity for the authentication of cell lines. Since the 

construction of customer specific mass profile libraries is straightforward, 

we propose this approach as an additional method for routine cell line 

authentication in biotechnology settings. 

Further we demonstrated that MALDI-TOF MS is also a fast and simple 

tool for the characterization of eukaryotic protein expression systems, 

especially for the determination of a precise mass. 

Reference 

1. Masters JR, et al: Short tandem repeat profiling provides an international 

reference standard for human cell lines. PNAS 2001, 98:8012-8017. 

P46 

DoE of fed-batch processes – model-based design and experimental 

evaluation 

Onur Sercinoglu 1 , Oscar Platas Barradas 1 , Volker Sandig 2 , An-Ping Zeng 1 , 

Ralf Pörtner 1* 

1 Institute of Bioprocess and Biosystems Engineering, Hamburg University of 

Technology, Hamburg, D-21073, Germany; 2 ProBioGen AG, Berlin, D-13086, 

Germany 

E-mail: poertner@tuhh.de 



Background: Experimental process-development and optimization is 

expensive and time-consuming. Real optimization by means of design of 

experiments involves data generation before optimization can be aimed 

for. This can make the way from process development to process 

establishment even harder, since academia or start-up research facilities 

might not have the possibility to generate these data. Furthermore, 

bioprocesses involving mammalian cells deal with many critical variables; 

processes are not only carried out batch wise, but increasingly in fedbatch 

mode with desired feeding profiles. 

The use of DoE tools in combination with an appropriate growth model 

might allow the experimenter to develop and to test fed-batch strategies 

in silico, before experiments are carried out in the laboratory. 

In our work, an unstructured model for mammalian cell culture was used 

for simulation. Kinetic parameters were derived from a small number of 

shake-flask experiments. The model was tested for data generation on 

common fed-batch strategies. By means of design of experiments 

strategies, relevant conditions were selected and experimentally tested. In 

this way, suitable fed-batch strategies for mammalian cell lines are 

evaluated in silico before bioreactor experiments are to be performed. 

This results in a significant reduction in the number of experiments 

during process development for mammalian cell culture. 

Concept – fed-batch Analyzer -: A tool was developed in Mathworks‘ 

Matlab for simulation of batch and fed-batch cultivations of mammalian 

cells. This program was created as a GUI (Guided User Interface), in which 

the user will be guided through the process of developing a fed-batch 

strategy. Growth models can be selected as well as different fed-batch 

modes. For a manageable comparison of fed-batch strategies, in silico 

experiments can be planned by design of experiments (DoE) and initial 

conditions as well as cell-line-specific kinetic parameters can be obtained 

from a reduced number of small-scale experiments e.g. shake flasks. The 

following steps will explain the procedure for in silico experimentation 

during the development of a fed-batch strategy for the human 

production cell line AGE1.hn (ProBioGen AG). Afterwards, one process 

condition will be chosen and tested in the laboratory. 

Step 1: Define the process model: Growth models [1,2] have been 

predefined for operation of the tool. However, the user can modify or 

include new models according to her/his expertise and knowledge of the 

cell line. For determination of model parameters, a reduced number of 

experiments in small scale (e.g. shake flasks), is to be performed. 

Step 2: Load the model: Step 3: Choose a feeding strategy: Simple 

feeding strategies have been predefined in order to make the transfer of 

in silico results into laboratory experimentation easier. Constant feed, 

linear feed, exponential feed and step feed can be selected. New feeding 

profiles can be added to the program to improve its possibilities. 

Step 4: Create design: The design (e.g. full factorial design) can be 

either planed within the program (MATLAB Statistics Toolbox) or loaded 

from other DoE tools (e.g. Design Expert). In order to define the central 

point for the design, data from batch experiments can be used. 

Step 5: Run design: The tool conduct the in silico experiments and 

delivers a response. This is normally a variable the user is interested in 

(IVCD, VCD or product concentration). 

Step 6: Obtain in silico results: For each set of conditions, the tool 

generates a sheet with virtual curves. For an overview of results and 

better comparison of the process conditions, a response curve can be 

generated (see Figure 1). 

Step 7: Implementation in the laboratory: Following experiment was 

planned and performed in a 1 L Bioreactor. (Table 1, Figure 2) 

Acknowledgements: Funding by the BMBF, Grand Nr. 0315275A is 

gratefully acknowledged.



Figure 1(abstract P46) Surface response plan for a feeding strategy with constant feed rate. 

References 

1. Jang JD, Barford J.P: An Unstructured Kinetic Model of Macromolecular 

Metabolism in Batch and Fed-Batch Cultures of Hybridoma Cells 

Producing Monoclonal Antibody. Biochemical Engineering Journal 

4(2):153-168. 

2. Zeng A-P, Deckwer W-D: Mathematical modeling and analysis of glucose 

and glutamine utilization and regulation in cultures of continuous 

mammalian cells. Biotechnology and Bioengineering 1995, 47:3. 

Table 1(abstract P46) Experimental conditions for 

validation of a contant feeding strategy 

Parameter Value 

Initial conditions (batch) 

Glucose 10 mM 

Glutamine 2 mM 

fed-batch strategy 

Mode constant 

Glucose in Feed 60 mM 

Glutamin in Feed 8 mM 

Feed Rate 0.059 mL min -1 

Feed Start 24 h 


Figure 2(abstract P46) Experimental results after culture of AGE1.HN 

cells in bioreactor with a constant feed rate.



P47 

Criteria for bioreactor comparison and operation standardisation 

during process development for mammalian cell culture 

Oscar Platas Barradas 1 , Uwe Jandt 1 , Linh Da Minh Phan 1 , Mario Villanueva 1 , 

Alexander Rath 2 , Udo Reichl 2 , Eva Schräder 3 , Sebastian Scholz 3 , Thomas Noll 3 , 

Volker Sandig 4 , Ralf Pörtner 1* , An-Ping Zeng 1 


Technology, Hamburg, D-21073, Germany; 2 Bioprocess Engineering, Max 

Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, 

D-39106, Germany; 3 Faculty of Technology, AG Zellkulturtechnik, Bielefeld 

University, Bielefeld, D-33501, Germany; 4 ProBioGen AG, Berlin, D-13086, 

Germany 



Background: Development of bioprocesses for animal cells has to deal with 

different bioreactor types and scales. Bioreactors might be intended for 

generation of cell inoculum and production, research, process development, 

validation or transfer purposes. During these activities, not only the difficulty 

of up- and downscaling might lead to failure of consistency in cell growth, 

but also the use of different bioreactor geometries and operation 

conditions. In such cases, the criteria for bioreactor design and process 

transfer should be carefully evaluated in order to avoid an erroneous 

transfer of cultivation parameters. 


In this work, power input, mixing time, impeller tip speed, and Reynolds 

number have been compared systematically for the cultivation of the 

human cell line AGE1.HN® within three partner laboratories using five 

differentbioreactorsystems.Acommonprocesswindowformixing 

time in the range of 8 – 13 s has been found in bioreactors having 

significant differences in their inner geometries. The obtained results 

are employed for process standardisation and transfer between 

research institutions. 

Cell culture in laboratory bioreactors with different inner geometries: 

Finding conditions for consistent cultivation of mammalian cells in 

bioreactors is not an easy task. For standard stirred tanks, correlations 

existing in literature can be used in order to predict operation conditions for 

process transfer purposes. However, if the inner geometry of two bioreactors 

cannot be compared within tolerance ranges, the characterization of the 

bioreactor hydrodynamics becomes necessary. 

For this work, five geometrically different bioreactors were used, which 

are operated within three partner laboratories for data generation 

during research on Systems Biology. Characterization of the bioreactor 

hydro-dynamics was performed with the main goal of finding a 

relationship between process transfer criteria and cell growth in the 

systems. 

Bioreactor characterization: Following criteria were considered for 

characterization of bioreactor hydrodynamics. 

(1) Power input (P/V): Power numbers Np were calculated from Np = f 

(Re) correlations available in literature [1-3]. Corrections for Np were 

Figure 1(abstract P47) Relationship between maximum specific growth rate μ max and values for process transfer during bioreactor culture: 

a) Power input, b) Mixing time, c) Impeller tip speed, and d) Reynolds number. Curve fitting for bioreactors 3 (Black square) and 5 (Purple circle).



considered due to geometry deviations from a standard configuration 

[4-7]. Volumetric power inputs were calculated according to Equation 1: 

P 

V 

= 

NpN d 

V 

3 5 r 

(2) Mixing time (Θ 94.5): This criterion was obtained from the 

decolourization of a I/KI solution of after addition of Na 2S 2O 3.Starchwas 

added previously to the bioreactor. Decolourization time course was 

video recorded. The resulting videos were computer analyzed, and Θ 94.5 

was obtained after gray-scale conversion and measurement of loss of 

saturation (MATLAB, MathWorks). 

(3) Impeller tip speed (u tip): calculated according to Equation 2. 

utip = p Ndi 

(2) 

(4) Reynolds Number at impeller tip (Re i): calculated according to 

equation 3: 

Re i 

Ndi 

= r 

2 

h 

The specific growth rate μmax was employed as indicator for comparison 

of bioreactor performance. The use of μmax made the comparison of the 

two cell line clones possible, despite the differences in initial cell 

densities during bioreactor culture. 

Relationship between cell growth and process transfer criteria: 

Figure 1 shows the dependency of the μmax on process transfer criteria. A 

process window for mixing time values between 8 and 13 seconds can 

be identified as common for all bioreactors, where the smallest deviation 

in µ max between different bioreactors can be observed. 

Conclusions: Criteria for process transfer were analyzed during the 

cultivation the human production cell line AGE1.HN. Growth was 

compared within ranges for power input, mixing time, impeller tip speed 

and Reynolds number. Maximum specific growth rates were observed for 

AGE1.HN cells at a common mixing time range of 8 - 13 seconds for all 

cultivation systems. This criterion was observed to be a reference for 

consistency of results within laboratory bioreactors with different internal 

geometry. 

Funding by the BMBF, Grand Nr. 0315275A is gratefully acknowledged. 

Nomenclature: 

di impeller diameter [m] 

M degree of mixing 

N agitation speed [rpm] 

P = NprN 3 5 

di power input [W m -3 ] 

Rei Reynolds number at impeller tip [-] 

utip impeller tip speed [m s -1 ] 

V working volume [m 3 ] 

Greek letters: 

Θ mixing time [s] 

μmax specific growth rate [d -1 ] 

r density [kg m -3 ] 

h dynamic viscosity [kg m -1 s -1 ] 

References 

1. Rushton JH: Power Characteristics of Mixing Impellers. Part I. Chem. Eng. 

Prog 1950, 46(8):395-404. 

2. Rushton JH: Power Characteristics of Mixing Impellers. Part II. Chem. Eng. 

Prog 1950, 46(9):467-476. 

3. Bates RL: An examination of some geometric parameters of impeller 

power. I&EC Process Design and Development 1963, 2(4). 

4. Markopoulos J, Pantuflas E: Power consumption in gas-liquid contactors 

agitated by double-stage rushton turbines. Chem. Eng. Technol 2001, 

24(11):1147-1150. 

5. Henzler H-J: Verfahrenstechnische Auslegungsunterlagen fur 

Rührbehalter als Fermenter. Chem.-Ing.-Tech 1982, 54(5):461-476. 

6. Hudcova V, Machon V, Nienow AW: Gas-Liquid dispersion with dual rushton 

turbine impellers. Biotechnology and Bioengineering 1988, 34:617-628. 

7. Hemrajani RR: Mechanically Stirred Vessels. Handbook of Industrial Mixing. 

Science and Practice Wiley-Interscience 2004. 

(1) 

(3) 


P48 

Cultivation strategies of a BA/F3 cell line for fundamental cell 

research 

Martin Schaletzky 1 , Oscar Platas Barradas 1 , Henning Sievert 2 , 

Stefan Balabanov 2 , An-Ping Zeng 1 , Ralf Pörtner 1* 


Technology, Hamburg, D-21073, Germany; 2 Cancer Center Hamburg, 

University Medical Center Hamburg-Eppendorf (UKE), Hamburg, D-20246, 

Germany 



Background: During the chronic myelogenous leukemia (CML) the Bcr-Abl 

oncoprotein is produced, which leads to unregulated cell proliferation. 

CML is treated with one of several targeting therapies such as imatinib, 

(formerly STI-571 [Glivec; Novartis, Switzerland]) a selective inhibitor that 

blocks tyrosin kinase activity of the Bcr-Abl oncoprotein. Apart from the 

second generation Bcr-Abl inhibitors, identifying novel direct or indirect 

downstream targets of Bcr-Abl could contribute significantly to the 

development of new synergistic treatment strategies against CML. The 

effects of imatinib on the protein expression of Bcr-Abl positive cells are 

being investigated [1]. A protein which is downregulated during treatment 

with imatinib (eukaryotic translation initiation factor eIF5A) was identified. 

This protein is a potentially promising target for single-agent and 

combined-treatment strategies for CML. For protein complex identification 

a high cell number is needed. This is difficult to be obtained reproducibly 

withflaskculturesorrollerbottles.Theaimofthisprojectwastodevelop 

and establish a reproducible bioreactor cultivation of murine suspension 

cell lines (BA/F3 p210), which yields a total cell number close to 1·10 10 cells 

required for analytics. Cells should be in exponential growth under 

constant culture conditions at the time of harvest. A small stirred tank 

bioreactor with a working volume of 150 mL was used to study and 

compare different operation modes: batch, fed-batch and continuous. Cell 

growth and glucose consumption were assessed as main culture 

parameters. 

Material and methods: Cell lines: BA/F3 p210 and BA/F3 p210 eIF5A-2 

(BA/F3 = mouse pro B cells, p210 = Bcr-Abl oncoprotein (210kDa), eIF5A- 

2 = isoform of the eukaryotic translation initiation factor eIF5A). 

In a first step, a working cell bank was established and cell growth was 

characterized in T-flasks. Afterwards, different cultivation modes were 

tested in a stirred tank bioreactor (Vario1000, Medorex, Germany) as 

follows: 

batch: Cultivation volume Vstart = 350 mL, duration: 40 h 

fed-batch: Cultivation volume V start = 345 mL, duration: 64 h, Feeding took 

place every time Glucose concentration fell below 2 mM. Feed medium 

consisted on a mixture of batch medium and higher concentrations of 

glucose and glutamine. 

Continuous: Cultivation volume V start = 115 mL, dilution rate D = 0.049 h -1 

duration: 118.5 h. 

Thescale-upexperimentwasperformedina5Lstirredbench-top 

bioreactor (Biostat B, Sartorius Stedim Biotech GmbH) with pH and DO 

control. 

Results and conclusions: In batch mode, the maximum viable cell 

density during exponential growth was VCD max = 14.7·10 5 cells mL -1 .In 

fed-batch mode VCD max = 22.6·10 5 cells mL -1 . This higher cell density is an 

advantage over the batch culture mode. It was not possible to obtain 

higher cell densities in this mode, since the feed medium consisted on a 

formulation for batch culture with further addition of glucose and 

glutamine. In continuous mode the highest possible cell density was 

maintained in the bioreactor, in order to produce continuously cells for 

further treatment. A maximum cell yield of 8.3·10 6 cells h -1 could be 

harvested from the bioreactor. After scale-up, this yield might be 

increased, so that the needed cell number could be harvested in only few 

days. A disadvantage of the continuous process with cell harvest was 

observed for the storage process, since cell lysis took place after storage at 

4 °C. 

A first approach for scale-up was performed in the 5 L bioreactor (Figure 1), 

where the maximum cell density during exponential phase allowed for the 

needed cell number. Regarding the required reproducibility for cultivation, 

the 5 L batch mode was preferred over T-flasks due to the possibility for 

control of process variables like pH and pO 2.



Figure 1(abstract P48) Schematic diagram of the final batch process in a 5 L bioreactor that yields a total cell number close to 1·10 10 . 

Figure 2(abstract P48) V start = 5090 mL, max. viable cell density in exponential growth after 39.5 hours VCD max = 18.1·10 5 cells mL - . 

Page 74 of 181



Compared to T-flasks, glucose uptake during bioreactor cultivation was 

much higher, which led to lower final-cell-density yields. fed-batch and 

continuous modes were firstly favored due the theoretical final cell 

numbers reached during culture. However, the difference in growth, 

limitation of bioreactor volume and the need of a special medium 

formulation for higher cell densities during fed-batch, limited the final 

yield. Continuous mode with temperature reduction of harvested cells 

allowed for constant cell production in exponential phase. On the other 

hand, storage of intact cells was limited probably due to protease action. 

The 150 mL batch cultivation was scaled up to 5 L in a stirred bench-top 

bioreactor (Biostat B, Sartorius Stedim Biotech GmbH). 

Reference 

1. Balabanov S, et al: Hypusination of eukaryotic initiation factor 5A (eIF5A): 

a novel therapeutic target in BCR-ABL-positive leukemias identified by a 

proteomics approach. Blood 2007, 109(4). 

P49 

Physical methods for synchronization of a human production cell line 

Oscar Platas Barradas 1 , Uwe Jandt 1 , Ralf Hass 2 , Cornelia Kasper 3 , 

Volker Sandig 4 , Ralf Pörtner 1 , An-Ping Zeng 1* 

1 

Institute of Bioprocess and Biosystems Engineering, Hamburg University of 

Technology, Hamburg, 21073, Germany; 2 Gynecology and Obstetrics. Medical 

University Hannover, Hannover, 30625, Germany; 3 Institute for Technical 

Chemistry, Leibniz University Hannover, Hannover, 30167, Germany; 

4 

ProBioGen AG, Berlin, 13086, Germany 

E-mail: aze@tuhh.de 


Background: The study of central metabolism and the interaction of its 

dynamics during growth, product formation and cell division are key tasks 

to decode the complex metabolic network of mammalian cells. For this 

purpose, not only the quantitative determination of key cellular molecules 

is necessary, but also the variation of their expression rates in time, e.g. cell 

cycle dependent gene expression. Thus, synchronization of cultured cells is 

a requisite for almost any attempt to elucidate these time dependent 

cellular processes. Synchronous cell growth can help to gain deeper 

insight into dynamics of cellular metabolism. 

In our work, physical methods for synchronization of the human production 

cell line AGE1.HN (ProBioGen AG) are experimentally tested. Cell-size 

distribution, DNA-content and the number of synchronous divisions are 

used for comparison of the methods. 

According to our results, the enrichment of an AGE1.HN cell population 

within a cell cycle phase is possible. Currently, the increase of cell yield and 

the improvement of conditions for cell-growth resumption after 

synchronization are being studied. 

Synchronization methods: Synchronization of cells can be defined as the 

enrichment of cells within a certain phase of the cell cycle. Many authors 

have pointed to the importance of further synchronous growth or even a 

narrow cell size distribution. Thus, a synchronized culture is one in which 

cells of similar age progress as a cohort through the division cycle [1]. 

Physical and chemical methods for cell synchronization are well described 

in literature. The choice depends not only on the cell type (suspension, 

adherent, growth medium, etc.), but also on the desired yield of cells and 

the degree of synchrony. For the analysis of cell-cycle dependent 

metabolic processes, the method of choice should not alter the rate of 

cellular reactions as they occur normally. The following physical methods 

are part of this study: (1) Temperature reduction: cell growth can be 

slowed down by means of a reduction of temperature during culture [2], 

which allows for enrichment of cell populations within the G 1 and early 

Table 1(abstract P49) Flow cytometry analysis of one elutriated fraction (ELU8) 

S-phase. Since yields obtained by using this method are low, temperature 

cycles can be used for yield improvement. (2) Gradient centrifugation: 

cells can be separated according to their density in a gradient by 

centrifugation. A sucrose gradient can be used for this purpose, with 

the advantage of being a simple and less expensive procedure. 

(3) Counterflow centrifugal elutriation: cells are separated in a 

centrifugal field; at the same time, a fluid in counterflow is used to 

separate the cells according to their mass within the centrifugal field. 

Depending on the mass of the cell, cell populations can be eluted out of 

the system by increasing the flow rate of the fluid. 

Materials and methods: (1) Temperature reduction: shake flasks were 

inoculated with 1·10 6 cells mL -1 and cultivated at 37 °C and 5% CO 2. After 

24 h the flasks were set at a reduced temperature. Sampling was 

performed at least every 24 h. Samples were treated for flow-cytometry 

analysis. For yield improvement a repeated-batch strategy with 

temperature cycles (37 °C ® 30 °C, 28 °C) was performed in a controlled 

bioreactor (1 L, pH = 7.15, DO = 25% air sat.). 

(2) Gradient centrifugation: discontinuous sucrose gradients were 

prepared in 50 mL centrifuge tubes. A first gradient considered 5 to 60% 

(w/w) of sucrose in culture medium. In order to avoid undesired mixing of 

the gradient layers during layer addition, the tubes were set every time at 

-80°C after each new addition. Phenol red was used for visual identification 

of the layers. A second gradient was produced (20 – 50% w/w sucrose), 

which approaches to the density range of AGE1.hn cells. 1.5·10 8 cells were 

centrifuged and added to the top of the gradient. Centrifugation was 

performed at 230 g for 10 min. After centrifugation samples from the 

gradient were washed in phosphate saline solution (PBS) and analyzed for 

cell-size distribution (Z2 Particle Counter, Beckman Coulter, Germany). 

(3) Counterflow centrifugal elutriation: 7·10 8 cells were centrifuged and 

resuspended in 5 mL PBS. This volume was inserted under sterile 

conditions into an elutriator (Beckman Coulter) and pumped at a minimal 

velocity into the separation chamber. Pumping rate of sterile PBS was 

increased stepwise in order to elute fractions of cells. Nine fractions were 

collected. The size distribution of the fractions and their DNA-content 

(flow cytometry) were analyzed. All fractions were resuspended in fresh 

medium and cultivated in an incubator. 

Results and conclusions: Pre-experiments for temperature reduction 

had shown previously an increase of up to 80% of S-Phase DNAcontent 

during shake-flask culture at 30 °C. Further temperature 

reduction (28 °C) was needed during repeated batch cultivation with 

temperature cycles for cell growth arrest. A viability decrease after 

temperature resumption (37°C) was observed after 50 h at 28 °C and 

might be attributed to apoptosis. Duration of the temperature cycles is 

being currently studied. 

High viability was observed in all samples after gradient centrifugation. 

Cell size distribution was reduced for one sample to almost 80% of cells 

between 12.5 and 15 µm. Gradient can be reduced in order to sharpen 

the separation of the cells. Further culture of a cell population and cell 

cycle analysis are needed to show the feasibility of this method. 

By using the Counterflow Centrifugal Elutriation, cells enriched in 

different phases of the cell cycle were obtained. The cell-cycle analysis and 

the growth curve of one of the fractions (ELU 8) are presented in Table 1 

and Figure 1. 

Funding by the BMBF, Grand Nr. 0315275A is gratefully acknowledged. 

References 

1. Cooper S: Reappraisal of G1-phase arrest and synchronization by 

lovastatin. Cell Biology International 2002, 26(8):715-727. 

2. Enninga IC: Use of low temperature for growth arrest and 

synchronization of human diploid Fibroblasts. Mutation Research 1984, 

130:343-352. 

Cell cycle phase Non-Sync. Elu8 

sub G1 1,5 0,6 

G0/G1 61,6 5,9 

S 17,8 20,8 

G2/M 14,2 52,6 

An increase of more than 35 % of cells in the G2/M-Phase was observed for this fraction. 

Page 75 of 181



Figure 1(abstract P49) 6-day culture of ELU8. Further culture was possible without noticeable perturbation of cell metabolism. 

P50 

Efficient production of recombinant IgG by the GLUT5 co-expression 

system 

Yuichi Inoue 1* , Aiko Inoue 2 , Hiroharu Kawahara 1 

1 The Cell Engineering Center, Kitakyushu National College of Technology, 

Kitakyushu, 802-0985, Japan; 2 KYURIN CORPORATION, Kitakyushu, 806-0046, 

Japan 

E-mail: inoue@kct.ac.jp 


A fructose containing cell culture medium has the advantage of low 

lactate production and a small pH change, leading to cell and product 

stability. But, not all cell lines grow well in the medium, and the fructose 

transporter, GLUT5, is related to it [1]. Thus, we developed an efficient 

production system of recombinant proteins by metabolic control and coexpression 

with GLUT5 in a fructose-based medium [2]. In this report, the 

availability of the GLUT5 co-expression system was indicated in CHO-K1 

and the human cell line, SC-01MFP [3]. 

As a model, an IgG and GLUT5 co-expression vector was constructed and 

transfected into cells. When the transfected CHO-K1 and SC-01MFP cells 

were cultured in the fructose-based medium, both IgG productions were 

increased up to about two-fold of that cultured in the glucose-based 

medium (Table 1). Our study may be useful for efficient production of 

recombinant proteins using the fructose-based cell culture. In particular, 

the production in SC-01MFP cells is valuable for functional analysis of 

recombinant proteins with a human glycosylation profile. 

References 

1. Inoue Y, Kawahara H, Shirahata S, Sugimoto Y: Retinoic acid improves a 

hybridoma culture in a fructose-based medium by up-regulation of 

Table 1(abstract P50) Proliferation and IgG production in the fructose-based medium 

fructose incorporation via retinoid nuclear receptors. Biosci Biotechnol 

Biochem 2006, 70:2248-2253. 

2. Inoue Y, Tsukamoto Y, Yamanaka M, Nakamura S, Inoue A, Nishino N, 

Kawahara H: Efficient production of recombinant IgG by metabolic 

control and co-expression with GLUT5 in a fructose-based medium. 

Cytotechnology 2010, 62:301-306. 

3. Kawahara H: Human cell stains for protein production, provided by 

selecting strains with high intracellular protein and mutating with 

carcinogens. UK Patent 2008, GB2426523. 

P51 

Innovative animal component-free surface for the cultivation of human 

embryonic stem cells 

Thomas Stelzer 1* , Tina Marwood 1 , Cindy Neeley 2 

1 Thermo Fisher Scientific Labware and Specialty Plastics, Roskilde, DK-4000, 

Denmark; 2 Thermo Fisher Scientific Labware and Specialty Plastics, Rochester, 

NY 14625, USA 

E-mail: Thomas.stelzer@thermofisher.com 


Background: The promise of pluripotent stem cells lies in their ability to 

form any cell or tissue in the body. However, this promise requires a 

stable and reproducible method to grow the cells. Current methods rely 

on feeder cells or extracellular matrix proteins to cover the cultureware 

growth surface, and either manual selection or enzymatic dissociation in 

cell passaging and harvesting. This study describes a novel and simple 

method to grow pluripotent stem cells without the use of feeder cells or 

extracellular matrix proteins. 

Materials and methods: Human ESC cultivation 

Cell line Relative cell proliferation Relative IgG production 

CHO-GLUT5/IgG 1.02 ± 0.19 1.77 ± 0.59 

SC-01-GLUT5/IgG 0.86 ± 0.01 1.84 ± 0.04 


Cells (1 x 10 5 cells/ml) were cultured in the glucose- and fructose-based media. After 3 days, cell proliferation and recombinant IgG production were compared 

between two media. Each value in the glucose-based culture is estimated as 1.00. Data represent relative values of means ± SD (n = 3).



Figure 1(abstract P51) A. Expression of pluripotency markers in human ESC as determined by qRT-PCR after 1 passage (p1), 4 passages (p4), and 8 

passages (p8) on the Nunclon Vita surface. B. Expression of pluripotency markers in human ESC as determined by flow cytometry after 4 passages (p4) 

and 11 passages (p11) on the Nunclon Vita surface. C. Expression of pluripotency markers in human ESC as determined by immunofluorescence staining 

after 11 passages on the Nunclon Vita surface. 

Cells: Passage-49 human ESC (H1 line from WiCELL, USA) were maintained 

in mouse embryonic fibroblast (MEF)-conditioned medium on Nunclon 

Delta surface (Thermo Fisher Scientific, USA) coated with a 1:30 dilution 

of growth-factor reduced Matrigel (Becton Dickinson, USA). Cells were 

dissociated from the surface for passage by treatment with 1 mg/ ml 

collagenase, and then seeded onto Nunclon Vita surface with or without 

Rho-kinase inhibitor in the medium, as described below. 

Cultivation without Rho-kinase inhibition: H1 ESC was grown for 4 passages 

in MEF-conditioned medium on Nunclon Vita surface. Cells were dissociated 

from the surface for passage by treatment with 1 mg/ml collagenase. Cells 

plated on the Nunclon Vita surface took 7 days of culturing before they 

were ready for passage. 

Cultivation with Rho-kinase inhibition: H1 ESC were grown in MEFconditioned 

medium supplemented with Rho-kinase inhibitor, Y-27632 (10 

µM unless otherwise indicated; Sigma-Aldrich, USA). Cells were dissociated 

from the surface for passage by treatment with 1 mg/ml collagenase. Cells 

plated in medium with 10 µm Y-27632 on the Nunclon Vita surface were 

ready for passage 4 days after plating. Cells were grown for the number of 

passages as indicated. 

Human ESC characterization: Colony presence and morphology were 

determined using phase-contrast microscopy, and by the naked eye after 

staining colonies with 0.5% crystal violet. 

Pluripotency was determined by the presence of pluripotency markers 

through the use of qRT-PCR for gene expression, flow cytometry for cell 

surface marker expression, and immunofluorescence for cell-surface and 

nuclear proteins. 

Karyotypic stability was determined by cytogenetic analysis of 20 Gbanded 

metaphase cells, and by fluorescent in situ hybridization (FISH) on 

200 interphase nuclei using probes for the ETV6 BAP (TEL) gene located on 

chromosome 12 and for chromosome 17 centromere. 

Ability to form embryoid bodies was determined by growing ESC in a lowbinding 

plate for 10 days in DMEM/F12 containing 10% FBS. 

Results: Human ESCs can be passaged a few times on the Nunclon Vita 

surface in MEF-conditioned media without Rho-kinase inhibition before 

the growth rate spontaneously declines. Such decline in growth rate of 

human ESC on the Nunclon Vita surface is prevented by supplementing 

the conditioned medium with Rho-kinase inhibitor Y-27632. 

Human ESCs grown on the Nunclon Vita surface in the presence of 

Y-27632 have normal karyotype, express pluripotency markers (Fig. 1), 

and can be differentiated into embryoid bodies. Non-enzymatic 

passaging of human ESCs on Nunclon Vita surface can be accomplished 

by removing Y-27632 from the culture media 15 to 30 minutes before 

sub-culturing. 

Conclusions: The Nunclon Vita surface support feeder cell- and 

extracellular matrix-free attachment, colony formation and growth of 

human ESC: 

For a few passages in media conditioned by mouse embryonic 

fibroblasts 

For over 10 passages in media conditioned by mouse embryonic 

fibroblasts and supplemented with Rho-kinase inhibitor Y-27632. The 

human ESCs expanded 11 passages on the Nunclon Vita surface 

maintained normal karyotype, pluripotency, and their ability to 

differentiate into germ layer cells. 

P52 

Avian cell line - Technology for large scale vaccine production 

Barbara Kraus, Simone von Fircks, Simone Feigl, Sabrina M Koch, 

Daniel Fleischanderl, Katherine Terler, Mitra Dersch-Pourmojib, 

Christian Konetschny, Leopold Grillberger, Manfred Reiter * 

Baxter Innovations GmbH, Uferstrasse 15, 2304 Orth/Donau, Austria 

E-mail: manfred_reiter@baxter.com 



Introduction: The establishment of an immortalized continuous cell line 

derived from quail cells was undertaken by Baxter in order to develop a 

new cell line platform for vaccine production that is free of genetically 

modifying sequences.



Important vaccines and viral vectors are still produced in embryonated 

chicken eggs or primary chicken embryo fibroblasts. However, the 

substitution of primary cells by a continuous cell line has several 

advantages. Although primary avian tissue for virus replication is 

provided by specific pathogen-free (SPF) production plants, sterility 

during vaccine production on embryonated eggs is difficult to guarantee, 

and the constant risk of contamination necessitates the addition of 

antibiotics. In addition, the supply of embryonated SPF eggs could be a 

limiting factor in vaccine production if increased amounts are demanded 

by the vaccine manufacturers, e. g. in case of a pandemic outbreak. 

Thesameistrueforapproacheswhere primary fibroblast monolayer 

cultures are used. Thus, avian cell lines have become a modern option for 

vaccine manufacturing and will definitely replace egg and primary 

fibroblast technology. 

Cell Line Development and Characterization: Here we describe the 

development of a continuous avian cell line from quail embryos into 

serum-free suspension culture and its manufacturing potential for several 

different vaccines. 

Briefly, embryos of Colinus virginianus virginianus were disintegrated and 

trypsinized to obtain a primary culture that grew adherently in serumcontaining 

medium. After UV treatment with a specific dose [1], immortalized 

cells were expanded and subsequently adapted to serum-free conditions 

(QOR2-sf) and growth in suspension using a chemically defined medium. 

Subcloning resulted in the isolation of clone QOR2/2E11, which was selected 

as the lead clone for development of vaccine production processes. Then, 

quality-controlled cell banks were established (Figure 1). The cell clone 

QOR2/2E11 is grown in a chemically defined, animal component-free 

medium (Baxter proprietary formulation). 

Quality control (QC) testing was performed at different stages during cell 

line development. Extended characterization according to relevant 

guidelines (Table 1) showed that the cells are free of adventitious agents 

and F-Pert negative. Tumorigenicity testing performed in BALB/c nude 

mice indicated no tumorigenic effect. Accordingly, the cells fulfill all the 

critical regulatory requirements. 

Figure 1(abstract P52) Schematic overview of the cell line history. 


Thus, the developed subclone QOR2/2E11 was considered to be qualified 

as source material for Good Manufacturing Practice (GMP) cell bank 

production. A pre master cell bank and cell bank derivates were produced 

in compliance with the current GMP regulations and are currently 

available. 

Scalable live virus production in QOR avian cells: For process 

development purposes, modified vaccinia Ankara (MVA) virus was used as 

a model virus. MVA virus is a highly attenuated strain of vaccinia virus 

belonging to the Poxviridae family that was produced by over 500 

passages in chicken embryo fibroblasts. MVA has lost about 10% of the 

vaccinia dsDNA genome and consequently cannot replicate in primate 

and human cells. Its complex genome of circa 200 kb allows the insertion 

of large exogenous DNA inserts. Therefore, MVA serves as a versatile live 

vector for the development of human vaccines against diverse disease 

targets, such as malaria and cancer, for which conventional approaches 

have so far failed [2]. 

Using the QOR cell line, high product titers can be achieved with a broad 

rangeofvirusessuchaswild-typeMVA, recombinant MVA (rMVA) strains, 

influenza and flaviviruses. Figure 2 shows the growth of virus in 200 ml 

spinner flask cultures using different rMVA constructs. The experiments 

were performed with a starting cell density of about 2 x 10 6 cells per ml 

and a multiplicity of infection between 1.0 and 0.1. Over the course of four 

days post infection (dpi), virus titers of ≥ 1x10 9 TCID 50 per ml were 

obtained with every rMVA construct tested. Constant cell performance up 

to 10L bioreactors (laboratory scale), where cell densities ≥ 2x10 6 cells per 

ml were achieved, has been confirmed and scalability to 100 - 1000L 

bioreactors is currently under evaluation. 

Furthermore, this avian cell line can serve as a control cell line with 

different model viruses in QC tests for adventitious agents. 

Conclusion: A scalable technology for the production of live and 

attenuated vaccines based on the qualified avian cell line QOR2/2E11 has 

been established. The markedly advantages are: 

Stable, continuous avian cell line established without the introduction of 

foreign genes



Table 1(abstract P52) List of guidelines according to which the avian cell line was tested 

Organization Number Title 

European Directorate for the quality of Medicines & 

HealthCare 

The International Conference on Harmonisation of 

Technical Requirements for Registration of 

Pharmaceuticals for Human Use (ICH) 

Post production cells are not tumorigenic in animal model 

Suspension growth in low-cost chemically defined medium 

TCID50 titers ≥ 1x10 9 for several rMVA constructs tested 

References 

1. Reiter M, et al: Method for producing continuous cell lines. US2009/ 

0215123 A1 (Patent Application Publication, United States, 2009). 

2. Clinical Trials Feeds. [http://clinicaltrialsfeeds.org]. 

P53 

Towards human central nervous system in vitro models for preclinical 

research: strategies for 3D neural cell culture 

Daniel Simão 1,2 , Inês Costa 1,2 , Margarida Serra 1,2 , Johannes Schwarz 3 , 

Catarina Brito 1,2* , Paula M Alves 1,2 

1 Instituto de Tecnologia Química e Biológica –Universidade Nova de Lisboa, 

2780-157 Oeiras, Portugal; 2 Instituto de Biologia Experimental e Tecnológica, 

European 

Pharmacopoeia. 

(EP) 2.6.16. 

Tests for extraneous agents in viral vaccines for human use 

EP 5.2.3. Cell substrates for the production of vaccines for human use 

Q5A (R1)-1999 Viral safety evaluation of biotechnology products derived from 

cell lines of human of animal origin 

Q5D-1997 Derivation and characterization of cell substrates used for 

production of biotechnological/biological products 

Q7A-2000 Good manufacturing practice guide for active pharmaceutical 

ingredients 

Center for Biologics Evaluation & Research (CBER) —— Points to Consider in the Characterization of Cell Lines Used to 

Produce Biologicals (1993) 

—— Guidance for Industry, Characterization and Qualification of Cell 

Substrates and Other Biological Materials Used in the Production 

of Viral Vaccines for Infectious Disease Indication (February 2010) 

Code of Federal Regulations (CFR) 9CFR113.47 Detection of extraneous agents viruses by fluorescent antibody 

technique 

21CFR610.18 General biological products standards 

Figure 2(abstract P52) Growth Kinetics of rMVA viruses on QOR2/2E11. 


2780-901 Oeiras, Portugal; 3 Department of Neurology, University of Leipzig, 

04103 Leipzig, Germany 


Background: The development of new drugs for human Central Nervous 

System (CNS) diseases has traditionally relied on 2D in vitro cell models 

and genetically engineered animal models. However, those models often 

diverge considerably from that of human phenotype (anatomical, 

developmental and biochemical differences) [1] contributing to a high 

attrition rate - only 8% of CNS drugs entering clinical trials end up being 

approved [2]. Human 3D in vitro models are useful complementary tools 

towards more accurate evaluation of drug candidates in pre-clinical stages, 

as they present an intermediate degree of complexity in terms of cell-cell 

and cell-matrix interactions, between the traditional 2D monolayer culture 

conditions and the complex brain and can be a better starting point for 

the analysis of the in vivo context. Aiming at developing novel 3D in vitro



models of the CNS, this work focus on the implementation of long-term 

cultures of human midbrain-derived neural stem cells (hmNSC) for the 

scalable supply of neural-subtype cells, with a focus on the dopaminergic 

lineage, following a systematic technological approach based on stirred 

culture systems. 

Materials and methods: Cell culture: hmNSC were isolated as previously 

reported [3] and routinely propagated in static conditions, on poly-Lornithine-fibronectin 

(PLOF) coated plates, in serum-free propagation 

medium, containing basic fibroblast growth factor and epidermal growth 

factor [3]. hmNSC were cultured in stirred systems in Cultispher S 

microcarriers (Percell Biolytica) without coating and coated with PLOF) or 

as neurospheres for 7 to 21 days, with media changes every 3-4 days. All 

experiments were performed in 125 mL shake flasks (20 mL working 

volume), with orbital shaking at 100 rpm. Cultures were maintained at 

37°C, in 3% O 2. Double stain viability test: aggregates were collected from 

stirred cultures, incubated with fluorescein diacetate (10 μg/mL) and 

propidium iodide (1 μg/mL) and observed on a fluorescence microscope 

(Leica DMI6000). Aggregate size was measured in pictures taken from 

each culture sample using Image J software (NIH), as previously reported 

[4]. Dissociation: For microcarrier cultures, Cultispher S was allowed to 

settle,washedwithPBSanddigestedwithTrypsin0.05%-EDTA(Gibco). 

Cells were collected by centrifugation and counted by trypan blue 

exclusion dye. Free cells were counted using the same aliquot. 

Aggregates were dissociated with Accutase (Sigma). 

Results: The feasibility of culturing hmNSC as 3D structures in stirred 

culture systems was assessed by testing two different approaches: 

microcarrier technology versus cell aggregated cultures (neurospheres). 

For the first strategy, Cultispher S, a collagen-based macroporous 

microcarrier was tested for its ability to support hmNSC attachment and 

growth. Microcarriers uncoated and coated with PLOF were tested. 

hmNSCs were labelled with PKH26 lipophilic dye (red) for detection 


purposes and inoculated at 1.5x10 5 cell/mL, in a carrier concentration of 

1g/L, corresponding to approximately 125 cell/microcarrier. Monitoring 

along 5 days of culture time revealed that the fraction of viable cells found 

on the microcarriers was less than 10% of the inoculum, for both uncoated 

and PLOF-coated microcarriers, indicating poor microcarrier colonization. 

Fluorescence microscopy analysis revealed that hmNSC aggregated in 

suspension rather than colonizing Cultispher S microcarriers (not shown). 

For the cell aggregate strategy, two inoculum concentrations were tested - 

2and4x10 5 cell/mL and aggregate size and number evaluated along 

culture time (Figure 1). 

The inoculum concentration of 2x10 5 cell/mL was as efficient as 4x10 5 

cell/mL in promoting cell aggregation (Figure 1A) whereas it allowed for 

lower mean diameters along culture time (362±32 μm at day 14) as 

compared to the higher inoculum concentration for which significantly 

higher mean diameter and also a wider range of aggregate sizes were 

observed (475±103 μm at day 14) (Figure 1B). Moreover, the lower 

inoculum concentration avoided the formation of necrotic centres, which 

were detected in cultures with an inoculum concentration of 4x10 5 cell/ 

mL (Figure 1C).Taken together the data presented indicates that 2x10 5 

cell/mL is the most favourable inoculum concentration for culture of 

hmNSC as aggregates in stirred culture systems. 

Conclusions: In this study the feasibility of culturing hmNSC as 3D 

structures in stirred culture systems was evaluated. Cell aggregates 

(neurosphere) culture, using an inoculum concentration of 2x10 5 cell/mL 

was selected as the best strategy, due to the higher cell viabilities and 

tightly control of aggregate diameter attained. The implemented 3D 

culture system will be applied in the optimization of differentiation of 

hmNSC into dopaminergic neurons, astrocytes and oligodendrocytes. 

Acknowledgments: The authors acknowledge the FP7 EU project 

BrainCAV (HEALTH-HS_2008_222992) and the FCT project PTDC/EBB-BIO/ 

112786/2009 for financial support. 

Figure 1(abstract P53) Effect of inoculum concentration on aggregate concentration (A), aggregate size (B) and viability (C), evaluated using a double 

stain viability test: fluorescein diacetate (live, green); propidium iodide (dead, red). Cells were cultured in shake flasks with inoculum concentrations of 2 

and 4x10 5 cell/mL. Error bars denote standard deviation of average of 3 independent experiments. *** indicates significant difference (P



References 

1. Gagliardi C, Bunnell BA: Large animal models of neurological disorders 

for gene therapy. ILAR J 2009, 50:128-143. 

2. Miller G: Is pharma running out of brainy ideas? Science 2010, 329:502-504. 

3. Milosevic J, Schwarz SC, Ogunlade V, Meyer AK, Storch A, Schwarz J: 

Emerging role of LRRK2 in human neural progenitor cell cycle 

progression, survival and differentiation. Mol Neurodegener 2009, 4:25. 

4. Serra M, Brito C, Costa E, Sousa MFQ, Alves PM: Integrating human stem 

cell expansion and neuronal differentiation in bioreactors. BMC 

Biotechnology 2009, 8:92. 

P54 

Evaluation of a disposable stirred tank bioreactor for cultivation of 

mammalian cells 

Alexander Hähnel 1 , Benjamin Pütz 3 , Kai Iding 2 , Tabea Niediek 2 , 

Frank Gudermann 3 , Dirk Lütkemeyer 1,2* 

1 Institute for Protein Characterisation, Faculty of Engineering and Mathematics, 

University of Applied Sciences, Bielefeld, Germany; 2 BIBITEC GmbH, Bielefeld, 

Germany; 3 Institute of Technical Analytics, Faculty of Engineering and 

Mathematics, University of Applied Sciences, Bielefeld, Germany 

E-mail: dirk.luetkemeyer@fh-bielefeld.de 


Introduction: Disposable bioreactors are increasingly gaining acceptance 

for cell culture applications due to a number of advantages including ease 

of use and reduced labour costs, less requirements for utilities such as steam 

and purified water. In addition, the system requires no cleaning or cleaning 

validation and only a reduced clean room footprint. Integrated ready to use 

pre-sterilised disposable sensors for pH and dO 2 monitoring are expected to 

reduce the risk of contamination compared to conventional electrodes. 

Accordingly, a disposable pre-sterilised and therefore ready-to-use 200 L 

bioreactor featuring optical sensors for dO 2 and pH has been evaluated for 

suspension culture of CHO cells in protein free media. 

Materials and methods: Several fed-batch cultivations with a maximum 

cultivation time of 9 days were performed as follows. 

Cells: Recombinant CHO cell lines producing monoclonal antibodies 

Media: MAM-PF2, Bioconcept, Allschwil, Switzerland 

Feed and medium from Teutocell, Bielefeld, Germany 

Both media are chemically defined and protein free. 

Feeds: Different commercial available amino acid concentrates, glucose 

and glutamine 

Bioreactor: BIOSTAT® CultiBag STR 200 L, Sartorius Stedim biotech, 

Göttingen, Germany 

Parameter control: Agitation of the culture was performed by a preinstalled 

magnetic driven stirrer with two 3-blade impellers at 80 rpm, pHadjustment 

via adding CO 2 to the overlay stream and dO 2 was controlled 

via aeration of pure oxygen through the ring-sparger. 

Results: Maximum cell densities between 5.0 x 10 6 and 1.3 x 10 7 cells/mL 

with viabilities between 85% and 100% were achieved. Results of one 

typical fed-batch cultivation are shown in figure 1. 

The cell densities depended more on the medium performance and feeding 

strategy than on the bioreactor-features itself. At a cell density of 1.3 x 10 7 

cells/mLthevolumeflowofpureoxygenwasatarateof0.85L/min. 

In regard to the system’s maximum flow rate of 20 L/min, the throughput of 

oxygen most likely would not become a bottleneck of even higher cell 

densities. Detailed comparison of the optical pH and dO 2 probes with their 

conventional counterparts revealed a reliable concordant performance. 

Conclusion: The disposable BIOSTAT® CultiBag STR 200 L bioreactor is a 

reliable cultivation system for high cell density operations above 10 7 cells/ 

mL with mammalian cells. The usually installed ring-sparger supplies a 

sufficient amount of gas into the liquid phase and still offers adequate 

reserves. In direct comparison the optical sensors correlated excellently with 

the conventional electrodes. External pH-testing and recalibration is 

mandatory to compensate for the drift of the pH-sensor. The reactor’s 

overall performance is convincing in this field of operation, which is mainly 

due to its ease of use combined with the low utilisation of space. The set up 

procedure can be done in less than three hours which is remarkable 

compared with stainless steel bio-reactors and the advantage in prevention 

of cross-contaminants during changeover of campaigns is preeminent. 

Acknowlegement: The supply of materials by Dr. Greller and Mrs. Noack 

(Sartorius Stedim Biotech GmbH) is greatly acknowledged. 

P55 

Controlled expansion and differentiation of mesenchymal stem cells in 

a microcarrier based stirred bioreactor 

Sébastien Sart 1,2* , Abdelmounaim Errachid 1 , Yves-Jacques Schneider 1,3 , 

Spiros N Agathos 1,2 

1 Université Catholique de Louvain / Institut des Sciences de La Vie, Belgium; 

2 Laboratory of Bioengineering (GEBI), Place Croix du Sud, 2/19, 1348 

Louvain-la-Neuve, Belgium; 3 Laboratory of Cellular Biochemistry, Place Croix 

du Sud, 4/5 box 3, 1348 Louvain-la-Neuve, Belgium 

E-mail: sebastien.sart@gmail.com 


Introduction: Cell based therapy requires great numbers of cells in a 

functional state permitting their in vivo implantation for the restoration of 

Figure 1(abstract P54) Cell growth and viability during a fed-batch cultivation of CHO cells in the 200 L disposable bioreactor. 

Page 81 of 181



tissue homeostasis. Three main parameters are believed to be essential 

for such a purpose: an appropriate cell population, a suitable scaffold and 

appropriate physical / biochemical factors enabling proper expansion and 

in vitro cell differentiation. In recent years, mesenchymal stem cells 

(MSCs) have been attracting a lot of interest in this field, because of their 

differentiation potential and their trophic factor secretion abilities. The 

aim of this work is to perform a rational analysis of key factors involved 

in the efficient proliferation and differentiation of MSCs, in the context of 

a stirred microcarrier (MC)-based bioreactor. 

Materials and methods: MSCs from external ear (E-MSCs) and bone 

marrow stroma (BM-MSCs) were extracted from Wistar rats, selected and 

cultivated on plastic dishes as previously described [1]. The differentiation 

potential of E-MSCs along the adipogenic, osteogenic and chondrogenic 

pathways was established and assessed by staining (respectively Oil red O, 

von Kossa, Alcian Blue) as well as by RT-PCR analysis on marker genes of 

differentiation (respectively, C/EBPa, osteocalcin, aggrecan) as previously 

described [1]. MCs (i.e. Cultispher-S, Cytodex-3, Cytopore-2) were prepared 

and cells were seeded as reported in [2]. Cell counting was performed as 

follows: (1) after a full digestion of Cultispher-S by trypsin and using trypan 

blue exclusion counting method, as in [2]; (2) by crystal violet staining and 

nuclei counting for Cytodex-3, as in [2]; or (3) cell counting on Cytopore-2 

was performed using MTT according to [3]. The multiplication ratio was 

calculated as defined elsewhere [2]. Cell cycle was analyzed by FACS, after 

cell staining with propidium iodide, as in [2]. The actin organization was 

assed by confocal microscopy after cell staining with phalloidin-rhodamine. 

Results: 

MSC and microcarrier screening: E-MSCs were compared to the “gold 

standard” BM-MSCs on the basis of their proliferative properties. E-MSCs 

bear characteristics of progenitor cells: expression of CD73, Sca-1 and 

Notch-1, and also in vitro differentiation potential into mesodermal cell 

types such as adipocytes, chondrocytes and osteoblasts (not shown). 

Thus, these cells are in vitro functionally analogous to BM-MSCs. This cell 

population was further selected on the basis of its high intrinsic 

proliferation potential in monolayer culture, a clear advantage in the field 

of MSC bioprocessing (Table 1). 

Next, we analyzed these cells’ behavior on various types of MCs. 

Interestingly, both cell types (E- and BM-MSCs) had similar proliferation 

profiles under all the conditions tested: Cultispher-S>Cytodex- 

3>Cytopore-2 (Table 1). This validated that E-MSCs are a valuable model 

for studying MSCs activities on MCs, given their faster growth and easier 

handling compared to BM-MSCs (Table 1). In addition, Cultispher-S turned 

out to be the most efficient MC for MSC expansion (Table 1). 

Maximization of MSC proliferation in MC-based stirred bioreactors: 

According to Table 1, a batch culture mode was not sufficient to 

promote efficient E-MSC propagation on Cultispher-S. Conversely, cyclic 

fed-batch increased E-MSC growth span (Table 1). In addition, the use of 


high levels of growth factors (using a pulsed culture composed of 40% 

FBS and 1 ng/mL of TGFb1)increasedgrowthspan(Table1).The 

beneficial effects of cyclic fed-batch and pulsed culture were linked to a 

sustainment of the percentage of cells in S-phase of the cell cycle 

compared to batch culture (Table 1). These results underline that the 

control of growth factor levels in the medium is the key to maximize 

E-MSC growth extent. 

Sequential proliferation and differentiation in MC-based stirred 

bioreactors, modulating actin organization: We have previously 

shown that the addition of differentiation media significantly diminished 

E-MSC proliferation. This indicated that the differentiation of E-MSCs on 

MCs must be performed sequentially, after an initial proliferation phase. 

According to Figure 1.b, after a first step of E-MSC expansion on MCs, it 

was shown that the repression of fibrillar actin (adding Y-27632 to the 

differentiation medium) maximized adipogenic differentiation on 

Cultispher-S, while the promotion of stress fibers (using lysophosphatidic 

acid, LPA) diminished it. In the same vein, Y-27632 improved E-MSC 

osteogenic differentiation, while LPA lowered the expression of 

osteocalcin (Figure 1.c). Cytopore-2, previously shown to promote 

disorganized actin form (similar to that of aggregate cultures and E-MSCs 

treated with cytochalasin-D), enabled an efficient E-MSC chondrogenic 

differentiation, in comparison to Cultispher-S (Figure 1.d). This latter MC 

was previously found to enable a proper E-MSCs actin organization 

(composed of mixed cortical and fibrillar actin) linked with their efficient 

propagation. These results indicate that a tight control of the E-MSC 

microenvironment leading to adapted actin shape is the key towards 

efficient MSC differentiation on MCs. 

Conclusions: According to these data, it emerges that a correct control 

of MSC microenvironment in terms of MC composition is necessary to 

promote these cells’ efficient proliferation via proper actin organization. 

An efficient MC system must also be combined with adapted biochemical 

signaling. Indeed, the growth factor content is an essential factor to 

monitor towards improved MSC growth yield. As we observed that the 

differentiation step could not be combined with expansion, sequential 

phases are required for the mass scale production of a given MSC 

differentiated phenotype. Similarly to the expansion phase, the microenvironment 

to which MSCs are exposed modulates the efficiency of 

their differentiation. According to our results, the promotion of an 

adequate actin organization is one of the essential parameters enabling, 

in association to biochemical signaling from the differentiation medium, 

efficient MSC differentiation on MCs. 

Taken together, these results open the way toward mass scale production 

of MSCs suitable for future in vivo applications. 

Acknowledgments: This work was supported by a FSR grant of 

Université Catholique de Louvain, and an IN.WALLONIA-BRUSSELS 

INTERNATIONAL (IN.WBI) grant. 

Table 1(abstract P55) Multiplication ratios of E-MSCs and BM-MSCs on various culture systems. Multiplication ratios of 

E-MSCs and percentage of cells in S-phase at day 5 of a 7 day run, under various modes of culture 

MSC and MC screening 

BM-MSCs Culture system Multiplication ratio 

T-Flasks 0.4 ± 0.2 

Cultispher-S 0.16 ± 0.1 

Cytodex-3 -0.1 ± 0.12 

Cytopore-2 -0.5 ± 0.04 

E-MSCs T-Flasks 2.4 ± 0.1 

Cultispher-S 2 ± 0.3 

Cytodex-3 0.7 ± 0.6 

Cytopore-2 

Maximization of MSC proliferation 

-0.6 ± 0.3 

E-MSCs on Cultispher-S Mode of culture Multiplication ratio % of cells in S-phase at day 5 

Batch 1.5 ± 0.3 1.4 ± 0.3 

Cyclic-fed-batch 2.6 ± 0.2 8 ± 1 

Pulsed culture 3 ± 0.04 15 ± 1



Figure 1(abstract P55) RT-PCR analysis of E-MSC differentiation. Spontaneous marker gene expression after MC expansion (control) (a); adipogenic 

differentiation (C/EBPa as a marker) after MC expansion, adding an actin modulator in the differentiation medium (Y-27632 or LPA) (b); osteogenic 

differentiation (osteocalcin as a marker) after MC expansion, adding an actin modulator in the differentiation medium (Y-27632 or LPA); chondrogenic 

differentiation (aggrecan as a maker) after MC expansion, comparing Cultispher-S to Cytopore 2 (d). 

References 

1. Sart S, Schneider YJ, Agathos SN: Ear mesenchymal stem cells: an efficient 

adult mutipotent cell population fit for rapid and scalable expansion. 

J Biotechnol 2009, 139(4):291-299. 

2. Sart S, Schneider YJ, Agathos SN: Influence of culture parameters on ear 

mesenchymal expanded on microcarriers. J Biotechnol 2010, 

150(1):149-160. 

3. Pabbruwe MB, Stewart K, Chaudhuri JB: A comparison of colorimetric and 

DNA quantification assays for the assessment of meniscal 

fibrochondrocyte proliferation in microcarrier culture. Biotechnol Lett 

2005, 27(19):1451-1455. 

P56 

3D6 and 4B3: Recombinant expression of two anti-gp41 antibodies as 

dimeric and secretory IgA 

David Reinhart 1,2* , Robert Weik 2 , Renate Kunert 2 

1 Department of Biotechnology, Institute of Applied Microbiology, University 

of Natural Resources and Life Sciences, Vienna, Austria; 2 Polymun Scientific 

Immunbiologische Forschung GmbH, Vienna, Austria 

E-mail: David.Reinhart@boku.ac.at 


Background: Sexually transmitted diseases are predominantly acquired 

via mucosal membranes of the rectal or genital tract during sexual 

intercourse. This major port of virus entry is naturally defended by the 

humoral immune response, with immunoglobulin A (IgA) as the primary 

antibody class to elicit mucosal immunity. Dimeric IgA (dIgA) reaches the 

luminal side of mucosal tissues by transcytosis through epithelial cells 

lining the mucosa. In a first step, dIgAs specifically bind to the basolaterally 

expressed polymeric immunoglobulin receptor (pIgR) on epithelial cells. 

For release of IgAs on the luminal side the extracellular portion, termed 


secretory component (SC), remains attached to the antibody to form 

secretory IgA (sIgA) [1,2]. 

One example of a sexually transmitted disease is the human immunodeficiency 

virus (HIV) which annually infects several million individuals on a 

global scale and potentially leads to the acquired immunodeficiency 

syndrome (AIDS). Although current therapies can reduce disease progression 

in infected individuals, no cure is yet available or within reach in near future. 

As a consequence increased attention is now being paid to develop drugs 

that could prevent virus acquisition. 

3D6 and 4B3 are two monoclonal antibodies (mAb) which have originally 

been isolated as IgG1 isotype from seroconverted HIV-1 patients and bind 

to the principal immunodominant domain of gp41. In the course of this 

project both mAbs were isotype switched to IgA1. Recombinant CHO cell 

lines were established for the production of 3D6 and 4B3 as dimeric as 

well as secretory IgA. While dIgA were expressed by a single cell line, sIgA 

are produced by a biochemical association of dIgA with SC. Both dIgA and 

sIgA variants were characterized and the contribution of the heavily 

glycosylated SC on IgA stability will be investigated. 

Antibody expression: MAbs 3D6 and 4B3 (IgG1) were developed at the 

Institute of Applied Microbiology [3,4]. Isotype switching was performed 

by substitution of the original heavy chain constant region with that for 

IgA1 (GenBank Accession Number 184743). For their recombinant 

expression as dIgA three plasmids were generated containing the coding 

region of either heavy chain, light chain or joining (J) chain. The latter 

one should increase dimerization of IgA monomers. Recombinant CHO 

clones were selected whereas high producers were isolated by applying 

the dihydrofolate reductase (dhfr) system. 

Assembly of 3D6 and 4B3 as sIgA is intended by an in vitro biochemical 

association of dimeric IgA with human secretory component (hSC). Thus, 

CHO cell lines which solely express hSC were established by cotransfection 

of two plasmids containing the coding region for hSC and 

dhfr, respectiviely.



Cultivation temperature greatly influences productivity: Mammalian 

cells are commonly cultivated at 37°C. In order to investigate the effect on 

mAb secretion, clones expressing 3D6-dIgA and 4B3-dIgA were propagated 

at sub-physiological temperatures. It was observed that temperature 

markedly impacts final IgA quantities in culture supernatants. Product titers 

of clones expressing 3D6-dIgA were almost 3-fold increased when 

incubated at 33 °C as compared to 37 °C. Furthermore, cultivation of 4B3dIgA-expressing 

clones at 33 °C, rather than at 37 °C, resulted in end titers 

which were approximately 13-fold increased. 

Purification of recombinant dIgA: The two established mAb-expressing 

cell lines secrete dIgA at different quantities: The best clone producing 3D6dIgA 

achieved a mean specific productivity of 59.1±14 pg/cell*day (pcd). 

Conversely, the highest 4B3-dIgA secreting clone reaches a mean specific 

productivity of 0.6±0.4 pcd. Hence, an increased amount of host cell 

proteins is present in cell culture supernatants of 4B3-dIgA as compared to 

3D6-dIgA. Therefore, we elaborated a purification protocol which allows the 

recovery of both dimeric IgAs at a greatly enhanced purity. The purification 

procedure is shown in table 1. 

Formation of secretory IgA: Secretory IgA can be assembled in vitro 

due to the natural affinity of dimeric IgA for secretory component and 

vice versa (5). Initially, supernatants from recombinant cell lines expressing 

hSC, 3D6-dIgA or 4B3-dIgA were buffer exchanged against PBS. 

Subsequently, sIgA formation was performed by incubation of hSC with 

dIgA at 25°C for 3 hours at different molar ratios. However, applying a 

molar ratio of 1:1 is commonly described as optimal [5]. The association 

of 3D6-dIgA or 4B3-dIgA with hSC was successfully verified by SDS-PAGE 

following Western blotting (data not shown). Conjugation of the 

immunoblot was performed with a mouse anti-human secretory 

component antiserum (Sigma) and detected via an anti-mouse IgG1 

antiserum (Sigma). 

Conclusions: The anti-HIV-1 mAbs 3D6 and 4B3 were isotype switched 

and could successfully be expressed as dIgA in stably transfected CHO 

cells. Furthermore, continuous product secretion was obtained for at least 

20 passages in spinner flasks. 

Cultivation of the recombinant cell lines at different temperatures revealed 

that their product expression can markedly be influenced: 3D6-dIgA 

expression was nearly triplicated by shifting the temperature from 37 °C to 

33 °C. Applying the same conditions, 4B3-dIgA product secretion was nearly 

13-fold increased. 

A purification protocol was developed which allows the recovery of the 

dIgAs 3D6 and 4B3 in highly pure fractions. Furthermore, the elaborated 

scheme enables the isolation of dimeric 3D6 from its various high 

molecular weight isotypes. 

Secretory IgA of both 3D6 and 4B3 can successfully be produced by 

mixing dimeric IgA with human secretory component. 

Acknowledgement: Funding from the European Community’s Seventh 

Framework Programme (FP7/2002-2013) under grant agreement N° 

201038, EuroNeut-41. 

References 

1. Snoeck V: The IgA system: a comparison of structure and function in 

different species. Vet Res 2006, 37(3):455-67. 


2. Bonner A, Perrier C, Corthésy B, Perkins SJ: Solution structure of human 

secretory component and implications for biological function. J Biol 

Chem 2007, 282(23):16969-80, Epub 2007 Apr 11. 

3. Buchacher A, Predl R, Strutzenberger K, Steinfellner W, Trkola A, 

Purtscher M, Gruber G, Tauer C, Steindl F, Jungbauer A, et al: 

Generation of human monoclonal antibodies against HIV-1 proteins; 

electrofusion and Epstein-Barr virus transformation for peripheral 

blood lymphocyte immortalization. AIDS Res Hum Retroviruses 1994, 

10(4):359-69. 

4. Kunert R, Rüker F, Katinger H: Molecular characterization of five 

neutralizing anti-HIV type 1 antibodies: identification of nonconventional 

D segments in the human monoclonal antibodies 2G12 and 2F5. AIDS 

Res Hum Retroviruses 1998, 14(13):1115-28. 

5. Crottet P, Corthésy B: Secretory component delays the conversion of 

secretory IgA into antigen-binding competent F(ab’)2: a possible 

implication for mucosal defense. J Immunol 1998, 161(10):5445-53. 

P57 

Creating new opportunities in process control through radio frequency 

impedance spectroscopy 

Daniel W Logan * , John P Carvell, Matthew P H Lee 

Aber Instruments Ltd., Aberystwyth, SY23 3AH, UK 

E-mail: daniel@aberinstruments.com 


Introduction: Process control systems are a valuable tool in the research, 

development, and production stages of biopharmaceuticals. They allow 

cell culture processes to optimise product production through monitoring 

and controlling of the inputs to the system (e.g. air, temperature, feeds). 

Process control systems usually read an online monitor and adjust the 

inputs of the system to keep that monitor at a set point. 

The challenge with many process control systems is the inability to 

monitor the most informative variables online. Typically, surrogate 

variables such as pH and dissolved oxygen are monitored online and 

assumed to track changes in cell growth and product production. 

Although the simplicity of these types of probes has led to their wide use 

in most bioreactors, their use as effective process control drivers is limited 

by the assumptions required to link, for example, oxygen uptake rate to 

product formation. 

The ideal process control variable would be a direct measure of the desired 

product which is often difficult to monitor. However, in biopharmaceutical 

applications only the cells in the bioreactor are directly responsible for the 

production of the desired products. Online monitors which measure the cell 

number (or volume) offer a superior method for tracking cell/product 

growth and can be used to control and optimise the process control. Radio 

frequency impedance spectroscopy is one such method that offers an 

opportunity to monitor cell growth directly online and adjust the 

fermentation process accordingly. 

Background of radio frequency spectroscopy: Radio frequency (RF) 

impedance spectroscopy is the most robust method for measuring viable 

Table 1(abstract P56) Purification scheme developed for isolation of 3D6 and 4B3 dIgA 

STEP EQUIPMENT PROCEDURE RECOVERY 

3D6 4B3 

dIgA dIgA 

Ultra/Diafiltration Kvick Start UDF Cassette, Harvested cell culture supernatant containing dIgA is concentrated and buffer >95 >95 

Millipore, 30 kD 

exchanged against PBS, pH 7.4 

% % 

Lectin Affinity Immobilized Jacalin, UDF retentate is immobilized onto a lectin affinity resin. After a washing step, 98.7 106.1 

Chromatography Thermo Scientific bound product is eluted by applying 1.5 M D-galactose in PBS, pH 7.4 % % 

Ultra/Diafiltration Kvick Start UDF Cassette, The eluate is buffer exchanged against 20 mM Tris, 10 mM NaCl, pH 8.5 >95 >95 

Millipore, 30 kD 

% % 

Anion Exchange DEAE Sepharose FF, GE The retentate is applied onto the anion exchanger. Post washing with 100mM 88.4 86.7 

Chromatography Healthcare NaCl product is eluted from the resin using 20 mM Tris, 200 mM NaCl, pH 8.5 % % 

Hydrophobic 

Interaction 

Phenyl-Sepharose 6FF 

low sub, GE Healthcare 

(NH4) 2SO4 is added to 0.75 M for 4B3-dIgA or 1.25 M for 3D6-dIgA to precipitate 

host cell proteins but not mAb itself. After a washing step 3D6-dIgA is eluted 

44.1 

% 

59.1 

% 

Chromatography 

with 0.4 M (NH4)2SO4, which allows to isolate dimeric IgA from other IgA 

isoforms. 4B3-dIgA desorbs at 0 M (NH4)2SO4



biomass online [1]. RF impedance spectroscopy measures the passive 

electrical properties of cells in suspension through the cells’ interaction 

with RF excitations; a technique commonly known as dielectric 

spectroscopy. 

Viable cells are composed of a conducting cytoplasm surrounded by a 

non-conducting membrane suspended in a conducting medium. When 

an alternating current is applied to the suspension, each cell becomes 

polarised and behaves electrically as a tiny spherical capacitor. The 

suspension’s reaction to the current is expressed as its permittivity can 

be measured by its capacitance and conductivity as a function of 

frequency. Viable cells possess intact membranes which prevent the free 

flow of ions and allow the cells to polarise. Dead, porous cells and 

debris lack an enclosing membrane and are unable to build up charge 

separation. Hence, dielectric spectroscopy only measures viable cells 

and is immune to both lysed cells and other debris (e.g. carriers) in the 

suspension. 

At low excitation frequencies the cells fully polarise and the capacitance of 

the suspension is maximised. As the excitation frequency increases, the cells 

lose their ability to fully polarise and the measured capacitance drops, 

eventually the cells have no polarisation at high frequencies. This relaxation 

is called the b-dispersion and has been modelled mathematically by the 

Cole-Cole function [2]. Analysis of the dispersion provides estimates of the 

biomass volume (proportional to cell number density x cell diameter 4 ), 

average cell diameter, and the internal conductivity of the cells. 

Futura biomass monitor: The Futura biomass monitor is an online, in-situ 

instrument that requires minimal setup and no user interaction during 

operation. The Futura range consists of biomass monitors tailored to fit 

bioreactors from 100mL to over 1000L as well as various disposable systems. 

Unlike dissolved oxygen probes which monitor cell growth indirectly 

through the oxygen uptake rate of the suspension, Futura measures the 

capacitance created directly from the cells. The modularity of the system 

allows a Futura to connect to a variety of probe lengths and styles for varied 

applications and its hub based system allows multiple instruments to 

connect to a single PC and/or PLC. 

Comparison of Futura results to offline measurements: The difficulty 

with offline measures of biomass growth is the sparse collection rate (often 


1 reading/day), the accuracy of the readings, and the difficulty in defining 

the difference between viable and non-viable cells. An online biomass 

monitor minimises many of these challenges and gives a more complete 

view of the suspension progress. 

In one example, a cell culture process with CHO cells was monitored online 

using a standard remote Futura with Futura SCADA. Additional offline 

monitoring including the cell diameter, cell number density, and oxygen 

uptake rate also was collected. The online monitoring of biomass shown in 

Figure 1 clearly illustrates the growth, stationary, and decline stages of the 

cell culture than the offline measurements alone. 

Because RF spectroscopy allows differentiation between biomass and cell 

size, additional analysis of the cell culture growth curve can be used to 

determine optimum parameters. In the figure, the calculated online cell 

diameter correlated well with the offline data up to 250 hours but 

thereafter there was a sudden dramatic drop in the on-line measurement. 

This change is an artefact due to a large shift in the critical frequency but 

it provides a key signpost of changes in the cell that are not easily 

picked up by the RFI signal alone. 

Once a correlation is determined between biomass or cell size and the 

production of the desired product, RF spectroscopy can be used to guide 

the timing of feeds or adjust the properties of the medium (e.g. oxygen 

content) to optimise the cell culture environment. 

Conclusions: Monitoring a cell culture process with online RF 

spectroscopy creates new opportunities to understand cellular changes 

throughout the length of the process. Monitoring the cells directly 

reduces the ambiguity in interpreting secondary monitors of both the 

cells and the medium as such monitors can lag the production 

mechanism or only be indirectly related to the production of the desired 

product. 

Acknowledgements: The authors gratefully acknowledge Gary Finka for 

data used in this paper. 

References 

1. Logan D, Carvel J: A biomass monitor for disposable bioreactors. 

BioProcess International 2011, 9:48-54. 

2. Cole K, Cole R: Dispersion and absorption in dielectrics I. Alternating 

current characteristics. J of Chem Phys 1941, 9:341-351. 

Figure 1(abstract P57) Online and offline measurements of a CHO cell culture process. Top: online capacitance (solid line) and normalised ViCell biomass 

volume (crosses). Bottom: estimated average cell diameter from online capacitance (solid line) and ViCell system (crosses).



P58 

Applications of nanoparticels in molecular and cellular biology and 

cancer research 

Katya Simeonova 1* , Ganka Milanova 2 

1 

Institute of Mechanics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria; 

2 

University of Architecture, Civil Engineering and Geodesy, 1000 Sofia, 

Bulgaria 

E-mail: katyas@bas.bg 


Background: Discovering of carbon nanotubes (CNTs), by S. Iijima in 1991, 

[1] was a revolution in nanoscience (nanomaterials) and nanotechnology. 

Moreover these nanoscale materials possess perfectly physicomechanicanical, 

electronic, optical, properties. They find applications in 

technique, engineering, electronics, optoelectronis, space and environments, 

[2]. Recently has been established that nanomaterials, play an 

important role in molecular and cellular biology and medicine. The aim of 

the work presented could be formulated as follows: to discuss some basic 

articles, devoted to applications of nanoparticles, nanotechnology based 

on gold nanoparticles for cancer research. 

Materials and methods: Synthesis methods for nanoparticles (nanoshells, 

nanorods, nanocrystals) have been analyzed. Application of gold 

nanoparticels for detection and therapy of cancer has been given too, [3]. 

Nanotechnology has been determined as an interdisciplinary science 

combining physics, mechanics, chemistry, materials science, engineering, 

biology becames a very good potential in many different fields of 

technique, for cancer therapy, in molecular and cellular biology. 

Methods for synthesis of nanoparticles: Some methods for synthesis of 

nanoparticles: by controlled different reducing agent; a two- phase method 

using other reductants; biocompatible bock polymers. Deposition process 

(DP), has been applied for synthesis also. It has been established that rods 

wires, multi-concentric shells, hollow tubes,capsules,monocrystasetc. 

possess exceptional optical and electronic properties. In [4], the Drude 

method (model) for description of optical properties is: 

2 

’ wp 

e = 1 − 

w + g 

2 2 

’’ wpg e = 1 − 

w w + g 

2 

2 2 ( ) 

Figure 1(abstract P58) Dependencies of extinction, versus wavelength for nanoshells with different core radius. 


(1) 

(2)



Here: ε′ and ε″ ; ω =2πc/l; l, c,g have been given in [5]. A correlation, 

available is: 

n 

g = gbulk 

+ 

r 

F 

eff 

Here g, gbulk, νF, roff are given in [5]. Both important characteristics for 

description of gold nanoparticles, absorption efficiency and scattering 

efficiency have been analyzed too. It must be pointed out as well, that 

these nanoparticles could employed in the many medical applications. 

Results: The paper presented could be considered as a recent review on 

application of nanoparticles and nanotechnology in cancer research. In 

the work [6], we could find basic research of American scientists in 

nanotechnology based for cancer research, (Figure 1). 

Conclusions: In conclusions we could say, that paper presented could be a 

successful tool for many medical scientists, physicians, molecular biology 

scientists, chemists etc. It’s give good knowledge, regarding nanotechnology 

based gold nanoparticles for cancer research. Also, some novel 

computational models, based on theoretical studies, analyzed here, could be 

developed in future inestigations. 

Acknowledgments: Authors would like to thank you very much to 

Professor Nicole Borth, personally for her cooperation of attending the 

ESACT Meeting, in Vienna, Austria from 15-18 May 2011 

References 

1. Iijima S: Nature. 1991, 395. 

2. Simeonova K, Milanova G: A review on the mechanical behavior of 

carbon nanotubes (CNTs), as semiconductors. ISCOM2007, Book of 

abstracts Peniscola, Spain 2007, 123-136. 

3. Weibo C, et al: Applications of gold nanoparticles in cancer nanotechnology. 

Review, Nanotechnology, Science and Applications 2008, 27-32. 

4. Lihua W, et al: Gold nanoparticle- based optical probes for targetresponsive 

DNA structures. Gold Bulletin 2008, 41(1):37-42. 

5. Harris N, et al: Tunable integrated absorption by metal nanoparticles: the 

case for gold rods and shells. Gold Bulletin 2008, 41(1):5-14. 

6. NCI Alliance for Nanotechnology in Cancer. nanoUtah, Piotr Grodzinski, 

PhD, Director 2007. 

P59 

Measurement of sialic acid content on recombinant membrane proteins 

Deniz Baycin-Hizal 1* , Sunny Mai 1 , Daniel Wolozny 1 , Ilhan Akan 2 , 

Noboru Tomiya 3 , Karen Palter 2 , Michael Betenbaugh 1 

1 Department of Chemical and Biomolecular Engineering, Johns Hopkins 

University, Baltimore, MD, 21218, USA; 2 Department of Biology, Temple 

University, Philadelphia, PA, 19122, USA; 3 Department of Biology, Johns 

Hopkins University, Baltimore, MD, 21218, USA 


(3) 


Background: Membrane proteins such as cell adhesion molecules, 

receptors, transporters and ion channel proteins all have essential roles in 

cell-growth, migration, and flow of information, cell-cell and cell-protein 

communication. Membrane proteins are targets of biopharmaceutical 

companies because they have diverse effects on the progression of many 

diseases [1]. Ion channels are membrane proteins that play critical roles in a 

number of cell functions including communication and neuromuscular 

activity. Treatment of channelopathy diseasessuchascancer,cardiac 

arrhythmia, ataxia, paralysis, epilepsy, memory and learning loss, requires a 

broad understanding of ion channel function. Sialic acid is a critical charged 

glycan that affects the action potential of potassium channels which leads 

to changes in the neuronal system of organisms. In order to understand the 

effect of the sialylation on channel function, the presence of the sialic acid 

on the protein of interest should be studied. In this study, a novel method 

for the quantification of sialic acid is described for a potassium channel 

membrane protein. 

Materials and methods: Cell lines: HEK293 cells were grown in DMEM 

media (Invitrogen, Carlsbad, CA) supplemented with 10% Fetal Bovine 

Serum (Invitrogen), 1% nonessential amino acids (Invitrogen) and 1% 

L-Glutamine (Invitrogen). 

Transfection: Lipofectamine TM 2000 Reagent (Invitrogen) was used to 

transfect potassium channel into HEK293 cells. Stable pools were formed 

after 15 days of antibiotic selection and 8 different clones were selected 

from the pools. 

Western blot analysis: Cells were lysed in RIPA buffer containing 

complete-mini EDTA free protease inhibitor cocktail (Roche Diagnostics, 

Mannheim, Germany). The protein concentrations of each sample were 

determined by using a BCA protein assay kit (Pierce, Rockford, IL). Equal 

total protein amounts were loaded onto 8% gels and separated by 

electrophoresis. The separated proteins were transferred from the gel to 

membrane. For detection of the channel protein, membrane was incubated 

in 1% milk in PBST solution containing primary antibody with a dilution of 

1:1000 overnight at 4°C. A secondary anti-rabbit IgG HRP conjugate 

antibody (Amersham, Louisville, CO) was used at a dilution of 1:5000 in 1% 

milk/PBST overnight at 4°C. 

Immunoprecipitation: The potassium channel protein was purified by 

using G-beads coupled antibody. 

HPLC analysis: The purified protein was run on the gel and protein band 

was cut from the gel. The cut band was acid hydrolyzed to release the sialic 

acid. The released sialic acid was derivatized with fluorescent 4,5- 

Methylenedioxy-1,2-phenylenediamine dihydrochloride (DMB) reagent and 

injected to HPLC [2]. 

Results: The transient expression of potassium channel was evaluated by 

western blot analysis. After 15 days of antibiotic selection, an expression 

level of the gene of interest was evaluated by Western blot. The stable pool 

expression is an average of the varied expression levels of the protein of 

interest in cells. In order to decrease the expression heterogeneity and 

Figure 1(abstract P59) Potassium channel sialylation. A) Western blot analysis of channel proteins B) Western blot of the channel protein before and 

after sialidase treatment C) Sialic acid presence in HPLC.



ensure that all the cells contain the same genetic content, eight different 

single clonal isolates were selected. The quality and quantity of the protein 

expression in these clonal isolates was compared with the protein 

expression of stable pool. Figure 1.A shows the western blot of 8 cell clones 

and stable pool of potassium channel. Both glycosylated and nonglycosylated 

bands of the specific protein can be distinguished in the 

Western blot. Although some of the clones and stable pool results showed 

non-glycosylated bands and poor expression, clone 1 and 3 showed high 

glycosylation quality and quantity. Forthisreason,clone3wasscaledup 

and purified using immunoprecipitation. The purified protein was treated 

with sialidase and the mobility shift of the protein before sialidase and after 

sialidase treatment was compared by running Western blot as shown in 

Figure 1.B. Due to the low molecular weight of sialic acid, the mobility shift 

detected by Western blot was minimal. In order to quantify the amount of 

sialic acid found on the protein, about 6 pmol of purified protein was run on 

the SDS-PAGE gel and was cut from the gel. The cut band was acid 

hydrolyzed to release the sialic acid and the released sialic acid was 

derivatized with DMB reagent and injected into HPLC. The sialic acid 

amount that is released from the gel was calculated by using a sialic acid 

calibration curve (data not shown). The amount of sialic acid was calculated 

to be 19 pmol depending on HPLC and calibration curve results. Since the 

potassium channel of interest may have 4 possible sites for the sialylation, 

and therefore 24 pmols sialic acid. For this reason, the estimated sialic acid 

occupancy efficiency of the channel was found as 79%. 

Conclusion: Membrane proteins are to be very difficult to produce and 

purify for structural or glycan analysis because of their low expression levels. 

In this study, HEK 293 cells were used to express a potassium channel 

protein and clonal isolates were picked from stable pools to evaluate the 

quality and quantity of the glycosylated protein. A highly expressing clone 

with glycosylation was then selected for the purification and sialic acid 

analysis. The results showed that the sialic acid occupancy efficiency of the 

channel of interest was around 80% when expressed in HEK293 cells. 

References 

1. O’Connor S, Li E, Majors BS, He L, Placone J, Baycin D, Betenbaugh MJ, 

Hristova K: Increased expression of the integral membrane protein ErbB2 

in Chinese hamster ovary cells expressing the anti-apoptotic gene BclxL. 

Protein Expr Purif 2009, 67:41-47. 

2. Hara S, Yamaguchi M, Takemori Y, Nakamura M, Ohkura Y: Highly sensitive 

determination of N-acetyl- and N-glycolylneuraminic acids in human 

serum and urine and rat serum by reversed-phase liquid chromatography 

with fluorescence detection. J Chromatogr 1986, 377:111-119. 

P60 

Influence of operating parameters of a settling-based perfusion process 

on expansion of VERO cells attached on microcarriers 

Amal El Wajgali 1 , Frantz Fournier 1 , Eric Olmos 1 , Cécile Gény 2 , Hervé Pinton 2 , 

Annie Marc 1* 

1 Laboratoire Réactions et Génie des Procédés, UPR-CNRS 3349, Vandoeuvrelès-Nancy, 

France; 2 Sanofi Pasteur, Marcy L’Etoile, France 

E-mail: annie.marc@ensic.inpl-nancy.fr 


Background: The growing demand for biologicals produced by animal 

cells motivates the development of more efficient and reliable culture 


production processes. In the particular case of the industrial production of 

viral vaccines by Vero cells adhered on microcarriers, the cell propagation 

phases are mainly devoted to reach high cell density in less time while 

maintaining a good cell physiological state. Several papers have reported 

the optimization of culture conditions for microcarrier cultures [1-4]. 

Otherwise, perfusion bioreactors, based on continuous medium renewal 

and cell retention, can be a good alternative to batch systems. Among the 

various technologies for cell retention, gravitational settler is a promising 

device for large-scale perfusion culture process. This simple device takes 

advantage of the difference between the cell settling rate and the medium 

harvesting flow rate. Moreover, in the particularcaseofcellsattachedon 

microcarriers, it is more suitable than in the case of single suspended cells. 

So, the aim of this work was to evaluate the performances of adherent 

Vero cell cultures performed inside a perfused bioreactor using a 

gravitational settler as cell retention device. The study focused on the 

influence of two operating parameters, such as microcarrier concentration 

(MCs) and initial cell density (C0), on the cell growth. 

Materials and methods: Vero cells were provided by Sanofi Pasteur and 

cultivated in a serum-free medium attached on Cytodex-1 microcarriers 

(GE Healthcare). Cultures were performed in a 2 L bioreactor (Pierre 

Guérin, France) controlled at pH: 7.2, temperature: 37°C, pO 2: 25% with an 

agitation rate of 90 rpm. After 48 h of batch culture, the perfusion 

medium flow rate was started at 0.5 vol.d -1 . The harvest flow rate was 

made through a settling glass tube. The concentration of adherent cells 

was measured by the crystal violet method. 

A Design of Experiment (DoE) was set up, with the objective to study the 

effect of two operating parameters (MCs and C 0) in order to reach high 

maximal cell density and rapid cell growth. The chosen criteria for DoE 

response were the maximal cell concentration, either per medium volume 

(cells.mL -1 ) or per microcarrier (cells.MCs -1 ), and the population doubling 

level (PDL). A D-optimal design was chosen because of the irregularity of the 

experimental region. Three levels were defined for each parameter. The 

Modde 7 software was used to generate the DoE: 7 experiments plus one 

repetition in order to assess the repeatability. Theoretical initial cell densities 

(C 0) were corrected by the experimental values. The table 1 gives the 

corresponding operating parameters of the various perfused cultures. 

Results: The settling tube used as cell retention device was observed to be 

not only easy-to-implement but also a reliable system for retention of cells 

adhered on microcarriers. Indeed, no microcarrier was found in the harvest 

flow rate, whatever the culture operating conditions. The evolution of cell 

density was studied for more than 10 days. A final stabilization of the 

maximal cell density was observed during several days for all experiments 

(Table 1). The repeatability was evaluated with experiments 7 and 8, 

performed with the same operating parameters (MCs: 2.5 g.L -1 and C 0:14 

200 cells.cm -2 ). Both experiments displayed similar cell kinetics and reached 

the same maximal cell density (2.5 x 10 6 cells.mL -1 ). 

On the one hand, the experimental kinetics results were compared on the 

basis of the maximal cell concentrations. The highest cell density per 

medium volume (3.9 x 10 6 C.mL -1 ) was reached for the culture performed 

with the highest MCs (5 g.L -1 , exp. 2). But the highest cell density per 

microcarrier (300 cells.MCs -1 ) was obtained with the lowest MCs of 1.2 g.L -1 

(exp. 3 and 5) whatever the C 0 value (6 000 and 23 500 cells.cm -2 ). It was 

also pointed out that, according to the microcarrier concentration used, 

the maximal cell density (in cells per medium volume) could depend on 

the initial cell-to-bead-ratio. Indeed, the two experiments performed with 

Table 1(abstract P60) Operating conditions and experimental results for perfused experiments 

Experiments Operating factors Experimental results 

MCs (g.L -1 ) C0 (cells.cm -2 ) Cmax (10 6 cells .mL -1 ) Cmax (cells.MC -1 ) PDL 

1 2.5 9100 1.9 114 4.2 

2 5 23000 3.9 116 2.9 

3 1.2 6000 2.5 311 6.3 

4 3.8 10100 2.7 106 3.9 

5 1.2 28500 2.2 275 3.8 

6 3.8 36000 3.2 125 2.4 

7 2.5 14200 2.5 149 3.7 

8 (7bis) 2.5 14200 2.5 147 4.2



2.5 g.L -1 MCs reached 1.9 x 10 6 cells.mL -1 (exp. 1) when C 0 was 9 100 cells. 

cm -2 , and 2.5 x 10 6 cells.mL -1 with C 0 of 14 200 cells.cm -2 (exp. 7). The 

same conclusion was obtained with 3.8 g.L -1 MCs (exp. 4 and 6). Therefore, 

the highest cell concentration was reached when a sufficient number of 

cells per microcarrier was respected at the beginning of culture. But, with 

1.2 g.L -1 MCs the maximal cell density was similar for the two C 0 used, 

suggesting that the maximal cell concentration was less dependent on 

initial cell density at very low carrier concentration. 

Furthermore, the statistical analysis of the influence of the operating 

parameters, on the basis of the three chosen criteria, showed that the best 

model was observed with the PDL values. The model predicted correctly 

experimental results and was described by a first-order polynomial. The 

regression showed satisfactory statistical qualities and neither interaction 

nor quadratic effects were found significant. The model indicated that low 

values for both operating factors MCs and C 0 favoured a higher PDL. 

Conclusion: The easy-to-implement settling tube was a reliable system for 

retention of cells adhered on microcarriers. The influence of two operating 

parameters (MCs and C 0) on the Vero cell growth was quantified according 

to three criteria related to cell growth (cells.mL -1 , cells.MCs -1 and PDL). 

While the highest microcarrier concentration led to the highest total 

amount of attached cells, the lowest MCs induced the best carrier recovery 

by the cells. Moreover both low MCs and C 0 values favored a high PDL. 

These results provide a preliminary screening of operating conditions 

before undertaking a rational scale-up of the perfusion process. 

References 

1. Clark JM, Hirtenstein MD: Optimizing culture condition for the production 

of animal cells in microcarrier culture. Ann. N. Y. Acad. Sci 1981, 369-33. 

2. Mendonça RZ, Prado JCM, Pereira CA: Attachment, spreading and growth 

of VERO cells on microcarriers for the optimization of large scale 

cultures. Bioprocess Eng 1999, 20:565-571. 

3. Sean P, Forestell SP, Kalogerakis N, Behie LA, Gerson DF: Development of 

the optimal inoculation conditions for microcarrier cultures. Biotechnol. 

Bioeng 2004, 39:305-313. 

4. Bock A, Sann H, Schulze-Horsel J, Genzel Y, Reichl U, Möhler L: Growth 

behaviour of number distributed adherent MDCK cells for optimization 

in microcarrier cultures. Biotechnol. Progr 2009, 25:1717-1731. 

P61 

3D-Bioreactor culture of human hepatoma cell line HepG2 as a 

promising tool for in vitro substance testing 

Christiane Goepfert 1 , Wibke Scheurer 1 , Susanne Rohn 1 , Britta Rathjen 1 , 

Stefanie Meyer 1 ,AnjaDittmann 1 , Katharina Wiegandt 2 ,RolfJanßen 2 ,RalfPörtner 1* 


Technology Hamburg, D-21073, Germany; 2 Institute of Advanced Ceramics, 

Hamburg University of Technology, Hamburg, D-21073, Germany 




Introduction: Future developments in pharmaceutical research and 

regulatory requirements such as the European REACH program require high 

numbers of animal experiments. As a result of ethical concerns, cell culture 

tests with human cell lines or primary cells are considered as an alternative. 

However, current testing protocols using 2D cell cultures in Petri dishes are 

not equivalent to animal trials. 3D tissue cultures may overcome 

fundamental obstacles in the development of new therapeutic agents. Many 

new candidates of therapeutic agents are intended as agonists or 

antagonists of specific receptors on human cells. For these substances, 

organ-like test systems based on human cells are mandatory. In some cases, 

new pharmaceuticals lead to unexpected adverse reactions even after 

successful animal trials. It is assumed that 3D test systems based on human 

cells might help to overcome these problems. 

Materials and methods: Human hepatoma HepG2 cell line was cultivated 

in monolayer culture and on two 3D carrier systems macroporous ceramic 

carrier Sponceram (Zellwerk, Germany) and Fibracel composed of polyester 

non-woven fiber on a polypropylene scaffold (New Brunswick Scientific, 

USA). 3D-carrier cultivation was performed in 24-well-plates, in a multi-well 

flow-chamber bioreactor or a fixed bed (Fig. 1) [1]. Cells were seeded at 

cell densities of 1*10 5 /ml. Medium was DMEM/Ham´s F-12 mixture 

supplemented with 10% FBS. Growth of cultures was determined using 

DNA measurement with H33528 after digesting the cells with Papain.An 

average DNA content of 14.8 pg DNA per cell was previously obtained 

using defined cell concentrations. Functional assays were carried out 

according to the method described in [2] using 7-ethoxyresorufin as a 

substrate. Induction of EROD activitiy was done using 3-methylcholanthrene 

or Ketoconazole. Cellular viability was monitored using 

Resazurin and live/dead staining (AO/PI). 

Cell growth on carrier systems: On Sponceram, cells spreaded initially 

but formed dense clusters after extended cultivation. On FibraCel, cells 

formed small aggregates after seeding. Later they grew within the whole 

carrier structure. In the flow chamber 3.83 · 10 5 ± 6.04 · 10 4 cells per carrier 

were reached within two compared to 1.45 · 10 6 ±3.68·10 6 in 24 well 

plates. For the fixed bed an average of approx. 1.35 · 10 6 cells per carrier 

were obtained. 

Liver specific EROD assay: The cultivation of Hep G2 cells in the two 

reactor systems was carried out for 7 days and for 14 days prior to induction 

of EROD activity.. Measurement of EROD activity found to be linear for at 

least for 1h (sampling every 15 min). Activities were similar to static cultures 

in the flow chamber [approx. 1.7 fmol Resorufin/(cell*h)]. Lower activities 

were detected in the fixed bed bioreactor after 14d [approx. 0.5 fmol 

Resorufin/(cell*h)] compared to 7d [approx. 0.15 fmol Resorufin/(cell*h)], 

possibly as a result of the formation of large cell clusters. 

Conclusions: In this study it was shown that 3D dynamic culture systems 

can be used to carry out functional assays such as the EROD assay with a 

human liver cell line. HepG2 cells could be cultivated for a longer time in 

dynamic 3D culture and showed a better viability compared to static 

monolayer cultivation. 

Figure 1(abstract P61) Cultivation systems for 3D-culture of HepG2-cells (A) Multi-well flow-chamber bioreactor (medorex) (B) 10 mL fixed bed reactor 

(medorex) [1].



References 

1. Pörtner R, Platas O, Fassnacht D, Nehring D, Czermak P, Märkl H: Fixed bed 

reactors for the cultivation of mammalian cells: design, performance 

and scale-up. Open Biotechnol J 2007, 1:41-46. 

2. Donato MT, Gómez-Léchon MJ, Castell JV: A microassay for measuring 

cytochrome P450IA1 and P450IIB1 activities in intact human and rat 

hepatocytes cultured on 96-well plates. Anal Biochem 1993, 213:29-33. 

P62 

“BioProzessTrainer” as training tool for design of experiments 

Ralf Pörtner 1* , Oscar Platas-Barradas 1 , Janosh Gradkowski 1 , Richa Gautam 1 , 

Florian Kuhnen 2 , Volker C Hass 2 


Technology Hamburg, D-21073, Germany; 2 Institute of Environmental and 

Bio-Technology, Hochschule Bremen, D-28119, Germany 



Concept: Design and optimization of cell culture processes requires 

intensive studies based on “Design of experiments”-strategies. In 

academia teaching of DoE-concepts is often insufficient, as in most cases 

only simple culture strategies (batch) can be performed, as time and 

money are limited. More complex tasks such as feeding strategies for fed 

batch culture can be discussed theoretically only. 

To close this gap the virtual “BioProzessTrainer”, a model based simulation 

tool, was developed. It supports biotechnological education with respect 

to process strategies, bioreactor control, kinetic analysis of experimental 


data and modeling. Along with a set of examples for different control and 

processstrategies(batch, fed batch, chemostat etc.) learners are prepared 

for real experiments [1,2]. 

The “BioProzessTrainer” (Figure 1) helps to improve the quality of 

education by using interactive learning forms and by transmitting 

additional knowledge and skills. Costs for practical experiments can be 

minimized by reducing plant operation costs. Here a concept for teaching 

DoE-concepts for batch- (optimization of e.g. substrate concentrations 

and inoculation cell density) and fed-batch-processes (evaluation 

and optimization of feeding strategy) using the “BioProzessTrainer” is 

shown. 

Example 1: DoE for impact of glucose and glutamine concentration during 

batch (1,5 L) on cell density and antibody concentration of a mammalian 

cell line 

Experimental design: 

➣ Seed concentration: 4E8 cells/L [±10%] 

➣ Glucose conc.: low 15 mmol/L; high 30 mmol/L 

➣ Glutamine conc.: low 1 mmol/L; high 4 mmol/L 

➣ Culture time: 24h 

To induce an experimental error, the seed concentration was varied by +- 

10%. Results see Table 1 

Analysis via statistical tools: 

➣ One-dimensional ANOVA with respect to glucose at high glutamine 

concentrations: glucose conc. not significant for cell conc. (p=0.1), 

significant for antibody conc. (p=0.044); level of significance 0.05 

➣ Two-dimensional ANOVA with repetition: interaction between glucose 

and glutamine conc. not significant for cell conc. (p=0.14); significant for 

antibody conc. (p=0.046); level of significance 0.05 

Figure 1(abstract P62) (A) Teaching material: theoretical back-ground, exercises, sample solution [1] (B) Screen of „BioProzessTrainer“ (C) Example: fedbatch 

process with fixed feed rate perfomed with the BioProzessTrainer.



Table 1(abstract P62) DoE performed with the BioProzessTrainer 

cell conc. [ 10 8 cells/L] antibody conc. [mg/L] 

seed conc. [10 8 cells/L ] glutamine [mmol/L] glucose [mmol/L] 

15 30 15 30 

set 4.0 1 7.71 8.01 11.8 12.6 

-10% 3.6 1 7.15 7.49 11.1 12.1 

+10% 4.4 1 8.23 8.49 12.3 13.0 

set 4.0 4 1.17 1.37 22.5 27.7 

-10% 3.6 4 1.06 1.24 20.6 25.2 

+10% 4.4 4 1.27 1.49 24.4 30.2 

Impact of glucose and glutamine concentration on cell density and antibody concentration. 

Table 2(abstract P62) Impact of feed rate for glucose and glutamine feed during fed-batch (constant feed rate) on cell 

density and antibody concentration 

cell conc. [10 9 cells/L] antibody conc. [mg/L] 

glutamine feed rate [mL/min] glucose feed rate [mL/min] 

0.02 0.08 0.02 0.08 

0.02 2.10 2.15 84.2 63.0 

0.08 2.95 3.10 67.0 133 

Example 2: DoE for impact of feed rate for glucose and glutamine feed 

during fed batch (constant feed rate) on cell density and antibody 

concentration of a mammalian cell line 

Experimental design: 

➣ Seed concentration: 8E8 cells/L 

➣ Glucose conc. in glucose feed: 180 mmol/L 

➣ Glutamine conc. in glutamine feed: 30 mmol/L 

➣ Start feed: 24h; start volume 1.5 L; final volume 3 L 

➣ Feed rate glucose / glutamine feed: low 0.02 mL/min; high 0.08 mL/min 

Results see Table 2 

Analysis via statistical tools: 

➣ Two-dimensional ANOVA without repetition: glucose feed rate not 

significant for cell conc. (p=0.295) and antibody conc. (p=0.699); 

glutamine feed rate significant for cell conc. (p=0.035) and not for 

antibody conc. (p=0.653); level of significance 0.05 

References 

1. Hass V, Pörtner R: Praxis der Bioprozesstechnik. Spektrum Akademischer 

Verlag978-3-8274-1795-4 2009. 

2. Pörtner R, Hass VC: Interactive virtual learning environment for 

biotechnology (eLearnBioTec). Chemie-Ingenieur-Technik 2005, 77(8):1256. 

P63 

ADCC potency assay: increased standardization with modified 

lymphocytes 

Laurent Bretaudeau * , Véronique Bonnaudet 

Clean Cells, Boufféré, 85600, France 

E-mail: lbretaudeau@clean-cells.com 



For few years now, the use of monoclonal antibodies represents a 

significant progress in different therapeutic applications. In addition to 

commercialization of new products, important efforts in research and 

development have been made to launch new therapeutic antibodies. 

Many antibodies act through a mechanism of Antibody-dependant cell 

cytotoxicity (ADCC). National Health Agencies recommend or require the 

use of biological activity assays (potency) in order to characterize those 

pharmaceutical products. The ADCC assay combines the 3 following 

elements: 

The antibody of interest that is specific to a given antigen; 

The targeted cells that express the antigen of interest at their surface; 

Table 1(abstract P63) Advantages and drawbacks of ADCC effectors for the validation of a standardized ADCC assay 

ADCC effector types Advantages Drawbacks 

Primary NK or PBMC, isolated from Representative from the genetic diversity Not convenient for standardization 

donors 

Requirement for donor genotyping 

Requirement to evaluate several donors for accurate comparison 

Exposure of operators to biohazard 

Significant ressources required (budget and time) for cell 

preparation 

Modified NK cell lines Increased suitability for standardization The NK activity may interfere with the measurement of the 

ADCC-related lysis 

Absence of functionally qualified batches of cells 

Absence of biosafety qualified batches of cells 

To be handled as a GMO 

CD16-transduced lymphocytes Increased suitability for standardization 

Qualified batches available in a ready-touse 

format 

Absence of NK activity-related background 

To be handled as a GMO



Figure 1(abstract P63) Characterization of ADCC function of CD16-transduced lymphocytes. Two models of antigen expressing cells were used to 

establish the inter-assay precision of the ADCC measurement in a standard chromium-release assay. Briefly, the cells were 51 Cr-labelled, washed, 

incubated with the antibodies and the release of 51 Cr in the supernatant was analyzed after 4h. 

The effector that can trigger the lysis the targeted cell when the 

antibody is linked to the antigen. 

As the usually met ADCC assays use Natural Killer cells, isolated from 

healthy donors, as effectors they are hardly reproducible (Table 1). Thus, 

those assays can barely be validated when lots of pharmaceutical 

products are released. Furthermore, of the rare NK cell lines established 

in culture don’t express the CD16 receptor needed for the ADCC function. 

In this context, the use of standardized effectors should improve 

significantly the ADCC assays. Previous work has highlighted that human 

lymphocytes, modified to express the CD16 receptor, have acquired the 

ADCC functions [1]. Thanks to our specific know-how, clones of CD16+ 

lymphocytes have been produced on a large scale (10 9 cellules). The 

results obtained have pointed out that cells stored in liquid nitrogen and 

used as soon as they were thawed, were usable for ADCC assays on a 

reproducible basis (Figure 1). Thanks to that approach, two models of 

ADCC measurement are characterized in the present presentation: CD20 

and Her2neu. 

The produced effector cells constitute a relevant alternative to replace the 

use of NK cells when the standardization of ADCC potency assay is 

needed. 

Reference 

1. Clémenceau B, Congy-Jolivet N, Gallot G, Vivien R, Gaschet J, Thibault G, 

Vié H: Antibody-dependent cellular cytotoxicity (ADCC) is mediated by 

genetically modified antigen-specific human T lymphocytes. Blood 2006, 

107(12):4669-77. 

P64 

Anti-idiotypic antibody Ab2/3H6 mimicking gp41: a potential HIV-1 

vaccine? 

Renate Kunert * , Alexander Mader 

Department of Biotechnology, Institute for Applied Microbiology, BOKU – 

University of Natural Resources and Life Sciences, A-1190 Vienna, Austria 

E-mail: renate.kunert@boku.ac.at 


Background and aims: Anti-idiotypic antibodies (Abs) represent an 

alternative vaccination approach in human therapy. This approach is based 

on the idiotype (Id) network theory postulated by Jerne describing the Ab 

(Ab1) – anti-idiotypic Ab (Ab2) – anti-anti-idiotypic Ab (Ab3) cascade 

stimulation. Specific anti-Id Abs serve as an “internal image” of the target 

antigen and can be used to induce Abs able to bind to the cognate 

antigen [1]. The anti-Id Ab Ab2/3H6 [2], developed at our Institute was 

generated in mouse and is directed against the human monoclonal Ab 

(mAb) 2F5, which broadly and potently neutralizes primary HIV-1 isolates 

[3]. Ab2/3H6 which has been characterized previously [4,5] is able to mimic 


the antigen recognition site of 2F5 and therefore it is suggested as a 

putative candidate for an HIV-1 vaccine. 

We investigated the potential of Ab2/3H6 by immunization of Fab 

fragments and fusion proteins with interleukin 15 (IL15) and tetanus toxin 

(TT) tags as immune modulators. After three prime/boost administrations 

rabbit sera were purified and analyzed for 2F5-like specific Abs. Further, 

the 2F5-like Abs from the sera were enriched by affinity purification and 

characterized for their binding affinity to 2F5. 

In an additional approach we applied different humanization methods to 

reduce the immunogenicity of the originally mouse derived Ab2/3H6. The 

mouse variable regions of Ab2/3H6 were subjected to three different 

humanization methods, namely resurfacing, CDR-grafting and superhumanization. 

Four differently humanized Ab2/3H6 variants were 

characterized for their binding affinity to 2F5 in comparison to the original 

Ab2/3H6. 

Results: To evaluate the humoral immune response of Ab2/3H6 we 

designed Ab2/3H6 Fab fusion proteins with IL15 and TT. Recombinant CHO 

cell lines were established and after protein purification New Zealand white 

rabbits were immunized with the Ab2/3H6 Fab variants. Ten days after the 

final boost sera were collected and analyzed for total rabbit IgG levels. After 

proteinA affinity purification of the sera the isolated rabbit IgGs were tested 

for Ab2/3H6 Fab and recombinant gp140 (UG37) specificity (Figure 1A). 

Further an affinity enrichment step using a UG37/ELDKWA column was 

performed and the obtained Ab3 fraction was tested on UG37 (Figure 1B) 

and additionally on the original 2F5 epitope ELDKWA (Figure 1C). Finally the 

Ab3 fraction was tested for binding affinity to the UG37 in a bio-layer 

interferometry assay which showed that the Ab3 fraction has a 6.6 fold 

reduced affinity towards UG37 compared to the mAb 2F5 (Table 1). 

For the humanization approach three different methods were chosen. The 

“resurfaced” variant (RS3H6) was developed by a computer model and 

surface exposed amino acids in the murine framework (FR) were substituted 

by residues usually found at equivalent positions in human Abs. The 

“superhumanized” form (SH3H6) was designed by structural homologies 

between the murine Ab2/3H6 CDRs and human germline CDRs. The most 

homologous human germline Ab was then used as acceptor FR. For the 

“CDR-grafted” variant two versions were expressed. An “aggressive” graft 

(GA3H6) harbouring less backmutations, making the grafted Ab more 

human-like and a “conservative” graft (GC3H6) with more backmutations. 

The different “reshaped” variable regions of Ab2/3H6 were expressed in 

CHO-cells as IgG1 molecules. The obtained “humanized” Ab variants were 

further characterized by competition ELISA (Figure 1D). 

RS3H6 and GC3H6 showed a nearly identical slope of a concentration 

dependent 2F5 inhibition compared to the chimeric (ch) 3H6. RS3H6, GC3H6 

or ch3H6 complexed 50% of mAb 2F5 with a five fold molar excess. GA3H6 

was able to bind 50% of 2F5 in a 15 fold excess. In contrast, SH3H6 did not 

interact with 2F5 even in an 80 fold excess which is comparable to the



Figure 1(abstract P64) (A) Binding of purified rabbit IgGs immunized with different Fab preparations and control (pre-immun IgG fraction) to Ab2/ 

3H6Fab and recombinant HIV-1 gp140 (UG37); (B) Binding of the Ab3 (2F5-like) Ab fraction purified from pooled rabbit IgG fractions to recombinant HIV- 

1 gp140 (UG37) and (C) synthetic 2F5 epitope (ELDKWA); (D) Competition ELISA. Biotinylated 2F5 (2F5-B) was allowed to complex with serial dilutions of 

Ab2/3H6 mutants and afterwards uncoupled 2F5-B was determined on an epitope pre-coated ELISA plate. The optical density resulting from binding of 

2F5-B to the synthetic epitope GGGELDKWASL is plotted versus the logarithm of the concentration of Ab2/3H6 mutants. 

negative control (unspecific IgG). The ELISA results were supported by 

binding affinity experiments with 2F5 in a bio-layer interferometry assay: 

RS3H6, GC3H6 and ch3H6 have similar binding affinity to 2F5 whereas 

GA3H6 has a 2-fold reduction in affinity. 

Discussion and conclusion: The Ab2/3H6 was generated to be used as 

an HIV-1 vaccine, based on the induction of 2F5-like Abs (Ab3s). First we 

did a “proof of concept” andimmunizedrabbitswithdifferentAb2/ 

3H6Fab fusion proteins to induce 2F5-like Abs. Immunochemical analyses 

showed that the use of IL15 and TT as immune modulators do not 


significantly enhance total rabbit IgG levels or the production of Ab2/ 

3H6Fab specific Abs. However it could be demonstrated that UG37 

binding Abs (Ab3s) were induced significantly (Figure 1A). Quantification 

of various purification steps revealed that only 0.7% of the rabbit IgG 

fraction contained UG37/ELDKWA binding Abs. This affinity enriched Ab3 

fraction shows significant binding to UG37 and reduced binding to the 

synthetic 2F5 epitope ELDKWA in ELISA (Figure 1B/C). Affinity binding 

studies showed that the obtained Ab3 fraction has only a 6.6-fold lower 

affinity to the recombinant UG37 protein compared to 2F5 (Table 1).



Table 1(abstract P64) Summary of k-values for binding to UG37 

Samples [c=400nM] kon [1/Ms] koff [1/s] KD [nM] 

mAb 2F5 1.66 x10 04 

3.09 x10 -04 

18.65 

Ab3 fraction 1.56 x10 04 

1.91 x10 -03 

122.90 

Control IgG fraction n.d. n.d. n.d. 

In a next step different humanization methods were evaluated. The 

“resurfaced” and the “conservative” grafted variants showed similar binding 

properties to 2F5 as the original Ab2/3H6 version, while the “aggressively” 

graftedAbhada2-foldloweraffinityandthe“superhumanized” form 

completely lost the ability to bind to 2F5. 

We postulate that after humanizing the framework regions of Ab2/3H6 the 

immune response will be focused towards the paratope of the Ab2 and 

therefore enhance elicitation of Ab3 when administered during human 

therapy. Therefore affinity to 2F5 is not the crucial factor in evaluating the 

best candidate for a clinical study. Our results show that the Ab2/3H6 

variants RS3H6 and GA3H6 would be suitable candidates for future studies. 

Acknowledgement: This work was funded by Austrian Science Fund, 

Vienna (P20603-B13) 

References 

1. Jerne N: Towards a network theory of the immune system. Ann Immunol 

(Paris) 1974, 125C:373-389. 

2. Kunert R, Weik R, Ferko B, Stiegler G, Katinger H: Anti-idiotypic antibody 

Ab2/3H6 mimics the epitope of the neutralizing anti-HIV-1 monoclonal 

antibody 2F5. AIDS 2002, 16:667-668. 

3. Wolbank S, Kunert R, Stiegler G, Katinger H: Characterization of human 

class-switched polymeric (immunoglobulin M [IgM] and IgA) anti-human 

immunodeficiency virus type 1 antibodies 2F5 and 2G12. J Virol 2003, 

77:4095-4103. 

4. Gach J, Quendler H, Weik R, Katinger H, Kunert R: Partial humanization and 

characterization of an anti-idiotypic antibody against monoclonal 

antibody 2F5, a potential HIV vaccine? AIDS Res Hum Retroviruses 2007, 

23:1405-1415. 

5. Gach J, Quendler H, Strobach S, Katinger H, Kunert R: Structural analysis 

and in vivo administration of an anti-idiotypic antibody against mAb 

2F5. Mol Immunol 2008, 45:1027-1034. 

Figure 1(abstract P65) Cell growth and cell viability. 


P65 

Application of hydrocyclones for continuous cultivation of SP-2/0 cells 

in perfusion bioreactors: Effect of hydrocyclone operating pressure 

Elsayed A Elsayed 1,2* , Roland Wagner 3 

1 Advanced Chair for Proteomics & Cytomics Research, Zoology Department, 

Faculty of Science, King Saud University, 11451 Riyadh, Kingdom of Saudi 

Arabia; 2 Natural & Microbial Products Dept., National Research Centre, Dokki, 

Cairo, Egypt; 3 Technology Development Department, Rentschler 

Biotechnology GmbH, D-88471 Laupheim, Germany 

E-mail: eaelsayed@ksu.edu.sa 


Background: Hydrocyclones (HCs) have been recently extensively evaluated 

for their application in the separation of mammalian cells in perfusion 

bioreactors. The high centrifugal force derived within hydrocyclones is the 

key mechanism for cell separation inside such small-sized simple devices. 

This is usually accompanied by a very short residence time of the cells inside 

the separating equipment, usually not more than 0.2 s. Moreover, they have 

other specific characteristics, which highly recommend their application for 

the production of pharmaceutical products in perfusion cultivation 

bioreactors. They are characterized by their high performance, robustness, 

lack of movable parts, ease of in situ sterilization and suitability for cleaningin-place 

processes. 

Materials and methods: The hydrocyclone HC 2520, specially designed for 

cell cultivation, was used for separation of the recombinant mouse lymphoid 

cell line SP-2/0. It has a 25-mm underflow and 20-mm overflow orifice. 

A pulsation-free pumphead (Watson Marlow 505L) was fitted to a WM 505DU 

pump and the perfusion was performed intermittently. Cell were cultured on 

serum-free ZKT-1 medium supplemented with 5% foetal bovine serum.



Figure 2(abstract P65) Hydrocyclone separation efficiency. 

Results: Effect of hydrocyclone operating pressure on cell growth 

and cell viability: The results obtained (Figure 1) showed that cells 

required an adaptation phase to adapt themselves to the HC operating 

pressure. Moreover, the length of the adaptation phase depends on the 

HC pressure. Generally, cells were able to grow with high viability after the 

operation of hydrocyclone at both tested pressure values (0.85 and 1.30 

bar). Maximal cell concentrations of about 8.2 × 10 6 and 6.0 × 10 6 mL -1 

were achieved at an operating pressure of 0.85 and 1.30 bar with a 

viability range from 92 to 98%, respectively. This corresponds to a 5- and 

3.5-fold increase than the batch cultivation, respectively. 

Effect of hydrocyclone operating pressure on separation efficiency 

and separated cell quality: Results (Figure 2) showed that increasing 

operating pressure from 0.85 to 1.30 bar increased the average total 

separation efficiency from 89 to 95%. Additionally, results also showed that 

hydrocyclone preferably separates more viable cells in the underflow, and 

hence, back to the bioreactor system. Thus, more dead cells are separated 

in the overflow leaving the bioreactor system, which will improve system 

viability and product quality. 

Conclusions: - Hydrocyclone can be successfully used for cell separation in 

continuous mammalian perfusion bioreactors. 

- Cells adapt themselves to the shear forces inside the HC without being 

adversely affected by the pressure. 

- Higher separation efficiencies up to 96% can be achieved depending on 

the operating HC pressure. 

- HCs separate preferably more living cells in the underflow, thus more 

dead cells are leaving the system, which will finally lead to the 

improvement of system viability and product quality. 

P66 

Improving volumetric productivity of a stable human CAP cell line by 

bioprocess optimization 

Ruth Essers * , Helmut Kewes, Gudrun Schiedner 

CEVEC Pharmaceuticals GmbH, Gottfried-Hagen-Str. 62, D-51105 Köln, 

Germany 

E-mail: essers@cevec.com 



Background: For the production of recombinant proteins, a human cellderived 

expression technology can offer significant advantages with 

respect to protein quality, serum half-life and safety. High volumetric 

productivity with first-class quality is the ultimate ambition during 

process development. 

CEVEC’s proprietary expression system based on human amniocytes offers 

significant advantages for the production of complex human proteins 

and antibodies. The key benefits are the stable and high expression of 

recombinant proteins with human type posttranslational modifications, the 

robust growth behaviour with competitive high cell densities and the easy 

handling in serum free suspension. CAP cells meet all regulatory 

requirements, they are of non-tumour origin and from an ethically accepted 

source. 

In order to test the performance of CAP cells for the production of very 

complex proteins, stable C1-inhibitor expressing CAP cells were developed. 

C1-inhibitor is a serine protease inhibitor (serpin) and one of the most 

heavily glycosylated plasma proteins bearing numerous complex N- and Oglycans. 

Material and methods: Cells: CAP cells stably expressing C1-inhibitor 

Base medium: Protein Expression medium PEM (Gibco #12661013) 

containing 4mmol*L -1 glutamine (Invitrogen #25030024) 

Supplements: Soy Peptone E110 (OrganoTechnie #AI885), Glucose (Sigma 

#G8679), Valproic acid VPA (Sigma #4543) 

ELISA: In-house C1-inhibitor ELISA using serum derived C1-inhibitor as 

standard 

SDS-PAGE/Western Blot: In-house Western Blot for C1-inhibitor 

To investigate the influence of different hydrolysates and supplements 

under controlled conditions we use DASGIP´s parallel bioreactor system for 

cell culture. See table 1 for standard physical process parameters. 

Results: Parental CAP cells were transfected with a plasmid containing 

expression cassettes for the human C1-inhibitor driven by the CMV 

promoter, and a Blasticidine resistancegeneforselection.Outofthree 

stable pools, one pool was selected for further single cell cloning by 

limiting dilution. From initial 251 single cell clones, the best 5 were 

chosen for further scale-up. Subsequent process development was 

carried out with one clone showing the best performance in growth 

and expression.



Table 1(abstract P66) Standard physical process 

parameters for DASGIP fermentation 

process parameter 

T 37°C 

V 0.6L 

stirring marine impeller, 120rpm 

aeration sparging, 0.03vvm 

pH 7.0 (CO2/ 0.5M NaOH) 

DO 40% (air saturation) 

starting cell density 3-5E5mL -1 

First we tested the influence of different media supplements shown 

before to improve cell growth and productivity in CAP cells. Either 

additional glucose, glutamine, pyruvate, soy peptone, tryptone plus, 

tryptone or an in-house mixture of R3-IGF, transferrin, SyntheChol and 

progesterone were added to the base medium. Cells were cultivated in 

shake flasks and cell densities, viabilities, metabolites and product 

concentration were monitored continuously. 

Whereas the in-house mixture lead to higher growth rates, the product 

yields increased upon the addition of glucose (1.2-fold), soy peptone or 

tryptone (1.5-fold). 

Based on these results, we tested additional hydrolysates from cotton seed, 

wheat gluten, rice protein and soy. Hydrolysates and extra glucose were 

added to the initial medium and cells were cultivated in shake flasks. 

Only medium supplemented with soy peptone II (up to 1.7-fold) and with 

cotton seed hydrolysate (up to 2.4-fold) showed better performance as 

compared to the control with extra glucose but without hydrolysate. 

In addition to medium supplementation we determined the optimal pH 

value for good growth rates, high product quantity and quality. The 

different batches (pH unregulated, pH 7.0, 7.2 or 7.4) did not differ in 

maximal product concentrations but in product qualities. This was 

monitored with SDS-PAGE and western blot. Due to additional specific 

bands at pH7.2 and pH7.4 either caused by degradation or incomplete 

glycosylation we decided to continue with pH7.0 for following process 

development steps. 

Under controlled conditions additional glucose alone or either with soy 

peptone I, soy peptone II or cotton seed hydrolysate were supplemented to 

the base medium. Parallel fermentations with different enriched media were 

started at pH7.0 and cell densities of 3.0*10 5 mL -1 under controlled 

conditions. Cotton seed hydrolysate increased growth, but highest cell 

density could be observed with soy peptone II or without hydrolysate 

addition. The maximum product concentration was higher compared to the 

control in all peptone-fed fermentations. Supplementing soy peptone II or 

cotton seed hydrolysate almost doubled maximum product concentration. 

As a next step, we examined the effect of valproic acid (VPA) on productivity. 

VPA is a short-chain fatty acid used as well established drug and classified 

histone deacetylase inhibitor. We set up two cultures with glucose and soy 

peptone II and two cultures with glucose and cotton seed hydrolysate. In 

each case, one of the duplicate cultures was additionally supplemented with 

4mmol*L -1 VPA. 

Initial cell densities were 3.5*10 5 mL -1 in all four parallel controlled cultures. 

The addition of VPA was carried out at viable cell densities of 3.0*10 6 mL -1 . 

Addition of VPA resulted in 1.6-2.0-fold increase in productivity compared 

to the corresponding control cultures without VPA 

To determine the product quality we performed Western blots with samples 

from the supernatant of the batches supplemented with VPA. The highest 

protein quality was obtained from the Soy peptone II-supplemented 

cultures. 

We confirmed the results with 8 (parallel) runs and observed consistent 

results for cell densities, growth behaviour, product titers, cell specific and 

volumetric productivities. 

Conclusions: The optimized fed batch strategy starting with 2g*L -1 extra 

glucose and 4g*L -1 soy peptone II and addition of 4mmol*L -1 VPA at a 

viable cell denstity of 3.5*10 6 mL -1 yields 4.3-fold higher volumetric 

productivity as compared to the batch culture (Fig.1). With the optimized 

fed batch we are able to produce 200-250 mg*L -1 C1-inhibitor. 

P67 

Development of a triculture based system for improved benefit/risk 

assessment in pharmacology and human food 

Alexandra Bazes, Géraldine Nollevaux, Régis Coco, Aurélie Joly, 

Thérèse Sergent, Yves-Jacques Schneider * 

Biochimie cellulaire, nutritionnelle & toxicologique, Institut des Sciences de la 

Vie & UCLouvain, Croix du Sud 5/3, 1348 Louvain-la-Neuve, Belgium 

E-mail: yjs@uclouvain.be 


Caco-2 cells are widely used for both mechanistic studies in molecular cell 

biology as well as for studies aiming at estimating, in vitro, the 

bioavailability of drug candidates, xenobiotics, food compounds …. This 

Figure 1(abstract P66) Process development C1-inhibitor expressing CAP cell line – Improvement of volumetric productivity. 

Page 96 of 181



Table 1(abstract P67) In vitro models characterisation: Cell monolayer integrity was determined by LY transport. LY 

was incubated with cell monolayers for 180 min at 37°C (n=3) and it was quantified by fluorimetry. The results were 

expressed as % of total fluorescent 

Caco-2 HT29-MTX Caco-2/HT29-MTX Caco-2/Raji Caco-2/HT29-MTX/Raji 

TEER (Ω.cm 2 ) 283±70 122±31 122±19 88±27 60±17 

Lucifer Yellow transport (% fluorescence) 0,40±0,02 6,42±0,09 3,25±0,06 7,16±0,23 5,07±0,22 

results largely from the spontaneous differentiation of this human colon 

adenocarcinoma line into enterocytes-like cells in classical culture 

conditions, as well as from the use of bicameral culture inserts including 

porosity calibrated filters. 

Although Caco-2 cells represent the current “golden standard” of in vitro 

models of the human intestinal barrier, there is a trend to develop new 

systems mimicking more closely the intestinal epithelium for pharmaceutical 

and toxicological studies. In particular, goblet cells may bring a main 

constituent of the intestinal barrier as secreting mucus and contribute to the 

permeability met in vivo. In addition, gut associated M cells offer a putative 

way for oral delivery of nanoencapsulated therapeutic peptides and mucosal 

vaccines. 

Material and methods: The triculture model was adapted by combining 

the in vitro model of the human follicle-associated epithelium (including Mlike 

cells) according to the protocol of des Rieux et al. (2007) and the coculture 

of Caco-2 and HT29-5M1 cells from Nollevaux et al. (2006). Briefly, 

Caco-2 cells with or without HT29-5M1 cells were seeded on 12-wells 

Transwell inserts (3.0 μm pore diameter, Costar, Elscolab, Kruibeke, BE) and 

cultivated for 3 days in DMEM supplemented with 10% foetal calf serum 


(FCS). Some inserts were then inverted and a piece of silicon rubber (Labo- 

Modern, Queveaucamps, BE) placed around the basolateral side. Then, 

inserts were transferred into a Petri dish pre-filled with culture medium and 

maintained for 10 days with the basolateral medium refreshed every 2 days. 

Raji-B cells, suspended in DMEM + 10% FCS, were added to the basolateral 

compartment of the inserts. The tricultures were maintained for 5 days. Cocultures 

were cultured under the same conditions, but without the addition 

of Raji-B cells or HT29-5M1 cells. 

Cell monolayer integrity, both in mono-, co- and tri-cultures, was controlled 

by measurement of the transepithelial electrical resistance (TEER, Millipore 

Millicell® ERS, Billerica, MA) and by evaluation of cell permeability to Lucifer 

Yellow (LY; 457 Da, Sigma-Aldrich, St-Louis, MO). 

After integrity experiments, cells were washed in PBS, fixed with acetone 

and permeabilised with 1% (v/v) Triton X-100. Cells were then washed with 

PBS, blocked with 1% BSA and washed again with PBS, and then incubated 

with mouse anti-MUC5AC at 1:100 dilution, rinsed in PBS and incubated 

with Alexa Fluor 488 goat anti-mouse 10µg/ml and Rhodamine-Phalloidin 

4U/ml (Invitrogen, Eugene, OR). Finally, cells were mounted with Ultracruz 

Mounting Medium (Santa Cruz Biotechnology, Santa Cruz, CA). The 

Figure 1(abstract P67) Immunofluorescent localization of different cell types. The nuclei of cells were stained in blue with the mounting medium. 

Enterocyte actin was stained with rhodamine-phalloidin (red) and goblet-like cells were MUC5AC-labelled (green). The M-like cells were detected by their 

lack of microvilli.



monolayers were viewed using a Zeiss LSM 710 confocal laser scanning 

microscope (Carl Zeiss MicroImaging GmbH, Jena, DE). 

Results and discussion: To validate the triculture system as an intestinal 

barrier model, transport studies were used to determine the culture 

integrity and the permeability capacity with TEER measurement and 

assessment of the passage of LY. Furthermore, a morphological analysis 

was initiated to evaluate the proportion of the M cells, goblet cells and 

enterocytes and their implication in the intestinal epithelium model. 

Cell monolayer integrity and permeability properties: Adecreaseof 

TEER was observed in the different co-culture systems. This should, most 

probably, be correlated to an increase of the barrier permeability, upon 

addition of HT29-MTX or/and Raji cells in the system (Table 1). 

The effect of the addition of goblet-like cells or of the conversionof 

Caco-2 cells into M-like cells suggests a role of these cells in the ability of 

the intestinal cells to increase their absorbtion of hydrophilic molecules, 

as observed by the increase of the permeability of LY in the co- and triculture 

systems in comparison with the control. 

The contribution of the different cell types in the triculture system further 

allows the integration of mechanisms such as transcytosis (M cells), the 

presence of mucus (goblet-like cells), and the effectiveness of the tight 

junctions in the culture system, as compared to a standard Caco-2 model. 

Localisation of the intestinal cell types: The intestinal cell types were 

detected by fluorescent labelling and confocal analysis (Figure 1). Actin 

(red) and MUC5AC (green) were restricted respectively on enterocyte and 

goblet-like cells. The M-like cells appeared within the enterocyte layer 

and were identified by their lack of microvilli (actin) at their apical 

surface. The mucus produced by goblet-like cells was on the surface 

(MUC5AC). In the triculture system, there was close contact between 

HT29-MTX, Raji and Caco-2 cells but the goblet-like cells have tendency 

to cluster together. 

Conclusion, perspectives: These results clearly indicate that the 

cocultivation of 3 different cell types allows to reconstitute a cell culture 

system that should better mimic the intestinal barrier, specially to 

investigate its interaction with nanoparticles. This should facilitate a 

better evaluation of pharmacological or toxicological properties of these 

materials in food and drugs. 

We are now developing additional co-culture systems involving f.i. 

RAW264.7 macrophages to further determine the interactions involved in 

inflammatory processes; HepG2 cells to estimate the intestinal and 

hepatic effects in presystemic biotransformations. 

References 

1. des Rieux A, Fievez V, Théate I, Mast J, Préat V, Schneider YJ: An improved 

in vitro model of human intestinal follicle-associated epithelium to 

study nanoparticle transport by M cells. Eur J Pharm Sci 2007, 

30(5):380-391. 

2. Nollevaux G, Deville C, El Moualij B, Zorzi W, Deloyer P, Schneider YJ, 

Peulen O, Dandrifosse G: Development of a serum-free co-culture of 

human intestinal epithelium cell-lines (Caco-2/HT29-5M21). BMC Cell Biol 

2006, 2:7-20. 

P68 

Daphnane diterpene hirsein B downregulates melanogenesis in B16 

murine melanoma cells by cAMP pathway inhibition 

Myra O Villareal 1 , Junkyu Han 1,2 , Kenjiro Ikuta 3 , Hiroko Isoda 1,2* 

1 Graduate School of Life and Environmental Sciences, University of Tsukuba, 

Tennodai 1-1-1, Tsukuba, Ibaraki, 305-8572, Japan; 2 Alliance for Research on 

North Africa (ARENA), University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki, 

305-8572, Japan; 3 Yokohama Corporate Research Laboratories, Mitsubishi 

Rayon Co., Ltd, Tsurumi-ku, Yokohama, Kanagawa, 230-0053, Japan 



Background: Skin pigmentation serves as protection against ultravioletinduced 

skin damage through melanin’s optical and chemical filtering 

properties [1]. Although melanin plays and important role in skin protection, 

excessive melanin production or hyperpigmentation may lead to skin 

cancer. Recently, the inhibition of melanogenesis has been considered as a 

valid therapeutic target for the management of advanced melanotic 

melanomas [2] which increases the need for melanogenesis inhibitors 

that are of plant origin and are not cytotoxic to mammalian cells. The 

biosynthesis of the pigment melanin is catalyzed by the melanogenic 

Table 1(abstract P68) Differentially-expressed genes in 

hirsein B-treated B16 murine melanoma cells as 

determined by DNA microarray 

Biological Process Differentially 

expressed genes 

Up- or Downregulated 

Melanin biosynthesis Mitf, Mc1r Downregulated 

Melanosome transport Rab27a, Mlph, 

Myo5A, Myo7A 

Downregulated 

Negative regulation of 

transcription from RNA 

polymerase II promoter 

Sorbs3 Downregulated 

Wnt signaling pathway Ppap2b, Wisp, 

Kremen1 

Upregulated 

Cell cycle regulation Gadd45b, Csnkl Upregulated 

Activation of MAPK signaling 

pathway 

Gadd45b , Pxn, 

Map2k3, Met, Avpi1, 

Spag9 


Upregulated 

Cytoskeleton organization Pxn Upregulated 

Protein phosphorylation Mapkapk3 Upregulated 

enzymes tyrosinase, tyrosinase related protein 1 and the dopachrome 

tautomerase, the transcriptional regulation of which is being regulated by 

the microphthalmia associated transcription factor (Mitf) [3]. Previously, we 

have reported that hirsein B (HB) or 5b-hydroxyresiniferonol-6a,7a-epoxy- 

12b-coumaroyloxy-9,13,14-ortho-decanoate from Thymelaea hirsuta [4] has 

antimelanogenesis effect (without cytotoxicity) on B16 murine melanoma 

cells by downregulating the expressions of the Mitf gene and the 

melanogenic enzymes’ genes [5]. The exact mechanism by which hirsein B 

inhibited the Mitf gene expression, however, has not yet been determined. 

In melanogenesis, the Mitf gene expression can be regulated through the 

cAMP pathway or the Wnt signaling pathway. This study aimed to 

determine the mechanism underlying the inhibitory effect of HB on Mitf 

gene in B16 murine melanoma cells. 

Materials and methods: Total RNA was isolated from B16 murine 

melanoma cells (Riken Cell Bank, Tsukuba, Japan) and used for DNA 

microarray analysis, using chips of 528 spots loaded with 265 genes prepared 

by Genopal (Mitsubishi Rayon Co., Ltd, Tokyo, Japan), to determine the 

expressions of genes for melanogenesis, membrane-bound receptors, 

tyrosine kinase regulation, melanosome transport, and other cell signal 

regulation-related genes (including the housekeeping and negative control 

genes). To validate the results, real-time PCR, using TaqMan FAST 7500 

(Applied Biosystems, Foster City, CA, USA) and specific TaqMan primers 

(Applied Biosystems, Foster City, CA, USA) for the differentially-expressed 

genes, was performed. 

Results: Results showed that the expressions of the Mitf gene and the 

melanogenic enzymes’ genes were downregulated, verifying our previous 

report [5]. In addition, the expression of the gene for melanocortin 1 

receptor (Mc1r) of the cAMP pathway was downregulated while most of 

the genes that were upregulated are those involved in the Wnt signaling 

pathway (Table 1). 

In mouse, peptide hormones from the pituitary gland bind to the MC1R 

and stimulate melanin production through the cAMP/PKA signalling 

pathway [6], by inducing changes in the protein phosphorylation and 

gene expression, through the MITF gene product. 

Conclusions: The results obtained suggest that the significant 

antimelanogenesis effect of hirsein B is through the inhibition of the 

expression of the Mc1r gene of the cAMP pathway. HB may therefore be 

used as a treatment for hyperpigmentation due to its significant 

melanogenesis downregulation effects in B16 cells or as a pretreatment 

for melanotic melanomas. 

Acknowledgment: This study was partially supported by the Science and 

Technology Research Partnership for Sustainable Development (SATREPS). 

References 

1. Ahene AB, Saxena S, Nacht S: Photoprotection of solubilized and 

microdispersed melanin particles. Melanin, Its Role in Human 

Photoprotection Overland Park, KS: Valendmar: Zeise L, Chedekel M, 

Fitzpatrick TB 1995, 255-269.



2. Slominski A, Zbytek B, Slominski R: Inhibitors of melanogenesis increase 

toxicity of cyclophosphamide and lymphocytes against melanoma cells. 

Int J Cancer 2009, 124:1470-1477. 

3. Slominski A, Tobin DJ, Shibahara S, Wortsman J: Melanin pigmentation in 

mammalian skin and its hormonal regulation. Physiol Rev 2004, 

84:1155-1228. 

4. Miyamae Y, Orlina-Villareal M, Isoda H, Shigemori H: Hirseins A and B, 

daphnane diterpenoids from Thymelaea hirsuta that inhibit 

melanogenesis in B16 melanoma cells. J Nat Prod 2009, 72:938-941. 

5. Villareal M, Han J, Yamada P, Shigemori H, Isoda H: Hirseins inhibit 

melanogenesis by regulating the gene expressions of Mitf and 

melanogenesis enzymes. Exp Dermatol 2010, 19:617-627. 

6. Busca R, Ballotti R: Cyclic AMP a key messenger in the regulation of skin 

pigmentation. Pigment Cell Res 2000, 13:60-69. 

P69 

The neuroprotective effects of electrolyzed reduced water and its 

model water containing molecular hydrogen and Pt nanoparticles 

Hanxu Yan 1 , Taichi Kashiwaki 2 , Takeki Hamasaki 2 , Tomoya Kinjo 1 , 

Kiichiro Teruya 1,2 , Shigeru Kabayama 3 , Sanetaka Shirahata 1,2* 

1 Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 

Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; 2 Department of Bioscience 

and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812- 

8581, Japan; 3 Nihon Trim Co. Ltd., 1-8-34 Oyodonaka, Kita-ku, Osaka 531- 

0076, Japan 

E-mail: sirahata@grt.kyushu-u.ac.jp 



Background: Human brain is the biggest energy consuming tissue in 

human body. Although it only represents 2% of the body weight, it 

receives 20% of total body oxygen consumption and 25% of total body 

glucose utilization. For that reason, brain is considered to be the most 

vulnerable part of human body against the reactive oxygen species (ROS), 

a by-product of aerobic respiration. Oxidative stress is directly related to a 

series of brain dysfunctional disease such as Alzheimer’s disease, 

Parkinson’s disease etc. Electrolyzed reduced water (ERW) is a functional 

drinking water containing a lot of molecular hydrogen and a small amount 

of platinum nanoparticles (Pt NPs, Table 1). ERW is known to scavenge ROS 

and protect DNA from oxidative damage [1]. We previously showed that 

ERW was capable of extending lifespan of Caenorhabditis elegans by 

scavenging ROS [2]. Molecular hydrogen could scavenge ROS and 

protected brain from oxidative stress [3]. Pt NPs are also a new type of 

multi-functional ROS scavenger [4]. 

Materials and methods: In this research, we used TI-200S ERW derived 

from 2 mM NaOH solution produced by a batch type electrolysis device 

and model waters containing molecular hydrogen and synthetic Pt NPs of 

2-3 nm sizes as research models of ERW to examine the anti-oxidant 

capabilities of ERW on several kinds of neural cells such as PC12, N1E115, 

and serum free mouse embryo (SFME) cells. We pretreated the ERW and 

200 μM H2O2 and examined the neuroprotective effects of ERW on PC12, 

N1E115 and SFME cells, using WST-8 method. We also examined the 

intracellular ROS scavenging effects of ERW on N1E115 cells after 

pretreated cells with ERW and H 2O 2 using DCFH-DA. We checked the 

protective effects of ERW on mitochondria and cytoplasm by Rh123 and 

Fuo-3 AM stain. We also examined the ATP production of SFME cells after 

pretreated with ERW and H 2O 2 by Bioluminescence Assay Kit. Finally, we 

Table 1(abstract P69) Characteristics of the water samples.The characteristics of water samples were determined 

immediately after the preparation of ERW. ERW, electrolyzed reduced water; CW, activated charcoal-treated water. 

The pH values were shown as average ± standard deviation (N = 5). The values of DH, DO and Pt NPs were shown the 

minimum and maximum values after 5 independent measurements 

MQ (NaOH) TI-200 ERW TI-9000 CW TI-9000 ERW 

pH 11.3 ± 0.1 11.6 ± 0.1 7.9 ± 0.1 9.6 ± 0.2 

Dissolved Hydrogen (mM) 0 0.2– 0.45 0 0.1– 0.25 

Dissolved Oxygen (μM) 0 3.1– 78.1 0 0– 21.9 

Pt NPs (nM) 0 0.5– 12.8 0 0– 3.6 

Redox potential value (mV) + 350 -659 - - 

Figure 1(abstract P69) Protective effect of ERW on H 2O 2-induced neuroblastoma N1E115 cells death. Cells were treated with water samples (ERW 

and control ultrapure water with same pH with ERW) and 200 μM H 2O 2 for 24 h. Cell viabilities were assayed by WST-8 method. N=3, * p < 0.05.



used dissolved hydrogen (DH) and Pt NPs as research models to examine 

their neuroprotective effects. 

Results: ERW significantly reduced the cell death induced by H 2O 2 

pretreatment (Figure 1). ERW also scavenged the intracellular ROS and 

prevented the decrease of mitochondrial membrane potential and ATP 

production induced by ROS. We also examined the neuroprotective 

effects of molecular hydrogen and Pt NPs and showed that both 

molecular hydrogen and Pt NPs contributed to the neuroprotective 

effects of ERW. 

Conclusion: The results suggest that ERW is beneficial for the prevention 

and alleviation of oxidative stress-induced human neurodegenerative 

diseases. 

References 

1. Shirahata S, Kabayama S, Nakano M, Miura T, Kusumoto K, Gotoh M, 

Hayashi H, Otsubo K, Morisawa S, Katakura Y: Electrolyzed-reduced water 

scavenges active oxygen species and protects DNA from oxidative 

damage. Biophys Biochem Res Commun 1997, 234:269-274. 

2. Yan H, Tian H, Kinjo T, Hamasaki T, Tomimatsu K, Nakamichi N, Teruya K, 

Kabayama S, Shirahata S: Extension of the lifespan of Caenorhabditis 

elegans by the use of electrolyzed reduced water. Biosci Biotech Biochem 

2010, 74:2011-2015. 

3. Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, 

Katsura K, Katayama Y, Asoh S, Ohta S: Hydrogen acts as a therapeutic 

antioxidant by selectively reducing cytotoxic oxygen radials. Nature Med 

2007, 13:688-694. 

4. Hamasaki T, Kashiwagi T, Imada T, Nakamichi N, Aramaki S, Toh K, 

Morisawa S, Shimakoshi H, Hisaeda Y, Shirahata S: Kinetic analysis of 

superoxide anion radical-scavenging and hydroxyl radical-scavenging 

activities of platinum nanoparticles. Langmuir 2008, 24:7354-7364. 

P70 

Development and fine-tuning of a scale down model for process 

characterization studies of a monoclonal antibody upstream production 

process 

Mareike Harmsen * , Jimmy Stofferis, Laetitia Malphettes 

Cell Culture Process Development Group, Biological Process Development, 

UCB Pharma S.A, Braine-l’Alleud, 1420, Belgium 

E-mail: Mareike.Harmsen@ucb.com 


Background: It has always been an objective of process development 

and more recently it has also become a regulatory expectation to build 

robustness into and demonstrate proper control of a manufacturing 

process, thus ensuring that the biological product meets consistently its 

quality attributes and specifications. This is achieved mainly through 

systematic process development and understanding. Once a process is 

locked and ahead of consistency runs at the intended commercial scale, 

process characterization studies (PCS hereafter) further contribute to the 

demonstration of process robustness and the justification of process 

Page 100 of 181 

control ranges. These studies characterize the relationship between 

process parameters and process performance as well as product quality 

attributes. For practical reasons PCS are performed in a scale down model 

(SDM hereafter) of the manufacturing process studied. Therefore, it is 

essential to establish a SDM that is representative of the commercial 

scale. 

Here we describe a road map for developing a scale down model of a cell 

culture process for recombinant protein production. The cell culture process 

modeled was a 12,000 L scale fed batch process producing a monoclonal 

antibody. 

Methods: In a first step, the SDM was defined such that volume- dependent 

parameters (medium and feeds volumes, working volumes) were scaled 

down linearly and volume-independent ones (temperature, dissolved 

oxygen, pH, cell age) were kept at the same set-points as in the commercial 

scale bioreactor. Some cell culture process parameters such as agitation and 

aeration are difficult to scale down linearly due to bioreactor and sparger 

geometry and differences in controller systems. Hence determination of 

these parameters was first done by engineering scale down considerations. 

These considerations determined a theoretical small scale model used for 

proof of concept experiments. Process performance in initial scale down 

runs was then compared to commercial scale production in terms of cell 

growth, key metabolites, product accumulation. 

Two main deliverables were defined for the SDM: 

1. The model should exhibit process performance comparable to the 

commercial scale process. This was necessary to study the effects of input 

parameter variations during PCS. 

A way of assessing process performance is by monitoring in-process 

parameters during the SDM runs and comparing them to commercial 

scale batch data. Here the following evaluation parameters were chosen: 

Viable cell density and viabilities 

Metabolism indicators (namely off-line pH, dCO 2, osmolality, glucose and 

lactate concentrations) 

Product titer 

2. The model should deliver material with product quality attributes 

comparable to the material produced at the corresponding manufacturing 

scale process step (post cell harvest). This was necessary to study the 

effects of parameter changes on upstream product quality. 

Here the evaluation parameters were based on selected product quality 

specifications for the commercial scale batches. Namely: 

Oligosaccharide profiles 

Monomer/aggregate/fragment proportions 

Monomer,- Heavy-and Light chain patterns 

Acidic peak group species proportions 

Results: Proof of concept bioreactor runs using the theoretical SDM showed 

good comparability between the model and the commercial scale regarding 

process performance; however it was observed that the dissolved CO 2 

values were lower than in commercial scale. We consider this to be a 

consequence of geometrical bioreactor differences. The liquid surface-tovolume 

ratio is significantly higher in small scale than it is in large scale. 

Furthermore in small scale bioreactors a higher maximum volumetric gas 

Table 1(abstract P70) Product quality attributes of purified harvest material 

Product quality attribute Commercial scale reference 

sample 

SDM verification run 1 SDM verification run 2 SDM verification run 3 

Oligosaccharide mapping Profile comparable to standard Profile comparable to Profile comparable to Profile comparable to 

standard 

standard 

standard 

Monomer [%] 99 

Purity by size exclusion HPLC 

100 99 99 

Aggregate [%] 1 0 0 1 

Fragment [%] 0 0 

Monomer pattern by capillary gel electrophoresis 

1 0 

Monomer [%] 97 97 97 97 

Light and heavy chain pattern by capillary gel electrophoresis 

Light chain + heavy chain 

[%] 

99 99 

Acidic species by cation exchange chromatography 

98 99 

Acidic species [%] 30 30 28 32



flow rate was employed to compensate for a reduced gas bubble residence 

time. These two main differences resulted in increased CO 2 stripping. 

An overlay flow containing 6% CO 2 was applied to the headspace of the 

2 L vessels to reduce the stripping (fine –tuning). 

After establishing the CO2 overlay as part of the aeration strategy, three SDM 

verification runs were carried out. Cell growth, cell metabolism and Mab 

titers were monitored throughout the cultures. Lab scale data resulting from 

daily sampling was mostly within the average ± 3 x standard deviation (SD) 

of the commercial scale data and therefore comparable. 

Scale down model harvest material was compared to harvest material from 

the commercial scale with regards to their product quality attributes. The 

clarified cell culture fluid from both scales was protein A purified in 

laboratory scale and product quality was assessed (Table 1). 

Conclusions: Good comparability regarding process performance of the 

production bioreactor between the SDM runs and large scale data was 

demonstrated. Growth data, product titer, pH, osmolality, glucose and 

lactate profiles all showed the same trends. The tested output parameters 

where mostly within ± 3 x SD of the average commercial scale data. 

Product quality attributes of scale down samples were comparable to 

commercial scale product quality as shownbycomparisonwithclarified 

cell culture fluid samples from commercial scale. 

The verification results demonstrate that the systematic scale down approach 

applied in this work provided a laboratory scale model that was representative 

of the commercial scale process in terms of process performance and 

product quality of the generated material and was therefore suitable to be 

used in process characterization studies. 

Acknowledgements: We would like to thank Robert McCombie and Rav 

Ganesh for protein purification and Simon Briggs and François Delcroix 

for product quality analysis. 

P71 

Insect cell lines and baculoviruses as effective biocontrol agents of forest 

pests 

Guido F Caputo * , Sardar S Sohi, Sharon V Hooey, Lawrence J Gringorten 

Natural Resources Canada, Great Lakes Forestry Centre, Sault Ste. Marie, ON 

P6A2E5 CANADA 

E-mail: gcaputo@nrcan.gc.ca 


Background: Canada is a nation of trees. Canadian forests cover 50% of 

Canada’s land area and represent 10% of world’s forestedarea.Canada 

exports more processed forest products than any other country. It is also a 

host to a variety of forest insects both native and non native that limit the 

economic, recreational and wildlife habitat use. Our research focuses on 

initiating insect cell lines, determining nutritional requirements of cells, 

and developing low cost media for large scale propagation of insect 

pathogenic viruses as ecologically sound alternatives to chemical 

pesticides. Cell lines are used for bioassay and strain selection of viruses 

and bacterial toxins and for production of foreign gene products using 

baculovirus- and entomopoxvirus expression vectors. They offer a cleaner, 

viable alternative to insect larvae for producing viral pesticides. 

Since 1969, over 150 continuous cell lines have been produced for forest 

insect pest research at GLFC. This collection represents one of largest single 

site repository of frozen insect cell lines in the world. Cell lines developed 

include tissues of the eastern spruce budworm (Choristoneura fumiferana), 

western spruce budworm (Choristoneura occidentalis), forest tent caterpillar 

(Malacosoma disstria), tobacco hornworm (Manduca sexta), white-marked 

tussock moth (Orgyia leucostigma), red-headed pine sawfly (Neodiprion 

lecontei), gypsy moth (Lymantria dispar), white pine weevel (Pissodes strobi), 

the tarnished plant bug (Lygus lineolaris) and the ash and privet borer 

(Tylonotus bimaculatus). These cell lines represent six tissues of origin, namely 

neonate larvae, pre-pupae, embryos, ovaries, hemocytes, and midgut and 

four Insect Orders namely Lepidoptera, Hymenoptera, Coleoptera and 

Hemiptera. 

Presently, cell lines developed at GLFC are being used by 41 researchers 

in 8 Canadian provinces, 42 researchers in 21 US States and 28 

researchers in 12 countries worldwide. 

Biological control: Although chemical pesticides are generally more 

reliable and effective control agents, they are not environmentally 

acceptable as their negative impact far out weigh their benefits. We have 

developed methodologies to duplicate the in vivo process of the insect 

by producing both baculovirus phenotypes, the occlusion derived virus 

Page 101 of 181 

(ODV) and the budded virus (BV) in vitro. During the in vivo infection 

process hemolymph (BV) is collected and cultures are infected producing 

both phenotypes. BV, released in the media is used to infect other cells; 

ODV produced in the nuclei to infect other insects. 

Lawn assay: Trees produce numerous compounds with toxic and growth 

regulating properties to protect themselves against insect attack. We have 

developed a rapid in vitro agarose lawn assay for testing the toxicity of 

freeze-dried ethanolic leaf extracts and secondary compounds. Foliage from 

sugar maple, trembling aspen and mulberry was assayed for possible 

insecticidal properties against cells from two lepidopteran defoliators, spruce 

budworm and fall armyworm. Cells were suspended in buffered agarose and 

spread in a petri dish. Leaf extracts solubilized in 50-70% DMSO were 

applied directly to the cells. Trypan blue staining was used as an indicator of 

toxicity. Quantitative comparisons were determined by threshold doses that 

elicited positive responses. Toxicity and validity of the assay were further 

confirmed by the presence of membrane disruption and cellular lysis. The 

activity of trembling aspen was markedly enhanced when the pH was raised 

from 7 to 10.5, a level similar to that of the larval midgut, but the effect was 

largely abolished in the presence of gut juice. Analysis of a crude mulberry 

extract treated with neat gut juice suggested that most of the active 

material in the lepidopteran leaf diet is either insoluble or precipitated in the 

larval midgut, while the activity of any solubilized material is suppressed 

through interaction with gut-juice proteins. 

Molecular entomology using insect cell lines: A spruce budworm 

midgut cell line, FPMI-CF-203 has been shown to respond to the molting 

hormone ecdysone, juvenile hormone and other chemicals. Not only was 

gene expression stimulated in these treated cells, but they allowed for the 

analysis of house keeping genes or molting gene promoters as well as the 

study of cell signaling pathways such as protein kinase C and its targets, 

steroid hormone and molting gene expression. In addition, GFP tag fused 

target proteins were readily visible in single living CF-203 cells. They were 

permissive for the production of active foreign gene proteins using 

recombinant baculoviruses vector systems. We have observed that while 

RNA interference (RNAi) technology for the study of gene function does not 

work well in vivo within the whole insect, CF-203 cells are an invaluable in 

vitro tool for this function. 

Challenges within insect tissue culture discipline: Developing insect 

cell lines suitable for a specific application is a very slow and challenging 

process. Primary cultures started may or may not develop into cell lines. 

Those that do might take months and the resulting cells may not be 

suitable for a desired function. Of the 4-6 M insect species, we currently 

have 800+ cell lines from 100 species. There are presently no cell lines from 

exotic or invasive species. Once established, insect cells, like insects, must be 

fed to be kept alive. Suitable culture medium for the growth of tissues of 

different insects is currently not available. Large scale production of insect 

viruses would require the media to be optimal for cell growth as well as for 

replication of the virus or other control agents. Each cell line/virus 

combination requires its own unique media composition. Two pressing 

concerns requiring immediate attention are cross and misidentification 

contamination of existing cell lines and retiring of key researchers. With few 

insect cell lines in public collections, private collections are often 

inaccessible after the principal investigator leaves. Retiring scientist equals 

lost insect cell cultures. 

Future requirements to achieve “cultural immortalization”: More 

dedicated researchers developing new lines from different species, 

routine and aggressive identification, characterization and verification of 

cells lines employing new molecular techniques and maximizing the 

potential of insect cells as viable commercial ventures are needed. 

P72 

Down-regulation of CD81 in human cells producing HCV-E1/E2 

retroVLPs 

Ana F Rodrigues 1 , Miguel R Guerreiro 1 , Rute Castro 1 , Hélio A Tomás 1 , 

Charlotte Dalba 2 , David Klatzmann 3 , Paula M Alves 1 , Manuel J T Carrondo 1,4 , 

Ana S Coroadinha 1* 

1 Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia 

Química e Biológica (IBET/ITQB-UNL), Apartado 12, P-2781-901 Oeiras, 

Portugal; 2 EPIXIS SA, 5 rue des Wallons, 75013 Paris, France; 3 AP-HP, Hôpital 

Pitié-Salpêtrière, Biotherapy, F-75013 Paris, France; 4 Faculdade de Ciências e 

Tecnologia/Universidade Nova de Lisboa (FCT/UNL), P-2825 Monte da 

Caparica, Portugal 

E-mail: avalente@itqb.unl.pt 

BMC Proceedings 2011, 5(Suppl 8):P72



Background: Retroviral based biopharmaceuticals are used in numerous 

therapeutic applications, from gene therapy to vaccination [1-3]. 

Nonetheless, retroviruses are known to incorporate proteins from the host 

cell which may increase the vector immunotoxicity [4]. CD81, a membrane 

protein belonging to the tetraspanin superfamily has been recently 

identified as one of the host cell proteins majorly incorporated into Moloney 

Murine Leukemia Virus (MoMLV) derived vectors[5]. Therefore, CD81 was 

chosen as a target for studying the effects of host cell protein incorporation 

in the immunotoxic profile of retroviral vectors (RV). A human cell line, 

293rVLP, producing retrovirus like particles (rVLPs) was used to prove the 

concept of customizing RV composition by manipulating cellular protein 

content, using the CD81 tetraspanin as a case study. rVLPs will be used as 

candidate vaccines for hepatitis C by displaying HCV-E1/E2 proteins. 

Materials and methods: Cell lines and culture media: 293rVLP is a HEK 

293 (ATCC CRL-1573) based cell line established by stable transfection of 

pCeB plasmid [6] driving MoMLV gag-pol expression as described in [7]. 

293T cells (ATCC CRL-11268) were used for producing and titer shRNA 

lentiviral vector stocks. All cells were maintained in Dulbecco’s modified 

Eagle’s medium (DMEM) (Gibco) 10% (v/v) Foetal Bovine Serum (FBS) 

(Gibco). 

Plasmids and lentivirus production: Short hairpin (sh) RNA lentiviral vectors 

based on pLKO.1-puro [8] containing a puromycin resistance gene and 

targeting five different sequences on the human CD81 mRNA or a nontarget 

(nt) shRNA vector were purchased from Sigma Mission® RNAi (Sigma). 

The lentiviral vector stocks were produced by transient co-transfection of 

293T cells with pSPAX2, pMD2G (Addgene) and the respective pLKO.1-puroshRNA. 

Flow cytometry for CD81 detection: The presence of CD81 on the cell 

membrane was detected by flow cytometry using mouse monoclonal 

antibody against human CD81 and an Alexa FluorR 488 dye conjugated 

secondary antibody (Invitrogen). 

Oxidative stress induction and cell viability: The susceptibility to oxidative 

stress injury was assessed by analyzing tert-butyl hydroperoxide induced 

death. Cell viability was quantified by the percentage of cells staining 

negative for PI (by flow cytometry). 

RT activity: To determine MoMLV reverse transcriptase (RT) activity the 

RetroSys C-Type RT activity kit (Innovagen) was used. 

Further details on Materials and Methods are in [9]. 

Results and discussion: 293rVLP cells producing rVLPs were used to 

evaluate the possibility of customizing vector composition by 

manipulating host protein content. CD81 tetraspanin was chosen to be 

down-regulated by shRNA, given that it is significantly incorporated into 

Page 102 of 181 

RV particles, conferring them a strong immunogenic character in mice. 

Each cell population, shCD81 8 to 12, as well as the non-target control, 

were analyzed for the presence of CD81 by flow cytometry (Table 1). 

CD81 down-regulation efficiency varied considerably among the five 

shRNA sequences, ranging from negligible in the case of shCD81 11 to a 

very strong silencing effect in shCD81 10 cell population, with more than 

90% of cells being negative for CD81. ntshRNA population stained almost 

totally positive (94%) for CD81. To increase CD81 down-regulation, the 

shCD81 10 cell population was doubly infected with shCD81 9 and 12 

(Table 1). Only 1.5% of the resulting cells were positive for CD81 which, 

considering the background values for the ntshRNA control (approx. 6%), 

suggested a totally CD81 silenced population. 

shCD81 9+10+12 cell population was cloned by limiting dilution and 30 

clones were isolated. Four of those, #14, #19, #22 and #30 were chosen as 

representatives of the highest CD81 silencing (≥85%) and #9 as a 

representative of intermediate CD81 silencing. The rVLP production was 

analyzed by reverse transcriptase (RT) quantification in the supernatant; the 

cellular response to CD81 down-regulation and/or RNAi pathway activation 

was assessed by evaluating cell growth and cell death susceptibility under 

oxidative stress conditions (Table I). Cell population ntshRNA was used as 

negative control to distinguish CD81 silencing from RNAi pathway activation 

effects. The results were compared to the parental cell line, 293rVLP. With 

the exception of clone #14, all silenced clones presented lower cell growth. 

This effect, seemed to be attributed to CD81 down-regulation, as it was not 

detected in the ntshRNA control. Indeed, CD81 has been previously 

described to be implicated in proliferation and cytostasis of various cell 

types, including lymphocytes, fibroblasts, epithelial cells and astrocytes 

[10-13]. Lower half maximal inhibitory concentration values (IC 50) under 

oxidative stress were observed for silenced cells, including the ntshRNA 

population, suggesting that the activation of RNAi pathway played a role in 

injury induced death susceptibility. From a bioprocessing point of view, 

increased susceptibility to stressful conditions, oxidative or other, should be 

a drawback. However, RNAi is faster and simpler than a knockout, and 

undeniably useful in preliminary stages. Additionally, for certain vital 

proteins, a total knock-out may not be possible. 

To assess rVLP productivity in shCD81 clones the RT in the viral supernatant 

was quantified. Three of the five clones presented lower RT productivities, 

whereas clones #14 and #22 showed identical values to the non-target 

silenced control or the parental cell line. This suggested that CD81 downregulation 

does not affect viral particles production. Finally, rVLP shCD81 

#14, was used to produce rVLPs, which were concentrated, purified and 

analyzed for the presence of CD81 by Western blotting. The rVLPs were 

Table 1(abstract P72) CD81 down-regulation in 293rVLP cells 

CD81 - cells 

(%) 

sh populations ntshRNA 5.8 

shCD81 8 20.4 

shCD81 9 88.4 

shCD81 10 92.8 

shCD81 11 10.0 

shCD81 12 31.0 

shCD81 9+10 

+12 

98.5 

CD81 - cells Specific cell growth 

(%) 

(h -1 IC50 (µM tert-butyl 

Specific RT productivity 

) 

hydroperoxide) 

(10 -6 ng/cell.h) 

293 rVLP 2.1 0.0201 ± 0.0002 3.3 ± 1.1 1.7 ± 0.3 

ntshRNA 0.8 0.020 ± 0.002 9.2 ± 1.4 2.3 ± 0.4 

shCD81 9+10+12 

clones 

shCD81 #9 86.4 0.0124 ± 0.0004 15.4 ± 1.0 0.9 ± 0.2 

shCD81 #14 97.1 0.021 ± 0.002 4.5 ± 1.1 2.0 ± 0.4 

shCD81 #19 92.9 0.015 ± 0.001 6.5 ± 1.1 1.2 ± 0.2 

shCD81 #22 88.9 0.018 ± 0.001 6.0 ± 1.1 1.8 ± 0.4 

shCD81 #30 82.3 0.017 ± 0.001 5.6 ±1.0 1.4 ± 0.2



shown to be absent of CD81 demonstrating that it could be eliminated. 

These results suggest that, although naturally incorporated on RV, CD81 is 

notessentialfortheassemblyorsecretion of fully functional particles. 

Hepatitis C envelope proteins E1/E2 were successfully expressed in CD81 

silenced cells and incorporated in rVLPs. 

Conclusions: In this work it was demonstrated that is possible to 

manipulate the composition of the host cell proteins incorporated into 

rVLPs. rVLps can be used as a epitope display platform and used in vaccine 

development therefore, CD81 was chosen since it is strongly immunogenic 

in mice. The results herein presented support that is possible to manipulate 

cellular protein expression, CD81 or others, as a tool to control RV 

composition and their immunogenic profile. Additionally, for gene therapy 

purposes, the removal immunotoxic proteins in the producer cell may 

improve the in vivo efficacy of retrovirus, lentivirus or other enveloped virus. 

Acknowledgements: The authors acknowledge the financial support 

received from the European Commission (Clinigene: LSHB-CT2006-018933) 

andfromFundaçãoparaaCiênciaeaTecnologia(FCT)—Portugal (PTDC/ 

EBB-BIO/102649/2008 and PTDC/EBB-BIO/100491/2008). A.F. Rodrigues 

acknowledge FCT for her PhD grant (SFRH/BD/48393/2008). 

References 

1. Dalba C, Bellier B, Kasahara N, Klatzmann D: Replication-competent vectors 

and empty virus-like particles: new retroviral vector designs for cancer 

gene therapy or vaccines. Mol Ther 2007, 15:457-466. 

2. Dalba C, Klatzmann D, Logg CR, Kasahara N: Beyond oncolytic virotherapy: 

replication-competent retrovirus vectors for selective and stable 

transduction of tumors. Curr Gene Ther 2005, 5:655-667. 

3. Edelstein ML, Abedi MR, Wixon J: Gene therapy clinical trials worldwide to 

2007–an update. J Gene Med 2007, 9:833-842. 

4. Arthur LO, Bess JW Jr., Urban RG, Strominger JL, Morton WR, et al: 

Macaques immunized with HLA-DR are protected from challenge with 

simian immunodeficiency virus. J Virol 1995, 69:3117-3124. 

5. Segura MM, Garnier A, Di Falco MR, Whissell G, Meneses-Acosta A, et al: 

Identification of host proteins associated with retroviral vector particles 

by proteomic analysis of highly purified vector preparations. J Virol 2008, 

82:1107-1117. 

6. Danos O, Mulligan RC: Safe and efficient generation of recombinant 

retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad 

Sci U S A 1988, 85:6460-6464. 

Table 1(abstract P73) Characteristics of the different single-use depth filters 

7. Cosset FL, Takeuchi Y, Battini JL, Weiss RA, Collins MK: High-titer packaging 

cells producing recombinant retroviruses resistant to human serum. 

J Virol 1995, 69:7430-7436. 

8. Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, et al: A lentiviral 

RNAi library for human and mouse genes applied to an arrayed viral 

high-content screen. Cell 2006, 124:1283-1298. 

9. Rodrigues AF, Guerreiro MR, Santiago VM, Dalba C, Klatzmann D, et al: 

Down-regulation of CD81 tetraspanin in human cells producing 

retroviral-based particles: tailoring vector composition. Biotechnol Bioeng 

2011, 108(11):2623-2633. 

10. Geisert EE Jr., Abel HJ, Fan L, Geisert GR: Retinal pigment epithelium of 

the rat express CD81, the target of the anti-proliferative antibody 

(TAPA). Invest Ophthalmol Vis Sci 2002, 43:274-280. 

11. Geisert EE Jr., Yang L, Irwin MH: Astrocyte growth, reactivity, and the target 

of the antiproliferative antibody, TAPA. J Neurosci 1996, 16:5478-5487. 

12. Oren R, Takahashi S, Doss C, Levy R, Levy S: TAPA-1, the target of an 

antiproliferative antibody, defines a new family of transmembrane 

proteins. Mol Cell Biol 1990, 10:4007-4015. 

13. Toledo MS, Suzuki E, Handa K, Hakomori S: Cell growth regulation through 

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fibroblast WI38 and its oncogenic transformant VA13. J Biol Chem 2004, 

279:34655-34664. 

P73 

Evaluation of disposable filtration systems for harvesting high cell 

density fed batch processes 

Antje Pegel * , Friedemann Übele, Sven Reiser, Dethardt Müller, Gregor Dudziak 

Rentschler Biotechnologie GmbH, 88471 Laupheim, Germany 

E-mail: antje.pegel@rentschler.de 


Introduction: In the underlying study we evaluated different single-use 

filtration systems for cell separation and harvest clarification in 1,000 L 

scale. A screening of different depth filters was carried out with various 

single-use filters from Pall, Cuno (3M), Millipore and Sartorius Stedim. In 

total, we included 85 depth filtrations in the screening. Out of that, two 

single-use filtration systems were chosen and further tested in 200 L 

Manufacturer Material* Filter type Retention range (µm)* Number of filter layers* 

PDK7 20 – 4 

Pall 

Seitz® HP-Series 

Cellulose, 

Diatomaceous earth, 

Resin 

PDK6 20 – 3 

PDK5 20 – 1 2 

PDH4 15 – 0.4 

PDE2 3.5 – 0.2 

Seitz® P-Series KS50P 0.8 – 0.4 1 

10SP02A 7 – 1 

Cuno 

Zeta Plus® 

Millipore 

Millistak+® 

Sartorius 

Sartoclear P® 

Cellulose, 


Perlite 

Cellulose, 

Diatomaceous earth 

Cellulose, 


Binding matrix 

30SP02A 5 – 0.8 2 

60SP02A 5 – 0.65 

60ZA05A 0.8 – 0.6 

D0HC 9 – 0.6 2 

C0HC 2 – 0.2 

PB1 11 – 4 2 

PB2 8 – 1 

* Data according to manufacturers [http://www.pall.com, http://www.cuno.com, http://www.millipore.com, http://www.sartorius-stedim.com]. 

Page 103 of 181



scale. Based on these results, a single-use filtration set-up for harvesting 

production scale fed batch processes was determined. 

Material and methods: High cell density fed batch cultivations of a 

monoclonal antibody (mAb) expressing Chinese Hamster Ovary (CHO) cell 

line were harvested by depth filtration and 0.2 µm filtration after 14 to 

Page 104 of 181 

19 days at viabilities ranging from 40 to 95%. For the screening in 10 L 

scale, single-use depth filters (23 to 26 cm 2 ) with different separation 

ranges were used (Table 1). Subsequently, two disposable depth filtration 

systems were tested in 200 L scale using filter capsules with a filter area of 

0.23 to 0.25 m 2 . Depth filtrates were 0.2 µm filtered with Pall EKV (20 cm 2 ). 

Figure 1(abstract P73) A) Maximum capacities of depth filters in small scale screening. B) Calculated filter areas for filtration of 1,000 L harvest based on 

results of large scale trials.



During filtration, a constant flow of 100 L·m -2 ·h -1 was applied. Maximum 

capacities of the filters were determined at a pressure of 1 bar. Filter 

performance was assessed with regard to filter capacity, filtrate turbidity 

and product yield. Furthermore, content of DNA and Host Cell Protein 

(HCP) in filtrates were measured. 

Results: 

Depth filter screening: The filters PDK7, PDK6 and PDK5 from Pall 

showed the highest maximum capacities with 161-167 L/m 2 (Figure 1 A). 

These double layered filters had identical first membranes and differing 

finer second membranes. Filtrate turbidity was below 7 NTU when 

applying PDK6, whereas in filtrates generated with the coarser filter PDK7 

turbidities up to 9 NTU were observed. Additionally, product loss with 

PDK6 (6%) was lower compared with PDK7 (8%) or PDK5 (13%). For that 

reason Pall PDK6 was selected for the scale-up experiments. 

Additionally, the depth filters 10SP02A and 60SP02A from Cuno were chosen. 

For filter 10SP02A a maximum capacity of 131 L/m 2 was obtained. However, 

the filtrate turbidity was higher than 10 NTU causing a fast blocking of the 

0.2 µm filter. Therefore, this filter was combined with the finer depth filter 

60SP02A. The filter 60SP02A was selected due to turbidity values below 10 

NTU and an acceptable capacity of 105 L/m 2 when applied stand-alone. 

Turbidity breakthroughs at pressures below 1 bar were observed for the 

depth filters Millipore D0CH and Sartorius Stedim PB1 (Figure 1 A). 

Consequently, these filters were not considered for the scale-up studies. 

Scale-up: The selected depth filters were applied in 200 L scale using the 

Stax Disposable Depth Filter System (Pall) and the Zeta Plus Encapsulated 

System (Cuno), respectively. Performance of depth filters was comparable in 

large scale and small scale. Maximum capacities in the large scale trials were 

174 L/m 2 for Pall PDK6, 152 L/m 2 for Cuno 10SP02A, and 137 L/m 2 for Cuno 

60SP02A. Product loss was below 10%. 

Based on maximum capacities filter areas were calculated for harvest in 

1,000 L scale (Figure 1 B). An optimal filtration set-up leading to the lowest 

total filter area (6.9 m 2 ) was found in the depth filter Pall PDK6 and a 

subsequent 0.2 µm filter. Insertion of a second depth filter after Pall PDK6 

reduced the area of the 0.2 µm filter but did not affect the total filter area. 

The depth filter area of Cuno 60SP02A (7.3 m 2 ) was comparable to that of 

the filter combination Cuno 10SP02A – 60SP02A (7.4 m 2 ). 

DNA content in the filtrate was reduced by 30% with Pall PDK6 and even 

by 70% with the additional depth filter PDE2. The Cuno depth filter 

combination 10SP02A – 60SP02A reduced DNA content by 99% 

compared to 90% when only applying 60SP02A. With the Pall depth 

filters a reduction of HCP by 30% was measured whereas no HCP 

removal was observed for the Cuno depth filters. 

Conclusion: In this concept study disposable filtration systems were 

successfully tested in terms of identifying suitable filtration set-ups for 

equipping a 1,000 L disposable manufacturing line. These single-use 

filtration systems can thereby replace conventional (stainless steel) disc 

centrifuge and filtration steps in industrial mammalian cell culture 

production processes. The Stax system from Pall equipped with the filter 

PDK6 followed by a 0.2 µm filtration was identified as the first choice singleuse 

filtration set-up offering high capacities and a low product loss. Addition 

of a finer second depth filter can further increase filtrate clarification 

resulting in a reduction of DNA and HCP in filtrates and a smaller area of the 

subsequent 0.2 µm filter, but is also combined with a higher risk of product 

loss and an increased time and handling effort. 

Acknowledgement: We thank Novartis Pharma AG for supporting this 

project. 

P74 

Osteogenic Differentiation of adipose mesenchymal stem cells with 

BMP-2 embedded microspheres in a rotating bed bioreactor 

Stefanie Boehm 1* , Yael Lupu 2 , Marcelle Machluf 2 , Cornelia Kasper 1 

1 Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 

30167 Hannover, Germany; 2 Faculty of Biotechnology and Food Engineering, 

Technion, Haifa, Israel 


Introduction: Bone tissue engineering aims at the generation of functional 

bone tissue for replacement of defect bone tissue in order to reestablish 

normal function. For this purpose mesenchymal stem cells (MSCs) are widely 

used since they can be isolated from different sources and can easily be 

differentiated in vitro into the mesenchymal lineages [1,2]. 

Page 105 of 181 

The aim of this work was to study the effect of growth factor BMP-2 on 

the osteogenic differentiation of human mesenchymal stem cells from 

adipose tissue. The cells were cultivated in a rotating bed bioreactor 

system (Z®RPD system, Zellwerk GmbH) on the aluminium oxide based 

ceramic Sponceram® for four weeks. The ceramic discs were loaded with 

PLGA microspheres releasing BMP-2. For this experiments a system was 

used which allows the parallel cultivation of four bioreactors. The used 

bioreactor containers were manufactured of disposable material for single 

use application. During the cultivation glucose and lactate concentrations 

were measured and after the experiments histological stainings were 

performed (DAPI, von Kossa and alizarine red). Furthermore the 

concentration of alkaline phosphatase was measured in medium samples 

and mRNA of the cells was isolated to perform RT-PCR to investigate the 

expression of different bone markers. 

Materials and methods: Biomaterial: The Sponceram® biomaterial 

consists of AlO 2. It has a macroporous structure (600 µm) with a 

microporous surface and a porosity of 85%. For the cultivations scaffolds 

with a diameter of 65 mm and a thickness of 3 mm were used. The PLGA 

microspheres were produced by solvent evaporation and at a size of about 

50 µm. The BMP-2 concentration in the microspheres was about 0.05 µg 

per mg PLGA. The total amount of BMP-2 in the dynamic cultivations was 

2.5 µg in each bioreactor. The BMP-2 release and the concentration in the 

medium was measured with a BMP-2 ELISA (Quantikine, R&D Systems) The 

BMP-2 concentration in medium samples of bioreactor 4 decreases almost 

linear from 875 pg/ml to zero pg/ml in the first 16 days of the dynamic 

cultivation. 

Cells: The adipose tissue derived mesenchymal stem cells (adMSC) were 

isolated by enzymatic treatment under GMP conform conditions in a 

laboratory of the Red Cross in Linz, Austria. For the dynamic cultivation the 

isolated cells were expanded in cell factories (Nunclon Δ Cell Factory) and 

the dynamic cultivations were performed with cells in passage 5. For the 

cultivations 5x10 6 cells were used in every bioreactor. 

Bioreactor: The Z®RPD bioreactor system consists of four disposable 

rotating bed bioreactors and is working in a perfusion mode. The reactor is 

equipped with a Sponceram® ceramic, which is served as a rotating bed. In 

the standard operation mode the bioreactor is half filled with media (50 mL) 

and the Sponceram® is rotating with 0.5 rpm. This way the cells on the 

ceramics have alternating contact to medium and gas-filled headspace 

improving cell nutrition and oxygen supply.Theaimoftheparallel 

cultivation of four bioreactors was, to test the reproducibility of the system, 

which is important for GMP standards in clinical application. The bioreactors 

were integrated in a GMP conform breeder with a control unit for GMP 

conform documentation and evaluation of pH, oxygen and temperature. 

Furthermore a sterile sampling of media during the cultivation is possible. 

The dynamic cultivations were performed over a time period of 28 days and 

the cells were cultivated in osteogenic medium with dexamethasone, 

l-ascorbate and b-glycerol phosphate. 

Results: During 28 days of dynamic cultivation about 200 mg glucose were 

consumed in each bioreactor. The glucose consumption increased during 

the cultivation indicating a continuous cell growth on the ceramics. Also 

DAPI staining of the cell seeded ceramics showed a high density of cells on 

the scaffolds. Furthermore the concentration of alkaline phosphatase was 

measured daily (Sigma fast TM , Sigma Aldrich). The amount of the secreted 

alkaline phosphatase decreased during the cultivation period which is an 

indication for advanced osteogenic differentiation. Also the expression of 

various genes involved in the osteogenic differentiation of cells was verified. 

Different intensities of the gene expression in the four bioreactors was 

observed; especially the gene expression in bioreactor 1 was obviously 

higher. SEM pictures of the cell seeded ceramics showed thick cell layers on 

Sponceram®. Small round beads and a fibrous structure were noticeable. 

The beads might be cells, which are embedded in the extracellular matrix 

and the fibrous structure could be a hint for Collagen reposition. To verify 

the osteogenic differentiation of the cells von Kossa- and alizarine red 

stainings were performed. Both stainings showed high amounts of calcified 

matrix on all four ceramics. Also the alkaline phosphatase staining (Sigma 

fast BCIP, Sigma Aldrich) was successful and showed high amounts of 

secreted alkaline phosphatase on all four scaffolds. 

Conclusion: The glucose consumption increased during the cultivation 

indicating a continuous cell growth and the consumption was almost 

the same in all bioreactors. Also the DAPI staining of the ceramics 

showed a homogenous spreading of the cells on the Sponceram® 

ceramics.But only in two bioreactors a typical alkaline phosphatase



Figure 1(abstract P74) ZRP® bioreactor system (Zellwerk GmbH) with the special setup for performing four parallel cultivations. 

concentration curve could be verified in collected medium samples. The 

histological staining showed matrix calcification of the cells on every 

ceramic. Also the expression of typical bone markers was shown. In 

summary, osteogenic differentiation of adMSC on Sponceram®, caused 

by BMP-2 released from PLGA microspheres, was demonstrated and the 

reproducibility of the osteogenic differentiation in four parallel 

cultivations was demonstrated. 

Acknowledgements: This project is sponsored by the state of lower 

Saxony. Moreover we gratefully thank Zellwerk GmbH for providing the 

Z®RPD system and Sponceram® ceramics. 

References 

1. Moretti P, Hatlapatka T, Marten D, Lavrentieva A, Majore I, Hass R, Kasper C: 

Mesenchymal stromal cells derived from human umbilical cord tissues: 

primitive cells with potential for clinical and tissue engineering 

applications. Adv Biochem Eng Biotechnol 2010, 123:29-54. 

2. Hass R, Kasper C, Bohm S, Jacobs R: Different populations and sources of 

human mesenchymal stem cells (MSC): A comparison of adult and 

neonatal tissue-derived MSC. Cell Commun Signal 2011, 9(1):12. 

P75 

Medium and feed optimization for fed-batch production of a 

monoclonal antibody in CHO cells 

Nadine Kochanowski * , Gaetan Siriez, Sarah Roosens, Laetitia Malphettes 

Cell Culture Process Development Group, Biological Process Development, 

UCB Pharma S.A., Braine L’Alleud, 1420, Belgium 

E-mail: Nadine.Kochanowski@ucb.com 


Background: Mammalian cells are used extensively in the production 

of recombinant proteins, and of monoclonal antibodies (MAbs) in 

particular. The trend towards avoiding animal-derived components in 

biopharmaceutical production processes has led to the extensive use of 

non-animal origin hydrolysates such as plant hydrolysates or yeast 

Page 106 of 181 

hydrolysates. The source of hydrolysates affects cell growth and productivity 

and may also affect product quality. Accordingly, careful consideration 

should be given during process and cell culture media development, in 

order to determine the appropriate type and amount of hydrolysates to be 

added, for the cell and product at hand. 

In this study, we assessed the impact of several hydrolysate additives and 

chemically defined (CD) commercial feeds on MAb titers, MAb average 

specific productivity (average Qp), cell viabilities and metabolite profiles 

in suspension cultures of recombinant CHO cells expressing a monoclonal 

antibody in shake flasks and 2 L bioreactors. 

Materials and methods: Initial experiments were performed using 

chemically defined culture medium in 125 mL shake flasks with 50 mL 

working volume. CHO cells were seeded at 0.3x10 6 viable cells/mL and 

incubated at 140 rpm, 36.5°C and 5% CO2. 2L stirred tank bioreactors 

(Sartorius) were carried out for 14 days in a fed-batch mode in a chemically 

defined medium supplemented with chemically defined feeds and 

hydrolysates. Glucose was maintained between 1 and 6 g/L. At the day of 

harvest the clarification was performed by depth filtration. Analysis of daily 

samples included determinations of cell viability, cell density, metabolites, 

osmolality and product titer. Product concentration of the supernatant 

samples was quantified using Octet QK and Protein A high performance 

liquid chromatography (HPLC). Protein characterization of Protein-A purified 

samples were profiled by reduced and non reduced SDS PAGE. Isoelectric 

focusing (IEF) analysis of Protein-A purified MAb was carried out using a 

iCE280 IEF Analyzer. Aggregates and monomers proportion were 

determined by using size exclusion chromatography. Acidic and basic 

species were characterized using anion exchange (AEX) HPLC. Oligosaccharides 

were cleaved enzymatically using N-Glycanase, then labeled 

with 2-aminobenzamide and analyzed by HPLC using an amide column and 

a fluorescent detector. 

Results: Several chemically defined feeds and hydrolysates were assessed 

on CHO cells expressing a monoclonal antibody in fed-batch mode. The 

performance of the developed process was compared to an existing inhouse 

platform process.



Table 1(abstract P75) Chemically defined feeds and hydrolysates tested 

Supplier Commercial feed name Feed name in the poster 

ThermoFischer Cell Boost 1 CD Feed 1 

ThermoFisher Cell Boost 2 CD Feed 2 

ThermoFisher Cell Boost 3 CD feed 3 




Life Tech CHO Feed A CD Feed 7 

Life Tech CHO Feed B CD Feed 8 

Life Tech CHO Feed C CD Feed 9 

BD Biosciences Yeast Extract Hydrolysate 1 

BD Biosciences Yeastolate Hydrolysate 2 

BD Biosciences Select Phytone Hydrolysate 3 

BD Biosciences Ultrapep Soy Hydrolysate 4 

Sheffield HyPep 1510 Hydrolysate 5 

Sheffield HyPep 4605 Hydrolysate 6 

BD Biosciences 3 g/L Yeast Extract + 3.25 g/L Yeastolate Hydrolysate combination 1 

BD Biosciences 3.5 g/L Yeast Extract + 2.75 g/L Yeastolate Hydrolysate combination 2 









Page 107 of 181 

Figure 1(abstract P75) Relative percentage of improvement on MAb titer. Note: Platform process in shake flask was used as the 100% reference for all 

the calculations of relative percentage of improvement on Mab titer measured the day of harvest.



Nine different chemically defined feeds were assessed and added at 

different concentrations (Table 1). Among the feeds tested, addition of 

CD Feed 8 and 9 brought a 150% improvement on MAb titer on the day 

of harvest compared to the platform process. All the MAb titers 

measured were ranging from 2 g/L to 6 g/L (Figure 1). 

Six different hydrolysates were assessed at different concentrations in fedbatch 

mode (Table 1). Among the feeds tested, addition of hydrolysate 1 

and hydrolysate 2 showed an improvement of 175% and 167% 

respectively on MAb yield. 

To identify potential synergies between hydrolysates, the best 

hydrolysates from previous experiments were selected and were tested in 

combination at different ratios on CHO cell cultures (Table 1). Antibody 

concentration at harvest was 290% higher with some of the hydrolysate 

combinations. 

Based on feed combination optimization results, the number of bolus 

feeds and the feed addition timing were then fine-tuned using the best 

hydrolysate combination. Reducing the number of bolus feeds enabled to 

reduce ammonia and osmolality while maintaining a high MAb titer 

(Figure 1). Moreover, under these conditions, cell viabilities were 

maintained above 80% throughout the culture (data not shown). 

Based on experimental results obtained in shake flasks, the best hydrolysate 

combination (3 feeds and 4 feeds) and CD feed were assessed on CHO cells 

cultured in 2 L stirred tank bioreactors. Cell growth and cell metabolism 

were monitored daily throughout the cultures in bioreactors. By feeding the 

cultures with hydrolysates, addition of 3 or 4 bolus feeds enabled to attain 

similar maximum viable cell count. Addition of chemically defined feed led 

to a 30% higher maximum viable cell count. Cell viabilities were maintained 

at acceptable values throughout the cultures in the established culture 

conditions. Lactate profiles were similar independently of the feeding 

regime. Decreasing the number of hydrolysate feeds enabled to maintain 

osmolality and ammonia at acceptable concentrations for CHO cell growth 

and product quality. Cell growth performance and metabolism profiles 

observed in 2 L bioreactors were comparable to those observed in shake 

flasks. 

Product titers have been measured throughout the fed-batch cultures with 

the Octet QK system. The MAb titers were in a 2-6 g/L range for all the 

tested feeding regimes at the day of harvest. A combination of 

hydrolysates and a chemically defined feed supplementation showed an 

improvement of 296% and 245% on MAb concentration at the day of 

harvest in comparison to the platform process (Figure 1). MAb average 

specific productivity (Qp) was increased by 700% and 360% by adding 4 

feeds of hydrolysates and chemically defined feed respectively. Decreasing 

the number of hydrolysate feeds showed a slight decrease on MAb titer 

and on Qp. 

Product quality attributes were determined on cell culture clarified fluids after 

Protein-A purification. Reduced and non reduced SDS electrophoresis, 

isoelectric focusing (IEF) analysis, gel permeation HPLC, size exclusion (SEC) 

chromatography, anion exchange HPLC (AEX) have been used to characterize 

the Protein-A purified MAb. Product quality data was comparable for all the 

feeding regimes tested in 2 L bioreactors. 

Conclusions: Hydrolysate combination additions significantly improved 

MAb production in comparison to single hydrolysate addition or 

chemically defined feeds. Number of bolus feeds and feeding timing 

optimization enabled us to improve the process robustness taking into 

account the impact of feeding strategy on cell metabolism and product 

quality. The feeding regimes established in shake flasks led to similar 

culture performance in 2 L bioreactors. 

Acknowledgments: We wish to acknowledge BD Biosciences, Sheffield, 

Life Tech and Thermofisher for providing media and feeds. We also wish 

to acknowledge Mari Spitali, Natasha Hinkel, Vanessa Auquier and Marc 

Speleers for Protein-A purification and product quality analysis. 

P76 

In-situ microscopy and 2D fluorescence spectroscopy as online methods 

for monitoring CHO cells during cultivation 

Sophia Bonk * , Marko Sandor * , Ferdinand Rüdinger, Bernd Tscheschke, 

Andreas Prediger, Alexander Babitzky, Dørte Solle, Sascha Beutel, 

Thomas Scheper 

Institute of Technical Chemistry, Leibniz University of Hanover, Germany 


Page 108 of 181 

Introduction: Typical methods for monitoring cultivation processes are 

offline analyses like cell counting, measurement of various substrates and 

products (e. g. glucose or lactate) as well as the online monitoring of 

several physical process parameters (temperature, pH-value or the 

concentration of dissolved oxygen). 

To improve cell cultivations detailed information about important analytes 

should be available online. Therefore new monitoring methods need to be 

established, preferably as in-situ methods to minimize the risk of 

contamination. 

Two different in-situ online-methods were used to monitor cultivations: Insitu 

microscopy and 2D fluorescence spectroscopy. Therefore CHO-K1 cells 

(provided by University of Bielefeld) were cultivated in a complex culture 

medium (TC 42, TeutoCell, Bielefeld, Germany) using a 2.5 L stainless steel 

reactor with a work volume of 2 L. A total of three cultivation runs were 

conducted. 

In-situ microscopy: The in-situ microscope (ISM), developed at our 

institute, offers the possibility to determine the cell density and to obtain 

further information about certain cell characteristics such as size, 

compactness and excentricity. In addition, it is possible to extract other 

information like cell clusters or microbial contaminations from the recorded 

images. In this way a general comprehensive overview of a cultivation can 

be obtained. 

The in-situ microscope was immersed directly in the culture liquid using a 

25 mm standard connection. During the cultivation process several images 

were obtained and subsequently evaluated by a self-developed algorithm 

which detects cells on the basis of grey scale values. 

It was necessary to correlate the cells per picture with offline data 

determined by a Neubauer counting chamber. This correlation gives a 

regression coefficient (R 2 )of0.988. 

By means of this correlation the cells per picture were calibrated in reference 

to the counted cells per mL (root mean square error of calibration; RMSEC: 

0.989). As a result it is possible to detect the cell density via ISM with an 

average standard error of 0.027 (pictures per cycle: 300) during further 

cultivations. 

2D fluorescence spectroscopy: The 2D fluorescence spectra were 

collected using a Bioview® sensor (Delta, Denmark). The fluorescence 

spectroscopy was used to monitor glucose, lactate und glutamate during 

cultivation. At first it was necessary to build a calibration model for each 

compound. Therefore two process runs were observed by collecting 2D 

fluorescence spectra every 15 min. Approximately every 6 hours a sample 

was taken and analyzed offline for glucose, lactate and glutamate. The 

offline reference data and the corresponding spectra were used to build a 

chemometric model for prediction of the most interesting variables during a 

further cultivation run. 

Utilizing multivariate analyzing software (Unscrambler X, vers. 10.1) a 

calibration model was built by partial least square regression (PLS1) for 

every variable. The number of factors necessary to built a model as well as 

the results for the regression coefficient (R 2 ), the root mean square error of 

calibration and validation (RMSEC and RMSEV) for every variable are shown 

in Tab. 1. 

The PLS-models were used to predict all three compounds in a third 

cultivation run. The standard errors of prediction (RMSEP) are also shown 

in Tab. 1. For glutamate the RMSEP is about 5% compared to a maximum 

glutamate concentration of 0.27 g/L. 

By applying the PLS-models to the fluorescence data the concentration 

gradients for every single compound can be predicted (Fig. 1). 

Conclusions: In biotechnology, especially in pharmaceutical 

biotechnology it is important to closely monitor a cultivation process. The 

in-situ microscopy together with digital image processing and the 2D 

fluorescence spectroscopy in combination with chemometric tools 

complement one another as online monitoring methods. 

It was demonstrated that the ISM can be used to monitor the cell density 

during a bioprocess with the same accuracy in comparison to a Neubauer 

counting chamber. After a successful calibration the cell density can be 

determined without the need for taking samples. This can reduce the risk 

of contamination, particularly when the cells are cultivated without the 

addition of antibiotics. 

The Bioview® sensor could be utilized to observe three important analytes 

during a cultivation run with an error of prediction under 10%. By 

monitoring these analytes in real time it is possible to observe sudden or 

unexpected changes during a cultivation and if necessary to react 

accordingly.



Table 1(abstract P76) PLS-model results for glucose, lactate and glutamate 

Compound factors R 2 

RMSEC [g/L] RMSEV [g/L] RMSEP [g/L] 

Glucose 3 0.967 0.381 0.468 0.524 

Lactate 3 0.972 0.368 0.461 0.494 

Glutamate 4 0.983 0.0036 0.0059 0.0155 

Figure 1(abstract P76) Predicted concentration gradients and their corresponding offline values. 

Both online methods were successfully applied to monitor the cell density 

as well as the glucose, lactate and glutamate concentration during 

cultivation. 

Outlook: Concerning the ISM further research is focused on a new 

prototype with precise linear stages makingitpossibletoadjusttoa 

certain sample volume. This should make a calibration with offline data 

unnecessary. 

The robustness of the calibration models for the 2D fluorescence 

spectroscopy will be tested by adding glucose during cultivation in order 

to determine if the model can be applied to a fed batch cultivation. 

Acknowledgement: This project was funded by the Federal Ministry of 

Education and Research within the project “SpektroSens” (spectroscopic 

sensor systems for the area of biotechnology and food). 

P77 

On-line and real time cell counting and viability determination for 

animal cell process monitoring by in situ microscopy 

Philipp Wiedemann 1,2* , Markus Worf 2 , Hans B Wiegemann 2 , Florian Egner 4 , 

Christian Schwiebert 4 , Jeff Wilkesman 5 , Jean S Guez 3 , Juan C Quintana 2 , 

Diego Assanza 2 , Hajo Suhr 2 

1 At present: School of Biotechnology and Biomolecular Sciences, University 

of New South Wales, Sydney NSW 2052, Australia; 2 Mannheim University of 

Applied Sciences, Paul-Wittsack-Str.10, D-68163 Mannheim, Germany; 

3 Laboratoire ProBioGEM, UPRES-EA 1024, Polytech-Lille / IUT A, Université 

des Sciences et Technologies de Lille, Avenue Paul Langevin, Villeneuve 

d’Ascq, F-59655, France; 4 InVivo BioTech Services, Neuendorfstr. 24a, D-16761 

Hennigsdorf, Germany; 5 University of Carabobo, Faculty of Sciences and 

Technology, Chemistry Department, Valencia, 2005, Venezuela 

E-mail: p.wiedemann@hs-mannheim.de 


Page 109 of 181 

Background: Two of the key parameters to be monitored during cell 

cultivation processes are cell concentration and viability. Until today, this is 

very often done off-line by sterile sampling and subsequent counting using 

a hemocytometer or an electronic cell counter. Cell biology lacks a 

measurable quantity by which single cells in suspension can be noninvasively 

diagnosed as dead or alive. However, it would be of significance 

for process monitoring and in the light of initiatives like PAT if cell density as 

well as viability could be determined directly and on-line. 

Optical measurement of cell density by in situ microscopy eliminates the 

need for sampling and allows for continuous monitoring of this key 

parameter; see e.g. [1,2]; Guez et al. [1] describe an in situ microscope (ISM) 

which does not use any moving mechanical parts within or outside the 

fermentation vessel. It transmits in real time images taken directly in the 

stirred suspension within the bioreactor. Image data is processed and 

evaluated to provide monitoring of cell-density and morphological 

parameters, e.g. cell size, by means of assessing the obtained in situ cellmicrographs. 

Previously, we have extended in situ microscopy towards viability 

assessment of suspended cells [3,5]. Now, we present new findings on 

this topic and show that in cultures of suspended cells, cell-death 

corresponds to measurable changes in morphometric parameters as e.g. 

variance, contrast or entropy of the greyvalues of in situ cell-micrographs. 

As an example, here we show viability determination via greyvalue 

dispersion. 

Material and methods: We use a custom built high resolution ISM (HS 

Mannheim) with water immersion objective, 40x magnification, numerical 

aperture 0.75 equipped with optical fiber illumination. Data acquisition is 

at 0.3 – 15 frames per second, frames have 1293x1040 pixels; primary 

data analysis results in cell micrographs (Figure 1a). We have applied the 

system to bench top and larger bioreactors (see e.g. [4]) and worked 

mainly with Jurkat, CHO and Hybridoma cells.



Figure 1(abstract P77) a: In situ micrographs of hybridoma cells. Left: Typical portrait taken in a cell culture with high viability (>90%) Right: Typical portrait 

taken in a cell culture with low viability (



Ethanol at 42 hours. Cell counts (not shown) and viability were 

determined by in situ microscopy and, as reference, by means of a ViCell 

cell viability analyser (Beckman Coulter) and Flow Cytometry (Partec) 

using Annexin V / FITC and PI staining. Jurkat cells (DSMZ ACC 282) were 

cultured in 90% RPMI 1640 + 10% FBS. 

Results: Figure 1a shows ISM-micrographs and corresponding greyvalue 

histograms during a hybridoma culture. The cell on the left side represents 

the typical image of cells during exponential growth at high viability. The 

cell on the right side represents the typical image of cells during the final 

stage of a culture at low viability. Cells at low viability are much less 

homogeneous and have a correspondingly wider dispersion of greyvalues 

as compared to cells at high viability. Therefore, the greyvalue-variance 

constitutes a random variable which is sensitive to the viability of the 

culture, meaning that the greyvalue-variances increase when viability drops. 

In order to define - on the basis of this effect - a measurable value 

analogous to viability, we compute the percentage of cells with greyvaluevariance 

below a certain threshold. The threshold is optimized in calibration 

experiments applying reference methods for viability determination. We call 

this value “percentage of cells at low variance” (PLV). The PLV value is a 

viability value with respect to inhomogeneity of in situ cell-micrographs. 

This is analogous to viability defined as percentage of living cells with 

respect to a specific ex situ diagnosis. It has to be conceded that there is no 

one to one correspondence between vital cells and cells with variance 

below the defined threshold. Both sets – vital cells on the one side and cells 

with low variance on the other side– are not perfectly identical since some 

cells may be dead with no strong inhomogeneity, and vice versa. Despite 

these statistical side-effects, the PLV signal constitutes an operative measure 

of “inhomogeneity” and as such it also reflects the viability of the culture 

(see Figure 1a/b). Therefore, a high correlation can be anticipated between 

the PLV-signal and the viability signals from e.g. counting via Trypan blue 

exclusion or Flow Cytometry with AV/PI assay. 

We perform online computation of PLV-values over the course of the 

culture to continuously monitor ISM-viability of the culture in addition to 

cell density (see [1]; [4]). Since data analysis and image processing 

happens continuously online, micrograph portraits of cells can also be 

assessed for other morphological characteristics as for example cell-size in 

real time. 

Figure 1c shows results of this viability determination in the case of a 

Jurkat culture as an example. Similar results have been obtained with CHO 

and hybridoma cells. The culture was insulted by addition of 3% Ethanol to 

assess responsiveness of the in situ microscope. Similar correlations of 

viability determination by in situ microscopy and reference methods were 

obtained after addition of Etoposide (final conc. 10µM), infliction of 

osmotic shock or no culture insult at all. A very good correlation between 

viability determination by ISM, ViCell (i.e. Trypan Blue) and Flow Cytometry 

is observed (Figure 1c). 

Conclusions and perspectives: We demonstrate that our ISM is suitable 

for determination of suspension cell density AND viability on-line and in 

real time. Future work will include investigating how the fine-structure in 

micrographs and variance or entropy histograms corresponds to specific 

apoptotic stages. 

For research and process applications, this work introduces non-invasive 

live/dead-classification of suspended mammalian cells in real time. 

Acknowledgements: P.W. and H.S. are funded by the Karl Völker 

Stiftung, Mannheim, and Land Baden-Württemberg. We gratefully 

acknowledge the support of Ariane Tomsche, HS Mannheim. 

References 

1. Guez JS, Cassar JPh, Wartelle F, Dhulster P, Suhr H: Real time in situ 

microscopy for animal cell-concentration monitoring during high 

density culture in bioreactor. J Biotechnol 2004, 111:335-343. 

2. Joeris K, Frerichs JG, Konstantinov K, Scheper T: In-situ microscopy: Online 

process monitoring of mammalian cell cultures. Cytotechnology 2002, 

38:129-34. 

3. Guez JS, Cassar JPh, Wartelle F, Dhulster P, Suhr H: The viability of Animal 

cell Cultures: Can it be Estimated Online by using In Situ Microscopy? 

Process Biochem 2010, 45:288-291. 

4. Wiedemann P, Egner F, Wiegemann HB, Quintana JC, Storhas W, Guez JS, 

Schwiebert C, Suhr H: Advanced in situ microscopy for on-line 

monitoring of animal cell culture. Proceedings of the 21st Annual Meeting 

of the European Society for Animal Cell Technology (ESACT), Dublin, Ireland, 

June 7-10, 2009 Dordrecht Heidelberg London New York: Springer: Jenkins 

N, Barron N, Alves P 2011. 

Page 111 of 181 

5. Wiedemann P, Guez JS, Wiegemann HB, Egner F, Asanza-Maldonado D, 

Filipaki M, Wilkesman J, Schwiebert C, Cassar JP, Dhulster P, Suhr H: In 

situ microscopic cytometry enables noninvasive viability assessment 

of animal cells by measuring entropy states. Biotechnol Bioeng 2011. 

P78 

Characterization of the human AGE1.HN cell line: a systems biology 

approach 

Sebastian Scholz 1 , Miriam Luebbecke 2 , Alexander Rath 3 , Eva Schraeder 1 , 

Thomas Rose 4 , Heino Büntemeyer 1 , Thomas Scheper 2 , Udo Reichl 3 , 

Thomas Noll 1* 

1 Institute of Cell Culture Technology, University of Bielefeld, Germany; 

2 Institute of Technical Chemistry, Leibniz University of Hannover, Germany; 

3 Max Planck Insitute for Dynamics of Complex Technical Systems, 

Magdeburg, Germany; 4 ProBioGen AG, Berlin, Germany 

E-mail: thomas.noll@uni-bielefeld.de 


Background: With the emergence of functional genomics approaches in the 

past decade, powerful tools for the discovery and understanding of cellular 

mechanisms are accessible. The main purpose of those analyses is to 

investigate the cell on the level of the transcriptome, proteome and 

metabolome to retrieve information on possible limitations for growth or 

productivity. In subsequent steps, these limitations could be addressed by 

genetic modifications (e.g. overexpression of genes coding for bottle neck 

enzymes) or supplementation of the media. 

In this work, we present a systems biology approach for the analysis of 

the human protein expression cell line AGE1.hn. The focal point of the 

analyses was on the central energy metabolism, namely glycolysis, TCA 

and oxidative phosphorylation. 

In addition to the metabolome analysis transcriptome and proteome data 

were obtained. Combining the above mentioned techniques we could 

gather valuable insights in the cellular processes of a human protein 

production cell line. 

Material and methods: The alpha1-antitrypsin expressing AGE1.HN.AAT 

cells were cultivated in a 20L STR reactor under controlled conditions 

(Temperatur 37°C; pH 7.15; 40% DO).The batch cultivation was carried out in 

the protein-free, chemically defined medium 42MAX-UB (Bielefeld 

University) with an addition of 5 mM Glutamine. 

For the metabolome analysis a targeted LC-MS method was established. 

Using a HILIC column for metabolite separation and a Triple-Quad ESI- 

MS for the detection, over 50 intracellular metabolites could be 

quantified. 

Changes in the transcriptome level were detected with a whole genome 

DNA microarray (Eurogentec Human HOA 4.3) and further analyzed using 

the EMMA software [1]. For the proteome analysis a 2D-DIGE approach 

was conducted and >100 differentially expressed protein spots were 

analyzed with the DELTA-2D software (DECODON GmbH, Germany). In 

subsequent analyses theses spots were identified via nanoLC-MS and 

MALDI-MS. 

Results: The AGE1.HN.AAT cells showed growth up to a viable cell density 

of 5·10 6 cells·mL -1 at day 6 of the cultivation. Total cultivation time was 8 

days. The average growth rate during the exponential phase was 0.41 d -1 . 

24 hours after inoculation daily sampling for polyomic analyses was 

started. Initial concentrations of the extracellular metabolites glucose, 

lactate, and pyruvate were 25 mM, 8 mM and 2 mM, respectively. 

Both glucose and pyruvate were depleted at day 5 of the cultivation. The 

lactate concentration increased to a maximum of 38 mM at day 5. 

Subsequent to the maximum concentration, lactate was consumed by the 

cells. 

Figure 1 shows the changes of the intracellular metabolite concentrations 

and gene expression in the TCA cycle during the batch cultivation. Most 

of the intracellular metabolites exhibit a concentration time course similar 

to glucose. 

Following the depletion of pyruvate at day 4 a majority of the TCA 

metabolites started to decrease until the end of the cultivation. Yet, the 

metabolites were not entirely consumed but remained at low 

concentrations. In contrast, malate and a-ketoglutarate concentrations 

diminished sharply after the first sampling at day 2 and persisted on these 

levels.



Figure 1(abstract P78) Viable Cell Density, extracellular (glucose&lactate) and intracellular TCA metabolite concentrations and corresponding gene 

expression profile of a batch cultivation of AGE1.HN cells. 

The transcriptome data suggest a strong gene expression for enzymes of the 

central metabolism in the first two days of the cultivation, corresponding 

with the elevated intracellular glucose and pyruvate concentrations. While 

for most genes the expression decreased over time, various genes showed a 

high expression throughout the cultivation, notably the malic enzyme, 

which is responsible for the conversion of malate into pyruvate under 

formation of NADH/NADPH. 

Despite the depletion of nutrients feeding glycolysis and the TCA cycle (e.g. 

glucose, glutamine) both the adenylate energy charge (AEC) and the 

catabolic reduction charge (CRC) (data not shown) remained on consistent 

levels. 

Conclusion: The presented results indicate a truncated connectivity 

between glycolysis and TCA cycle via the conversion of pyruvate to acetyl- 

CoA. A recent publication showed similar results for the AGE1.HN cells 

using metabolic flux analysis [2]. A possible explanation for the observed 

truncation is the relatively abundant gene expression of the PDK4 gene in 

the exponential growth phase. The PDK4 enzyme is known to inhibit the 

pyruvate dehydrogenase complex thus limiting the conversion of pyruvate 

to acetyl-CoA [3]. As a result, a major fraction of the pyruvate is directly 

converted into lactate. Thus, a highly inefficient metabolism is favored in 

Page 112 of 181 

the beginning of the cultivation as long as the levels of glucose and 

pyruvate are high enough. 

Yet, although the glycolytic flux is directed towards the energetic less 

favorable lactate formation, the amount of ATP generated during 

glycolysis seems to be sufficient to sustain the energy requirements (AEC, 

CRC) of the cell. 

In addition with yet unpublished data, these findings offer certain points 

of action for further cell line or media optimization. Among others, the 

elevated gene expression makes the PDK4 gene a possible target for cell 

line engineering. 

In conclusion, the integration of polyomics techniques allows a deeper 

insight in the metabolism of mammalian cells to identify possible targets 

for modifications of cells, media formulation or process strategies. 

References 

1. Dondrup M: EMMA: a platform for consistent storage and efficient 

analysis of microarray data. J Biotech 2003, 106(2-3):135-146. 

2. Niklas J, Schräder E, Sandig V, Noll T, Heinzle E: Quantitative 

characterization of metabolism and metabolic shifts during growth of 

the new human cell line AGE1.HN using time resolved metabolic flux 

analysis. Bioprocess. Biosyst. Eng 2010, 533-545.



3. Sugden MC, Holness MJ: Mechanisms underlying regulation of the 

expression and activities of the mammalian pyruvate dehydrogenase 

kinases. Arch.Physiol.Biochem2006, 112(3):139-49. 

P79 

Influence of the nickel-titanium alloy components on biological 

functions 

Akiko Ogawa 1* , Ryota Akatsuka 1 , Hidekazu Tamauchi 2 , Katsuya Hio 3 , 

Hideyuki Kanematsu 1 

1 Suzuka National College of Technology, Suzuka, Japan, 510-0294; 2 Mie 

Prefecture Industrial Research Institute Metal Science Branch, Kuwana, Japan, 

511-0937; 3 Kitasato University School of Medicine, Sagamihara, Japan, 225-8555 

E-mail: ogawa@chem.suzuka-ct.ac.jp 


Background: Nickel-titanium alloy is applied to biomaterials such dental 

wire, stents and so on, because they have outstanding capacities for 

corrosion-resistant, adequate mechanical strength and shape-memory [1,2]. 

On the other hand they have a risk of metal allergy because of leaking nickel 

ions from them in aqueous conditions. It is not clear the reason why nickel 

causes allergy and what the rate of components is suitable for biomaterial 

therefore we have to design biocompatible nickel-titanium alloys based on 

experiences. In order to create biological friendly nickel-titanium alloys more 

effectively, it is necessary to make the relationship clear between characters 

of nickel-titanium alloys and biological reactions. In this study, we 

investigated 1) the material properties of nickel-titanium alloys, 2) the effect 

of these alloys on cellular function and 3) the allergy test for these alloys. 

Materials and methods: Titanium particles were mixed with nickel 

particles to become 5%, 15%, 25% or 50% titanium by weight and the 

nickel-titanium alloys were made by arc melting. The components of nickeltitanium 

alloy were determined by X-ray fluorescence analysis. Nickel plate 

was used as a control. Nickel plate and nickel-titanium alloys were cut into 

determined surface area by sharing machine then soaked in 70% ethanol 

due to sterilize. The sterilized ones were utilized for cellular assay. First, they 

were soaked in PBS and heated at 36.5 °C for 25 hours in order to make 

extracts. Next, each extract was added to the culture supernatant of MOLT-3 

cells (Riken bioresource center cell bank, Japan) and the MOLT-3 cells were 

Page 113 of 181 

cultured with 10% FBS containing RPMI for 5 days at 36.5 °C in humidified 

air containing 5% of CO 2. After that, the viable cell numbers were 

determined by the trypan blue exclusion assay and counting in a 

hemacytometer under a phase contrast microscope. Both nickel and 

titanium concentrations of the extracts were determined by ICP atomic 

emission spectrometer (Shimadzu, Japan). Nickel-titanium alloys were 

shaped into round columns ( : 10 mm, depth: 5 mm) then sterilized and 

transplanted to dorsal subcutaneously of mice. After 28 days, a certain 

amount of nickel solution was injected into the auricularis skin of the mice 

and the degree of turgid auriculae was measured. 

Results: In cellular assay with MOLT-3, the viable cell number of the cells 

cultured with the extract of nickel-5% or 50% titanium alloy was significantly 

more than that of nickel plate (Figure 1). In allergy test, transplanting nickel- 

50% titanium alloy indicated the lower degree of turgid auriculae than 

transplanting nickel plate in a mouse but nickel-5% or 15% titanium alloy 

did not. About nickel concentration of the extract, nickel-50% titanium alloy 

was the lowest and nickel-25% titanium alloy was the highest. 

Conclusions: We studied the relationship between characters of nickeltitanium 

alloys and biological reactions. Four kinds of nickel-titanium 

alloys having different titanium contents were tested. Except nickel-50% 

titanium alloy, the nickel concentration of the extract of nickel-titanium 

alloy increased with the content rate of titanium. This result suggests that 

increasing the content rate of titanium will promote leaking nickel from 

nickel-titanium alloys. Otherwise the viable cell number decreased with 

the content rate of titanium. The extract of nickel-50% titanium alloy 

indicated not only the largest viable cell number among all extracts but 

also the lowest nickel concentration. These results indicate that the 

amount of nickel would affect cell survival of MOLT-3 cells, and that 

nickel concentration of the extracts was not correlated with the titanium 

content rates of nickel-titanium alloys but it was done with the structural 

states of them. When nickel-titanium alloys were transplanted into mice, 

only nickel-50% titanium alloy affected the allergic reaction for the better. 

This result suggests that cellular assay will be more sensitive to detect 

the material differences of nickel-titanium alloys than animal test. 

References 

1. Fukushima O, Yoneyama T, Doi H, Hanawa T: Corrosion resistance and 

surface characterization of electrolyzed Ti-Ni alloy. Dental Materials 

Journal 2006, 25:151-160. 

Figure 1(abstract P79) Effect of extract of nickel-titanium alloys on cell survival of MOLT-3 cells. MOLT-3 cells were seeded in 12-well plate at 96,800 

cells/well (n =4). Next day, each extract of nickel-titanium alloy or nickel plate was added to the culture supernatant.



2. DiBari GA: Nickel alloys. The properties of electrodeposited metals and alloys 

Florida: The American electroplaters and surface finishers society: Safranek 

W H , 2 1986, 327-364. 

P80 

Metabolomics - a useful tool for prediction of protein production and 

processing? 

Jennifer Kronthaler 1,2* , Timo Schmidberger 2 , Christine Heel 2 

1 2 

University of Innsbruck, Innsbruck, Austria; Sandoz Biopharmaceuticals, 

Langkampfen, Austria 

E-mail: jennifer.kronthaler@sandoz.com 


Introduction: Although CHO cells are widely used as hosts for recombinant 

protein production, still only little is known regarding the interaction of 

metabolic alterations and protein production and processing. Therefore, a 

more sophisticated look into the cellular metabolism might lead to a more 

efficient use of the production medium resulting in high quantities of 

the protein with the desired product quality. For intracellular metabolite 

quantification the sample preparation including quenching of cells is a very 

critical step strongly affecting subsequent results. A simple and straightforward 

protocol, not needing special equipment, was investigated focusing 

on the efficiency of stopping metabolic activities and the potential metabolic 

leakage due to losses in membrane integrity. In a next attempt, the 

comparison between the producer cell line and the corresponding mock cell 

line using targeted mass spectrometry approaches for intra- and extracellular 

metabolite quantification was performed. The influence of the increased 

protein production capacity and the need of a complex glycosylation 

machinery on metabolite profiles was assessed. In order to find a link 

between the determined output parameters, detailed data evaluation 

including multivariate data analysis tools was implemented. 

Materials and methods: CHO producer cell line as well as corresponding 

mock cell line (transfected with an empty plasmid) were cultivated as a fedbatch 

process in serum-free, chemically defined in-house medium, 

comprising insulin. For the experiments 15L bioreactors were used. 

Conditions were 36,5°C and ph 6,9. Samplingforsubsequentmetabolite 

quantification (intra- as well as extracellular) took place at various time 

points throughout cultivation. 

Targeted metabolomic analysis was performed for cell culture supernatants 

as well as for cell lysates using freeze/thaw cycles after resuspension in 

phosphate buffer for extraction. For the quantification of amino acids, 

hexose and biogenic amines commercially available AbsoluteIDQ KIT 

Figure 1(abstract P80) Intracellular nucleotide sugar concentrations. 

Page 114 of 181 

plates were used. This fully automated assay was based on PITC 

(phenylisothiocyanate)-derivatization in the presence of internal standards 

followed by LC-MS/MS detection using a AB SCIEX 4000 QTrap mass 

spectrometer with electrospray ionization [1]. For the quantitative analysis 

of energy metabolism intermediates (glycolysis, citrate cycle, urea cycle), a 

hydrophilic interaction liquid chromatography (HILIC)-ESI-MS/MS method 

in highly selective negative MRM detection mode was used. Intracellular 

amounts of nucleotides and nucleotide sugars were analyzed by liquid 

chromatography-electrospray ionization-mass spectrometry on surfaceconditioned 

porous graphitic carbon as described by Pabst et al. [2]. 

Results: The comparison between a CHO producer cell line and the 

corresponding mock cell line enabled the assessment of increased protein 

production capacity and the need of a complex glycosylation machinery 

influencing metabolite profiles. Multivariate data analysis using a PLS (partial 

least square) model on all quantified compounds showed similar progress 

throughout fermentation for all four cultivations (producer and mock in 

duplicates). Changes between different sampling points, which represent 

different stages of cultivation showed the strongest impact. Thus, 

differences between tested cell lines have to be evaluated for each phase 

individually as no overall alteration is detectable. 

Nucleotide sugars which play important key roles within the mammalian 

glycosylation pathway showed the major variance between producer and 

mock cell line, as shown in figure 1: Early phase represents data within 

exponential phase (up to and including day 7) whereas late phase shows 

averaged data of day 10 and 12. The increased glycosylation processing in 

producer cells seemed to lead to higher intracellular concentrations of 

required precursors due to increased stimulation of the overall 

glycosylation machinery. Otherwise, an increased consumption by 

producer cells should have led to lower concentrations or even to limiting 

bottlenecks. The increased amounts of intracellular nucleotide sugars at 

late stage for both cell lines were evident, but an adequate explanation 

with respect to cell- and process knowledge is still missing. 

Moreover, within exponential phase (sampling on day 3-7) specific 

consumption/production rates of divers metabolites were different 

between producer and mock cell line. Mostly, amino acids were affected. 

Mock cells showed an increased energy demand reflected by higher 

specific consumption rates. Thus, especially the slightly higher growth rate 

of mock cells seemed to be of relevance resulting in a differentiation 

between proliferating and producing cells. However, detailed investigation 

of impacts of involved pathways on recombinant protein production and 

processing is still ongoing. 

Conclusion and outlook: Within this study the application of detailed 

metabolite data to mammalian process development was evaluated



preliminarily. Just marginal differences between producer and mock cells 

were detectable. However, further pathway-based investigations are still 

ongoing. 

References 

1. Freepatentsonline.com: 2007 [http://www.freepatentsonline.com/ 

20070004044.html]. 

2. Pabst M, Grass J, Fischl R, Leonard R, Jin C, Hinterkorner G, et al: Nucleotide 

and nucleotide sugar analysis by liquid chromatography-electrospray 

ionization-mass spectrometry on surface-conditioned porous graphitic 

carbon. Anal Chem 2010, 82:9782-9788. 

P81 

Accelerating process development through analysis of cell metabolism 

Dirk Müller * , Florian Kirchner, Stefanie Mangin, Klaus Mauch 

Insilico Biotechnology AG, D-70563 Stuttgart, Germany 

E-mail: dirk.mueller@insilico-biotechnology.com 


Background: Biopharma process development accumulates growing 

collections of physicochemical product information and fermentation data, 

partly in response to initiatives like Process Analytical Technology (PAT) 

and Quality by Design (QbD) backed by regulatory authorities. The key 

goal of these initiatives is to ensure robust processes and consistent 

product quality based on a scientific and mechanistic understanding of the 

product compound itself and of the manufacturing process. Biopharma 

companies gather much high-quality data during fermentations including 

online measurements and omics data such as transcript and metabolite 

measurements. Typically, these data will be stored for documentation 

purposes only whereas data evaluation and interpretation lags behind and 

often is sporadic at best. 

Cellular network models can tap this underused resource for predicting 

fermentation outcomes and for analyzing why certain fermentations failed 

or succeeded based on a mechanistic representation of cell physiology. 

In particular, network models of cell metabolism upgrade metabolomics 

data by enabling predictions of cell behavior from concentration time 

series of extracellular and – if available – intracellular metabolites. Model 

simulations can be used for rapid hypothesis testing, e.g. to evaluate the 

impact of changes in feeding on intracellular metabolism, growth, or 

product formation. Identifying suitable metabolic target genes for cell line 

engineering represents another application area of such models. Here, we 

illustrate this approach using the prediction of optimal media compositions 

for a Chinese hamster ovary (CHO) cell line employing a genomebased 

CHO network model as example. 

Methods: The CHO stoichiometric metabolic network was reconstructed 

using information from public databases as well as from primary literature 

and accounts for the specific amino acid composition and glycoform 

structure of the product molecule. In a first step, we applied the network 

model to a comprehensive metabolic characterization of the existing 

fermentation process. Rates of cellular nutrient uptake, growth, and 

product formation in physiologically distinct process phases were 

determined from concentration time series of extracellular metabolites 

during a fermentation run. These cell-specific rates served to compute 

intracellular flux distributions using the CHO network model. Comparing 

flux distributions for different process phases provided insight as to when 

and where in intracellular metabolism significant changes occur during the 

fermentation. This is often not obvious from inspection of concentration 

time series alone. Especially for fed-batch processes, multiple feed streams 

and volume changes due to pH control and sampling impede 

interpretation of raw data. If desired, further information about the usage 

of alternative intracellular pathways and in vivo reaction reversibilities can 

be obtained from labeling experiments combined with transient 13 C- 

Metabolic Flux Analysis [1,2], which is applicable to industrial fed-batch 

settings. 

Intracellular flux distributions also provide an ideal starting point for process 

optimization. Distinct optimal media compositions were computed for 

different fermentation phases based on the observed nutrient demand of 

the clone inferred from flux distributions. The chosen optimization approach 

combines stationary and dynamic model simulations on high-performance 

computing clusters. For dynamic simulations, the stoichiometric CHO 

network representation was transformed into a kinetic model. Model 

parameters were determined using evolutionary strategies and cluster 

computing based on the observed metabolite time series and considering 

thermodynamic constraints on reaction directionality. Integration of 

intracellular metabolite data into this workflow is easy and can further 

increase the predictive capabilities of the resulting model. The dynamic 

model also comprised a description of the fermenter including feeds and 

sampling. In this way, it can be predicted how changes in medium 

composition and feed flows impact rates of cell growth, productivity, and 

byproduct formation as well as intracellular metabolite profiles. Finally, 

media were optimized to maximize final product titer and specific 

productivity by varying the concentrations of glucose and individual amino 

acids in two continuous feed streams using evolutionary strategies on highperformance 

computing clusters. 

Results: The resulting optimized media were tested experimentally in a fedbatch 

process. The improved feeding resulted in a 50% increase of final 

product titer and in an increased integral of viable cells already in the first 

iteration (Figure 1). Simultaneously, ammonium release declined markedly. If 

desired, the procedure can be repeated to further optimize cellular growth 

and/or productivity profiles using data collected from the first evaluation 

fermentation as input. Considering replicate fermentations aids in assessing 

and improving the robustness of the predicted media compositions, but is 

not a prerequisite. The mechanistic model captures stoichiometric couplings 

between observed substrate uptake and resulting growth, product synthesis 

and byproduct formation. Consequently, the present approach requires 

much fewer fermentations runs as input for media optimization compared 

Figure 1(abstract P81) Optimized fed-batch media maintained (a) high viable cell counts and (b) resulted in a 50% increase in product titer. 

Page 115 of 181



to standard Design of Experiment (DoE) techniques, thus saving time 

and resources. Presently, the prediction focuses on amino acids and carbon 

sources, but the extension to further compounds is technically 

straightforward. 

Conclusions: The combination of metabolomics data and network models 

not only improves our quantitative understanding of cell physiology, but 

can also support and accelerate multiple steps of rational process 

development strategies: 

Tailor media compositions to specific clones and curtail the time and 

experimental effort required for medium optimization compared to 

standard DoE techniques 

Identify highly productive and robust clones for scale-up through 

comprehensive metabolic characterization during selection at small scales 

Devise cell line engineering strategies for overcoming metabolic 

bottlenecks in cell growth and product formation by incorporating 

intracellular metabolite measurements and other omics data (proteins, 

transcripts) 

Employ metabolic network models for controlling feed additions at the 

production scale. 

The above methodology enables biologics manufacturers to add value to 

data collected in PAT and QbD initiatives and to harness metabolomic data 

for quantitatively predicting and optimizing fermentation outcomes. This 

approach is not restricted to CHO cells, but is readily transferable to other 

cell lines and also to microbial production strains. 

References 

1. Schaub J, Mauch K, Reuss M: Metabolic flux analysis in Escherichia coli by 

integrating isotopic dynamic and isotopic stationary 13 C labeling data. 


2. Maier K, Hofmann U, Reuss M, Mauch K: Identification of metabolic fluxes 

in hepatic cells from transient 13 C-labeling experiments: Part II. Flux 

estimation. Biotechnol Bioeng 2008, 100:355-370. 

P82 

Evaluation of sampling and quenching procedures for the analysis of 

intracellular metabolites in CHO suspension cells 

Judith Wahrheit * , Jens Niklas, Elmar Heinzle 

Biochemical Engineering Institute, Saarland University, 66123 Saarbrücken, 

Germany 

E-mail: j.wahrheit@mx.uni-saarland.de 


Background: Metabolomics, aiming at the quantification of all extracellular 

and intracellular metabolites, is a valuable tool for characterizing, 

understanding and manipulating the physiology of mammalian cells. While 

extracellular metabolite analysis is well established, required quenching and 

extraction procedures for intracellular metabolite analysis in mammalian 

suspension cells are not yet routinely available. In this study a simple 

sampling and quenching protocol using ice-cold 0.9% saline as quenching 

solution [1] was tested on CHO cells. Quenching efficiency, preservation of 

cell integrity as well as cell separation and the necessity of washing steps 

were evaluated and possible sources of error are discussed. 

Materials and methods: The antithrombin-III producing CHO cell line 

T-CHO-ATIII was cultivated in serum free CHO-S-SFM II medium (Gibco/ 

Invitrogen, Carlsbad, CA, USA). Cells were kept in baffled shake flasks 

(Corning, Corning, NY, USA) in a shaking incubator (2 in. orbit, Innova 4230, 

New Brunswick Scientific, Edison, NJ, USA) at 37°C with a constant 5% (v/v) 

CO 2 supply at 185 rpm. Quenching was performed by mixing 5 ml of cell 

suspension with 45 ml ice-cold 0.9% ice-cold saline as quenching solution 

(QS) [1]. Unless otherwise stated, sampling was done by centrifuging for 

1 min at 1000g followed by shock-freezing of cell pellets in a CO 2-acetone 

bath. Intracellular metabolites were extracted from freeze-dried cell pellets 

using ice-cold 50% acetonitrile [1]. Survival of the cells in the QS at 0°C was 

tested after sampling at different relative centrifugal forces (RCF, 1000g – 

2000g – 3000g – 4000g). Cell number, viability and cell morphology of cells 

re-suspended in QS were checked during incubation at 0°C using an 

automated cell counter (Countess, Invitrogen, Karlsruhe, Germany). 

Carryover of extracellular metabolites from the culture medium was 

investigated without washing and after applying different washing 

strategies. Cell pellets were either re-suspended in 50 ml QS or rinsed with 

50 ml QS followed by another centrifugation step. Cell numbers and 

extracellular metabolites were analysed and compared to the initial sample. 

Page 116 of 181 

Quenching efficiency at 0°C was investigated by measuring enzyme activity 

and by monitoring intracellular metabolite amounts at 0°C. Lactate 

dehydrogenase activity was determined in quenched cells (0°C) and in cells 

without quenching (37°C) after cell permeabilization with 0.05% Triton-X-100 

[2]. Intracellular citrate, a-ketoglutarate, pyruvate, succinate, lactate and 

fumarate were quantified in cell extracts after incubation of quenched cell 

suspensions at 0°C for 0, 5 or 10 min. 

Results: Cell integrity was maintained in ice-cold 0.9% saline for at least 

30 min. Centrifugation at 1000g for 1 min led to 50% cell loss during 

sampling. Sampling at higher RCF increased cell yield to 65%. However, 

sampling at RCF higher than 2000g did not further increase cell yield but 

seemed to affect cell viability. Unchanging cell diameter and morphology 

before and after quenching and during incubation at 0°C indicated that no 

osmotic stress and no biased selection of cells during centrifugation 

occurred. Contamination with culture medium was found relatively low 

even without any washing steps. Less than 0.25% pyruvate and lactate and 

less than 0.1% glucose compared to the amounts measured in the 

extracellular medium of the cell suspension were found after quenching. 

Citrate found in the initial sample was not detected after quenching in any 

of the samples with or without washing. Furthermore, after washing by 

rinsing or re-suspending the cell pellets no glucose and less than 0.2% 

pyruvate and lactate could be detected. Washing by resuspending did not 

yield better results than rinsing the cell pellet but can possibly lead to cell 

damage and leakage of intracellular metabolites as indicated by traces of 

fumarate detected in the sample. The inclusion of washing steps further 

decreases cell yield. Cell loss after rinsing the cell pellet was very low (48% 

cell recovery compared to 56% without washing). In contrast, washing by 

re-suspending the pellet led to a tremendous cell loss; only 19% of the 

initial cell number could be recovered. In quenched cells enzyme activity 

was efficiently stopped as shown by comparing lactate dehydrogenase 

activity in quenched cells (specific activity 0.33 ± 1.1 pmol/(cell×h)) and 

control cells kept at 37°C (15.17 ± 0.19 pmol/(cell×h)). Increasing 

concentrations of pyruvate, lactate and fumarate after 10 min incubation 

of quenched cell suspensions at 0°C indicate that metabolism is not 

completely stopped at 0°C. However, metabolic activity seemed to be 

sufficiently slowed down within the first 5 min of incubation where no 

significant change of intracellular metabolites was observed. 

Conclusions: Ice-cold 0.9% saline proved to be a suitable QS maintaining 

cell integrity and cell morphology. Sampling via centrifugation at RCF higher 

than 2000g seemed to affect cell viability and should be avoided. By using a 

tenfold excess of QS compared to the sample volume rapid cooling of the 

sample could be achieved and contamination with culture medium was 

found relatively low even without any washing steps. Carryover of 

extracellular metabolites can be further reduced by rinsing the cell pellet 

with an excess of QS. Washing by re-suspending the cell pellet diminished 

cell yield tremendously and can possibly lead to cell damage resulting in 

leakage of intracellular metabolites. It is therefore not suitable. In quenched 

cells enzyme activity was efficiently halted within the first 5 min of 

incubation at 0°C indicating sufficient quenching of metabolic activity for 

the analysis of intracellular metabolites with lower turnover. Sampling and 

washing should therefore be completed within the first 5 min after 

quenching. 

References 

1. Dietmair S, Timmins NE, Gray PP, Nielsen LK, Krömer JO: Towards 

quantitative metabolomics of mammalian cells: Development of a 

metabolite extraction protocol. Anal Biochem 2010, 404:155-164. 

2. Niklas J, Melnyk A, Yuan Y, Heinzle E: Selective permeabilization for the 

high-throughput measurement of compartmented enzyme activities in 

mammalian cells. Anal Biochem 2011, doi:10.1016/j.ab.2011.05.039. 

P83 

Increasing productivity of hybridoma cell lines by sorting by side 

scattering light 

Daniel Landgrebe * , Cornelia Kasper, Thomas Scheper 

Institute for Technical Chemistry, Leibniz University of Hannover, 30167 

Hannover, Germany 


Introduction: The side scattering light of a mammalian cell is caused, 

among other things, by the membranes of cell organelles. We suggest that 

cells with a high side scatter contain a large amount of mitochondria and a



large endoplasmatic reticulum. In a simple approach we separate cells with 

a high side scatter via fluorescence activated cell sorting (FACS) to create 

sub populations with a higher productivity. We obtain cells with a high 

amount of mitochondria and a large endoplasmatic reticulum which could 

cause strong energy metabolism and high protein productivity. The 

advantage of this technique is that no staining dye or complex procedure 

is needed to reach the goal of increasing the productivity of a cell line. 

Materials and methods: For the sorting procedure the SC-71 murine 

hybridoma cell line (DSMZ, Braunschweig) is used. The cells produce an IgG 1 

specific for rat myosin. Cultivation of the cells before and after the sorting 

steps was performed at the same conditions. The cells were grown in 100 

ml Erlenmeyer shacking flasks with 20 ml medium (DMEM (Sigma-Aldrich, 

Steinheim),10% FCS, 4mM glutamine, 1% Penicillin/Streptomycin solution 

(PAA, Cölbe) ; conditions: 37°, 5% CO 2, 110 rpm) with an inoculation density 

of 0.45*10 6 cells/ml. During the cultivation samples were taken twice a day 

for the acquisition of the cell number and offline analysis (product 

concentration via ELISA (Roche, Mannheim), glucose and lactate via YSI 2700 

Biochemistry Analyzer (Yellow Springs Instruments) and amino acids via 

HPLC (Agilent Technologies, Waldbronn)). The product concentration is used 

to calculate the specific productivity rate of the cell lines as described by 

Brezinsky et al. [1]. 

For each sorting 5*10 6 cells were taken from the exponential phase of a 

preparatory culture. After a washing step with PBS the cells were stained 

with Propidium iodide (5 µl [1 mg/ml] (Sigma-Aldrich, Steinheim)). Only 

Propidium iodide negatives cells were sorted to exclude dead cells. 

Aggregates were excluded by analyzing the pulse heights and their pulse 

integrals. For the separation cells with the maximal 3% of the side scatter 

were sorted into a 6 well plate with 1ml of DMEM using a FACSVantage (BD 

Biosciences, Heidelberg) equipped with pulse processing. After an expansion 

time of 4 weeks the cells were used to repeat the procedure and to 

investigate growth and productivity qualities. The procedure were repeated 

three times to generate altogether four cell lines, one initial cell line and 

three sub cell lines (named population I – III according to the repeated 

sorting procedures). 

Subsequent flow cytometric analyses were used to verify the constancy of 

the sorting effect. Therefore the four populations were cultivated for two 

month. Passages were performed by medium changes every third or fourth 

day. Samples were taken after the twenties passage. 0.5*10 6 cells of each 

cell line were washed with PBS and the side scatter was analyzed using an 

Epics XL-MCL flow cytometer (Beckman Coulter, Krefeld). 

Figure 1(abstract P83) Specific productivity rates of initial population and sub populations. 

Page 117 of 181 

Results: The calculation of the specific productivity rates show that a higher 

specific productivity is achieved earlier in the sub populations than in the 

initial population (Fig. 1). Nevertheless the total productivity is the same. Sub 

populations I and III show a maximal specific productivity already after 36 h 

(resp. after 60 h for sub population II). Compared with the initial population 

which reaches their max. after 72 h, it is a considerable increase of a 

important process parameter. The sub populations show also an increased 

cell density of 10 to 30%. This effect is retrograding in the later sub 

populations. The cytometric analyses of the long term cultivation show that 

the separated cells have kept an increased side scatter. This effect is stable 

for at least 2 month of cultivation. 

Conclusions: The results suggest that the sub populations have a modified 

metabolism. This could be a result of the accumulation of mitochondria. To 

confirm this further experiments are necessary. These experiments should 

include staining of mitochondria and endoplasmatic reticulum and 

subsequent flow cytometric analysis to determine the amount of these 

organelles. Further investigations for the consumption of the main 

metabolites glucose and glutamate and the production of lactate could 

explain the changes in the growth behavior and productivity of the cells. 

Acknowledgement: This work was performed within the project 

“SysLogics: Systems biology of cell culture for biologics” and is founded 

by the Bundesministerium für Bildung und Forschung (FKZ 0315275F) 

Reference 

1. Brezinsky SC, Chiang GG, Szilvasi A, Mohan S, Shapiro RI, MacLean A, Sisk W, 

Thill G: A simple method for enriching populations of transfected CHO cells 

for cells of higher specific productivity. J Immunol Methods 2003, 277:141-155. 

P84 

Compartmentation and channelling of metabolites in the human cell 

line AGE1.HN® 

Jens Niklas 1* , Volker Sandig 2 , Elmar Heinzle 1 

1 Biochemical Engineering Institute, Saarland University, 66123 Saarbrücken, 

Germany; 2 ProBioGen AG, 13086 Berlin, Germany 

E-mail: j.niklas@mx.uni-saarland.de 


Background: A thorough knowledge of the metabolism and its 

compartmentation in mammalian cells is desirable to enable rational 

design and optimization of producing cell lines and production processes



for biopharmaceuticals. In this study we focused on acquiring a detailed 

understanding of metabolite channelling and the metabolic flux 

distribution during overflow metabolism in the human cell line AGE1.HN® 

(ProBioGen AG). This metabolic phenotype characterized by energy spilling 

as well as waste product formation is commonly observed in the 

beginning of the cultivation [1]. 13 C tracer experiments and 13 Cflux 

analysis as applied in this investigation represent methods offering indepth 

insights into cellular physiology [2,3]. 


Cultivation and analysis: The human neuronal cell line AGE1.HN® 

(ProBioGen AG, Berlin, Germany) was used. Cultivations were carried out in 

shake flasks (Corning, NY, USA) or bioreactor filter tubes (TPP, Trasadingen, 

Switzerland). 13 C-labeling experiments using the tracers [1,2- 13 C 2] glucose , 

[U- 13 C 5]glutamine,[U- 13 C 3] alanine, [1- 13 C 1] lactate (Cambridge Isotope 

Laboratories, Andover, MA, USA) and [U- 13 C 6] glucose (Euriso-Top, 

Saarbrücken, Germany) were conducted. Extracellular metabolites were 

quantified using different HPLC methods [4,5]. 13 C-labeling of metabolites 

was analysed using GC-MS [6]. Carbon mass isotopomers were determined 

from the analyte mass isotopomer distribution [7]. 

Carbon atom transition model: A carbon atom transition model was set 

up using the Kyoto Encyclopedia of Genes and Genomes (www.kegg.com) 

pathway database for Homo sapiens. 

13 C metabolic flux analysis: Fluxes were estimated using the method of 

Yang et al. [8] applying Matlab R2008 (The Mathworks, Natick, MA, USA). 

Results: Experiments applying 13 C-labelled glucose, glutamine, alanine and 

lactate tracers were carried out to identify active pathways and channelling 

of metabolite carbons in the central metabolism of AGE1.HN®. It was 

observed that almost 80% of glucose consumed was detected in lactate. 

Smaller amounts were channeled to alanine (5%) and serine (3%). Glucose 

carbons were additionally entering the tricarboxylic acid (TCA) cycle which 

can be deduced from an increase in fractional labelling of glutamate and 

proline in glucose tracer experiments. Reflux from TCA cycle metabolites to 

glycolytic metabolites was also detected since labelling in lactate as well as 

alanine was measured when using [U- 13 C 5] glutamine as tracer. Decrease in 

labelled extracellular lactate as well as an increase in labelled extracellular 

alanine as observed in the lactate tracer experiment shows that lactate was 

not only produced but also taken up during overflow metabolism. 

Furthermore, the lactate and alanine tracer experiments indicated that 

alanine and lactate and subsequently the pyruvate pools used for their 

synthesis are connected. Alanine taken up was mainly transaminated 

entering the cytosolic pyruvate pool, converted to lactate and secreted. 

Fluxes were calculated for the growth phase between day 1 and day 4 of 

the cultivation in which the cells exhibit overflow metabolism characterized 

by production of waste metabolites [1]. 

For flux calculation extracellular and anabolic rates as well as the labelling 

information stored in extracellular lactate resulting from three different 

parallel tracer experiments using [1,2- 13 C 2]glucose,[U- 13 C 6]glucoseand 

[U- 13 C 5] glutamine was included. In theory one could also use the labelling 

information stored in the building blocks of the cellular macromolecules, 

namely proteins, carbohydrates, lipids or nucleic acids as it is widely applied 

in microbial 13 C flux analysis. However, the time that is needed to 

incorporate the labelling in these molecules as well as the presence of huge 

amounts of unlabelled species caused by high seeded cell densities needed 

in mammalian cell culture processes is a huge disadvantage. Another 

possibility would be using the labelling patterns of intracellular metabolites. 

This requires, however, sampling methods that are still not well established 

in mammalian suspension cell culture. A main problem represents hereby 

the quenching procedure that remains still a huge challenge for suspension 

cell culture and it cannot be guaranteed that the measured labelling or 

intracellular concentration is really representing intracellular values not 

corrupted with extracellular species. The labelling of extracellular amino 

acids, which could also be used theoretically, is however not directly 

representing the labelling of intracellular species. This is caused by the fact 

that the amino acids are provided in the medium and the reversibility of 

production and uptake of these and their conversion dilutes the labelling 

which is then not representing the labelling of metabolites in central 

metabolism of the cell. This could only be solved by detailed knowledge 

and modelling of reaction reversibilities. In this study solely lactate labelling 

was therefore included in the metabolic flux estimation since it is not 

present in the beginning of the cultivation representing at steady state 

directly the labelling of cytosolic pyruvate which is probably the most 

important metabolic hub in the cell. 

Page 118 of 181 

It was observed that the flux through the oxidative branch of the pentose 

phosphate pathway was very low being around 2% of the glycolytic flux 

which might be compensated in AGE1.HN® by a high flux through malic 

enzyme additionally producing NADPH. The cytosolic pyruvate pool was fed 

mainly by the glycolytic flux, however, pyruvate taken up was also 

significantly contributing to intracellular pyruvate. Cytosolic pyruvate was 

almost exclusively converted to lactate and alanine and secreted. Flux 

between cytosolic oxaloacetate and cytosolic pyruvate was found to be 

reversible with similar fluxes in both directions. Pyruvate transport between 

mitochondria and cytosol was very low. Mitochondrial oxaloacetate pool 

was fed by cytosolic oxaloacetate. The flux from mitochondrial oxaloacetate 

to mitochondrial pyruvate was relatively high. The tricarboxylic acid cycle 

was fed mainly via a-ketoglutarate and oxaloacetate. 

Conclusions: In order to improve metabolic efficiency in AGE1.HN® 

engineering strategies focussing on improved metabolite transfer between 

cytosolic and mitochondrial pyruvate pools could be applied accompanied 

by improved control and targeted reduction of substrate availability and 

uptake. The 13 C flux analysis method that was applied in this study allows a 

detailed determination of metabolic fluxes in the central metabolism of 

mammalian cells and can thus be applied for studying related biological 

questions. The presented data is an important step for an improved system 

level understanding of the AGE1.HN cell line. 

References 




analysis. Bioprocess Biosyst Eng 2011, 34(5):533-545. 

2. Niklas J, Schneider K, Heinzle E: Metabolic flux analysis in eukaryotes. Curr 

Opin Biotechnol 2010, 21(1):63-9. 

3. Niklas J, Heinzle E: Metabolic Flux Analysis in Systems Biology of 

Mammalian Cells. Adv Biochem Eng Biotechnol 2011, DOI: 10.1007/ 

10_2011_99. [Epub ahead of print].. 

4. Niklas J, Noor F, Heinzle E: Effects of drugs in subtoxic concentrations on 

the metabolic fluxes in human hepatoma cell line Hep G2. Toxicol Appl 

Pharmacol 2009, 240(3):327-336. 

5. Krömer JO, Fritz M, Heinzle E, Wittmann C: In vivo quantification of 

intracellular amino acids and intermediates of the methionine pathway 

in Corynebacterium glutamicum. Anal Biochem 2005, 340(1):171-173. 

6. Wittmann C, Hans M, Heinzle E: In vivo analysis of intracellular amino acid 

labelings by GC/MS. Anal Biochem 2002, 307(2):379-382. 

7. Yang TH, Bolten CJ, Coppi MV, Sun J, Heinzle E: Numerical bias estimation 

for mass spectrometric mass isotopomer analysis. Anal Biochem 2009, 

388(2):192-203. 

8. Yang TH, Frick O, Heinzle E: Hybrid optimization for 13C metabolic flux 

analysis using systems parametrized by compactification. BMC Syst Biol 

2008, 26:2-29. 

P85 

Producer vs. parental cell – metabolic changes and burden upon a1antitrypsin 

production in AGE1.HN® 

Jens Niklas 1* , Christian Priesnitz 1 , Volker Sandig 2 , Thomas Rose 2 , 

Elmar Heinzle 1 

1 Biochemical Engineering Institute, Saarland University, 66123 Saarbrücken, 

Germany; 2 ProBioGen AG, 13086 Berlin, Germany 

E-mail: j.niklas@mx.uni-saarland.de 


Background: The human designer cell line AGE1.HN® represents a 

promising production system for biopharmaceuticals, particularly for those 

needing human-type post-translational modifications [1,2]. For further 

rational improvement of the cell line and the cultivation process a detailed 

understanding of the metabolism and metabolic changes during 

glycoprotein production is desirable. Metabolism and metabolic burden 

upon production of a 1-antitrypsin (A1AT) were analyzed by comparing 

parental cells and a derived clone, AGE1.HN.AAT. The questions addressed 

were (i), which changes occur in cell growth and metabolism upon A1AT 

production and (ii), what are specific cellular properties distinguishing the 

producer clone AGE1.HN.AAT from the parental cell population. 


Cultivation and analysis: Cultivations of the human neuronal cell line 

AGE1.HN® (ProBioGen AG, Berlin, Germany) were carried out in shake



flasks (Corning, NY, USA). Extracellular metabolites were quantified using 

different HPLC methods [3,4]. A1AT was quantified using an activity assay. 

Identification of extracellular proteins was done using MALDI-ToF/ToF MS 

(Applied Biosystems). Biomass components were quantified using classical 

biochemical methods. 

Model for A1AT production: Model for A1AT production was set up 

using the Kyoto Encyclopedia of Genes and Genomes (http://www.kegg. 

com) pathway database for Homo sapiens. 

Metabolic flux analysis: Fluxes were estimated using standard methods 

[1,5] by incorporating extracellular and anabolic rates using Matlab 

R2007b (The Mathworks, Natick, MA, USA). 

Results: Cell growth was similar in the first 5 days. After 5 days AGE1.HN.AAT 

cells were growing less than the parental cell line and the viability dropped 

faster. After 2 days dry weight was slightly higher in the parental cell line. 

A1AT production was low in the first 2 days and then increasing up to a final 

concentration of ~0.4 g/l in the end of the cultivation. The total cell mass 

was increasing similarly in the first 4 days. After 4 days AGE1.HN.AAT cell 

mass remained constant whereas the extracellular protein concentration was 

increasing. This was different in the AGE1.HN parental cells where the cell 

mass was still increasing after 4 days and the increase in total extracellular 

protein mass was less. Intracellular protein concentration was decreasing in 

AGE1.HN.AAT after 4 days. Specific cellular proteins might be degraded upon 

nutrient deprivation to maintain the production of the recombinant 

glycoprotein A1AT. This was clearly different in the parental cell line. 

Fractions of the biomass constituents RNA, lipid and phosphatidylcholine 

Figure 1(abstract P85) Schematic presentation of the glycoprotein production process in mammalian cells. 

Page 119 of 181 

were increased in AGE1.HN.AAT. The total mass of the analyzed components 

in the end of the cultivation was similar in the cultivations of both cell lines 

being around 2.5 g/l. The final A1AT concentration which was 0.4 g/l was 

~30% of the total protein in the culture. 

Similar time courses for most extracellular metabolites were observed. 

Glycine and glutamate production was higher in AGE1.HN.AAT whereas 

uptake of arginine and aspartate as well as alanine production were lower. 

Glutamine was consumed in the cultivations of both cell lines at ~4 days 

which resulted also in reduced growth. Especially the increase in glycine and 

glutamate production points to differences in C1 metabolism and cellular 

nucleotide demand. 

The differences that can be seen in the metabolism upon production of the 

glycoprotein were explained by using an appropriate model of the anabolic 

demand needed to produce active A1AT. The whole cellular production 

process of a glycoprotein includes its expression (transcription), synthesis of 

the amino acid chain (translation), posttranslational modification in 

endoplasmic reticulum (ER) and Golgi apparatus (Golgi) and secretion of the 

mature protein (Figure 1). Metabolite demand for A1AT production was 

finally simulated. These results indicate that one could expect an increase in 

the production of glutamate and glycine with increasing intracellular 

nucleotide demand which was observed for AGE1.HN.AAT; this is caused by 

differences in cellular composition of the producer, partly originating from 

recombinant A1AT production. 

The differences that were found in the biomass composition between both 

cell lines were included in a metabolic network model and intracellular



metabolic fluxes were calculated for both cell lines. Fluxes in central energy 

metabolism (glyolysis, TCA cycle) were similar. Differences were observed 

in C1 and glutamate metabolism including changes in activities of several 

transaminases. The observed changes in metabolic flux reflect the anabolic 

differences between producer and parental cells. 

Conclusions: This improved understanding of the metabolism and cellular 

changes during glycoprotein production supports the identification of 

targets for further improvement of (i) cell line, e.g., by genetic modifications 

and (ii) cultivation process, e.g., by improved feeding strategies. The 

presented data indicate that nucleotide and lipid metabolism might be 

interesting targets for further engineering of the AGE1.HN® cell line. 

References 




analysis. Bioprocess Biosyst Eng 2011, 34(5):533-545. 

2. Blanchard V, Liu X, Eigel S, Kaup M, Rieck S, Janciauskiene S, Sandig V, 

Marx U, Walden P, Tauber R, Berger M: N-glycosylation and biological 

activity of recombinant human alpha1-antitrypsin expressed in a novel 

human neuronal cell line. Biotechnol Bioeng 2011, 108:2118-2128. 

3. Niklas J, Noor F, Heinzle E: Effects of drugs in subtoxic concentrations on 

the metabolic fluxes in human hepatoma cell line Hep G2. Toxicol Appl 

Pharmacol 2009, 240(3):327-336. 

4. Krömer JO, Fritz M, Heinzle E, Wittmann C: In vivo quantification of 

intracellular amino acids and intermediates of the methionine pathway 

in Corynebacterium glutamicum. Anal Biochem 2005, 340(1):171-173. 

5. Niklas J, Heinzle E: Metabolic Flux Analysis in Systems Biology of 

Mammalian Cells. Adv Biochem Eng Biotechnol 2011, DOI: 10.1007/ 

10_2011_99. [Epub ahead of print]. 

P86 

Analysis of the mitochondrial subproteome of the human cell line 

AGE1.HN – a contribution to a systems biology approach 

Eva Schräder 1* , Sebastian Scholz 1 , Volker Sandig 2 , Raimund Hoffrogge 1 , 

Thomas Noll 1 

1 Institute for Cell Culture Technology, University of Bielefeld, Bielefeld, 

Germany; 2 ProBioGen AG, Berlin, Germany 

E-mail: esc@zellkult.techfak.uni-bielefeld.de 


Background: In Systems Biology approaches a good amount of reliable data 

is essential for effective modelling of distinct biological processes. Hence the 

focus of the SysLogics-Project was on modelling of the central metabolism 

using different functional genomic techniques. For displaying the involved 

proteins, it is crucial to enrich them using subproteomic fractions. As many 

enzymes being involved in central metabolism are located in the 

Page 120 of 181 

mitochondria, we investigated the expression dynamics of mitochondrial 

proteins during batch cultivations of human AGE1.HN cells (ProBioGen, 

Berlin, Germany). 


Cultivation and isolation of mitochondria: Cultivations were performed 

as batch-processes with chemically-defined and animal-component-free 

media 42-MAX-UB (Teutocell, Bielefeld, Germany) with addition of 5 mM 

glutamine. The processes were performed in a 20 L-stainless steel vessel 

(Sartorius-Stedim, Germany). After disintegration of cells by addition of glass 

beads (0,1 mm diameter) and vortexing, the isolation of mitochondria was 

performed with sucrose density-centrifugation in an ultracentrifuge 

(Beckman Coulter, Krefeld, Germany). Protein extraction from mitochondria 

was performed with lysis-buffer, which contains Tris-HCl, NaCl, EDTA, SDS, 

PMSF and NP40. 

Proteomics: The first dimension of 2D-electrophoresis was performed with 

Ettan IPGPhor3-system*, using pH 3-11 (NL) IPG-strips*. For generating the 

mitochondrial master-map, six gels with 0.45 mg protein extract were 

prepared. For DIGE-approaches 0.15 mg were applied, each sample with 

four replicates. Second dimension was accomplished with Ettan Dalt six 

Electrophoresis System*. 2DE-gels with DIGE-staining were processed with 

Ettan Dige Imager*. Software evaluation was implemented with Delta2D 

(Decodon, Germany). 

After isolation and tryptic digest protein-identification was performed with 

MS/MS using MALDI-ToF/ToF (UltrafleXtreme, Bruker, Germany), followed 

by database-searching. 

*(GE Healthcare, Sweden) 

Results: For proteomic analyses, mitochondria were isolated and 

successfully confirmed by staining with Rhodamine 123, a mitochondriaspecific 

fluorescent dye. 2DE-gels of mitochondrial fraction led to 519 

separated spots. 280 proteins could be reliably identified, out of which more 

than 50% are exclusively mitochondrial. This 2D-proteome map is our basis 

for approaches of analysing different protein expression in mitochondria. 

Two standardised batch cultivations in a 20 L-stirred tank stainless steel 

vessel were carried out with daily sampling for metabolomics, transcriptomics, 

metabolic flux and proteomics, in order to generate reliable and 

comparable samples. Batch cultivation in 20 L-scale of AGE1.HN.AAT shows 

cell densities and duration as expected. Glucose- and lactate-concentrations 

developed also as estimated in standard batch culture (see fig. 1a+b). 

Subproteomic DIGE-analysis of mitochondrial proteins in those 20 Lbatch 

cultivations resulted in 114 proteins that were significantly 

differently expressed in the first and 206 proteins in the second process 

during cultivation time (p < 0.05, factorial Anova). More than 40 

proteins were found to be significantly regulated in both experiments. 

DIGE-approaches of mitochondrial fraction of both batch cultivations led 

to expression profiles of identified proteins. Four examples of expression 

profiles of typical proteins of TCA and respiratory chain are shown 

below in table 1. 

Figure 1(abstract P86) a (left): VCD (solid) and viability (dashed) during batch process 1 (open squares) and 2 (closed triangles) Fig. 1b (right): 

Concentrations of glucose (solid) and lactate (dashed) during batch process 1 (open squares) and 2 (closed triangles).



Table 1(abstract P86) Examples of expression profiles of TCA and respiratory chain enzymes. Expression normalised 

on sample point 1 

ZE1 (2,6 d) ZE1 (4,8 d) ZE1 (6,5 d) ZE2 (2,9 d) ZE2 (4,2 d) ZE2 (5,3 d) ZE2 (6,9 d) 

Pyruvate dehydrogenase E1a 1,53 1,6 0,83 1,51 1,56 1,09 0,87 

L-lactate dehydrogenase B chain 0,95 0,87 5,18 0,98 1,53 2,4 3,26 

Dihydrolipoamide S-succinyltransferase 2,37 2,18 1,2 2,06 1,92 1,73 0,89 

Electron transfer flavoprotein alpha 2,57 2,24 1,38 2,29 1,88 1,99 1,45 

Apart from these examples it was possible to observe important 

biological processes with our 2DE-approach, as for instance: 

- tricarboxylic acid cycle 

- respiratory chain 

- anti-apoptosis and apoptosis 

- signal transduction 

- chaperone/protein folding and processing 

- fatty acid-/lipid-metabolism 

- amino acid-degradation 

- inhibition of DNA-synthesis 

- ketone-metabolism 

- protein-biosynthesis 

- redox-regulation 

- ubiquinone-biosynthesis 

Hence, we see this data as a valuable contribution to the Systems Biology 

approach for modelling central metabolism events. 

Conclusions: Reproducible methods for isolation of mitochondrial proteins 

from cultured cells for further analysis, such as proteomic 2DE-approaches, 

have been established. DIGE-analysis of mitochondrial subproteome of 

AGE1.HN.AAT cells result in the identification of more than 280 identified 

proteins, of which more than 40 are regulated during batch cultivation. 

Proteomics data in conjunction with the other “omics"-results offer a 

promising basis for characterisation of central metabolism during Systems 

Biology approaches. 


This work is part of the SysLogics Project: Systems biology of cell culture for 

biologics, founded by German Ministry for Education and Research (BMBF). 

P87 

Characterisation of cultivation of the human cell line AGE1.HN.AAT 

Eva Schräder 1* , Sebastian Scholz 1 , Jens Niklas 5 , Alexander Rath 2 , 

Oscar Platas Barradas 3 , Uwe Jandt 3 , Volker Sandig 5 , Thomas Rose 5 , 

Ralf Pörtner 3 , Udo Reichl 2 , An-Ping Zeng 3 , Elmar Heinzle 4 , Thomas Noll 1 

1 Institute for Cell Culture Technology, University of Bielefeld, Germany; 2 Max 

Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, 

Germany; 3 Institute for Bioprocess and Biosystems Engineering, Hamburg 

University of Technology, Germany; 4 Biochemical Engineering Institute, 

Saarland University, Saarbruecken, Germany; 5 ProBioGen AG, Berlin, Germany 

E-mail: esc@zellkult.techfak.uni-bielefeld.de 


Background: Human cell lines are an interesting alternative to CHO cells for 

the production of recombinant proteins and monoclonal antibodies, 

Page 121 of 181 

because of their ability to produce genuine human posttranslational 

modifications. The human cell line AGE1.HN.AAT (ProBioGen, Berlin, 

Germany), that originated from human neural precursor tissue, has been 

adapted to serum-free conditions and cultivated in many different systems. 

Here we present our results using this cell line in a scale-up of batch 

cultivation from 50 mL vented polypropylene tube on a shaking platform, 

polycarbonate shakeflask (cultivation volume from 50 mL up to 300 mL), a 2 

L-glass vessel stirred tank reactor and a 20 L-stainless steel stirred tank 

reactor (both Sartorius Stedim, Goettingen, Germany). 

Materials and methods: Cultivations were performed with chemicallydefined 

and animal-component-free media 42-MAX-UB (Teutocell, Bielefeld, 

Germany). Batch-cultivations were performed in 50 mL-bioreactor tubes (TPP, 

Switzerland) shakeflasks (Corning Life Sciences, Netherlands), 2 L-glass vessel 

and 20 L-stainless steel vessel (both Sartorius-Stedim, Germany). Chemostatcultivation 

was done in 0.5 L-bioreactor (DASGIP, Juelich, Germany) with a 

media exchange rate of 7 mL/h. As a further cultivation system a dialysisreactor 

(Bioengineering, Wald, Switzerland) was established, with a 1.3 L cellcontaining 

inner chamber and a 4 L media reservoir in the outer chamber, 

separated by a semipermeable dialysis membrane. 

Results: 

Proliferation in controlled and uncontrolled systems: The AGE1.HN. 

AAT cells show similar growth in different unregulated vessels and 

culture volumes. The used systems ranged from 50 mL-bioreactor tubes 

with a culture volume of up to 18 mL, 125 mL-shakeflasks with a culture 

volume of up to 50 mL- to 250 mL-shakeflasks, in which a culture volume 

of 100 mL can be used (Results shown in figure 1a). 

Cultivation in 2 L-glass vessel is as well feasible for batch process as well as 

preculture for 20 L-vessel. Cultivation at a 20 L-scale resulted in delayed cell 

growth but did not affect the final cell concentration (refer to figure 1b). 

AGE1.HN.AAT cells show a strong growth-coupled productivity as shown in 

figure 1c. 

No difference between 2 L- and 20 L-vessel concerning spec. productivity 

and spec. growth rate were observed. A scale-up of cultivation-volume in 

batch process is definitely possible. 

Cultivations with other systems: Cultivation of AGE1.HN.AAT cells in 

dialysis-reactor is also possible and batch cultivation without mediaexchange 

in the outer chamber showed maximum cell density up to 1.6 E7 

viable cells per milliliter in the inner chamber after eight days of cultivation. 

Chemostat-cultivation in 0.5 L-glass vessel shows constant cell density at 

2.5 E6 cells/mL for more than 14 days. 

Conclusions: Cultivation of AGE1.HN cell line is possible in different 

regulated and unregulated systems and at different scales. Cell specific 

productivity and titer of the AGE1.HN.AAT producer cell line depend mainly 

on cell growth. The cells are easily scalable from 2 to 20 L batch cultivation 

Figure 1(abstract P87) 1.1 (left): viable cell density and viability during shake flask batch-cultivation. open squares: bioreactor tube, open circles: 125 mLshakeflask, 

open diamonds: 250 mL-shakeflask. 1.2 (middle): viable cell density and viability during bioreactor batch-cultivation. open squares: 2 L-glass 

vessel, open circles: 20 L-stainless steel reactor. 1.3 (right): specific growth rate μ of bioreactor cultivation vs. corresponding specific productivity qP. open 

diamonds: 20 L- stainless steel reactor, open squares: 2 L-glass vessel.



in STR. Other cultivation strategies have been established successfully (incl. 

chemostat and dialysis-bioreactor) documenting the potential of the AGE1. 

HN cell line. 

Acknowlegments: The work presented was part of SysLogics (Systems 

biology of cell culture for biologics), which is funded by German Federal 

Ministry of Education and Research. Cultivation of 125 mL shakeflasks and 

bioreactor tubes were done in Saarbruecken, dialysis cultivation in Hamburg, 

chemostat-process was done in Magdeburg, while 250 mL shakeflask, 2 

L-glass vessel and 20 L-stainless steel bioreactor-cultivation were performed 

in Bielefeld. 

P88 

Strategies in umbilical cord-derived mesenchymal stem cells expansion: 

influence of oxygen, culture medium and cell separation 

Antonina Lavrentieva 1* , Tim Hatlapatka 1 , Ramona Winkler 1 , Ralf Hass 2 , 

Cornelia Kasper 2 

1 Leibniz University Hannover, Institute of Technical Chemistry, Callinstr. 5, 

D-30167 Hannover; 2 Hannover Medical School, Klinik für Frauenheilkunde 

und Geburtshilfe, AG Biochemie und Tumorbiologie, Carl-Neuberg-Str. 1, 

D-30625 Hannover 

E-mail: kasper@iftc.uni-hannover.de 


Background: Mesenchymal stem cells (MSC) from different sources attract 

tremendous interest in cell-based therapies for their ability to differentiate 

into different cell lineages. MSC are already used in clinical trials as cell 

therapy [1]. In comparison to other “classical” sources of MSC (e.g. bone 

marrow and adipose tissue), the umbilical cord (UC) matrix has great 

potential, since there are no ethical limitations, risks for the donor or 

problems with variety of donor age [2]. However, a large numbers of cells 

(about 10 6 cells per 1 kg body weight) are required which may still limit the 

implant preparation for clinical applications. Thus, strategies for the 

expansion of MSC must be well defined and controlled conditions need to 

be developed and established for reproducible production of cells under 

GMP conform conditions. Conventional in vitro cell cultivation is carried out 

under ambient oxygen concentration (21% of O2) whichisdefinedas 

“normoxic”. MSCin vivo usually are not exposed to such a high concentration 

of oxygen. In our work we studied the influence of oxygen 

concentration on the long-term as well as glucose and oxygen concentration 

on the short-term cultivation and expansion of UC-MSC. 

Materials and methods: UC-MSC were isolated from whole human 

umbilical cords using an explant culture approach and characterised as 

described earlier [3,4]. For the long-term cultivation, MSC from the same 

donor were isolated and subsequently cultivated in two different oxygen 

Page 122 of 181 

concentrations (5% and 21%). After isolation, cells were seeded at a density 

of 2000 cells/cm 2 in 25cm 2 cell culture flasks (Corning, Germany) and subcultivated 

every 3-4 days over 25 passages. Cell numbers were estimated at 

the end of each passage and cumulative population doublings were 

calculated for each culture conditions. MSC were cultivated in aMEM 

containing 1 g/l glucose (Biochrom, Germany), 10% allogenic human serum 

(provided by the Institute of Transfusion Medicine, Medical University 

Hannover, Germany) and 50 µg/ml gentamicin (PAA Laboratories GmbH). To 

reveal the influence of glucose concentration on the proliferation capacities 

of UC-MSC under different oxygen concentrations, cells were seeded in 6well 

plates (Sarstedt, Germany) at a density of 700 cells/cm 2 in aMEM (1 g/l 

glucose) and DMEM (4.5 g/l glucose) and cultivated over 7 days until full 

confluency was reached. Cell number and viability in all experiments were 

determined by trypan blue exclusion (n=4). 

Counterflow centrifugal elutriation (CCE) was performed for the separation 

of the cells according to their physical size (Beckmann J6-MC with the JE- 

5.0 rotor; 5 ml-standard elutriation chamber, Beckman Coulter, Germany). 

Small-sized MSC were separated from the primary heterogeneous 

population. After cell separation, proliferation activity of the cells was 

estimated as described above. 

Results: Long-term cultivation of UC-MSC under hypoxia revealed higher 

proliferation activity of the cells without changes in morphology when 

compared to ambient (21%) oxygen concentration (Fig 1A). 

High glucose concentration inhibited cell growth under ambient oxygen 

concentration. Hypoxic conditions, however, significantly increased 

proliferation of UC-MSC both, in high- and low-glucose culture medium 

(Fig.1B). 

Conclusions: Application of MSC in regenerative medicine as cell 

suspensions or as a part of tissue engineered implant requires large amount 

of cells of high quality. MSC expansion under physiological conditions allows 

obtaining higher cell numbers in a shorter period of time. Also the glucose 

concentration in medium should be close to that in vivo. Highamountsof 

glucose inhibit cell proliferation in ambient oxygen concentrations. In 

hypoxic conditions MSC proliferate better and retain their ability to 

differentiate. We conclude that GMP-conform cultivation of MSC with 

allogenic human serum under physiological oxygen concentrations as well 

as isolation of actively dividing cells may increase the efficiency of the cell 

expansion for further therapeutic applications. 


This work was supported by the state of Lower Saxony, DFG Project 

“GMP-Model Lab Tissue Engineering”, BMWi Project KF22501101SB9 

“Frozen Cells” 

References 

1. Ma T: Mesenchymal stem cells: From bench to bedside. World J Stem 

Cells 2:13-17. 

Figure 1(abstract P88) (A) Influence of different oxygen concentrations (5 % and 21%) on the long-term UC-MSC cultivation: cells were counted at the 

end of each passage and cumulative population doublings were calculated. (B) Influence of glucose concentration on the short-term proliferation of the 

MSC: cell growth in high-glucose medium is inhibited under 21% oxygen.



2. Fong CY, Richards M, Manasi N, Biswas A, Bongso A: Comparative growth 

behaviour and characterization of stem cells from human Wharton’s 

jelly. Reprod Biomed Online 2007, 15:708-718. 

3. Majore I, Moretti P, Stahl F, Hass R, Kasper C: Growth and differentiation 

properties of mesenchymal stromal cell populations derived from whole 

human umbilical cord. Stem Cell Rev 7:17-31. 

4. Lavrentieva A, Majore I, Kasper C, Hass R: Effects of hypoxic culture 

conditions on umbilical cord-derived human mesenchymal stem cells. 

Cell Commun Signal 8:18. 

P89 

Abstract withdrawn 



P90 




P91 

Discerning key parameters influencing high productivity and quality 

through recognition of patterns in process data 

Huong Le 1† , Marlene Castro-Melchor 1† , Christian Hakemeyer 2 , Christine Jung 2 , 

Berthold Szperalski 2 , George Karypis 3 , Wei-Shou Hu 1* 

1 Department of Chemical Engineering and Materials Science, University of 

Minnesota, Minneapolis, MN 55455, USA; 2 Roche Diagnostics GmbH, 82377 

Penzberg, Germany; 3 Department of Computer Science and Engineering, 

University of Minnesota, Minneapolis, MN 55455, USA 

E-mail: wshu@umn.edu 


Page 123 of 181 

Background: The adoption of Quality by Design (QbD) approach to 

biologics manufacturing requires fundamental understanding of complex 

relationship between the quality of the product, especially critical quality 

attributes (CQAs), and various parameters of the manufacturing process 

[1]. This can be approached through multivariate analysis of historical cell 

culture bioprocess data [2]. In this study, process parameters and raw 

materials data obtained from 51 runs with final titer varying from 0.8 to 

2.0 units and Gal0 glycan ranging from 47.5 to 67.5% was investigated. 

The aim was to discover prominent patterns which may cause the spread 

of final process outcome. 

Materials and methods: Offline and online data were processed using 

linear interpolation and a moving window average method, respectively as 

described previously [3]. Data from the 1,000 L scale was organized into six 

cumulative datasets corresponding to days 3, 6, 8, 10, 13, and 15. Euclidean 

distance for each process parameter between all pairs of runs was 

calculated and normalized to 0-1. The similarity measure was determined 

using exponential transformation of the negative of the corresponding 

distance, and organized into a matrix form. The overall similarity matrix was 

computed as the weighted combination of all individual similarity matrices. 

The weight of each reflects how well it correlates to the deviation in final 

process outcome. A support vector regression (SVR) model was constructed 

using the overall similarity of all process parameters to predict the final 

product titer and glycosylation profiles for each cumulative dataset. 

Prediction accuracy was assessed using the Pearson’s correlation coefficient 

(r) between the predicted and the actual values. 

Results: Final recombinant antibody concentration (final titer) was predicted 

with reasonable accuracy using process data at 1,000 L scale. At up to day 3, 

online and offline data can be indicative of the final titer with an accuracy of 

r = 46%. Inclusion of data at up to day 6 improved the prediction accuracy 

markedly to 80%. A modest increase in SVR models predictability was 

observed when data from day 8 and day 10 was incorporated with r = 83% 

and 87%, respectively. Online and offline data from days 13 and 15 of the 

1,000 L biorectors improved model predictability further to 90%, and 92%, 

respectively. 

Critical process parameters with significant contribution to SVR model 

predictability were weighted using a non-linear Spearman’s correlation 

coefficient between its similarity for all pairs of runs and the deviation in 

their final titer. Parameters with weights (w) greater than 0.2 included 

Figure 1(abstract P91) Process runs in three dimensional space of Gal0, Gal1, and Gal2. Low-titer runs are colored in red, and high-titer runs are in blue.



stirrerspeed,VCD,glucose,LDH,ammonia,andlactate.Eachbears 

significant contribution to prediction of the final titer at different time 

periods at the 1,000 L scale. Stirrer speed (w = 0.464) appeared to be 

critical throughout whereas VCD (w = 0.445) and LDH (w = 0.300) 

became important only after day 3. The contribution of glucose (w = 

0.416) and ammonia (w = 0.297) began at day 6, followed by titer values 

(w = 0.765) at day 8 and lactate (w = 249) at day 10. 

Similar results were obtained when Gal0 was used as the objective 

function for the SVR models in place of the final titer. A marked increase in 

prediction accuracy was also observed when data from day 6 was included 

compared to day 3 (from 61% to 85%). After a modest increase to 88% at 

day 8, almost no further improvement was made using data from later 

days (10, 13, and 15). 

Parameters with high correlation to Gal0 content profile were stirrer speed, 

VCD, glucose, LDH, ammonia, CO 2, lactate, temperature, and viability. 

Among those parameters, six were in common to when final titer was used 

as the objective function. Three parameters that were critical to prediction 

of Gal0 but not final titer included CO 2, temperature, and viability. The time 

periods in which each parameter had significant correlation to Gal0 content 

also varies. VCD (w = 0.536), stirrer speed (w = 0.517), and CO 2 (w = 0.354) 

were critical from the beginning of the 1,000 L scale. The contribution of 

LDH (w = 0.417), temperature (w = 0.236), and lactate (w = 0.293) became 

important at day 3. Day 6 marked the emergence of titer values at previous 

time points (w = 0.475), glucose (w = 0.424), and ammonia (w = 0.397), 

followed by viability (w = 0.226) at day 10. 

As final titer and Gal0 content were predicted with similar accuracy using 

similar parameters, a possible relationship between them was explored. 

Furthermore, other measures of glycosylation profiles such as NG, Gal1, 

and Gal2 that are possibly relevant to product quality [4] were also 

included in the analysis. k-means clustering (k = 2) was performed to 

separate runs into two clusters using each of these measures. Regardless 

of the measure, the two resulting clusters are reasonably well separated in 

final titer. Cluster 1 mostly corresponds to high final titer whereas cluster 2 

to low final titer. In a three dimensional space of Gal0, Gal1, and Gal2, 

process runs are also clustered according to their final titer Figure 1). Thus 

this observation further confirms an intrinsic correlation between product 

quantity and product quality. 

Conclusions: A strong correlation between productivity (final titer) and 

product quality (Gal0) was observed. Each was predicted with similarly high 

accuracy using support vector regression models built upon process data 

from the 1,000 L bioreactors. Predictability increased significantly when data 

up to day 6 was analyzed as compared to day 3. Prediction accuracy 

continued to increase with additional data inclusion but at a slower rate. 

Several parameters contributed significantly to the deviation of product 

quantity and quality across runs, including stirrer speed, LDH, lactate, 

glucose, and VCD. Among those, stirrer speed and VCD appeared to exert 

the most critical impact on final process outcome from early stages of the 

1,000 L scale. This approach represents an important step towards 

understanding process characteristics for enhanced process robustness, and 

thus contributes to the advance of bio-manufacturing. 

Acknowledgement: The authors would like to thank the Minnesota 

Supercomputing Institute (MSI) for computational support. 

References 

1. Rathore AS, Winkle H: Quality by design for biopharmaceuticals. Nat 

Biotech 2009, 27:26-34. 

2. Charaniya S, Hu W-S, Karypis G: Mining bioprocess data: opportunities 

and challenges. Trends in Biotechnology 2008, 26:690-699. 

3. Charaniya S, Le H, Rangwala H, Mills K, Johnson K, Karypis G, Hu W-S: 

Mining manufacturing data for discovery of high productivity process 

characteristics. Journal of Biotechnology 2010, 147:186-197. 

4. Hossler P, Khattak SF, Li ZJ: Optimal and consistent protein glycosylation 

in mammalian cell culture. Glycobiology 2009, 19:936-949. 

P92 

Proteomic and metabolomic characterization of CHO DP-12 cell lines 

with different high passage histories 

Tim Beckmann * , Tobias Thüte, Christoph Heinrich, Heino Büntemeyer, 

Thomas Noll 

Institute of Cell Culture Technology, Bielefeld University, 33615 Bielefeld, Germany 

E-mail: tbe@zellkult.techfak.uni-bielefeld.de 


Page 124 of 181 

Background: For industrial pharmaceutical protein production fast 

growing, high producing and robust cell lines are required. To select more 

pH shift permissive and fast growing sub-populations, the CHO DP-12 

(ATCC clone #1934) cell line, an anti-IL8 antibody producing CHO K1 

(DHFR - ) clone, was continuously subcultured at high viability (>90%) for 

more than four hundred days in shaking flasks using a chemically defined 

medium. During this long-term cultivation there was a repeated shift in pH 

and most robust and fast growing cells became accumulated [1]. Cell 

samples were cryopreserved at four different time points, after 21, 95, 165 

and 420 days (in the following named sub-populations (SP)). The effects of 

long-term passaging before cryopreservation correlating with an increase 

in specific growth rate as well as changes in product formation and 

metabolism were examined in parallel bench-top bioreactor cultivation of 

SP21, SP95, SP165 and SP420 sub-populations. During exponential growth 

phase samples were taken for the analyses of differences in intracellular 

metabolites and protein expression (Please consider article “Growth 

characterization of CHO DP-12 cell lines with different high-passage 

histories” by Heinrich et al. in this issue for a detailed discussion of longterm 

cultivation, changes in specific growth rate, product formation and 

metabolic shifts). 

Material and methods: The CHO DP-12 cell line was cultivated in 

chemically defined, animal component-free medium TC 42 (TeutoCell AG) 

with addition of 8 mM glutamine and 200 nM methotrexate. For first steps 

of suspension adaption PowerCHO-2 (Lonza AG) medium was used. Parallel 

bioreactor cultivations were performed in 2 L bench-top vessels with an 

initial working volume of 1.2 L. Cultivation parameters were closed-loop 

controlled and set to 37 °C, pH 7.1 and 40% DO of air saturation. Cell 

counting and determination of viability was performed using a CEDEX 

system (Innovatis-Roche AG). Samples for metabolome and proteome 

analysis were taken from parallel bioreactor cultivations of the four subpopulations 

during exponential growth phase. Changes in the protein 

expression were analyzed by differential two-dimensional gel 

electrophoresis (GE Healthcare) with four technical replicates inclusive dye 

swap, respectively. Image processing and statistical evaluation was carried 

out with Delta 2D 4.2 software (Decodon GmbH). All differentially expressed 

protein spots were successfully identified using an ultrafleXtreme MALDI- 

TOF-TOF (Bruker Daltonics). For the analysis of intracellular metabolites an 

in-house developed fast-filtration quenching procedure was used to 

generate samples for GC-MS and LC-MS measurements [2]. For GC-MS 

analyses keto- and aldehyde functions were converted into their oxime 

derivatives using methoxyamine hydrochloride. Other relevant functions like 

amines, carboxylic acids or hydroxyls were masked with trimethylsilyl groups 

resulting in volatile derivatives. The nucleotide concentrations were 

determined by a HILIC-MS method using a MonoChrom diol column 

(Varian). 

Results: Based on the expression of 1377 detected proteins spots the four 

sub-populations could be clearly separated from each other in a principle 

component analysis. An ANOVA analysis (a ≤ 0.05, false significant 

proportion ≤ 0.01) revealed 43 protein spots with significantly different 

abundance. Hierarchical clustering and examination of fold-changes of 

significantly different expressed proteins showed that the quantity of 

different proteins and the degree of change increased with the number of 

passages before cryopreservation. Between SP21 and SP95 only three protein 

spots were detected with an at least two-fold change. For SP196 and SP420 

sevenand41spotsshowedaratiowithanabsolutevalueoftwoin 

comparison with SP21, respectively. In a follow up analysis the correlation of 

protein expression and the number of passages was examined. A template 

matching approach (R ≥ 0.98) [3] revealed 23 proteins whose abundance was 

linearly correlated with the number of days before cryopreserving the cells. 

Among these spots anti-stress proteins, candidates involved in protein 

folding, glycolytic enzymes and also proteins participating in transcription 

regulation, mRNA processing, cytoskeleton formation, protein biosynthesis as 

well as folate and purine metabolism were identified. In addition, there was a 

set of 10 unique proteins which showed an inverted correlation with the 

number of passages. The results from proteomic analysis indicate that the 

four subpopulations not only differ in protein abundances directly related to 

cell growth, but also show differences in diverse aspects of cellular protein 

expression. 

The analysis of intracellular metabolites revealed a positive correlation 

between the uptake rates of extracellular metabolites and their 

intracellular pool size. Interestingly, this trend is not fulfilled for a couple of 

the investigated metabolites: For example, the aspartate pool increases



while the uptake of extracellular aspartate decreases although it is not at 

limiting concentrations. This indicates that the replenishment by 

intracellular pathways is favored over the uptake of extracellular substrate 

in this case. Furthermore, an increase of the adenine as well as guanine 

energy charge was observed for an increasing number of passages. Both, 

adenosine- and guanosine-5’-triphosphate, are necessary for anabolic 

reactions as well as protein synthesis. They may also play a role in 

apoptosis by inhibiting the formation of the apoptosome [4] and therefore 

might be one factor for the observed increase in specific growth rate. 

By merging the proteomic findings with the measurements of intracellular 

metabolite pools we gained a better understanding of factors which let a 

production cell line grow faster and be more robust against pH shifts. This 

example shows that the analysis of protein expression combined with 

measurements of intracellular pool sizes may give additional hints for cell 

line, process and media development in addition to classical approaches. 

References 

1. Heinrich C, Wolf T, Kropp C, Northoff S, Noll T: Growth characterization of 

CHO DP-12 cell lines with different high-passage histories. BMC 

Proceedings 2011, 5(Suppl 8):P29. 

2. Volmer M, Northoff S, Scholz S, Thute T, Buntemeyer H, Noll T: Fast 

filtration for metabolome sampling of suspended animal cells. Biotechnol 

Lett 2011, 33:495-502. 

3. Pavlidis P: Using ANOVA for gene selection from microarray studies of 

the nervous system. Methods 2003, 31:282-289. 

4. Chandra D, Bratton SB, Person MD, Tian Y, Martin AG, Ayres M, 

Fearnhead HO, Gandhi V, Tang DG: Intracellular nucleotides act as critical 

prosurvival factors by binding to cytochrome C and inhibiting 

apoptosome. Cell 2006, 125:1333-1346. 

P93 

A method for metabolomic sampling of suspended animal cells using 

fast filtration 

Martin Volmer, Julia Gettmann * , Sebastian Scholz, Heino Büntemeyer, 

Thomas Noll 

Institute of Cell Culture Technology, Bielefeld University, D-33615 Bielefeld, 

Germany 

E-mail: Julia.Gettmann@uni-bielefeld.de 


Background: For intracellular metabolomic analyses it is of utmost 

importance to rapidly stop the organism’s metabolism during sampling. This 

Page 125 of 181 

is to avoid false data due to residual enzymatic activity during sampling. 

Unlike for bacteria and yeast, there is not one generally approved protocol 

for metabolome sampling of suspended animal cells. A number of sampling 

procedures have been developed and described in literature but have not 

been compared in depth. 

Here we describe a sophisticated sampling method for metabolome 

analysis of suspended animal cells using a fast filtration protocol. The main 

requirements for the fast filtration method were to reduce the time for 

quenching and cell-medium separation while reducing cell disruption to a 

minimum. 

Additionally, the fast filtration method is compared to the other sampling 

methods described in literature. 

Methods: CHO DP12 cells were cultured in shaker flasks and 2 L Bioreactors 

to generate sample cell suspensions for the experiments. 

The different quenching methods were used according to the literature. 

Methods tested included sampling with fast filtration [1], sampling in cold 

saline solution [2], sampling in cold methanol/ammonium bicarbonate 

(AMBIC) [3], and sampling with a microstructure heat exchanger [4]. As a 

reference sampling of cells using centrifugation without dedicated 

quenching was used. 

For 13 C-labeling experiments the cells were transferred to saline solution 

prior to sampling. 13 C-labeled glucose was added to the cells followed by 

immediate sampling. The ratio of labeled and unlabeled glycolysis 

metabolites was then determined. 

LDH and ATP release from the cells was measured using plate tests. The 

intracellular energy metabolism was investigated using HILIC columns on 

a LC-MS system. 

Results: The cell disruption during sampling was investigated by 

measurement of LDH release from the cells. It was low at 5% and 2.5% for 

fast filtration and centrifugation, respectively. No influence of the cell 

disruption on the measured intracellular metabolite levels was observed. 

The intracellular amino acid content per cell was constant for filters loaded 

with up to 6x10 7 cells. Thus, indicating a good extraction efficiency with the 

tested cell numbers. Furthermore this suggests that no residual medium 

components remained on the filter. 

The adenylate concentrations were measured in extracts from all tested 

sampling methods. These showed good correlation of ATP concentrations 

for all sampling methods tested. The same was true for ADP and AMP 

concentrations. Thus, fortifying the observation of a good extraction 

efficiency of cells on filters. 

The adenylate energy charge (AEC) was used as an indicator of quenching 

efficiency for the comparison of the proposed method with those from 

Figure 1(abstract P93) Percentage of 13 C-labeled hexose 6-phosphate after feeding the cells with 13 C-glucose immediately prior to sampling (N=4).



literature. A significant advantage over sampling using a microstructure 

heat exchanger or a simple centrifugation protocol was observed. 

Comparison to quenching in cold methanol with ammonium bicarbonate 

and cold saline solution exhibited AEC values similar to quenching with 

fast filtration. Furthermore, experiments with isotope labeled glucose 

(Figure 1) exhibited only small amounts of labeled glycolysis intermediates 

in extracts of samples taken with fast filtration and cold methanol/AMBIC 

compared to saline solution quenching and centrifugation. This is most 

likely due to the lower temperature when sampling with methanol/AMBIC 

and the shorter overall sampling time when using fast filtration. 

Metabolite leakage of the two methods with the best quenching efficiency 

was done by investigating the ATP concentration in spent washing 

solutions after sampling. Sampling with methanol showed ATP leakage of 

up to 14% whereas no leakage of ATP was observed when sampling with 

the fast filtration method. 

Conclusion: The results show that the fast filtration method is well 

applicable for metabolome sampling of suspended animal cells. The two 

major concerns with this method were that medium components remained 

with the cells after filtration and that extraction of metabolites from the filter 

would not be efficient [2]. Both of which could be disproved in this study. 

The comparison of the different sampling methods described in literature 

showed that fast filtration and quenching in cold methanol/AMBIC offer the 

best quenching efficiency. The drawback of sampling using methanol is the 

metabolite leakage caused by the contact of methanol with the cell 

membrane. This leaves the fast filtration method as the best suited method 

for reliable metabolome sampling for suspended animal cells. 

References 

1. Volmer M, Northoff S, Scholz S, Thüte T, Büntemeyer H, Noll T: Fast 

filtration for metabolome sampling of suspended animal cells. Biotechnol 

Lett 2011, 33:495-502. 

2. Dietmair S, Timmins NE, Gray PP, Nielsen LK, Krömer JO: Towards 

quantative metabolomics of mammalian cells: development of a 

metabolite extraction protocol. Anal Biochem 2010, 404:155-164. 

3. Sellick CA, Hansen R, Maqsood AR, Dunn WB, Stephens GM, Goodacre R, 

Dickson AJ: Effective quenching processes for physiologically valid 

Page 126 of 181 

metabolite profiling of suspension cultured mammalian cells. Anal Chem 

2009, 81:174-183. 

4. Wiendahl C, Brandner JJ, Küppers C, Luo B, Schygulla U, Noll T, Oldiges M: 

A microstructure heat exchanger for quenching the metabolism of 

mammalian cells. Chem Eng Technol 2007, 30:322-328. 

P94 

Analysis of glycolytic flux as a rapid screen to identify low lactate 

producing CHO cell lines with desirable monoclonal antibody yield and 

glycan profile 

Rachel Legmann 1* , Julie Melito 1 , Ilana Belzer 2 , David Ferrick 1 

1 2 

Seahorse Bioscience, N. Billerica, MA, USA; ProCognia, Ashdod, IL, USA 

E-mail: rlegmann@seahorsebio.com 


Background: In CHO cell lines currently selected for the production of 

recombinant antibody, approximately 80% of the metabolized glucose is 

converted into lactic acid. These cells with a glycolytic phenotype exhibit 

significantly higher rates of proton production (extracellular acidification 

rate, ECAR) from lactate production than cells using oxidative phosphorylation 

(oxygen consumption rate, OCR). Therefore, shifts in the cell’s 

metabolism can be detected conveniently and dynamically through 

simultaneous detection of ECAR and OCR. Such measurements can 

characterize the metabolic programming of individual cell types and 

forecast the quality potential of their produced glycoproteins. In this study, 

we utilized an XF96 analyzer to measure glycolysis and mitochondrial 

respiration simultaneously, and in real-time. This allows one to determine 

the response of these two pathways to ATP demand, and indirectly, 

biosynthetic needs. A rapid screen was performed to determine the desired 

lactic acid production by exposing the cells to alternate sources of 

substrates, such as galactose or fructose. Specific metrics of the study 

included cell growth, product yield, and glycan profile. Higher titer, viable 

cell density, and viability along with glycol-similarity were observed for 

galactose and glutamine feeding strategies during the production phase. 

Figure 1(abstract P94) Seahorse XF analyzer measure the two ATP generating pathways of the cell. The cell is consuming oxygen, producing CO 2 and 

excreting (H+) protons that acidify the media.



We believe this rapid, cell based metabolic screen that is label-free and noninvasive 

can be used to identify low lactic acid CHO mAb cell producers in 

both batch and fed-batch systems. This selection is accomplished without 

compromising clone productivity and product quality. 

Material and methods: Suspended CHO cells producing a recombinant 

IgG monoclonal antibody (MAb) were maintained in high glucose serum 

free chemically-defined (CD) CHO supplemented with 0.2μM methotrexate 

(MTX). The low-buffered DMEM assay medium was used for XF96 ECAR 

screening and CD CHO medium was used for fed-batch flasks for lactic acid 

screening. Mitochondrial function and cellular bioenergetics were measured 

in intact CHO_mAb using a Seahorse Bioscience extracellular flux analyzer 

(XF96) as described previously (1, 3) and demonstrated in Figure 1. The 

sensor cartridge is embedded with 96 dual florescent biosensors (O and H+). 

Each sensor cartridge is also equipped delivery ports for injecting agents 

during an assay. Cells were maintained in a 5% CO2 incubator at 37 °C 

and 1 h before the experiment, cells were washed and incubated in 

nonbuffered (without sodium carbonate) DMEM (sugar-free) pH 7.4, at 37 °C 

in a non-CO2 incubator. Real time cellular ECAR and OCR were measured 

simultaneously before and after the substrate source injection. The 

metabolic rate of the cell population was measured repeatedly over about 

30 min. Lactic acid concentrations in the flasks were measured after 

different carbon sources were added to the cells after 5 days of growth and 

glucose depletion during the production phase using a NOVA BioProfile 

100 Plus. MAb concentrations in samples from flasks were quantified using 

Octet QK instrument with protein A biosensors. Glycan profile in crude 

harvest samples from flasks were quantified using lectin based array by 

Procognia Glycoscope instrument described previously(2). 

The illustration demonstrates how the XF96 measures changes in the 

microenvironment (3μl) surrounding live cells in a 96-well microplate. The 

O2 and proton concentration is determined by a quenching reaction of a 

fluorophore specific for O2 (blue) or protons (red) that is embedded into 

a single polymer martix on the biosensor cartridge. 

Results: Mammalian cells in culture have inefficient glucose metabolism 

where most of the glucose consumed is converted into lactate, while 

very little is oxidized in the tricarboxylic acid cycle (TCA).One of the 

interpretation for this phenomenon of high glycolysis rate even under 

fully aerobic condition is that lactate generation from pyruvate may be 

used by these cells as a way to re-equilibrate their redox potential. The 

high glycolysis rates would generate NADH at high rates, which should 

be reduced to NAD in order to keep this pathway functional. In Figure 2 

(A), metabolic analyses from this study demonstrate that different carbon 

and nitrogen sources added during the product production phase can 

promote or diminish aerobic glycolysis as indicated by ECAR. The data 

show that ECAR correlates well with glycolysis driving lactic acid synthesis 

in the recombinant CHO_mAb cells. The highest lactic acid concentration 

and ECAR were obtained when glucose was present in the medium as 

thesolecarbonsource.ThelowestlacticacidandECARwereobtained 

when galacose or fructose was present in the media during the 

glycoprotein production phase. Therefore, adding galactose or fructose 

when glucose becomes depleted at the end of the growth phase would 

Page 127 of 181 

provide an alternative carbon source to drive low-waste lactic acid 

production. The rapid method developed in this study identified the 

optimum carbon and nitrogen source that achieved less than 77% proton 

production compared to glucose while maintaining optimal and 

consistent protein glycosylation. Metabolic shift to aerobic glycolysis was 

observed when glucose or mannose was injected into the CHO_mAb cell 

cultures as shown in Figure 2B. XF analyses demonstrated that cells 

consuming glucose or mannose are divertedtowardglycolysisevenin 

the presence of sufficient levels of dissolved oxygen. Good correlation 

was observed between ECAR and accumulation of lactic acid in 

Erlenmeyer flasks (Figure 2C). 

Conclusions: In this study, we developed and validated a rapid assay, 

employing the XF96 analyzer, to screen for low ECAR producing cells to 

predict low lactic acid production. The experiments show that there is a 

good agreement between ECAR and lactic acid and therefore ECAR can 

be rapidly measured as an index of glycolytic activity and serve as an 

accurate surrogate of lactate production. The data show that alternating 

carbon sugars during the production phase ,such as galactose and 

fructose, can help to control glycolysis and, therefore, reduce the lactate 

accumulation in mammalian cell cultures without compromising on 

recombinant glycoprotein productivity and desired glycoform profile. 

References 

1. Ferrick DA, Neilson A, Beeson C: Advances in measuring cellular 

bioenergetics using extracellular flux. Drug Discovery Today 2008, 13(5- 

6):268-274. 

2. Rosenfeld R, Bangio H, Gerwig GJ, Rosenberg R, Aloni R, Cohen Y, Amor Y, 

Plaschkes I, Kamerling JP, Maya RB: A lectin array-based methodology for 

the analysis of protein glycosylation. J Biochem Biophys Methods 2007, 

70(3):415-426. 

3. Wu M, Neilson A, Swift AL, Moran R, Tamagnine J, Parslow D, Armistead S, 

Lemire K, Orrell J, Teich J, Chomicz S, Ferrick DA: Multiparameter 

metabolic analysis reveals a close link between attenuated 

mitochondrial bioenergetic function and enhanced glycolysis 

dependency in human tumor cells. Am J Physiol Cell Physiol 2007, 292(1): 

C125-36. 

P95 

Influence of cell specific productivity on product quality 

Ruchika Srivastava, Lavanya Rao, Kriti Shukla, Sunaina Prabhu, 

Saravanan Desan, Dinesh Baskar * , Ankur Bhatnagar, Anuj Goel, Harish Iyer 

Cell Culture Lab, Biocon Limited, Bangalore, India 

E-mail: Dinesh.baskar@biocon.com 


Introduction: The micro heterogeneity or quality of a protein has been 

shown to have a significant impact on its physical, chemical and biological 

properties both in vitro and in vivo [1]. Micro heterogeneity is evaluated in 

terms of post translational modifications such as glycosylation, charge 

variants, aggregates and fragments profile. The biggest challenge in 

Figure 2(abstract P94) The effect of different carbon sources on ECAR from lactic acid production and on metabolic shift in CHO_mAb. Change of 

baseline ECAR of CHO_mAb cells after Carbon/nitrogen source injection versus time (A). Glucose and mannose preference for aerobic glycolysis(B) 

Comparison of maximum ECAR in XF96 with lactic acid in shake flasks showed an excellent correlation, R 2 =0.91 (C).



Figure 1(abstract P95) Plot of N.PCD vs. N.GL % for Ab1 & Ab3 suggest that there are some clones which have very different PCDs but similar product 

quality. 

process development is to find a balance between increasing productivity 

while maintaining product quality. 

In our study we focus on protein glycosylation, which is a process in which 

oligosaccharides are added to the protein during synthesis. There are 

multiple possible reactions in the pathway and it takes a long time for a 

glycosylated protein to be fully processed. If some protein molecules have a 

shorter residence time in the ER and Golgi, the glycan may be only at an 

intermediate stage [1]. For recombinant glycoprotein, increase in cell specific 

productivity (amount of product produced per cell per unit time) which may 

result in shorter residence time in the ER and Golgi, must be weighed 

against possible changes in product quality attributes like glycosylation [2]. 

Ourstudyconcludesthatitispossible to produce a protein with desired 

product quality profile with high specific productivity. Two different clones 

with the same productivity can have different product quality profiles; 

alternatively, the same clone with different specific productivity can be 

manipulated to produce the same desired product quality by altering the 

cell culture parameters or addition of supplements. This observation also 

influences the acknowledged methodology for selecting clones with higher 

productivity while still maintaining their product quality profile. Various 

process manipulations were evaluated as an attempt to improve on the 

product quality profiles without compromising the productivity. 

Materials and methods: Three CHO cell lines (A, B & C) expressing three 

different Antibodies (Ab1, Ab2 & Ab3) were cultured in commercially 

available animal component free media in 125 ml Erlenmeyer shake flasks 

and BIOSTAT B-DCU lab bioreactors. Cell Count and Viability were analyzed 

by Cedex Hires (Innovatis) and heamocytometer using Trypan blue dye 

exclusion. The product concentration was determined by Affinity 

Figure 2(abstract P95) a: Profiles of Process 1, 2 & 3 for Ab1 Figure 2b: Profiles of Process A, B & C for Ab3. 

Page 128 of 181 

chromatography and characterization (Glycan profiling) by Normal phase 

HPLC. 

Results and discussion: Clone Selection program: Figure 1 shows the 

plot of N.PCD (normalized specific productivity – picogram per cell per 

day) vs. N.GL% (normalized values of one type of glycosylated species) of 

different clones for the antibodies Ab1 & Ab3. Both show a similar 

general trend indicating an increase in N. GL (%) with increasing specific 

productivities. There are however some exceptions where clones with 

significantly different specific productivity show very similar glycosylation 

profile, which suggest the role of process conditions in affecting the 

product quality. 

Case study 1: Ab1: As seen in (Figure 2a), the desired N. GL (%) for Ab1 

was comparable to the product obtained from the high PCD clones in 

Process 1. However, when the process was run in a different reactor 

configuration, a decrease in N.GL (%) was observed. Experiments were 

done to understand the impact of changes in the reactor conditions by 

varying the reactor dependent parameters (aeration, agitation etc) and 

the feeding strategy. These results were used to modify the Process 2 

andmadeasamorerobustProcess3.TheProcess3wasabletogivea 

higher value of N.GL (%) while still retaining the high PCD. 

Case study 2: Ab2: All the high producing clones for Ab2 were giving 

significantly higher N.GL (%) compared to the desired quality. A study 

was conducted to evaluate the possibility of choosing the high producing 

clone and manipulate the glycan profiles to be able to meet the product 

quality requirements. Intermittent samples were taken from the Fed 

batch runs and analyzed for product conc. and glycan profiles. Both PCD 

and N.GL (%) vary during the course of the run with a general trend of



higher N.GL(%) with increase in PCD. However there were exceptions like 

day 8 vs. day 12 where the PCD of day 8 was significantly lower than day 

12 however the N.GL(%) value was higherfortheday8.Thefeeding 

strategy and the process parameters (controlled and measured) around 

day 8 were estimated and compared to other days. 

The results of the investigation showed that a feed added at around day 8 

during the batch, helped in reducing the N. GL (%) values. The quantity of 

this feed as well as its feeding strategy were changed which helped in 

getting the desired product quality. The results from the study done above 

were implemented in the process. 

Case study 3: Ab3: The desired N. GL (%) for the product was significantly 

lower than the range obtained from most of the clones. The process used 

here is referred to as Process A. Feeding concepts implemented in the Ab2 

improved process were tested in the Ab3 which is referred as Process B. 

The feed manipulations helped in bringing down the N.GL (%) values. 

However they were still much higher than the desired range. The time 

course analysis of the Process B batches was done. The analysis indicated 

some days during the run where the level of N.GL (%) were much lower 

compared to other days. Detailed analysis of the culture conditions during 

these days was done. The outcome of these analysis were implemented in 

the process by changing the culture parameters during the run. This 

process is referred as Process C. The profiles of batches run with Processes 

A, B and C are shown in (Figure 2b). Significant decrease in the level of N. 

GL (%) was observed in Process C without affecting the PCD of the cells. 

Conclusion: It was generally observed that the clones with high specific 

productivity also have high levels of N.GL (%). Since some of the 

products required lower levels of N.GL (%); these high producing clones 

Page 129 of 181 

were found to be unsuitable. However, the results of this study show that 

with modified conditions of process parameters and feeding strategies, it 

was possible to alter the N.GL (%) levels without impacting the cell line 

specific productivity. These factors when taken into consideration during 

clone selection may still allow the selection of high producing clones 

with a possibility to alter the product profiles during development. 

Acknowledgement: Cell Culture group: Chandrashekhar Kuruvangi, 

Janani Kanakrajan, Rohit Diwakar 

References 

1. Hu WS, Betenbough M: Technology for Secretory Therapeutic Proteins. 

Cellular Bioprocess technology 2007. 

2. Hossler P, Khattak SF, Li Zheng: Optimal and consistent protein 

glycosylation in mammalian cell culture. Glycobiology 2009, 19(9):936-949. 

P96 

A novel peptide to enhance recombinant BMP-2 production in 

mammalian cell cultures 

Aileen J Zhou 1* , Cameron ML Clokie 1,2 , Sean AF Peel 1,2 

1 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, University 

of Toronto, Toronto, Ontario Canada, M5G 1G6; 2 Induce Biologics Inc, 

Toronto, Ontario, Canada, M5R 3N8 

E-mail: aileen.zhou@dentistry.utoronto.ca 


Background: Due to their osteoinductive properties, recombinant human 

bone morphogenetic proteins (rhBMPs) have been used successfully for 

Figure 1(abstract P96) After 24 h incubation with or without IND-1, the amount of proBMP-2 and mature BMP-2 proteins in (A) CHO and (B) HEK 

conditioned media were measured by proBMP-2 and BMP-2 ELISA. Data are presented as mean ± SEM (n = 3 experiments) (**P



bone regeneration and replacement. However, the yields rhBMPs yields in 

mammalian expression systems are very low, resulting in their high cost. 

BMPs are synthesized as a precursor, proBMP, which undergoes enzymatic 

cleavage by proprotein convertases (PCs) to form the mature BMP [1]. Furin, 

an enzyme of the PC family, has shown to cleave BMP-4 [2] and BMP-2 

(Zhou et al., unpublished data). This study investigated the effect and 

mechanism of action of polyarginine furin inhibitor, IND-1, on rhBMP-2 

production in mammalian cell lines overexpressing rhBMP-2. 

Materials and methods: Two stable cell lines expressing the hBMP2 gene, 

CHO-BMP2 and HEK-BMP2, were cultured in the presence of IND-1 in shortterm 

(24 h, multi-well) and long-term (two-month, perfusion flasks) 

cultures. The rhBMP-2 produced was characterized by Western blot and its 

activity assessed using the C2C12 cell-based assay. The amount of proBMP- 

2 and mature BMP-2 produced was quantified by ELISA. The mRNA level of 

BMP-2 and furin in cells treated with or without IND-1 was compared by 

real-time RT-PCR. Cellular uptake of IND-1 was estimated by measuring the 

fluorescence of cell lysates following incubation with FITC labeled IND-1. 

Cellular PC activity post IND-1 incubation was measured using the Boc- 

RVRR-AMC substrate. Furin-specific siRNA was used to knock down the 

furin expression in CHO-BMP2 cells and its effect on the rhBMP-2 

production was determined. 

Results: Stably transfected CHO-BMP2 cells secreted 36 kDa rhBMP-2 

dimers that were biologically active. In 24 h cell cultures, IND-1 treated 

cells produced significantly greater amounts of proBMP-2 (≥ 10-fold, 

P



Table 1(abstract P97) Viable cell density (VCD) and cell viabilities of CHO cells cultured in a fed-batch bioreactor 

process 

Process time (d) VCD mean (cells/mL) STDV CV (%) Viability mean (%) STDV CV (%) 

Cedex 0.8 7.33E+05 1.35E+04 1.8 96.39 0.92 1.0 

3.8 4.18E+06 5.94E+04 1.4 97.01 0.33 0.3 

MACSQuant Analyzer 0.8 6.47E+05 9.42E+03 1.5 95.30 0.03 0.0 

3.8 4.41E+06 2.87E+04 0.7 96.06 0.06 0.1 

Vi-Cell 0.8 7.11E+05 1.06E+05 14.9 96.31 0.73 0.8 

3.8 5.03E+06 9.92E+04 2.0 96.98 0.06 0.1 

Figure 1(abstract P97) (A) The time course of an unstable MTX amplification is presented. A decrease in the fraction of IgG-producing cells (F P) is caused 

by resistance to increasing MTX concentrations (c MTX). (B) Dot plots for the samples at 307 h (160 nM MTX) and 687 h (640 nM) are shown. IgG-producing 

cells appear in the upper right gates. 

The MACSQuant Analyzer allows the determination of cell density and 

viability with high reproducibility. The mean data were comparable to 

common cell-counting systems. The results obtained with the MACSQuant 

Analyzer, however, showed better accuracy as reflected by the lower CV. 

Moreover, through its FSC, SSC, and seven fluorescence channels, as well as 

its three lasers it allows sophisticated, multiparametric flow cytometric 

analysis. 

References 

1. Cacciatore , Chasin , Leonard : Gene amplification and vector engineering to 

achieve rapid and high-level therapeutic protein production using the 

Dhfr-based CHO cell selection system. Biotechnol Adv 2010, 28(6):673-681. 

2. Jun , Kim , Baik , Hwang , Lee : Selection strategies for the establishment of 

recombinant Chinese hamster ovary cell line with dihydrofolate reductasemediated 

gene amplification. Appl Microbiol Biotechnol 2005, 69(2):162-169. 

P98 

Bioreactor cultivation of CHO DP-12 cells under sodium butyrate 

treatment – comparative transcriptome analysis with CHO cDNA 

microarrays 

Sandra Klausing *† , Oliver Krämer † , Thomas Noll 


Germany 

E-mail: skl@zellkult.techfak.uni-bielefeld.de 


Page 131 of 181 

Background: Sodium butyrate (NaBu) is not only known to inhibit 

proliferation but also to increase the specific productivity in cultivation of 

Chinese hamster ovary (CHO) cells [1] – the most commonly used 

mammalian cell line for pharmaceutical protein production [2]. So far, 

little is known about the underlying mechanisms and genes that are 

affected by butyrate treatment. Besides the proteomic approach to 

unravel proteins involved in the processes, the analysis of transcriptomes 

presents another promising method. Here we show an application of our 

CHO cDNA microarray to identify genes associated with increased 

productivity during cultivation of CHO cells under sodium butyrate 

treatment. 

Materials and methods: Four batch cultivations of CHO DP-12 cells 

(clone # 1934, ATCC CRL-12445) were performed in 2 L bioreactor systems 

under pO 2- and pH-controlled conditions. In the exponential growth 

phase, 67 hours after inoculation, 2 mM sodium butyrate was added to 

three processes. The fourth was left untreated to function as control 

culture. Samples were taken before and then repeatedly after the addition 

of butyrate. RNA was isolated from cell pellets of 5·10 6 cells using TRIzol® 

Reagent (Invitrogen). For subsequent cDNA labeling, the Agilent Low-Input 

QuickAmp Labeling Kit (Agilent Technologies) was used. The custom 

designed 2 x 105 k cDNA microarray (Agilent Technologies) was spotted 

with 94,580 probes designed from CHO cDNA sequenced in-house. 38,310 

of 41,039 sequenced contigs were used for the microarray, each covered 

by 2-4 probes [3]. Data analysis was done with ArrayLims, EMMA, and 

SAMS, three CeBiTec based software tools [4]. The raw data gathered by



Figure 1(abstract P98) (A): Concentration of viable cells and cell viabilities for the time course of CHO DP-12 batch processes. Error bars represent the 

standard deviation of triplicate measurements with the Cedex system (Roche Diagnostics). Red and orange lines represent biological replicates of cultures 

treated with 2 mM NaBu, the control process is shown in green. Dashed lines show viabilities. The grey arrow indicates the addition of NaBu, the grey 

circles show the sample points compared later in the microarray analysis (72 h of NaBu Treatment). (B): Number of up- and downregulated genes of 

selected KEGG pathway categories with a detailed view of four pathways from the Cell Growth and Death KEGG category. Results show only those found 

as differentially expressed after filtering. Red: downregulated in NaBu cultures; green: upregulated in NaBu cultures (compared to control culture). 

the microarray experiments were processed by standard Agilent 

background normalization and subsequent lowess normalization. 

Results: The control culture reached a maximum viable cell density of 

1·10 7 cells/mL while NaBu treated cells reached a plateau at about 6·10 6 

cells/mL and retained a viability above 90% four days longer than 

untreated cells (Figure 1A). The three biological replicates of NaBu cultures 

yielded results with similar general trends. The maximum antibody 

concentration of the control culture was 110 mg/L whereas cells treated 

with NaBu reached a maximum of 175 mg/L antibody. 72 hours after 

addition of NaBu the specific antibody production rate was increased by 

a factor of 3.6 (NaBu culture: 4.5 pg/(cell·d)) compared to control culture 

(1.2 pg/(cell·d)). 

Of this time point, samples were analyzed in microarray experiments. A 

significance test with FDR control (a=0.05) was carried out for the four 

technical replicates (including two dye-swaps) of the microarray. For 

analysis, the following filtering settings were chosen to identify differentially 

expressed genes: adjusted p-value ≤ 0.05, log-ratio < -1 or > 1 (equals fold 

change < -2 or > 2) and log-intensity ≥ 6 (equals ≥ 64 raw intensity). From a 

total of 1461 genes found to be differentially expressed under NaBu 

treatment, 771 genes were upregulated and 690 genes were downregulated 

Table 1(abstract P98) Fold change of selected genes from microarray analysis 

(derived from EC numbers in KEGG pathways, Figure 1B). Many differentially 

expressed genes from pathways involved in carbohydrate, lipid, amino acid 

and glycan metabolism are upregulated which is most likely linked to higher 

productivity. A large portion of genes from pathways associated with cell 

growth and death are downregulated and most of these genes originate 

from cell cycle processes. This correlates with reports of cell cycle arrest 

under NaBu treatment [1]. Some examples of regulated genes are shown in 

Table 1. 

Conclusions: Microarray analysis revealed a high number of regulated 

genes under sodium butyrate treatment in pathways like carbohydrate 

metabolism, cell cycle and signal transduction. Some of the regulated 

genes are promising targets for overexpression or knockdown/knockout 

experiments and we will further investigate the knockdown effect of 

selected genes using a siRNA approach in CHO cells. Our in-house 

microarray is suitable for further transcriptomic analysis of CHO cells 

under various conditions. 

Acknowledgements: We would like to thank E. Schulte-Berndt (Institute 

for Genome Research and Systems Biology, CeBiTec, Bielefeld) for help 

with microarray hybridization and O. Rupp (Bioinformatics Resource 

Facility, CeBiTec, Bielefeld) for help with microarray data analysis. 

KEGG pathway category Gene symbol Description Mean fold change 

in NaBu culture 

compared to 

control culture 

Page 132 of 181 

# of probes 

CA150 Transcription factor CA150b -3.31 ↓ 3 

Transcription & Translation Ccdc12 Coiled-coil domain containing 12 -2.01 ↓ 1 

Y14 RNA-binding protein 8A 2.14 ↑ 2 

Pkmyt1 Protein kinase, membrane associated tyrosine/threonine 1 -2.04 ↓ 2 

Cell Cycle c-Myc Myc proto-oncogene protein -3.44 ↓ 3 

Ink4c Cdkn2c cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) 3.04 ↑ 2 

CycD Cyclin D1 (Ccnd1) 2.49 ↑ 1 

Pdcd4 Programmed cell death 4 3.89 ↑ 2 

Apoptosis Casp6 Caspase 6 3.06 ↑ 3 

PI3K Phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1 2.44 ↑ 2



References 

1. Kumar N, Gammell P, Clynes M: Proliferation control strategies to improve 

productivity and survival during CHO based production culture: A 

summary of recent methods employed and the effects of proliferation 

control in product secreting CHO cell lines. Cytotechnology 2007, 53(1- 

3):33-46. 

2. Jayapal K, Wlaschin K, Yap M, Hu W-S: Recombinant protein therapeutics 

from CHO cells - 20 years and counting. Chem. Eng. Prog. 2007, 

103(10):40-47. 

3. Becker J, Hackl M, Jakobi T, Rupp O, Timmermann C, Szczepanowski R, 

Borth N, Goesmann A, Grillari J, Noll T, Pühler A, Tauch A, Brinkrolf K: Next- 

Generation Sequencing of the CHO cell transcriptome. BMC Proceedings 

2011, 5(Suppl 8):P6. 

4. Center for Biotechnology, IFB - Institute for Bioinformatics: Computational 

Genomics, Software. [http://www.cebitec.uni-bielefeld.de/cebitec/ 

computational-genomics/software.html]. 

P99 

Characterization of chromatographic yeast extract fractions promoting 

CHO cell growth 

Mathilde Mosser 1,2 , Romain Kapel 1 , Arnaud Aymes 1 , Laurent-Michel Bonanno 2 , 

Eric Olmos 1 , Iris Besançon 2 , Dominique Druaux 2 , Isabelle Chevalot 1 , Ivan Marc 1 , 

Annie Marc 1* 

1 Laboratoire Réactions et Génie des Procédés, UPR CNRS 3349, Nancy- 

Université, Vandœuvre-lès-Nancy, France; 2 Bio Springer, Maisons-Alfort, 

France 



Background: Many studies underline the great benefits of yeast extracts 

(YE), used as supplements in animal free culture media, on cell growth and 

recombinant protein production [1,2]. Nevertheless, their unknown 

composition and batch-to-batch variability of commercial YE remain 

constraints for industrial processes [3,4]. Consequently, the identification of 

bioactive YE molecules is challenging for process reliability. The main 

strategy to respond upon this problem is to fractionate the extract and then 

to characterize the fractions. So far, several fractionation processes such as 

ultrafiltration [5], gel filtration chromatography [6] or alcoholic precipitation 

[7] have been investigated. These studies suggested that the active 

components were mainly small molecules (< 1000 Da). But, the other 

physico-chemical properties of YE components have been poorly studied 

until now. In regards of this statement, it is proposed to get better 

knowledge of the YE molecules leading to an improvement of CHO cell 

growth, by implementing various chromatographic fractionation processes. 

Material and methods: CHO-AMW cell line producing anti-human RhD- 

IgG1 was provided by Dr. F. Wurm. Cell growth assays were performed in 

96-well plates with 200 μL of working volume inside an incubator at 37 °C 

and 5% CO 2. Cells were cultivated in a protein-free chemically defined 

medium RPMI 1640/BDM (90/10) supplemented with 4 mM of glutamine 

(Eurobio, France) and supplemented or not by 1 g.L -1 of YE fraction. Cell 

concentration was in-situ monitored by Cellscreen® analyser (Innovatis, 

Germany). 

Raw YE, the soluble fraction of yeast autolysate, was provided by 

BioSpringer. A nanofiltration step using Nanomax 50 membrane (Millipore, 

Bedford, MA) was used to remove the very small molecules such as salts 

and amino acids. YE was first fractionated by three one-step preparative 

chromatographies according to the described conditions (Table 1). 

Fractions containing salts and buffers were purified and concentrated by 

nanofiltration. In another way, a complete chromatographic fractionation 

was achieved by sequentially separating YE with anion-exchange, 

hydrophobic interaction and size exclusion chromatography. All fractions 

were lyophilized for further analyses and cell culture assays. 

Concentration of total amino acids, polysaccharides and nucleic acids 

were determined in all fractions by HPLC methods after total hydrolysis. 

Molecular weights were evaluated by size-exclusion HPLC [8]. 

Results: Firstly, the YE was separately fractionated either by anion 

exchange (AEC), hydrophobic interaction (HIC) or gel filtration 

chromatography (GFC). The AEC allowed isolating in one fraction the 

nucleic acids strongly bound to the gel, while peptides were distributed 

among all fractions depending on their electrical charges. In the HIC 

fractions, important quantities of buffer salts still remained despite the 

desalting process. Finally, the GFC led to three fractions containing various 

proportions of nucleic acids, polysaccharides and peptides. Then, the 

influence of the fractions on the CHO cell growth was evaluated in 96-well 

microplates (Figure 1-A). All GFC fractions presented similar results than 

raw YE. The HIC fractions did not stimulate the cell growth, probably due 

to residual buffer salts. Furthermore, only one fraction from AEC, devoid of 

nucleic acids but enriched in positive-charged peptides, presented a 

similar impact than YE. 

Secondly, YE was divided in 18 fractions by a global process including the 

three chromatographic methods in a sequential mode: AEC, HIC, GFC. 

The final GF step presented the advantage to remove the residual salts. 

The cell growth monitoring pointed out two fractions, which led to similar 

results than the YE (Figure 1-B). These fractions contained peptides, mainly 

positively charged and polar, low quantities of polysaccharides but no 

nucleic acids. The amino acids quantification of fraction A.1-H.1-W.3 

underlined that peptides were mainly composed of lysine and arginine. 

This result was consistent with a recent patent claiming that C-terminal 

arginine containing tripeptides were at least partially responsible for the 

growth promoting activity of a peptone (9). 

Conclusion: A one step-fractionation process by anion exchange 

chromatography led to a simplified composition of raw YE without 

compromising its stimulating effect on CHO cell growth. Furthermore, the 

three-step fractionation process allowed a better knowledge of the physicochemical 

properties of the molecules involved in cell growth stimulating 

effects: polar positively charged and mainly composed of peptides enriched 

in arginine and lysine residues. 

References 

1. Sung YH, Lim SW, Chung JY, Lee GM: Yeast hydrolysate as a low-cost 

additive to serum-free medium for the production of human 

thrombopoietin in suspension cultures of Chinese hamster ovary cells. 

Appl Microbiol Biot 2004, 63:527-536. 

2. Kim DY, Lee JC, Chang HN, Oh DJ: Development of serum-free media for 

a recombinant CHO cell line producing recombinant antibody. Enzyme 

Microb Tech 2006, 39:426-433. 

3. Butler M: Animal cell cultures: recent achievements and perspectives in 

the production of biopharmaceuticals. Appl Microbiol Biot 2005, 

68:283-291. 

4. Grillberger L, Kreil TR, Nasr S, Reiter M: Emerging trends in plasma-free 

manufacturing of recombinant protein therapeutics expressed in 

mammalian cells. Biotechnol J 2009, 4:186-201. 

5. Chun BH, Kim JH, Lee HJ, Chung N: Usability of size-excluded fractions of 

soy protein hydrolysates for growth and viability of Chinese hamster 

ovary cells in protein-free suspension culture. Bioresource Technol 2007, 

98:1000-1005. 

6. Mendonça RZ, Oliveira EC, Pereira CA, Lebrun I: Effect of bioactive 

peptides isolated from yeastolate, lactalbumin and NZCase in the insect 

cell growth. Bioprocess Biosyst Eng 2007, 30:157-164. 

7. Shen CF, Kiyota T, Jardin B, Konishi Y, Kamen A: Characterization of 

yeastolate fractions that promote insect cell growth and recombinant 

protein production. Cytotechnology 2007, 54:25-34. 

Table 1(abstract P99) Operating conditions of preparative chromatographic fractionations 

Separation Gel / Column Fractions 

Anion-exchange Q Sepharose, H 10 cm, j 10 cm A.1, A.2, A.3 

Hydrophobic interaction Phenyl Sepharose , H 10 cm, j 5 cm H.1, H.2 

Size-exclusion G-15 Sephadex, H 75 cm H, j 5 cm W.1, W.2, W.3 

Sequentially three step process same than one step processes A.x-H.y-W.z 

Page 133 of 181



Figure 1(abstract P99) Maximal concentration of CHO cells with or without 1 g.L -1 of YE fractions obtained (A) by one-step and (B) three-step 

chromatography. The control culture was performed in the chemically-defined medium. 

8. Chabanon G, Alves da Costa L, Farges B, Harscoat C, Chenu S, Goergen JL, 

Marc A, Marc I, Chevalot I: Influence of the rapeseed protein hydrolysis 

process on CHO cell growth. Bioresource Technol 2008, 99:7143-7151. 

9. Wilkins J: Biologically active c-terminal arginine-containing peptides. 

United states patent application 2009, US 2009/0143248 A1. 

P100 

Study of expansion of porcine bone marrow mesenchymal stem cells 

on microcarriers using various operating conditions 

Caroline Ferrari 1 , Frédérique Balandras 1 , Emmanuel Guedon 1 , Eric Olmos 1 , 

Nguyen Tran 2 , Isabelle Chevalot 1 , Annie Marc 1* 

1 Laboratoire Réactions et Génie des Procédés, UPR-CNRS 3349, Nancy- 

Université, Vandœuvre-lès-Nancy, France; 2 École de Chirurgie, Faculté de 

Médecine, Vandœuvre-lès-Nancy, France 



Background: Bone marrow mesenchymal stem cells (BM-MSCs) represent 

promising source for tissue engineering and cell therapy, due to their 

multipotency, immunoregulation and self-renewal properties [1,2]. The 

expansion phase of these cells prior to differentiation and/or injection to the 

patient remains a critical step. BM-MSCs are classically expanded in small 

scale culture systems, with low control of culture conditions. However, tissue 

engineering and cell therapy require very large quantities of cells that 

cannot be easily achieved using processes in static flasks [3]. Microcarriers, 

classically used for industrial large scale culture of continuous cell lines, 

could be advantageously applied to stem cells expansion such as BM-MSCs 

[4-6]. Our objective was to study the influence of some operating 

parameters (agitation rate, microcarrier feed) on expansion and organization 

(adhesion, aggregation) of porcine BM-MSCs cultivated on collagen-free 

microcarriers, in order to improve cell expansion while maintaining their 

multipotency. 

Materials and methods: BM-MSCs were extracted from the iliac crest of 

bone marrow of three month old pigs, and purified by adherence and selfrenewal 

properties. Cells were expanded in T-flask and on 1.2 g/L Cytodex 

1 carriers (GEHealthcare) in spinner flasks. Initial cell density was 6000 cell/ 

cm 2 and 30 000 cell/mL (total surface of 1000 cm 2 and medium volume of 

200 mL). Culture medium was a modified a-minimal essential medium (a- 

MEM, Sigma) supplemented with 10% fetal bovine serum (FBS). All cultures 

were performed inside an incubator (37 °C; 5% CO2). Medium was 

exchanged (50% volume) every two days starting from day three. Cell 

nuclei were counted by flow cytometry (Guava Easycyte) after cell lysis by 

citric acid. Cell adhesion and aggregation were observed by optical 

microscopy (x 40) after methylene blue staining. Cell multipotency was 

assayed by using differentiation kits (Invitrogen). 

Results: Prior to study the cell expansion phase, the adequate operating 

conditions of the seeding phase were determined such as the cell to bead 

ratio and the medium composition. Then, kinetics of cell expansion was 

compared between T-flask and spinner flask with cells on mirocarriers. 

Page 134 of 181 

After 8 days, BM-MSCs reached the same maximal total cell nuclei in both 

culture systems until cells remained as monolayer in T-flask but 

aggregated on microcarriers. As a consequence, cell density seemed to 

decrease on microccariers due to cell aggregation (Figure 1). 

In a second part, growth kinetic studies of porcine BM-MSCs attached on 

Cytodex 1 carriers were performed at different agitation rates (0, 25, 75 

rpm) in spinner flasks. Under stirred conditions, BM-MSCs cell population 

reached a maximal cell concentration (1.5 x 10 5 cell/mL; x 5 multiplication 

factor) before to decline whatever the agitation rate used. Small 

aggregates was observed on microcarrier surface in the early stage of the 

cultures. Once those aggregates reached a critical size, they left from the 

microcarriers and remained in suspension, where no more growth could 

be observed. However, culture without agitation reached a similar maximal 

cell density but a longer steady phase was observed during 300 hours until 

cell/microcarrier clusters occurred at day 13. As cell/cell aggregation was 

notobservedat0rpm,itcouldbeassumed that Cytodex 1 surface was 

not directly involved in the cell aggregation phenomenon, which seemed 

promoted under agitation. 

To verify if the aggregates were able to dissociate when exposed to new 

surfaces, cells were cultivated on agarose in static mode to favor 

aggregates. Those aggregates dissociated once exposed to static surfaces 

such as T-flasks or fresh Cytodex 1 carriers. Based on these observations 

and to allow homogeneous and controlled stirred cultures, the addition 

of fresh carriers during stirred cultures was further evaluated. 

Firstly, fresh microcarriers were added in stirred spinner flask at day 10, 

when cell aggregates were already formed and cell density decreasing. 

Cells were able to colonize the new added surface, and to grow again, 

showing that cell aggregation can be reversed by adding fresh carriers. In 

a second time, fresh carriers were sequentially added at day 4, 8 and 12. 

As a result, after 12 days, total cell nuclei reached values 3 times higher 

than in the culture without carrier addition, suggesting that cell 

aggregation could be prevented by early addition of fresh carrier in the 

stirred culture. 

Following expansion in T-flask or on microcarriers, BM-MSCs were 

harvested by trypsination and tested for their multipotency. Their similar 

ability to differentiate in adipocytes, chondrocytes and osteocytes 

indicated that the multipotency was preserved after cell expansion on 

Cytodex 1 carriers. 

Conclusion: BM-MSCs culture on Cytodex-1 collagen-free carriers in 

stirred systems allowed the cell expansion while maintaining their 

multipotency. By adding fresh microcarriers, cell aggregation could be 

prevented and the expansion phase duration extended. This culture 

process is expected to be transferred to a larger controlled culture 

system, in order to fulfill the need of high quantities of multipotent 

BM-MSCs. 

References 

1. Caplan A: Why are MSCs therapeutic? New data: new insight. J Pathol 

2009, 217:318-324. 

2. Chamberlain G, Fox J, Ashton B, Middleton J: Concise review: 

mesenchymal stem cells: their phenotype, differentiation capacity,



Figure 1(abstract P100) Porcine BM-MSCs growth on 1000 cm 2 curface of Cytodex 1 stirred at 25 rpm. Addition of fresh carriers at day 10 ; 50 % 

medium change every 2 days. 

immunological features, and potential for homing. Stem Cells 2007, 

25:2739-2749. 

3. Godora P, McFarland C, Nordon R: Design of bioreactors for mesenchymal 

stem cell tissue engineering. J Chem Technol Biotechnol 2008, 83:408-420. 

4. Frauenschuh S, Reichmann E, Ibold Y, Goetz P, Sittinger M, Ringe J: A 

microcarrier-based cultivation system for expansion of primary 

mesenchymal stem cells. Biotechnol Prog 2007, 23:187-193. 

5. Schop D, Janssen F, Borgart E, de Bruijn J, van Dijkhuizen-Radersma R: 

Expansion of mesenchymal stem cells using a microcarrier-based 

cultivation system: growth and metabolism. J Tissue Eng Regen Med 2008, 

2:126-135. 

6. Sart S, Schneider Y-J, Agathos S: Ear mesenchymal stem cells: an efficient 

adult multipotent cell population fit for rapid and scalable expansion. 

J Biotechnol 2009, 139:291-299. 

P101 

Growth and death kinetics of CHO cells cultivated in continuous 

bioreactor at various agitation rates 

Frédérique Balandras, Eric Olmos * , Caroline Hecklau, Fabrice Blanchard, 

Emmanuel Guedon, Annie Marc 

Laboratoire Réactions et Génie des Procédés, UPR CNRS 3349, Nancy- 

Université, Vandœuvre-lès-Nancy, France 

E-mail: eric.olmos@ensic.inpl-nancy.fr 


Background: Mammalian cells are known to be sensitive to hydrodynamic 

stresses. Therefore, soft agitation and aeration are generally recommended 

in culture bioreactor to prevent cell damages. Nevertheless, at the industrial 

scale, this may induce CO 2 accumulation, high mixing times, poor air 

dispersion and concentration gradients. In batch reactor, we have previously 

found that growth kinetics of CHO cells could be enhanced by increasing 

stirring frequencies from 80 to 600 rpm. However, these studies were 

limited in time due to depletion of substrates leading to a cell death which 

is not associated to hydrodynamic constraints. To overcome the effects on 

cell death from those nutritional limitations, in this work, longer stationary 

Page 135 of 181 

cultures of CHO cells were carried out in continuous mode in a 2-liter 

reactor operating with various stirring profiles. 

Materials and methods: CHO 320 cells were grown in a serum-free 

medium PF-BDM in a sparged and stirred tank reactor (1.4 L working 

volume; pO2: 50%; pH: 7.4; temperature: 37°C). Continuous culture was 

started in mid-exponential phase (D=0.02 h -1 ) and two agitation rate profiles 

were applied : 300-600-300-600 rpm and 600-300-600-800-900-1000 rpm. 

Viable and necrotic cell densities were estimated according to the Trypan 

blue exclusion method. Lysed cells were measured via the LDH release 

assay, whereas apoptotic cells were analysed by using Guava Easycyte 

cytometer. Glucose, lactate and glutamine concentrations were assayed with 

enzymatic commercial kits (Ellitech, Biomerieux, R-Biopharma) and ammonia 

concentration with a selective probe (Orion). Bioreactor hydrodynamics was 

numerically simulated by using Computational Fluid Dynamics to calculate 

the power dissipation rates and the velocity fields at each agitation rate 

(Fluent 6.3). The experimental validations were performed by Laser Doppler 

Velocimetry. 

Results: 

Continuous culture performed with the first agitation rate profile : 

300-600-300-600 rpm: Based on previous batch culture results [1], the 

batch phase was started at 300 rpm during 48 h in order to obtain the 

mid-exponential cell growth phase before starting the continuous mode 

(Figure 1A). Viable cell concentration increased to 30.10 5 cells.mL -1 at the 

steady state, while dead and lysed cells reached an average level of 5.10 5 

cells.mL -1 . A step of agitation rate from 300 to 600 rpm induced a 70% 

decrease in viable cells and a doubled dead cell concentration. The same 

variation was obtained for the second step of the experiment (300 rpm to 

600 rpm) but with higher dead and lysed cell concentrations of 13.10 5 

cells.mL -1 . Kinetics of glucose and glutamine consumption were monitored 

during the culture (Figure 1B). After 100 h of continuous culture, glutamine 

concentration decreased down to a limiting level around 0.03 g.L -1 and 

remained constant all over the experiment. Following the step from 300 to 

600 rpm, glucose concentration increased from 0.1 to 0.6 g.L -1 along with 

the viable cell concentration decrease from 28.10 5 to 8.10 5 cells.mL -1 . 

Consequently, no glucose limitation occurred at 600 rpm. Under 

hydrodynamic stresses, cell death mayoccureitherbynecrosisor 

apoptosis mechanisms [2,3]. Necrosis is a sudden phenomenon followed



Figure 1(abstract P101) Kinetics of continuous cultures performed with two different profiles of agitation rates (—): (A, C) viable cells (-⃟-), dead cells 

(-⃞-), lysed cells (-Δ); (B, D) glucose (-⃞-) and glutamine (-Δ-). 

by a rapid cell lysis, whereas apoptosis involves enzymatic cascades 

reactions leading to molecular, enzymatic and structural properties 

modifications. In our study, analysis of dead cell populations by using flow 

cytometry revealed that cells mostly died by apoptosis for the first step 

from 300 to 600 rpm, while necrosis became the predominant death 

mechanism after the second step (Table 1). 

Continuous culture performed with the second agitation rate profile 

: 600-300-600-800-900-1000 rpm: For the second run, the batch phase 

was started at 600 rpm since such an agitation rate was shown not to 

be deleterious to CHO cells cultivated in batch mode [1]. In this case, 

the cells reached an average concentration of 30.10 5 cells mL -1 at the 

steady state of the continuous culture (Figure 1C). However, lysed cells 

accumulatedupto8.10 5 cells.mL -1 after 200 h of culture. The first 

change from 600 to 300 rpm did not significantly influence the viable 

cell density, while a slight decrease of lysed cell concentration was 

observed. It has to be noted that the increase in the stirring rate from 

600 to 1000 rpm did not lead to massive cell death. While the viable 

Page 136 of 181 

cell density decreased and remained at 20.10 5 cells.mL -1 ,thelysed 

cell concentration gradually increased to reach 10.10 5 cells.mL -1 and 

the dead cell density (Trypan blue) remained negligible at 2.10 5 cells. 

mL -1 . In these conditions, no glucose and glutamine limitation was 

observed (Figure 1D). Dead cell analysis by Guava cytometry indicated a 

maximal apoptotic cell level around 5 to 10% of total cells throughout 

the culture (Table 1). Necrotic cells appeared only at 800 rpm and 

accounted to 40% of the total cells at 1000 rpm. Then, cell death 

occurred essentially by lysis and necrosis during this second continuous 

culture. 

Discussion: When CHO cells were cultivated in a continuous mode and 

subjected to an increase in power dissipation rates from 0.32 (300 rpm) 

to 2.5 W.kg -1 (600 rpm), massive cell necrosis and apoptosis occurred, 

while this cellular response was not observed in batch mode for the 

same levels of agitation. Therefore, the CHO cell death could be 

attributed to the observed glutamine limitation known to induce 

apoptosis [4]. Furthermore, cells initially cultivated with a higher



Table 1(abstract P101) Percentages of apoptotic and necrotic cells measured by using a GUAVA cytometer 

Time (h) RPM Necrotic cells (%) Apoptotic cells (%) 

Culture 1 Culture 2 Culture 1 Culture 2 Culture 1 Culture 2 Culture 1 Culture 2 

50 50 300 600 7 4 2 6 

100 150 300 300 19 2 2 4 

150 340 600 300 9 1 15 4 

180 480 600 600 2 2 31 7 

280 600 300 600 4 2 34 8 

320 675 300 800 23 8 8 5 

380 745 300 800 21 10 5 8 

430 825 600 900 18 30 6 10 

450 865 600 1000 49 40 16 13 

dissipation rate (600 rpm, 2.5 W.kg -1 ) revealed a better ability to resist to 

very high dissipation rates (1000 rpm, 11 W.kg -1 ), suggesting cell 

adaptation or selection. In conclusion, the growth and death 

mechanisms of CHO cells cultivated at high dissipation rates strongly 

depend on the sequence of power dissipation levels and on the 

availability of nutrients. Chemostat could be thus an appropriate system 

to better understand the complex cellular response to hydrodynamic 

stresses. 

References 

1. Barbouche N: Réponse biologique de cellules animales à des contraintes 

hydrodynamiques: simulation numérique, expérimentation et 

modélisation en bioréacteurs de laboratoire. PhD thesis, Nancy-University, 

Nancy, France 2008. 

2. Al-Rubeai M, Singh RP, Goldman MH, Emery AN: Death mechanisms of 

animal cells in conditions of intensive agitation. Biotechnol. Bioeng 1995, 

45:463-472. 

3. Mollet M, Godoy-Silva R, Berdugo C, Chalmers JJ: Acute hydrodynamic 

forces and apoptosis: A complex question. Biotechnol. Bioeng 2007, 

98:772-788. 

4. Hwang SO, Lee GM: Nutrient deprivation induces autophagy as well as 

apoptosis in Chinese hamster ovary cell culture. Biotechnol. Bioeng 2007, 

99:678-684. 

Page 137 of 181 

P102 

Increasing antibody yield and modulating final product quality using 

the Freedom TM CHO-S TM production platform 

Michelle Sabourin 1* , Ying Huang 1 , Prasad Dhulipala 3 , Shannon Beatty 1 , 

Jian Liu 1 , Peter Slade 2 , Shawn Barrett 3 , Shue-Yuan Wang 1 , Karsten Winkler 4 , 

Susanne Seitz 4 , Thomas Rose 4 , Volker Sandig 4 , Peggy Lio 1 , Steve Gorfien 3 , 

Laurie Donahue-Hjelle 3 , Graziella Piras 1 

1 Life Technologies TM , Frederick, MD, 21704, USA; 2 Life Technologies TM , 

Eugene, OR, 97402, USA; 3 Life Technologies TM , Grand Island, NY, 14072, USA; 

4 ProBioGen AG, Goethestrasse 54, 13086 Berlin, Germany 

E-mail: michelle.sabourin@lifetech.com 


Background: Cell line development (CLD) is a critical step in the 

generation of biotherapeutics, but it is still hindered by several pain 

points, including the lengthy and labor-intensive workflow needed to 

isolate desirable clones, lack of reproducibility, as well as potential 

protein quality issues. Over the last decade, antibody titers in 

mammalian cell culture systems in excess of 3 g/L have been achieved 

through the use of novel media and feeds. However, it is still a 

challenge to consistently and rapidly create a stable cell line and a cell 

Figure 1(abstract P102) Cloning medium choice does not impact clone titer distribution. The same Molecule 1-producing stable pools were seeded for 

limiting dilution cloning in either a lean cloning medium prototype or CD FortiCHO TM Medium. Following clone scale-up, the top clones from each 

process were assessed in shake flasks using a simple fed-batch process; day 7 data are shown. While there are differences in the absolute numbers, 

especially for the top 3 clones, overall the clones produced similarly whether they were isolated from lean cloning medium prototype or from CD 

FortiCHO TM Medium.



Table 1(abstract P102) Molecule 1 clone productivity during scale-up from shake flask to bioreactor 

Molecule 1 clone Shake flask simple fed-batch (g/L) Shake flask fed-batch (g/L) Bioreactor fed-batch (g/L) 

1 0.59 0.87 2.2 

2 0.55 1.06 1.5 

3 0.52 0.96 3.3 

culture process capable of supporting both high antibody yield and 

acceptable post-translational modifications while managing the effort 

required for execution of the workflow. The goal of the study was to 

develop a robust and reproducible stable cell line workflow to generate 

scalable high-producing clones in less than 6 months, with industrystandard 

titers and desirable product quality using minimal effort. 

Using CHO-S as the host cell line, we first evaluated if a single medium 

could be used for the entire CLD workflow, therefore avoiding the issues 

and complications of changing media during this process. We 

investigated if a formulation previously shown to increase titer as a 

production medium could in fact be used for all CLD steps, from 

transfection to stable pool isolation all the way through to clone 

productivity, without compromising titers or performance. The same rich 

production medium was used in limiting dilution cloning and compared 

to a lean cloning medium prototype. Furthermore, robustness of the 

workflow was verified by testing multiple molecules. We also explored 

reducing effort by streamlining all the steps of the workflow. Finally, we 

assessed top clone scalability and expressed product quality. We tested 

whether clones chosen only by titers responded well to scale-up and 

process development in a model bioreactor setting. In addition, product 

glycosylation from these clones was compared to the same molecule 

produced in CHO DG44 cells, a well-characterized production platform. 

Results: Our results show that cell growth during selection and 

productivity assessment were affected by both the basal medium and 

nutrient feed strategy. Stable pools generated in either CD OptiCHO TM 

Medium or CD FortiCHO TM Medium gave the most desirable outcomes 

in terms of recovery time and productivity. We also found that CD 

FortiCHO TM Medium can be used for every step of the CLD workflow, 

including limiting dilution cloning. When we performed a limiting 

dilution cloning using the same pool seeded in either a lean cloning 

medium prototype or in CD FortiCHO TM Medium, we found that the top 

10 clones isolated from each medium showed similar average titer 

trends (Figure 1). We also established the robustness of the selection 

scheme by demonstrating that all tested molecules show an increase in 

cell pool titer during a two-stage selection scheme that uses 

simultaneous puromycin and methotrexate treatment. In addition, the 

resulting clones can readily be scaled up to bioreactors: clones that 

were producing ~0.5 g/L in simple fed-batch (glucose feed only) using 

shake flasks readily scaled-up to a DasGip bioreactor and produced up 

to 3.2 g/L under fed-batch mode with minimal process development 

(Table 1). Finally, we demonstrated that the majority of clones isolated 

are stable for productivity, and that the glycosylation pattern of the 

same molecule produced in either CHO DG44 or CHO-S TM cells was 

indistinguishable. 

Conclusions: Theuseofasinglemediumfortheentireworkflowis 

revolutionary, and has the distinct advantage of avoiding any need for 

media adaptation at any point in the workflow, whether it be preceding 

or following cloning, or for productivity assessment. This in turn avoids 

any undesirable genetic selection that may occur during such adaptation 

steps, and facilitates streamlining the workflow for ease of use and 

efficiency. Clones are easily scaled from shake flask to bioreactor and 

produce 2-3 g/L with minimal process development. Product 

glycosylation in top CHO-S TM clones was comparable to historical data 

from CHO DG44-derived clones expressing the same molecule. In 

addition, the establishment of clone stability and acceptable glycosylation 

patterns are key attributes required for regulatory approval of 

biotherapeutic production. Together, these results demonstrate the 

capabilities of the Freedom TM CHO-S TM Kit as an efficient and robust 

stable CLD platform, which can be accessed without the burden of 

milestone or royalty payments. 

Page 138 of 181 

The top 3 clones expressing Molecule 1 were cultured in CD FortiCHO TM Medium in shake flasks using a simple fed-batch or fed-batch process, and in a DasGip 

bioreactor using a fed-batch process. Clone titers increased 3- to 6-fold from simple fed-batch shake flask culture to fed-batch bioreactor culture. 

P103 

Single use bioreactors for the clinical production of monoclonal 

antibodies – a study to analyze the performance of a CHO cell line and 

the quality of the produced monoclonal antibody 

Sonja Diekmann * , Constanze Dürr, Alexander Herrmann, Ingo Lindner, 

Daniela Jozic 

Roche Diagnostics GmbH, Pharma Biotech Penzberg, 82377 Penzberg, 

Germany 

E-mail: Sonja.Diekmann@roche.com 


Background: In recent years, the use of disposables in the pharmaceutical 

industry has increased extensively. Disposables can be used in many areas 

of biopharmaceutical production. The use of disposables not only reduces 

investment costs, but also requires less manpower to operate, since time 

consuming change-over procedures are significantly reduced. In addition, 

disposables enable a high flexibility by reducing unit operation times such 

as cleaning and sterilization as well as the validation of these procedures [1]. 

Disposable bioreactors can be subdivided into two main groups, static and 

dynamic systems. The dynamic systems differ with regard to the power 

input in stirred, vibromixed or wave-induced systems [2]. Since the power 

input, the mixing time, the tip speed and the oxygen transfer coefficient 

may influence the cell culture process and the product quality itself, a 

thorough characterization of the system used is necessary [3]. SSB (stainless 

steel bioreactors) and SUB (single use bioreactors) differ in terms of their 

physical design. Usually, an SSB is equipped with two or three stirrer blades. 

In contrast, stirred SUBs usually have just one stirrer blade. In addition, 

the design and the position of the stirrer differ significantly. These 

distinctions lead to different physical characteristics regarding the power 

input, mixing time, and tip speed. The SUBs are characterized by a 

significantly lower power input and tip speed and a significantly higher 

mixing time. In order to maintain a sufficient oxygen transfer coefficient, the 

single use bioreactor is equipped with a micro sparger with a pore size of 

25 µm. Furthermore, pure oxygen can be used to achieve higher dissolved 

oxygen concentrations in the cell cultivation medium. This study describes 

the influence of these technical differences on the performance of a CHO 

cell line and the product quality of a monoclonal antibody. 

Results: After inoculation, the cells were cultured in both systems in a fed 

batch mode with two continuous feeds for twelve days. Figure 1a shows the 

viable cell density as a function of time. Whereas the overall growth 

characteristic of the cells in both systems are comparable over the whole 

cultivation time, from 92 h until 188 h, the cells in the SSB are characterized 

by a significantly (*) faster growth (p< 0.05). The viability of the cells in both 

systems remains between 90% and 100% over the whole cultivation time 

(data not shown). Figure 1b shows the LDH activity in both systems as a 

function of time as an indicator of cell lysis. Up to 68 h cultivation time the 

LDH activity in both systems is comparable. Thereafter, the LDH activity 

measured in the SUB is significantly (*) higher in comparison to the SSB (p < 

0.05). Figure 1c shows the product titer [%] produced by the cells in the SSB 

in comparison to the SUB and Figure 1d shows the specific productivity. 

Between 140 h and 250 h the product titer in the SSB is slightly higher in 

comparison to the SUB, whereas the titer at harvest is comparable. Since the 

growth of the cells in the SSB is faster in comparison to the cells in the SUB, 

the specific productivity of the cells cultivated in the SUB is higher. Figure 

1d shows the IEC data of the antibody produced by the CHO cells in the 

SUB and the SSB. Whereas the acidic region of the antibody of the SSB is 

slightly higher in comparison to the antibody of the SUB the difference is 

not significant. The main peaks are comparable. The basic region of the 

antibody of the SSB is slightly lower in comparison to the SUB, balancing



Figure 1a(abstract P103) Viable cell density. 

Figure 1b(abstract P103) LDH activity. 

Figure 1c(abstract P103) Product titer. 

Page 139 of 181



Figure 1d(abstract P103) IEC pattern. 

Table 1(abstract P103) SEC pattern 

Bioreactor-type Monomer 

[%] 

SSB 99.7 ± 0 0.267 ± 0.047 0.1 ± 0 

SUB 99.7 ± 0 0.3 ± 0 0.1 ± 0 

Figure 1e(abstract P103) Glycopattern. 

the slight increase in acidic region observed for the antibody derived from 

the SSB. 

The SEC patterns of both products are almost identical (Tab. 1). The 

glycopatterns of the mAB produced in the SUB and of the mAB produced 

in the SSB shows no significant differences (Fig. 1e). The G0 fraction of the 

mAB produced in the SSB is slightly higher in comparison to the G0 

fraction of the mAB produced in the SUB whereas the G1 fraction of the 

mAB produced in the SSB is slightly lower compared the mAB produced in 

the SUB. The G2 fraction is very similar. The G0-Fucose value of the mAB 

produced in the SSB is higher than for mAB produced in the SUB, which 

leads to a higher overall a-fucose value for the SSB derived product 

compared to the SUB derived product. However, all these differences are 

not significant. All other fractions are comparable between both 

antibodies. To investigate the influence of both bioreactor types on the 

impurity profile, the DNA and HCP values of the harvested supernatant 

were compared. Figure 1f shows the specific DNA concentration (DNA 

HMW 

[%] 

Page 140 of 181 

LMW 

[%] 

concentration divided by viable cell density) of the harvested supernatant 

of the SSB and the SUB. The specific DNA concentration of the harvested 

supernatant of the SSB is slightly higher compared to the SUB. However, 

theses differences are not significant. Figure 1g shows the specific HCP 

concentration measured in the harvested supernatant of the SSB and the 

SUB. The specific HCP concentration (HCP concentration divided by viable 

cell density) of the SUB is slightly higher compared to the specific HCP 

concentration of the SSB. Again, these differences are not significant. 

Conclusions: The present study describes the influence of a single use 

bioreactor on the performance of a production CHO cell line, on the 

product quality of the produced antibody and the process related 

impurities in comparison to a commercial stainless steel bioreactor. It 

seems that the microsparger of the SUB lead to a cell damage, which is 

measured by LDH activity. Nevertheless, our findings indicate that this 

cell damage has no influence on the productivity, the concentration of 

DNA and HCP and, most importantly on the quality of the antibody.



Figure 1f(abstract P103) DNA concentration. 

Figure 1g(abstract P103) HCP concentration. 

References 

1. Brecht R: Disposable bioreactors: maturation into pharmaceutical 

glycoprotein manufacturing. Adv Biochem Eng Biotechnol 2009, 115:1-31. 

2. Eibl R, Eibl D: Application of Disposable Bag Bioreactors in Tissue 

Engineering and for the Production of Therapeutic Agents. Adv Biochem 

Eng Biotechnol 2008. 

3. Hanson MA, Brorson KA, et al: Comparisons of optically monitored smallscale 

stirred tank vessels to optically controlled disposable bag 

bioreactors. Microb Cell Fact 2009, 8:44. 

4. Hacker DL, De Jesus M, et al: 25 years of recombinant proteins from 

reactor-grown cells - where do we go from here? Biotechnol Adv 2009, 

27(6):1023-1027. 

P104 

Development of live cultural pandemic influenza vaccine Vector-Flu 

Elena A Nechaeva 1* ,TatyanaYSen’kina 2 , Alexander B Ryzhikov 2 , Irina F Radaeva 2 , 

Ol’ga G P’yankova 2 ,Natal’ya V Danil’chenko 2 , Tatyana M Sviridenko 2 , 

Marina P Bogryantzeva 2 ,Natal’ya V Gilina 2 , Nikolay A Varaksin 2 , 

Tatyana G Ryabicheva 2 , Irina V Kiseleva 3 , Larisa G Rudenko 3 

1 Department of Cell Culture Technology, SRC VB VECTOR, Novosibirsk, 630559, 

Russia; 2 SRC VB VECTOR, Novosibirsk, 630559, Russia; 3 Institute of Experimental 

Medicine, Russian Academy of Medical Sciences, St. Petersburg, 197376, Russia 

E-mail: nechaeva@vector.nsc.ru 


Page 141 of 181 

Background: The threat of pandemic A/H1N1 influenza remains a matter 

of considerable public concern. Recent influenza outbreaks underline the 

importance of rapid production of a reserve of vaccine sufficient for 

pandemic and interpandemic periods. Traditional vaccines intended for 

seasonal influenza do not satisfy this demand, because they are unable to 

induce cross-reactive antibodies to pandemic flu. 

Live influenza vaccines made in the cell culture offer potential advantages 

because it is possible to produce a considerable amount of these vaccines 

over a short period. Their use eliminate concerns about allergic reactions to 

egg proteins, and they allow the antigenic composition of the vaccine to be 

closer to the circulating influenza virus strains. 

The goal of this work was to develop a live attenuated vaccine against 

pandemic influenza, Vector-Flu, derived from cold-adapted strain A/17/ 

California/2009/38(H1N1) and to study the immunogenic and protective 

properties of the vaccine using ferrets and mice. 

Materials and methods: Cell culture: MDCK from Cell Culture Collection 

of SRC VB VECTOR. Cells were passed in serum-free SFM4MegaVir 

medium (USA). The characteristics of the MDCK cell line were studied in 

accordance with WHO [1]. 

Viruses: The vaccine strain A/17/California/2009/38(H1N1) was generated 

at the Institute of Experimental Medicine (St. Petersburg, Russia) by 

reassortment of the cold-adapted attenuated A/Leningrad/134/17/57 

(H2N2) master donor virus with the pandemic strain A/California/7/2009 

(H1N1). The A/Chita/3/2009(H1N1) influenza virus was obtained from 

VECTOR’s Collection of Microorganisms.



Table 1(abstract P104) The titers of ferret blood serum to A/H1N1 influenza virus strains before and after 

immunization with the Vector-Flu vaccine determined by HAI and microneutralization assay 

Serological method HAI Microneutralization 

Influenza virus strain A/Chita/3/2009 A/California/7/2009 A/Chita/3/2009 A/California/7/2009 

Number of vaccinations 0 1 2 0 1 2 0 1 2 0 1 2 

GMT a over the group



Figure 1(abstract P105) Viable cell density (●, ○), viability (▲, △) and perfusion rate (■, □) in perfusion processes using ATF system (A) and TFF system (B). 

Closed symbols indicate the ATF9 perfusion experiment, whereas the open symbols represent the ATF15 and TFF10 perfusion experiments. 

Supplementations of glucose or glutamine were performed according to cell 

need. The pH was controlled by adding 0.5 M Na 2CO 3 or pulsing CO 2 into 

the headspace. The cell density, viability, pH, pCO2, concentrations of 

glucose, lactate, glutamine and ammonia were measured by Bioprofile FLEX 

(Nova Biomedical). Antifoam C (Sigma Aldrich, US) was added up to 50 ppm 

concentration in the bioreactor either by boost addition or by continuous 

pumping. The mAb concentration was measured by protein A HPLC. 

Results: Cell density, viability and perfusion rate: Two perfusion 

experiments (ATF9 and ATF15) were conducted using ATF (Figure 1A) and 

one perfusion culture (TFF10) using TFF device (Figure 1B). In experiments 

ATF9 and TFF10, cell density was maintained between 20-30 x 10 6 cells/ 

mL by daily cell bleeds for 2 weeks using one HF. Interestingly, in 

experiment ATF9, the use of an ATF flow rate of 0.7 L/min was not 

sufficient to remove bubbles entrapped in the HF resulting in a decrease 

in viability (93.5% vs. 97%). Therefore, from day 9, the flow rate was 

increased to 1 L/min, i.e. shear stress of 3400 s -1 . Experiment ATF15 was 

performed at 1 L/min. 

In experiment ATF15, a cell density of 132 x 10 6 cells/mL was reached after 

10 days of culture of exponential growth. Reaching this density coincided 

with interruption of ATF function due to insufficient pressure to push the 

highly viscous cell broth, showing the limit of this system at this very high 

cell density when using a non-pressurisable disposable bioreactor. A batch 

culture was then carried out between days 13 and 20 (data not shown) 

after which perfusion was re-started. A maximal cell density of 123 x 10 6 

cells/mL was obtained confirming the first observed maximal cell density. 

The culture was then continued for 4 days at cell density around 100 x 10 6 

cells/mL by performing daily cell bleeds showing that healthy high cell 

density culture could be obtained somewhat under 130 x 10 6 cells/mL in 

the same culture. 

Using the TFF, exponential cell growth was obtained at similar rate as with 

ATF, the cell density was then maintained around 100 x 10 6 cells/mL by 

daily cell bleeds for 2 weeks. Then, higher cell densities were achieved, up 

to more than 200 x 10 6 cells/mL for 2 days. Reaching 214 x 10 6 cells/mL 

coincided with the limit of this system due to high pressure in re-circulation 

Table 1(abstract P105) Comparison of mAb production using ATF or TFF system at day 17 with cell densities 

maintained at 20-30 x 10 6 cells/mL 

ATF TFF 

Production in harvest (g) 9 7 

Yield (production in harvest/total production) (g/g in %) 75 55 

Total removal of mAb in cell bleeds/total production (g/g in %) 19 30 

Residual mAb mass in bioreactor/total production (g/g in %) 6 15 

Page 143 of 181



loop (1 bar) related to the viscosity, oxygenation limitation and CO 2 

accumulation (31 kPa). During these runs, CHO cells were cultivated at high 

and very high cell densities with a cell viability maintained above 90% 

(mostly around 95%). 

Total mAb production: Over an 17-day period of culture with cell 

densities maintained at 20-30 x 10 6 cells/mL, a total of 9 g and 7 g of mAb 

were harvested in the ATF9 and TFF10 processes respectively (Table 1). 

The lower yield obtained using the TFF was mainly due to mAb removed 

in cell bleeds (30% in TFF vs. 19% in ATF). In both cultures, the cell specific 

production rates of mAb were similar and around 10-15 pg/cell/day (data 

not shown). 

Conclusions: A very high cell density of 100 x 10 6 cells/mL was stably 

maintained in growing phase and at high viability by performing cell 

bleeds in a perfused WAVE Bioreactor using TFF or ATF cell separations. 

With the settings used here, the maximal cell density was limited to 

214 x 10 6 cells/mL using TTF and 132 x 10 6 cells/mL using ATF. Using 

TFF, the cell density could not be further increased due to the limitations 

of membrane capacity (for the encountered high viscosity), oxygenation 

and CO 2 level, and using ATF due to pressure limitation to push highly 

viscous fluid and use of non-pressurisable disposable bioreactor. Thus, the 

TFF system allowed reaching higher cell densities. To our knowledge, it is 

the first time that a CHO cell density of more than 200 x 10 6 cells/mL 

was achieved in a wave-agitated bioreactor. 

Higher retentions of mAb were observed using the TFF system than using 

the ATF system. In perfusion, the major effect of this retention was loss of 

mAb in the cell bleeds. Consequently, ATF system was more favourable 

for production at stable cell density maintained by cell bleeds. 

The use of a disposable bioreactor equipped with a disposable separation 

device offers a solution alleviating technical and sterility challenges 

occurring in perfusion processes. 


This work was financed by GE Healthcare. Thank you to Eric Fäldt, 

Christian Kaisermayer and Craig Robinson from GE Healthcare. We 

acknowledge The Swedish Governmental Agency for Innovation Systems 

(VINNOVA), which supported the lab activity. We thank IMED, Sweden, for 

their kind permission of using their research cell line. We would like also 

to acknowledge Refine Technology for helpful inputs. 

P106 

Utilizing Roche Cedex Bio analyzer for in process monitoring in biotech 

production 

Dörthe Druhmann * , Sabrina Reinhard, Felizitas Schwarz, Christina Schaaf, 

Katrin Greisl, Tim Nötzel 

Roche Diagnostics GmbH, Pharma Biotech Penzberg 


Introduction: One task during development and control of bioprocesses 

used for production of recombinant proteins is to deliver fast, accurate 

and reliable analytical process data. 

Monitoring of nutrients and metabolites in animal or bacterial cell culture is 

essential to avoid limitations or toxic accumulation during fermentations 

that can effect cell growth, viability, product yield and quality. Within certain 

fermentation processes low levels of nutrients trigger in-process actions 

such as feed. Other parameters are used to track culture reproducibility and 

to gain process understanding for process development and validation. 

Typical parameters in mammalian cell culture are glucose, lactate, 

glutamine, glutamate, ammonia, sodium and potassium. Therefore it is 

common to use multi-analyzer systems based on enzymatic-dependent 

biosensors and ionselective potentiometry. 

Furthermore the release of lactate dehydrogenase (LDH) into the 

fermentation media is used as an indicator of cell death during fermentation 

processes therefore a 96-well based photometric assay is used . The 

measurement of product titer like IgG is needed to track productivity or 

yield during fermentation and protein purification processes and is mostly 

done by HPLC methods. 

One major motivation for this new Bioprocess analyzer lies within the 

eminent issues of enzyme membrane based analytical technology such 

as: short life cycles as well as poor or changing quality of the enzyme 

membranes, high material costs, non-linearity of measurements and in 

particular insufficient sensitivity and accuracy. 

Additionally the laborious maintenance required to operate different 

analytical devices and labor intensive sample management as well as 

manual steps to avoid operator dependent variability needed to be reduced. 

Applying diagnostic knowledge to bioprocess care: Combining the 

knowledge of 25 years of diagnostic healthcare with expert knowledge in 

fermentation processes a new Bioprocess analyzer (Cedex Bio analyzer*) was 

developed. The new Cedex Bio analyzer stands out with increased sensitivity 

plus enlarged measurement ranges compared to currently used methods 

and analyzers. 

An automated dilution increases the measurement ranges and reduces 

operator variability because no manual dilution is needed before 

applying the sample to the Cedex Bio analyzer. 

The aim of this evaluation and method validation was to compare this 

new device to common analytical systems. 

Validation and correlation studies: Reference standards were analyzed 

at least once a day before and after each tray of samples to control the 

performance of the membrane analyzer. Day to day and side by side 

comparability as well as accuracy and linearity of the Cedex Bio analyzer are 

superior to the membrane analyzer as shown in Figure 1. 

An increased sensitivity allows to operate nutrient limited fermentations 

and likewise to detect remote changes within the metabolite profile of 

the fermentation process. 

Monitoring of fermentations processes using a membrane analyzer 

required laborious maintenance, a lot of calibrations and permanent 

control of recovery. 

However correlation studies conducted with samples of a fed-batch 

mammalian cell fermentation show that the Cedex Bio analyzer easily 

replaces the membrane analyzer with comparable batch data (Figure 2). 

Furthermore additional parameters like LDH or IgG can be analyzed from 

the same sample. 

Summary: ο The Cedex Bio analyzer combines three devices to one with 

the possibility to measure up to 9 parameters in fermentation browth at 

once and from the same sample within a few minutes. 

ο Automated dilution reduces operator dependent manual steps and 

variability. 

Table 1(abstract P106) measurement ranges current methods versus Cedex Bio 

Parameter Current methods Cedex Bio Cedex Bio 

w/o dilution automated dilution 

glucose [mg/L] 200 – 15.000 1) 

20 – 7.500 20 – 75.000 

lactate [mg/L] 200 – 5.000 1) 

18 – 1.400 18 – 14.000 

ammonia [mg/L] 3,6 – 450 1) 

0.48 – 12 0.48 – 480 

glutamine [mg/L] 30 – 877 1) 

60 – 1.500 60 – 7.500 

glutamate [mg/L] 30 – 882 1) 

15 – 1.500 15 – 7.500 

sodium [mg/L] 920 – 5.006 2) 

460 – 5.750 460 – 5.750 

potassium [mg/L] 39,1 – 978 2) 

39 – 1.170 39 – 1.170 

LDH [U/L] 20 – 150 3) 

20 – 1.000 20 – 10.000 

IgG [mg/L] 35 – 1.500 4) 

10 – 1.600 10 – 16.000 

1) Membrane analyzer, 2) Ion selective electrode 3) 96-well-plate assay, 4) HPLC method. 

Page 144 of 181



ο Methods based on wet chemical photometric assays, ionselective 

electrodes and turbidity assays are easy to handle, reliable and 

robust. 

ο The compact analyzer helps to save laboratory space and costs for 

different instruments. 

ο Sensitive, precise and accurate analytical data allow a tight control of 

cell culture processes. 

ο The assay portfolio will also be suitable for microbial fermentation with 

additional parameters like acetate or ethanol. 

ο The Cedex Bio analyzer combines three devices to one with the 

possibility to measure up to 9 parameters in fermentation browth at once 

and from the same sample within a few minutes. 

* CEDEX is a trademark of Roche 

Page 145 of 181 

Figure 1(abstract P106) Comparison of recovery values of the Cedex Bio analyzer versus a membrane analyzer. Range of recovery of reference standards 

(quality control material, n ~ 250, t = 6 months). 

Figure 2(abstract P106) Field Data with samples from mammalian cell culture: Cedex Bio analyzer versus a membrane analyzer. The slope < 1 due to a 

constant overestimation of the glucose concentration by the membrane analyzer. 

P107 

LDH-C can be differentially expressed during fermentation of CHO cells 

Berthold Szperalski 1* , Christine Jung 1 , Zhixin Shao 1 , Anne Kantardjieff 2,3 , 

Wei-Shou Hu 2 

1 Pharma Biotech, Roche Diagnostics GmbH, 82377 Penzberg, Germany; 

2 University of Minnesota, Minneapolis, MN 55455, USA; 3 Alexion 

Pharmaceuticals, Cheshire, CT 06410, USA 

E-mail: berthold.szperalski@roche.com 


Methods: CHO-cells producing a recombinant human antibody were 

cultivated in a proprietary proteinfree medium and inoculated in 4 x 2L



Table 1(abstract P107) 

Correlation to PC Gene set Number of genes in gene set Nominal p-value 

Positive to PC 1 Cell cycle 26 0 

DNA replication 24 0 

Cytoskeleton 67 0.02 

Microtubule organizing center 24 0.04 

Negative to PC 1 Golgi apparatus 50 0 

Cell-cell signaling 48 0 

Positive to PC 2 RNA processing 43 0.01 

Proteolysis 46 0.05 

Negative to PC 2 DNA replication 32 0.04 

stirred tank bioreactors. Bioreactors were controlling pH, pO2 and 

temperature. A fixed feeding protocol was used to overcome the 

limitation of consumed medium components. Temperatures of 2 cultures 

were shifted at day 4 from 37°C to 34°C. Daily samplings of the cultures 

were performed to monitor cell density and viability by using an 

automated Cedex cell counter and the trypan blue exclusion method. 

The supernatant of the culture was monitored for product concentration, 

glucose, glutamine, lactate, ammonium. Measurement of LDH (lactate 

Figure 1(abstract P107) 

Page 146 of 181 

dehydrogenase ) in cell culture supernatant was used as an indicator of 

cell lysis. Sedimented cells of cell culture samples were prepared and 

cRNA was processed according to Affymetrix standard procedures.[1] 

and hybridized with custom CHO Affymetrix arrays from the 

Consortium for Chinese Hamster Ovary (CHO) Cell Genomics [2]. 

Results: The comparison of temperature shifted and control cultures 

showed significant differences in the growth curves of the experiment. 

Temperature shift induced an early shift to the plateau phase. It reduced



the cell death. Cell specific productivity was slightly higher. Lactate 

consumption was higher and started earlier than in control cultures (data 

not shown). PCA (principal component analysis) was used to compare 

expression ratios at different temperatures. PC 1 showed that most 

expression changes are onset at day 6 and maintained throughout the 

rest of the culture. Transcriptome analyses showed several significant 

changes after the temperature shift (Table 1). One outstanding result is 

the upregulated RNA of LDH-C (Figure 1). LDH-A RNA expression showed 

no significant change after temperature shift. 

Discussion: LDH-C is known to be present in sperm cells , testis cells 

and some tumors [3] but is not reported to be regulated in CHO-cell 

lines. In sperm cells LDH-C is known to have different kinetic properties 

compared to A and B isoforms of LDH preferring lactate as substrate 

[4]. LDH-C is localized in cytoplasm and in specific “sperm type 

mitochondria” and seems to be integrated in a shuttle system for the 

transfer of reducing activity into the mitochondrial matrix [7][8]. An 

pseudogene association with mitochondrial cyclophilin D is reported in 

thegenebankofmousegenome[9].TheroleofLDH-CinCHO-Cellsis 

still unclear. The influence of temperature shift under normal body 

temperature seems to induce a special situation for sperm cell 

migration. LDH-C helps sperm cells to survive in lactic acid containing 

micro milieus of the oviduct. It allows lactic acid to be an energy 

source. These functions could be mimicked in a high lactate containing, 

temperature shifted fermentation process with CHO cells. LDH-C can 

also be regulated by hormonal mechanisms. They are known to have 

slight regulatory influence on the transcriptional expression [5]. 

Selective inhibitors of LDH isoforms are described [6]. Specific inhibitors 

for LDH -C are proposed as antifertilizing drugs [6]. Inhibitors to LDH-A 

and -B could help to favor LDH-C and so reduce lactate production. 

LDH-C is an interesting target for engineering manufacturing processes 

with cell lines like CHO cells for shifting these cells to aerobic lactate 

metabolism and improving growth performance. 

References 

1. Kantardjieff A, Jacob NM, Yee JC, Epstein E, Kok YJ, Philp R, Betenbaugh M, 

Hu WS: Transcriptome and proteome analysis of Chinese hamster ovary 

cells under low temperature and butyrate treatment. J Biotechnol 2010, 

145:143-159. 

2. Consortium for Chinese Hamster Ovary (CHO) Cell Genomics. 2007 

[http://hugroup.cems.umn.edu/CHO/cho_index.html]. 

3. Koslowski M, Tureci O, Bell C, Krause P, Lehr HA, Brunner J, Seitz G, 

Nestle FO, Huber C, Sahin U: Multiple splice variants of lactate 

Figure 1(abstract P108) Curves, reflecting change of probability, versus stiffness parameter, at different values of k. 

Page 147 of 181 

dehydrogenase C selectively expressed in human cancer. Cancer Res 

2002, 62:6750-6755. 

4. Blanco A, Burgos C, Gerez de Burgos NM, Montamat EE: Properties of the 

testicular lactate dehydrogenase isoenzyme. Biochem J 1976, 

153:165-172. 

5. Ohsako S, Kubota K, Kurosawa S, Takeda K, Qing W, Ishimura R, Tohyama C: 

Alterations of gene expression in adult male rat testis and pituitary 

shortly after subacute administration of the antiandrogen flutamide. J 

Reprod Dev 2003, 49:275-290. 

6. Yu Y, Deck JA, Hunsaker LA, Deck LM, Royer RE, Goldberg E, Vander 

Jagt DL: Selective active site inhibitors of human lactate dehydrogenases 

A4, B4, and C4. Biochem Pharmacol 2001, 62:81-89. 

7. Burgos C, Maldonado C, Gerez de Burgos NM, Aoki A, Blanco A: 

Intracellular localization of the testicular and sperm-specific lactate 

dehydrogenase isozyme C4 in mice. Biol Reprod 1995, 53:84-92. 

8. Gladden LB: Lactate metabolism: a new paradigm for the third 

millennium. J Physiol 2004, 558:5-30. 

9. Mouse DNA sequence from clone RP23-313I15 on chromosome 13 

Contains a peptidylprolyl isomerase D (cyclophilin D) (Ppid) pseudogene 

and a sperm specific lactate dehydrogenase 3C (Ldh3) pseudogene, 

complete sequence. GenBank: AL606965.20 2011 [http://www.ncbi.nlm. 

nih.gov/nucleotide/AL606965.20]. 

P108 

Stem cell: from basic theoretical assumptions and mathematical 

concepts to the computational models 

Katya Simeonova 1* , Ganka Milanova 2 

1 

Institute of Mechanics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria; 

2 

University of Architecture, Civil Engineering and Geodesy, 1000 Sofia, 

Bulgaria 

E-mail: katyas@bas.bg 


Background: Stem cells (SC) and their therapeutic applications have a 

great promise for treatment of many human diseases, [1]. Characteristics 

of Embryonic Stem Cells (ESC) have been presented in [2]. The aim of 

the paper presented has been formulated as follows: to give some 

classical mechanics and mathematics theories for SC studies. 

Mathematical models for cancer diseases and neurological disorders have 

been discussed too.



Materials and methods: Models, including a viscoelastic continuum, a 

combination of viscoelastic fluid have been developed as well, [3]. By 

relatively modern (for that time) microscopic techniques, developed 

during the eighteenth have been observed the images of living cells. 

Later using imaging mechanics, have been discovered AFM, imaging for 

study of living cells. High resolution of AFM imaging “has provided 

information on the structure. It has been proved as well, [4] that blood, 

blood vessels and nerves, could be tested mechanically. 

Mathematical models of SC for neuroscience: Theoretical concepts 

and mathematical models have been proposed for explanation 

quantitatively biological mechanisms. Mechanisms and models of 

cellular State Transition have been discussed too. Other periodic 

hematological diseases involve oscillations in all of the blood cells. In 

the paper [5], has been developed a simple mathematical model0. The 

probabilities for CSC to reach levels, compatible with the diagnosis of 

acute leukemia, by CSC, as well as probability for extinction have been 

computed analytically. 

An analytical model: The probability “that at least once, the population 

will have M 1CSC, approximately given by: 

1 r 

pM ( 0, M1) 

= 

1 r 

− 

− 

−M0 

−M 

Here M 1CSC, at time t =0,r is the relative fitness parameter of CSC. The 

general expression for the fixation probability could be given as follows: 

( ) = 

p M , M 

0 1 

q 

q 

( M0 

) 

( M ) 

1 

For the birth-exports process authors in [11], considered the transition 

probability T*(k, NSC ), that numbers of CSC increasing from k to k+1, 

given by formula: 

p k 

CSC ( ) = 

kr 

kr + N − k 

( ) 

A computational model, results: Solving the equation (4) we obtained 

the next formula for k: 

pN 

k = 

p + r − pr 

Here all parameters: p, r and N have been presented in the work [5]. So 

we investigate the effect of parameter k, on the probability p CSC(k). 

Numerical FORTRAN programs designed by authors have been used. By 

numerical experiments have been obtained dependencies at different k. 

Results, have been analyzed and shown (Figure 1). 

Conclusions: Future problems and current studies on SC are: SC is very 

promising tool for various biopharm applications, Future technologies 

will be enabled fuller understanding of SC, Future directions for human 

S Cell Culture optimization, ES could be used as vechile for gene 

therapy. 

Acknowledgments: We thank you to Professor Nicole Borth, and to 

Organizers of ESACT2011, for their great cooperation to attend ESACT 

Meeting in Vienna, 15-18 May, 2011 and to present both poster reports. 

References 

1. Yanhong S: Stem Cell Research and therapeutics: Advances in 

Biomedical Research. SPRINGER: Deunis O 2008. 

2. Simeonova K: Nucleic Acids Res. ESF -UB Conference Rare Diseases II: 

Hearing and Sight Loss Costa Brava, Spain 2009, 105[http://www.esf.org/ 

conferences/09295]. 

3. Satin BD, et al: Nucleic Acids Res.32:4876-4879. 

4. Ingo R, Radtke F: Stem cell biology meets system biology. Meeting Review, 

Development 136 2009, 3525-3530. 

5. Dingli D, et al: Stochastic dynamics of hematopoietic tumor stem cells. 

Cell Cycle 2007, 6:e1-e6. 

(1) 

(2) 

(3) 

(4) 

Page 148 of 181 

P109 

Comparison of the activity and pluripotency maintaining potential of 

human leukemia inhibitory factor (LIF) produced in E.coli and CHO cells 

Claas Haake 1 , Sophia Bonk 1 , Jana Parsiegla 1 , Magda Tomala 1 , Komal Loya 2 , 

Malte Sgodda 2 , Tobias Cantz 2 , Axel Schambach 3 , Cornelia Kasper 1 , 

Thomas Scheper 1* 

1 Institute for Technical Chemistry, Leibniz University of Hanover, 30167 

Hannover, Germany; 2 Junior Research Group Stem Cell Biology, Cluster of 

Excellence REBIRTH; MPI Münster and Hanover Medical School, Hannover 

Germany; 3 Department of Experimental Hematology, Hanover Medical 

School, 30625 Hannover, Germany 

E-mail: scheper@iftc.uni-hannover.de 


Introduction: Leukemia inhibitory factor (LIF) is a polyfunctional cytokine 

with numerous regulatory effects in vivo and in vitro. In murine stem cell 

cultures it is the essential media supplement for the maintenance of 

pluripotency of embryonic and induced pluripotent stem cells. To explore 

if the glycosylation and/or other post-translational modifications are 

affecting this activity, we produced and isolated LIF from either 

eucaryotic cells (Chinese hamster ovary (CHO) cells) or procaryotes (E.coli) 

and compared their biological activities in this study. 

Results: 

Production in E.coli: To increase the solubility the LIF is expressed 

together with thioredoxin as a fusion protein. The genes for the fusion 

protein are separated on the vector (pET32b) by a TEV (Tobacco Etch 

Virus) protease cleavage site, in addition thioredoxin is expressed with a 

his-tag. The resulting construct was transformed into E.coli BL21(DE3), 

which were afterwards cultivated at 23°C. This relatively low temperature 

leads to increased solubility and yield of the target protein. The protein 

was afterwards purified from the fermentation broth by metal chelate 

affinity chromatography through the utilization of Zn 2+ ions immobilized 

on IDA membrane adsorbers. To cleave the thioredoxin from the fusion 

protein the obtained protein fractions were directly treated with 

recombinant TEV protease. The released hLIF was purified from the 

protein mixture using ion exchange chromatography [1]. 

Production in CHO cells: The utilized CHO SFS cell line (CCS, Hamburg, 

Germany) was transducted using a lentivirus with a pRRL vector, which 

coded for his-tagged humane LIF. Verification of the expression, as well 

as of the extracellular localization was carried out by intracellular flow 

cytometric staining and by western blot.Thecultivationinserumfree 

medium (ProCHO5, Lonza, Basel, Switzerland) was initially performed in 

spinner flasks. Afterwards the scale-up to 1.5 L bioreactor scale was 

accomplished. The cultivation was carriedoutat37°C,200rpm,andpH 

7.2. The first step of the purification of the concentrated supernatant was 

performed by metal chelate affinity chromatography, where Ni 2+ ions 

were immobilized. Afterwards the pooled protein fractions were purified 

by a polishing step by heparin affinity chromatography. 

Effects on murine iPS suspension cells: Cell growth: The cells were 

cultivated as cell spheres in suspension in DMEM medium supplied with 

10 ng LIF per ml. As positive control ESGRO LIF, which is produced in 

E.coli, was used. For each of the three LIFs used as well as for the 

negative control two cultures of 2 ml each were harvested using trypsin 

every day and the cell count was determined manually using trypan blue. 

For the long-term cultivation the cells were passaged twice a week. The 

obtained values for maximal cell density and the viabilities show the 

activity of the produced LIFs when compared with the positive and 

negative control but no discrepancy in their activities. 

>Pluripotency maintaining potential: Via flow cytometry the expression of 

the pluripotency markers SSEA-1 and Oct4 was determined using an anti- 

SSEA-1-PE antibody and measuring the expression of GFP, which stands 

under the control of an Oct4 promoter respectively. For the negative 

control an isotype control antibody was used for SSEA-1. For GFP, the 

results achieved were compared to the GFP expression of the cells at the 

start of the cultivation. The results show a slight decrease in SSEA-1 (FL-2) 

but not GFP (FL-1) expression over the duration of the cultivation, but no 

differences regarding the activities in accordance to the source of the LIFs. 

Effects on adherent growing murine ESC: Murine ESC, carrying an 

Oct4-GFP reporter construct (OG2-ESC) [2] were seeded on C3H irridatred 

mouse embryonic fibroblast cells in 6-well-plates with LIF concentrations 

of 40, 10, and 2.5 ng/ml. As positive control an E.coli-derived LIF was



Figure 1(abstract P109) Living cell count and viability against the cultivation time for the cultivation of the ES cells with 10 ng LIF/ml. 

used, which has been produced at the MPI Münster and has previously 

been tested. The media were changed every 2 days and the cells were 

passaged twice a week for 5 passages. After 24 days the cells were 

harvested and used for qRT-PCR. Within the qRT-PCR b-Actin was used as 

the housekeeping gene. The expression of the pluripotency markers Oct4 

and Nanog as well as marker proteins for the three germ layers (Trp63 

for ectodermal, AFP for endodermal and Brachyury for mesodermal 

differentiation) were quantified. The results show that the mesodermal 

marker is downregulated, which indicates spontaneous differentiation of 

the negative control into the mesodermal germ layer. Between the used 

LIFs no significant differences could be determined. 

Effects on murine ES suspension cells: Cell growth: The procedure was 

in accordance to the approach with the iPS cells and the three LIFs were 

also used in concentrations of 100 and 1 pg each. Supplementary the 

amount of apoptosis was measured after four days of culture using the 

Annexin-V Kit (Invitrogen, Carlsbad, CA, USA) for flow cytometry. The results 

clearly show activity for the concentrations of 10 ng (figure 1) and 100 pg 

but no considerably difference from the negative control for the 

concentration of 1 pg and also no differences for the LIFs regarding their 

sources were found. 

Pluripotency maintaining potential: ThemarkerproteinSSEA-1was 

measured flow cytometrically once a week for the culture containing 

10 ng/ml LIF as well as once after 14 days for the one containing 1 pg. The 

SSEA-1 expression remains at a level of above 97% when 10 ng LIF/ml 

were used, but is broadly decreased for the negative control. For the 

concentration of 1 pg LIF/ml SSEA-1 expression levels of about 90% after 

14 days were achievable although this amount of LIF showed no 

differences from the negative control in regards of cell viability. From 

these results we can state that with regard to the pluripotency maintaining 

potential the different LIF did not show detectable differences. 

Conclusion: The production and isolation of LIF from E.coli as well as 

from CHO cells was successfully established. The bioactivity of both 

proteins was demonstrated in various experiment using different cells 

and methods of detection. We conclude from our results that LIF from 

mammalian cells and LIF from prokaroytes are equally effective. 

Acknowledgement: This work was performed within the activities of the 

JRG “large scale cultivation” of the DFG cluster of excellence “Rebirth” 

(EXC 62/1) 

References 

1. Tomala M, Lavrentieva A, Moretti P, Rinas U, Kasper C, Stahl F, 

Schambach A, Warlich E, Martin U, Cantz T, Scheper T: Preparation of 

bioactive soluble human leukemia inhibitory factor from recombinant 

Escherichia coli using thioredoxin as fusion partner. Protein Expr Purif 

2010, 73(1):51-7. 

Page 149 of 181 

2. Szabó PE, Hübner K, Schöler H, Mann JR: Allele-specific expression of 

imprinted genes in mouse migratory primordial germ cells. Mech Dev 

2002, 115:157-160. 

P110 

Disulphide bond reduction of a therapeutic monoclonal antibody 

during cell culture manufacturing operations 

Brian Mullan 1* , Bryan Dravis 2 , Amareth Lim 3 , Ambrose Clarke 4 , Susan Janes 3 , 

Pete Lambooy 2 , Don Olson 2 , Tomas O’Riordan 1 , Bruce Ricart 3 , 

Alexander G Tulloch 2 

1 Manufacturing Science and Technology, Eli Lilly & Co, Kinsale, Cork, Ireland; 

2 Bioprocess R&D, Eli Lilly & Co, Indianapolis, Indiana, USA; 3 Bioproduct 

Analytical Chemistry, Eli Lilly & Co, Indianapolis, Indiana, USA; 4 Analytical 

Technical Operations, Eli Lilly & Co, Kinsale, Cork, Ireland 

E-mail: mullan_brian@lilly.com 


Background: Disulphide bonding is critical to maintaining immunoglobulin 

(IgG) tertiary and quaternary structure for therapeutic monoclonal 

antibodies (MAb). Both inter- and intra-chain disulphide bonds are formed 

intracellularly in the expression host prior to secretion and purification 

during MAb production processes. Disulphide bond shuffling has 

previously been reported for IgG 2[1,2] and disulphide-mediated armexchange 

for IgG 4[3,4], reflecting innate behaviour of these IgG classes. 

However, atypical and significant reduction of disulphide bonds has been 

recently observed in IgG1[5,6] that present significant issues for 

manufacturing of therapeutic MAbs. 

During manufacturing of preliminary lots of a recently transferred MAb 

manufacturing process (IgG 1), gross disulphide bond reduction following 

affinity capture chromatography of clarified production bioreactor 

material was observed. Investigations leading to the identification of the 

nature of this reduction process, and process steps to mitigate against its 

future occurrence, are described here. The MAb was co-developed with 

MacroGenics, Rockville, MD. 

Methods: Production Bioreactor material for downstream processing was 

supplied from a 16 day, fed-batch, GS-CHO culture [7]. The Production 

Bioreactor was a single-use (Wave System) with a 100L (full scale) or 10L 

(lab model) working volume. Clarified harvest intermediate (CHI) hold 

studies were performed in either 560L LevMix units (full scale) or 5L 

Braun benchtop bioreactors (lab model). Purification to produce Affinity 

Capture chromatography eluted mainstream was performed using a 

20cm x 20cm MAbselect Protein A resin (GE Healthcare) column, and an 

AKTA Process skid (GE Healthcare).



Figure 1(abstract P110) IgG disulphide bond reduction under various conditions for cell-settled and immediately clarified harvest material. CHI, Clarified 

harvest intermediate; DiS, Disulphide; LoC, Lab-on-a-Chip (Agilent). 

LC-MS analysis was performed on a Polymer Laboratories PLRP-S HPLC 

column and analyzed using an Agilent 1100 HPLC system coupled to 

an Applied Biosystems QSTAR XL mass spectrometer, following sample 

preparation. CE-SDS analysis was performed using a Beckman Coulter 

PA800 capillary electrophoresis instrument fitted with bare-fused silica 

capillary and UV detection at 220 nm, following sample preparation. 

Microchip CE-SDS analysis was performed using a Lab-on-chip 

microanalyser (Agilent). Free thiols were quantified using Ellman’s 

reagent. Metabolic analysis was conducted by Metabolon (Durham, 

NC). 

Results: 

Identification of disulphide reduction of IgG during Primary Recovery: 

The IgG manufacturing process as transferred from the co-developing 

partner included a cell culture settling step following the Production 

Bioreactor and prior to Primary Recovery. 

Disulphide bond reduction was first detected during initial development 

runs by routine Non-reduced (NR) CE-SDS in-process analysis after Affinity 

capture chromatography (data not shown). NR-CE-SDS analysis identified 

elevated levels of free light chain and half antibody molecules, when 

compared to Reference Standard. 

Additional analysis, employing microchip-based NR-CE-SDS methods 

indicated that the antibody reduction occurred during the primary recovery 

cell settling step (results not shown). This was confirmed by LC-MC analysis 

(results not shown). Assessment of disulphide bonding pattern and 

intactness by LC-MS peptide mapping identified both inter- and intra-chain 

disulphide scrambling (results not shown). 

Delineation of events leading to IgG reduction: Initial investigations to 

understand process behaviour during primary recovery identified that 

reducing species, including free thiols (which increase over the course of 

the Production Bioreactor, up to 1mM), were present at the end of the 

Production Bioreactor (Figure 1a). Dissolved oxygen was also shown to 

deplete during the cell settling phase following harvest (data not shown). 

From this, an initial working hypothesis was formed that reducing 

Page 150 of 181 

species, including free thiols, became reactive at low dissolved oxygen 

concentrations and led to IgG 1 disulphide bond reduction. 

A revised process control strategy was implemented (see below) to 

prevent oxygen depletion and maintain dissolved oxygen levels above a 

minimum level. This involved including an aerated and agitated hold for 

Clarified Harvest Intermediate (CHI) in the process. 

Further studies identified that O 2 is critical to maintaining a stable 

environment for oxidised (i.e., normally disulphide bonded) IgG 1 in CHI. 

When O 2 was present, IgG 1 remained intact under all conditions 

evaluated. Only when O 2 was deliberately absent, or stripped away, 

would the harvest material or CHI demonstrate potential for reduction 

(Figure 1b). 

Metabolic behaviour of reducing intermediates: The working hypothesis 

was that by maintaining sufficient levels of dissolved oxygen in the CFM, the 

thiol species could be reacted out (oxidised) and a stable environment for 

oxidised IgG 1 created (Figure 1a). However, the relationship between IgG 

reduction and thiol redox state is not first order (Figure 1c), and the rate of 

thiol oxidation was found to be dependent on the source of Production 

Bioreactor material (i.e., varied with different harvest lots). This indicated the 

involvement of an additional component, potentially catalytic, which has not 

yet been identified in our studies. Thioredoxins have been identified as such 

a catalytic component by others [5,6], and these need to be recycled after 

one redox cycle via Thioredoxin Reductase / NADP(H). 

Metabolic analysis of cell and media material from Production Bioreactors 

indicated high levels of oxidised homocysteine and cysteine (both reactive 

redox molecules), which correlated with decreasing levels of folate (B6) and 

cobalamine (B12), both of which are involved in recycling homocysteine. 

Overall, this analysis identified numerous options for media optimisation to 

mitigate against IgG reduction. However, given the success of process 

controls (described below), and the late stage of process development (prevalidation) 

these media optimisation options were not pursued. 

Process controls to mitigate disulphide reduction of IgG: Aprocess 

control strategy was implemented including:



Establishing a minimal dissolved oxygen level in the Production 

Bioreactor prior to harvesting 

Immediately clarifying the Production Bioreactor material (i.e., 

eliminating the cell settling step) 

Holding the CHI in a hold vessel (LevMix container, agitated hold) that 

had been partially pre-filled with process air. 

Conclusions: Gross disulphide bond reduction was observed during late 

stage development of an IgG 1 monoclonal antibody being 

commercialised for a therapeutic indication; 

Disulphide bond reduction had a second, or higher, order link to low 

dissolved oxygen levels in process intermediates, and the involvement of 

a catalytic factor was also indicated; 

Implementation of an appropriate control strategy (and associated 

process analytics) informed by process development has ensured no 

recurrence of this issue (for n=15 full scale lots). 

References 

1. Wypych J, et al: Human IgG2 antibodies display disulfide-mediated 

structural isoforms. J Biol Chem 2008, 283:16194-16205. 

2. Liu YD, et al: Human IgG2 antibody disulfide rearrangement in vivo. 

J Biol Chem 2008, 283:29266-29272. 

3. van der Neut Kolfschoten M: Anti-inflammatory activity of human IgG4 

antibodies by dynamic Fab arm exchange. Science 2007, 317:1554-1557. 

4. Labrijn AF, et al: Therapeutic IgG4 antibodies engage in Fab-arm 

exchange with endogenous human IgG4 in vivo. Nature Biotechnology 

2009, 27:767-773. 

5. Trexler-Schmidt M, et al: Identification and Prevention of Antibody 

Disulfide Bond Reduction During Cell Culture Manufacturing. 

Biotechnology and Bioengineering 2010, 106:452- 461. 

6. Kao Y-H, et al: Mechanism of Antibody Reduction in Cell Culture 

Production Processes. Biotechnology and Bioengineering 2010, 107:622-632. 

7. Mullan B, et al: Transfer, Implementation and Late Stage Development of 

an End-To-End Single-Use Process for Monoclonal Antibody 

Manufacture. American Pharmaceutical Review 2011, 58-64. 

P111 

Preliminary evaluation of microcarrier culture for growth and 

monoclonal antibody production of CHO-K1 cells 

M Elisa Rodrigues, Pedro Fernandes, A Rita Costa, Mariana Henriques * , 

Joana Azeredo, Rosário Oliveira 

IBB-Institute for Biotechnology and Bioengineering, Centre of Biological 

Engineering, University of Minho, Braga, Portugal 

E-mail: mcrh@deb.uminho.pt 


Background: The large-scale production of biopharmaceuticals commonly 

relies on the use of suspension cell cultures, since they provide higher yields 

than adherent cultures. However, most mammalian cells grow in adherent 

mode and therefore need to go through a process of adaptation to 

suspended growth, which is not always simple or feasible. In this context, 

microcarrier cultures have introduced new possibilities, allowing the 

practical culture of anchorage-dependent cells, in suspension systems, 

achieving high yields. In these systems, cells grow as monolayers on the 

surface of small spheres – the microcarriers, which are usually kept in 

suspension in the culture medium by gentle rocking. The aim of the present 

study was to evaluate, compare and optimize the use of microcarrier culture 

for the growth and monoclonal antibody (mAb) production of CHO-K1 cells. 

Material and methods: Two types of microcarriers were assessed and 

compared in this study: macroporous and microporous. For this, cultures of 

mAb-producing CHO-K1 cells were performed in vented conical tubes, at 

37 °C and 5% CO 2. CHO-K1 culture assessment was divided in two phases: 

the initial cell adhesion phase; and the cell proliferation phase. A set of 

different conditions was tested, namely: initial cell concentration (2x10 5 

cells/ml and 4x10 5 cells/ml), microcarrier concentration (1 g/L for 

macroporous and 3 g/L for microporous), type of rocking during the initial 

phase of adhesion (continuous and pulse) and during the cell proliferation 

phase (continuous). Medium was renewed on a daily basis and the 

concentration and viability of cells adhered to the microcarriers were 

periodically assessed (hourly for the adhesion phase, and daily after that). 

Furthermore, samples were taken for antibody quantification by enzymelinked 

immunosorbent assay (ELISA). 

Page 151 of 181 

Results: Concerning the phase of initial cell adhesion to the microcarriers, 

it was observed that cell adhesion to the microporous microcarriers is 

favored by the use of a higher initial cell concentration (4x10 5 cells/ml) 

with both pulse and particularly continuous rocking methodologies. On 

the other hand, cell adhesion to the macroporous microcarriers is favored 

by a higher initial cell concentration, but only with continuous rocking. 

For a lower initial cell concentration, a pulse rocking methodology is 

recommended. For both microcarriers, the majority of cell adhesion 

occurs within the first 3 hours. 

Regarding the cell proliferation phase, the results showed that it is affected 

by the inoculum concentration only for the microporous microcarriers, with 

4x10 5 cells/ml providing the best cell proliferation. Comparing the two types 

of microcarriers in terms of cell growth, it was observed that the 

microporous provided higher cell proliferation than the macroporous. 

Additionally, the microporous microcarriers demonstrated a higher durability 

than the macroporous, which starts to disintegrate after two weeks. 

Concerning the results of mAb production, it was observed that in 

microporous cultures it is favored by the use of a pulse rocking 

methodology in the initial phase of adhesion, for both inoculum 

concentrations evaluated. This was also observed in macroporous cultures 

but only for the lowest concentration of cells. With 4x10 5 cells/ml the use 

of a continuous rocking methodology proved to be advantageous. 

Indeed, the highest level of production and productivity was achieved in 

these conditions – 4x10 5 cells/ml and continuous rocking. Furthermore, 

the results show that the cells have higher productivities when cultured 

in macroporous microcarriers than inmicroporous,inspiteofhaving 

better levels of proliferation in the last one. Indeed, with fewer cells, the 

macroporous carrier was able to provide levels of total mAb production 

similar and even greater than the microporous. 

Conclusions: This study demonstrated that microcarrier cultures are a 

viable alternative to suspended cultures for the growth and antibody 

production of CHO-K1 cells. For this purpose, the use of higher inoculum 

concentrations during the initial phase of cell adhesion is particularly 

favorable if continuous rocking is used. 

The comparison of the two different types of microcarriers assessed 

indicated that, in general, higher levels of cell adhesion and proliferation are 

obtained with microporous microcarriers, while higher mAb productivity 

and total production are achieved with the macroporous. Therefore, the 

microporous microcarriers assessed is recommended for purposes of cell 

growth while the macroporous is indicated for purposes of production. 

Among the culture conditions tested, the most favorable for the purpose of 

mAb production is the use of the macroporous microcarriers cultured at 

4x10 5 cells/ml under continuous rocking. 

P112 

Strategies for adaptation of mAb-producing CHO cells to serum-free 

medium 

A Rita Costa, M Elisa Rodrigues, Mariana Henriques * , Rosário Oliveira, 

Joana Azeredo 

IBB-Institute for Biotechnology and Bioengineering, Centre of Biological 

Engineering, University of Minho, Braga, Portugal 

E-mail: mcrh@deb.uminho.pt 


Background: The large-scale production of biopharmaceuticals, such as 

monoclonal antibodies (mAbs), commonly requires the use of serum-free 

medium, for both safety and economical reasons. However, because serum 

is such an essential supplement of the growth medium of most mammalian 

cells, its removal demands a very time-consuming process of cell adaptation. 

In this process, cells are usually subjected to a gradual, step-wise, decrease 

of serum concentration in the medium. With the purpose of alleviating cell 

adaptation, other medium supplements such as insulin and trace elements 

can be used, either isolated or in combination. Thus, the aim of this study 

was to assess strategies for the adaptation of CHO cells to serum-free media, 

using different supplement combinations, as well as to identify the most 

critical steps of the process. 

Materials and methods: The study was divided in two experiments. In the 

first one, cultures of mAb-producing CHO-K1 cells were initiated in 24 well 

plates, using Dulbecco’s Modified Eagle Medium (DMEM) supplemented 

with 10% serum. The effect of five combinations of supplements that could



support cells during adaptation was tested. These supplements included 

insulin and eight different trace elements. A methodology of gradual 

adaptation was followed, consisting of sequential steps of serum reduction. 

At the level of 0.625% serum the medium was gradually switched from 

DMEM to the chemically-defined serum-free EX-CELL CHO DHFR - medium. 

The second experiment of this study was performed in order to overcome 

the problems identified in the first assay. The adaptation methodology 

was similar, with the following changes: cell cultures were initiated in 25 

cm 2 T-flasks, and only three of the initial five combinations of 

supplements were tested. 

Results: In the first experiment, cells growing in medium supplemented 

with the two combinations containing a trace element in common died at 

2.5% serum, while for the remaining combinations cell death occurred at a 

later stage, 0.625% serum. Cell death was attributed to problems with the 

procedure of adaptation used in the first experiment, which were identified 

and corrected in the second experiment. The problems found and the 

procedure modifications implemented included the use of higher initial cell 

concentrations to allow the survival of an increased number of cells during 

the process; avoiding procedures that can be harsh to the cells, such as 

centrifugation and the use of enzymes (i.e. trypsin) due to a higher 

sensitivity of cells during adaptation; and allowing enough time in each step 

of the process for a complete cell adaptation. 

After these modifications, in the second experiment, it was possible to 

observe that cells required a long time to adapt to each level of serum 

concentration, particularly below 0.625%. At the level of 0.31% serum, cells 

start to detach, and become fully detached at 0.15%, growing in suspension 

from this point on. The 0.31% of serum was the most critical step of the 

process, demanding more time for cell adaptation and causing the death of 

cells growing in two of the combinations assayed. Indeed, only cells 

growing in medium supplemented with one of the combinations were able 

to survive the whole process. 

It should also be noted that adaptation of cells to EX-CELL is easily achieved 

as long as some serum supplementation is maintained. 

Conclusions: This study demonstrated that the process of adaptation to 

serum-free medium is very challenging to the cells. They become extremely 

sensitive to common cell culture procedures, such as centrifugation or the 

use of enzymes, and consequently extra care should be taken when 

developing the adaptation procedure. Furthermore, it was shown that cell 

adaptation to serum-free conditions is affected by the medium supplements 

used, as well as the time given to each step of the process. 

P113 

Impact of different influenza cultivation conditions on HA 

N-Glycosylation 

Jana V Roedig 1 , Erdmann Rapp 1* , Yvonne Genzel 1 , Udo Reichl 1,2 

1 Max Planck Institute for Dynamics of Complex Technical Systems, 

Sandtorstraße 1, Magdeburg, Germany; 2 Otto-von-Guericke-University, Chair 

of Bioprocess Engineering, Magdeburg, Germany 

E-mail: rapp@mpi-magdeburg.mpg.de 


Background: Influenza virus is a highly contagious human and animal 

pathogen causing infections of the respiratory track. Prevention such as 

high standard hygiene and vaccination still represent the best measures 

for protection. Beside the traditional egg-based influenza vaccine 

production, numerous cell culture-based processes are currently being 

established. Due to its ability to induce strong and protective immune 

responses, the highly abundant glycoprotein hemagglutinin (HA) 

represents the major component in influenza vaccines. Since variations 

in N-glycosylation of glycoproteins such as HA can alter quality 

characteristics of antigens, the impact of cell lines and process 

parameters for vaccine manufacturing needs to be addressed. This 

study investigates the impact of virus adaptation and different harvest 

time points on HA N-glycosylation. Therefore, the HA of influenza A 

virus Uruguay/716/2007 (H3N2, high growth reassortant), in the 

following referred to as IVA-Uruguay, was purified and N-glycans 

analyzed by capillary gel electrophoresis with laser-induced fluorescence 

(CGE-LIF). 


Cell culture and virus production: IVA-Uruguay (H3N2, #07/360, NIBSC, 

South Mimms, UK) was produced in either adherently growing MDCK 

Page 152 of 181 

(No. 84121903) or Vero (No. 88020401) cells purchased from ECACC 

(Salisbury, UK). For cell growth GMEM (Invitrogen, #22100-093, Darmstadt, 

Germany) was supplemented with 5.5 g/L glucose (Roth, #X997.3, 

Karlsruhe, Germany), 2 g/L peptone (IDG, #MC33, Lancashire, UK), 10% 

FCS (Invitrogen, #10270-106) and 4 mg/mL NaHCO3 (Roth, #6885.3). 

Infections were performed in the same medium without addition of FCS 

but supplemented with trypsin (Invitrogen, #27250-018) at a final 

concentration of 5 U/mL. Virus was quantified by a hemagglutination 

assay according to Kalbfuss et al. [1] and is expressed in HAU (log HA/ 

100 µL). 

HA N-glycosylation pattern analysis: Virus was harvested and processed 

for HA N-glycosylation pattern analysis according to Schwarzer et al. [2] 

applying an optimized work-flow [3] and data evaluation [4]. Finally, the 

samples were separated by CGE-LIF using an ABI PRISM 3100-Avant 

genetic analyzer (Applied Biosystems, Foster City, California, USA). For data 

processing and evaluation the x-axis of capillary electropherograms was 

normalized using an internal standard, resulting in N-glycosylation patterns, 

in which each peak corresponds to at least one distinct N-glycan structure. 

This allowed a direct qualitative comparison regarding N-glycan structure 

presence in different samples. For quantitative comparison, the relative 

peak height (RPH: the ratio of peak height to the total height of all peaks) 

was determined for each peak and sample. Low abundant peaks were 

defined with RPH < 5%. 

Results: MDCK cell-derived virus seed, exhibiting 2.6 HAU at 24 hours post 

infection (hpi; data not shown), was used to infect five consecutive 

passages of Vero cells. Adaptation of the virus to Vero cells resulted in 

increased virus yields within shorter time frames in the new host system: 

in the first passage of Vero cells 2.1 HAU were obtained at 96 hpi, whereas 

in the fifth passage a titer of 2.7 HAU at 72 hpi was reached (data not 

shown). The HA N-glycosylation pattern of the MDCK cell-derived IVA- 

Uruguay seed exhibited 25 different characteristic peaks in the range of 

160 bp to 400 bp. Of these a total number of 11 peaks representing large 

glycans (275 bp - 400 bp; #12, 13, 18 - 22, 24, 26 - 28) were unique to 

MDCK cell-derived virus (figure 1A). In contrast, the HA N-glycosylation 

pattern changed significantly with the first passage in Vero cells. Here, 15 

different peaks between 150 bp and 380 bp characterize the Vero cellspecific 

HA N-glycosylation pattern. Four peaks (#3, 5, 23 and 25) were 

unique to Vero cell-derived virus (figure 1A). In comparison to MDCK cellderived 

HA, the Vero cell-derived antigen showed a tendency towards 

smaller glycan structures. The relative abundance of each peak over all 

Vero passages only varied marginally with standard deviations (SD) ≤ 2.1% 

(table 1). The increase in virus titers within shorter time frames suggests 

increased viral fitness, during adaptation from MDCK to Vero cells. An 

impact of HA N-glycosylation on properties of the virus, i.e. virus 

replication, has already been descried [8-12]. Interestingly, the glycan 

pattern stabilized soon after the first passage in Vero cells. This clearly 

indicates that further increase in HA titer did not depend on changes in 

the HA N-glycosylation pattern. 

The impact of harvest time on the HA N-glycosylation pattern of MDCK 

cell-derived IVA-Uruguay is shown in figure 1B. Virus harvested at either 

24 hpi or 72 hpi exhibited the 25 different MDCK cell-specific peaks 

between 160 bp and 400 bp. At 72 hpi one additional, but very low 

abundant peak was detected (numbered 23). Overall, differences in 

relative structure abundance were rather small with a maximal difference 

of 1.7% RPH (table 1). This indicates that HA of virus particles released in 

the supernatant is rather stable over the time window relevant for 

influenza virus production [5,6]. 

However, there are minor variations in RPH from passage to passage 

during adaptation and between harvesting time points. Possible 

explanations are varying ratios either of completely/incompletely 

processed or of intact/degraded N-glycan structures or a combination of 

both. In 2009, Schwarzer et al. [7] characterized the MDCK cell-derived HA 

N-glycosylation pattern of a H3N2 influenza virus subtype as a mixture of 

complex N-glycan structures with terminal a- andb-galactose and high 

mannose type structures. In contrast, the Vero cell-derived HA was 

characterized by complex N-glycans with exclusively terminal b-galactose 

and structures of the high mannose type. For final evaluation of the 

results presented here, determination of the N-glycan structure of all 

peaks would be required. 

Conclusion: In this study, the impact of adaptation and harvesting time 

point on HA N-glycosylation of IVA-Uruguay was investigated. So far, it is 

not clear whether differences in the HA N-glycosylation have an impact



Page 153 of 181 

Figure 1(abstract P113) HA N-glycosylation patterns of Influenza A virus Uruguay/716/2007 (H3N2) – high growth reassortant. Relative 

fluorescence units (RFU) are plotted over the migration time (t mig) in base pairs (bp). (A) MDCK cell-derived virus (seed) was consecutively passaged in 

Vero cells (1 st passage to 5 th passage). Peaks annotated in blue are present in MDCK as well as Vero cell-derived HA; peaks annotated in black are MDCK 

cell-specific, and red annotation indicates Vero cell-specific peaks. (B) MDCK cell-derived virus harvested either 24 or 72 hours post infection (hpi). MDCK 

cell-specific peaks are annotated (1, 2, 4, 6 – 24, 26-28).



Table 1(abstract P113) Overview of relative peak heights (RPH) of 28 HA-glycan peaks during virus adaptation from 

MDCK to Vero cells, and of two harvest time points in a cultivation with MDCK cells. For virus adaptation, MDCK cellderived 

virus seed was consecutively passaged in Vero cells (pass. 1 to pass. 5). Two harvest time points, 24 and 72 hours 

post infection (hpi), compared by absolute values of RPH and percentage difference |ΔRPH|. Maximal SD and maximal |ΔRPH| 

are highlighted in bold 

Peak Virus Adaptation Time Course 

seed pass. 1 pass. 2 pass. 3 pass. 4 pass. 5 pass. 1 to 5 24 hpi 72 hpi |ΔRPH| 

No. Relative Peak Height (RPH) [%] SDRPH* [%] RPH [%] [%] 

1 7,8 0,9 1,7 0,9 2,2 4,8 1,6 3,9 4,5 0,6 

2 3,9 5,7 4,6 5,1 6,1 4,3 0,7 2,4 2,8 0,4 

3 0,0 2,2 1,9 3,4 1,4 1,4 0,8 0 0 0 

4 8,1 6,6 5,9 6,1 6,8 6,8 0,4 5,3 5,2 0,1 

5 0,0 4,0 2,7 2,5 2,5 1,9 0,8 0 0 0 

6 1,4 2,5 1,9 1,5 1,5 1,7 0,4 2,1 1,7 0,4 

7 6,2 4,4 4,8 4,1 4,7 4,8 0,3 4,0 3,6 0,4 

8 6,5 10,0 9,6 8,1 7,4 7,6 1,2 5,3 5,2 0,1 

9 1,7 4,3 3,4 3,4 4,1 5,6 0,9 1,1 1,1 0 

10 13,7 25,8 23,5 27,2 28,6 24,3 2,1 17,6 16,3 1,3 

11 6,8 11,8 12,3 12,2 11,7 9,5 1,2 5,5 4,9 0,6 

12 6,2 0 0 0 0 0 0 2,5 2,5 0 

13 2,0 0 0 0 0 0 0 4,6 4,4 0,2 

14 3,1 0,6 1,1 1,9 1,3 1,5 0,5 3,7 3,6 0,1 

15 6,3 4,1 4,1 4,4 3,8 3,5 0,3 5,0 4,7 0,3 

16 2,2 7,7 7,4 7,4 7,8 8,9 0,6 5,9 6,0 0,1 

17 1,6 3,3 4,4 3,3 3,1 4,3 0,6 2,3 2,8 0,5 

18 1,3 0 0 0 0 0 0 1,9 2,3 0,4 

19 4,5 0 0 0 0 0 0 3,6 4,1 0,5 

20 2,0 0 0 0 0 0 0 8,0 7,0 1,0 

21 2,6 0 0 0 0 0 0 1,7 2,2 0,5 

22 3,3 0 0 0 0 0 0 3,9 3,8 0,1 

23 0,0 4,7 7,9 6,2 5,0 6,4 1,3 0 1,7 1,7 

24 1,9 0 0 0 0 0 0 1,6 1,7 0,1 

25 0,0 1,5 3,0 2,3 1,9 2,6 0,6 0 0 0 

26 2,4 0 0 0 0 0 0 2,2 2,1 0,1 

27 1,1 0 0 0 0 0 0 2,3 2,3 0 

28 3,1 0 0 0 0 0 0 3,7 3,4 0,3 

* standard deviation (SDRPH) of all characteristic peaks in MDCK and Vero cells during virus adaptation. 

on immunogenicity or other properties of influenza vaccines. Other 

factors, e.g. differences in cell culture media, cell density, etc. may also 

contribute to variations in HA N-glycosylation. Nevertheless, monitoring 

N-glycosylation patterns during vaccine production processes allows not 

only to evaluate antigen quality and the impact of process modifications 

on lot-to-lot consistency but also to critically assess consequences of 

unwanted process variations or process failure. 

References 

1. Kalbfuss B, Knochlein A, Krober T, Reichl U: Monitoring influenza virus 

content in vaccine production: precise assays for the quantitation of 

hemagglutination and neuraminidase activity. Biologicals 2008, 

36(3):145-161. 

2. Schwarzer J, Rapp E, Reichl U: N-glycan analysis by CGE-LIF: profiling 

influenza A virus hemagglutinin N-glycosylation during vaccine 

production. Electrophoresis 2008, 29(20):4203-4214. 

3. Rödig J, Rapp E, Hennig R, Schwarzer J, Reichl U: Optimized CGE-LIF-Based 

Glycan Analysis for High-Throughput Applications. Proceedings of the 21st 

Annual Meeting of the European Society for Animal Cell Technology (ESACT) 

Dublin, Ireland: Springer Science+Business Media B.V. 2009 in press. 

4. Ruhaak LR, Hennig R, Huhn C, Borowiak M, Dolhain RJ, Deelder AM, Rapp E, 

Wuhrer M: Optimized workflow for preparation of APTS-labeled N- 

Page 154 of 181 

glycans allowing high-throughput analysis of human plasma glycomes 

using 48-channel multiplexed CGE-LIF. J Proteome Res 2010, 

9(12):6655-6664. 

5. Tree JA, Richardson C, Fooks AR, Clegg JC, Looby D: Comparison of largescale 

mammalian cell culture systems with egg culture for the 

production of influenza virus A vaccine strains. Vaccine 2001, 19(25- 

26):3444-3450. 

6. Aggarwal K, Jing F, Maranga L, Liu J: Bioprocess optimization for cell 

culture based influenza vaccine production. Vaccine 2011, 

29(17):3320-3328. 

7. Schwarzer J, Rapp E, Hennig R, Genzel Y, Jordan I, Sandig V, Reichl U: 

Glycan analysis in cell culture-based influenza vaccine production: 

influence of host cell line and virus strain on the glycosylation pattern 

of viral hemagglutinin. Vaccine 2009, 27(32):4325-4336. 

8. Tsuchiya E, Sugawara K, Hongo S, Matsuzaki Y, Muraki Y, Li ZN, Nakamura K: 

Effect of addition of new oligosaccharide chains to the globular head of 

influenza A/H2N2 virus haemagglutinin on the intracellular transport and 

biological activities of the molecule. J Gen Virol 2002, 83(Pt 5):1137-1146. 

9. Deshpande KL, Fried VA, Ando M, Webster RG: Glycosylation affects 

cleavage of an H5N2 influenza virus hemagglutinin and regulates 

virulence. Proc Natl Acad Sci U S A 1987, 84(1):36-40.



10. Wang CC, Chen JR, Tseng YC, Hsu CH, Hung YF, Chen SW, Chen CM, 

Khoo KH, Cheng TJ, Cheng YS, et al: Glycans on influenza hemagglutinin 

affect receptor binding and immune response. Proc Natl Acad Sci U S A 

2009, 106(43):18137-18142. 

11. Klenk HD, Wagner R, Heuer D, Wolff T: Importance of hemagglutinin 

glycosylation for the biological functions of influenza virus. Virus Res 

2002, 82(1-2):73-75. 

12. Wagner R, Heuer D, Wolff T, Herwig A, Klenk HD: N-Glycans attached to 

the stem domain of haemagglutinin efficiently regulate influenza A 

virus replication. J Gen Virol 2002, 83(Pt 3):601-609. 

P114 

Effect of iron sources on the glycosylation macroheterogeneity of human 

recombinant IFN-g produced by CHO cells during batch processes 

Marie-Françoise Clincke 1,2,3 , Emmanuel Guedon 1* , Frances T Yen 2 , 

Virginie Ogier 3 , Jean-Louis Goergen 1 

1 Laboratoire Réactions et Génie des Procédés UPR-CNRS 3349, ENSAIA-INPL, 

Nancy-Université, 54505 Vandoeuvre-lès-Nancy, France; 2 Lipidomix (EA4422), 

ENSAIA-INPL, Nancy-Université, 54505 Vandoeuvre-lès-Nancy, France; 3 Genclis 

SAS, 54505 Vandoeuvre-lès-Nancy, France 

E-mail: Emmanuel.Guedon@ensaia.inpl-nancy.fr 


Background: In the biopharmaceutical industry, the control of 

glycosylation to satisfy the quality consistency of recombinant proteins 

produced during a process has become an important issue. Indeed, the 

glycosylation pattern of recombinant proteins could be influenced by 

different factors including the cell line used, environmental factors such 

as oxygenation, temperature, shear stresses, extracellular pH... and the 

availability of nutrients. 

As previously reported [1,2], the BDM medium is able to support a 

better CHO cell growth and a higher IFN-g production compared to 

RPMI medium supplemented with serum. In addition, when BDM 

medium is used, CHO cells are capable to maintain a high percentage 

of doubly-glycosylated glycoforms of recombinant IFN-g produced 

during the whole process. Conversely, mono-glycosylated and nonglycosylated 

IFN-g forms increased during batch cultures performed 

with RPMI serum medium. 

Iron is an important nutrient and is reported to support essential 

functions in cells. In this study, the impact of different iron sources on 

the CHO cell growth, as well as on the production and the glycosylation 

of a human recombinant IFN-g were investigated. 

Materials and methods: CHO cell cultures producing human recombinant 

IFN-g (CHO 320: dhfr+, a2.6 ST-) were performed in Erlenmeyer flasks (37°C, 

pH 7.2, 70 rpm) and in two different media, namely RPMI and BDM [3]. 

Whereas RPMI is a classical medium containing serum (5%), BDM is 

Page 155 of 181 

a chemically defined medium without any proteins or serum addition. 

BDM was supplemented with 0.1% pluronic F-68, 750 µM ethanolamine 

and 500 µM iron citrate. 

IFN-g was quantified using an Elisa test. Glycosylation macroheterogeneity 

of IFN-g was characterized by Western Blotting (Amersham Biosciences) 

and each glycoform was quantified by densitometry as previously 


Results: CHO cell growth, IFN-g production and quality: 

CHO cell cultures producing human recombinant IFN-g were cultivated in 

Erlenmeyerflasks,inbothRPMIsupplementedwith5%SVFandBDM 

media. Kinetics performed in BDM medium resulted in a higher maximal 

viable cell density and IFN-g production compared to RPMI medium (data 

not shown). In fact, the higher IFN-g production by CHO cells observed in 

BDM medium was mostly due to a higher cell density, since the specific 

rate of IFN-g production ( qIFN-g) was slighly higher in the BDM medium 

than in the RPMI serum medium. 

In both media, three major molecular weight variants (2N, 1N, 0N) were 

detected during the process with a majority of IFN-g doubly-glycosylated 

(2N) whatever the medium used (figure 1). 

However, the quality of IFN-g remained constant during the CHO cell 

cultures performed in BDM medium, contrasting with the increase in the 

proportion of non-glycosylated (0N) IFN-g to the detriment of the doublyglycosylated 

form (2N), during the time course of the culture of CHO cells 

performed in RPMI serum medium. 

Addition of iron citrate in RPMI serum strongly affects the cell 

growth, the production and the quality of IFN-g: 

Supplementation of RPMI serum medium with iron citrate had a strong 

effect on kinetics of CHO cells producing IFN-g. Indeed, a higher maximal 

viable CHO cell density as well as a high IFN-g specific production rate 

were measured, compared to kinetics of CHO cells performed in RPMI 

serum medium without any supplementation (data not shown). In 

addition, supplementation of RPMI with iron citrate resulted in a constant 

glycosylation pattern of IFN-g whereas an increase of non-glycosylated 

IFN-g to the detriment of the doubly glycosylated form was observed 

when CHO cells are were cultivated in RPMI serum (Figure 1). 

Addition of other bioavailable iron sources such as ammoniacal ferric citrate 

or selenium iron citrate, in RPMI serum medium resulted in the same 

phenomenon. However, when RPMI serum medium was supplemented 

with ferric-EDTA, a negative effect on the production of IFN-g was observed 

(0.03 mg/10 8 cellules vs. 0.07-0.08 mg/10 8 cellules using iron citrate, 

ammoniacal ferric citrate or iron citrate complexed to selenium) despite the 

IFN-g macroglycoforms was maintained constant in this condition (data not 

shown). 

Conclusions: Addition of iron citrate to RPMI serum medium improved 

cellgrowth,aswellasIFN-g production. Furthermore, the glycosylation 

pattern of IFN-g remained constant when iron citrate was added in the 

medium. 

Figure 1(abstract P114) Proportion of IFN-g macroglycoforms produced by CHO cells cultivated in various media; RPMI serum, BDM and RPMI serum 

supplemented with iron citrate.



Addition of ammoniacal ferric citrate, iron citrate complexed to selenium 

or ferric-EDTA to RPMI serum medium also allowed to maintain a 

constant macroglycosylation pattern of IFN-g produced during batch 

cultures. 

However, using ferric-EDTA in supplement of RPMI serum, the specific 

production rate of IFN-g was lower compared to values obtained when 

other iron sources as named above were used. 

Thus, the addition of a bioavailable iron source to culture media could 

improve the physiological cell properties as well as the quality of a 

recombinant protein expressed. 


This work was financed by the Agence Nationale pour la Recherche 

Technique (ANRT) and Genclis SAS (Vandoeuvre-lès-Nancy, France). 

References 

1. Mols J, Burteau CC, Verhoeye FR, Ballez JS, Agathos SN, Schneider Y-J: 

Fortification of a protein-free cell culture medium with plant peptones 

improves cultivation and productivity of an interferon-g producing CHO 

cell line. In Vitro Cell Dev Biol Anim 2003, 39:291-296. 

2. Kochanowski N, Blanchard F, Cacan R, Chirat F, Guedon E, Marc A, 

Goergen J-L: Influence of intracellular nucleotide and nucleotide sugar 

contents on recombinant interferon-g glycosylation during batch and 

fed-batch cultures of CHO cells. Biotechnol Bioeng 2008, 100:721-733. 

3. Schneider Y-J: Optimization of hybridoma cell growth and monoclonal 

antibody secretion in chemically defined serum- and protein-free 

medium. J Immunol Methods 1989, 116:67-77. 

P115 

Characterization of metalloprotease and serine protease activities in 

batch CHO cell cultures: control of human recombinant IFN-g 

proteolysis by addition of iron citrate 

Marie-Françoise Clincke 1,2,3 , Emmanuel Guedon 1* , Frances T Yen 2 , 

Virginie Ogier 3 , Jean-Louis Goergen 1 

1 Laboratoire Réactions et Génie des Procédés UPR-CNRS 3349, ENSAIA-INPL, 

Nancy-Université, 54505 Vandoeuvre-lès-Nancy, France; 2 Lipidomix (EA4422), 

ENSAIA-INPL, Nancy-Université, 54505 Vandoeuvre-lès-Nancy, France; 

3 Genclis SAS, 54505 Vandoeuvre-lès-Nancy, France 

E-mail: Emmanuel.Guedon@ensaia.inpl-nancy.fr 


Background: During the production of any recombinant proteins, an 

evaluation of the product quality is crucial. Proteolytic events may 

occur during the process and could influence the product quality. 

Indeed, proteolysis is an unpredictable process and relatively little is 

know regarding the proteolytic enzymes produced and released by 

mammalian cells. In fact, proteases originating from the host cell line 

cannot be avoided in cell culture. Due to regulatory and safety 

prospects, the addition of serum, fetuin or albumin that usually limit 

protease activities, is not desirable. Thus, in serum-free cultures of 

mammalian cells, control of protease activity constitutes a major 

challenge. 

In the present work, the presence of proteases and their effect on quality 

of IFN-g produced by a recombinant CHO cell line cultivated in a stirredtank 

bioreactor were studied. Whereas the quality of IFN-g remained 

constant during the CHO cell cultures performed in BDM medium, IFN-g 

proteolysis was observed when cultures were carried out in RPMI 

medium with serum [1,2]. 

Materials and methods: IFN-g producing CHO cell lines (CHO 320: dhfr + , 

a2,6 ST - ) were grown in RPMI supplemented with 5% serum and in BDM 

medium [3]. Whereas RPMI is a classical medium containing serum, BDM 

is a chemically defined medium without any proteins or serum addition, 

but supplemented with 0.1% pluronic F-68, 750 µM ethanolamine and 

500 µM iron citrate. 

Batch cultures were performed in stirred-tank bioreactor (Inceltech, SGI). 

Dissolved oxygen concentration was set at 50% of air saturation. 

Agitation rate used was 50 rpm; pH and temperature were set at 7.2 and 

37°C respectively. 

Glycosylation macroheterogeneity of IFN-g was characterized by Western 

Blot (Amersham Biosciences). 

Gelatinase and caseinase activities were performed using zymography. 

Cell-free culture supernatants were concentrated 2-fold on a 10-kDa 

Page 156 of 181 

cutoff filter. Then, the concentrated samples were instantly mixed 3:1 

with nonreducing electrophoresis sample buffer (4.8 mL H 2O; 1.2 mL Tris- 

HCl0.5MpH6.8;2mLSDS10%;1mLglycerol;0.5mLbromophenol 

blue) and loaded on the zymogram gels. The SDS-PAGE gels (10% 

acrylamide) contained either 0.05% caseine or 0.1% gelatin. The gels were 

runat25mAfor1h.ToremoveSDS,thegelsweresoakedtwicefor30 

min in 2% Triton X-100 on a shaker, then washed in distilled H 2O 

followed by an 24h incubation in developing buffer (0.5 M Tris, pH 7.4, 1 

µM Zn 2+ and 5 mM Ca 2+ ). Inhibitor supplementations were also 

performed using EDTA a , PMSF b and Complete inhibitor Cocktail c . 

Visualization of protease activity was carried out by incubation for around 

3h in a Coomassie blue solution. 

a EDTA (ethylenediaminetetraacetic acid) = inhibitor of metalloprotease 

activities 

b Complete, Mini, EDTA-free (Roche) = serine and cysteine proteases 

inhibitor cocktail 

c PMSF (phenylmethylsulfonyl fluoride) = inhibitor of serine protease 

activities 

Results: CHO cell cultures producing human recombinant IFN-g were 

cultivated in stirred-tank bioreactor in both RPMI supplemented with 5% 

serum and BDM media. In both media, three major molecular weight 

variants (2N, 1N, 0N) were detected during the process with a majority of 

IFN-g doubly-glycosylated (2N) whatever the medium used. However, 

using the RPMI medium with serum, IFN-g proteolysis was observed 

during the whole culture (Figure 1). 

To determine the protease activities during CHO cell cultures performed 

in both media, zymogram gels containing either gelatin or casein were 

performed. Among the 5 caseinase activities detected when CHO cell 

cultures were performed with serum (Table 1), only the protease activities 

present all over the process could be potentially involved in the IFN-g 

proteolysis (220, 90 and 85 kDa). In addition, 2 gelatinase activities were 

detected during the whole process (90 and 65 kDa). When CHO cell 

cultures were carried out using protein-free BDM medium, 2 caseinase 

activities (90 and 85 kDa) and 1 gelatinase activity (90 kDa) were 

detected. To determine the type of protease activities present, different 

inhibitors were used in zymogram gels containing either casein or 

gelatin. EDTA inhibited all the gelatinase activities, identifying these 

enzymes as metalloproteases, whereas PMSF and Complete inhibitor 

Cocktail inhibited all the caseinase activities, classifying these enzymes as 

serine proteases (data not shown). 

Compositions of both BDM and RPMI with serum were compared and 

3 components which are present in BDM but completely absent in 

RPMI were identified. These 3 components are pluronic F-68 (PF-68), 

iron citrate and ethanolamine. Interestingly, addition of iron citrate in the 

developing buffer of zymogram gels allowed to inhibit the 

metalloprotease activities, most likely by blocking the Zinc atom in the 

catalytic domain (data not shown). CHO cell cultures were then 

performed in RPMI serum supplemented with iron citrate. Addition of 

iron citrate to RPMI serum allowed to minimized IFN-g proteolysis (Figure 

1). Furthermore, when CHO cell cultures were performed in BDM medium 

without iron citrate during the first 30 hours of the culture, IFN-g 

proteolysis was detected (data not shown). Therefore, IFN-g proteolysis 

has at least one cellular origin and the protease responsible for IFN-g 

proteolysis is most likely a metalloprotease (Molecular Weight close to 

90 kDa). 

Conclusions: Using zymogram analysis, gelatinase and caseinase 

activities in CHO batch cultures performed with or without serum were 

detected, and belong most likely to the metalloprotease and serine 

protease families. When cultures were carried out in RPMI with serum, a 

degradation of recombinant IFN-g was observed, while no IFN-g 

proteolysis was detected in culture performed with BDM medium. 

Furthermore, our results showed that despite the medium used (RPMI, 

BDM, with or without serum), addition of iron minimized IFN-g 

proteolysis, probably due to the inhibition of a 90 kDa metalloprotease 

activity. Thus, we demonstrated that the addition of iron citrate can be 

advantageously considered for industrial processes to prevent the 

proteolysis of a recombinant protein, in particular if one or several 

metalloproteases are present in the culture. 


This work was financed by the Agence Nationale pour la Recherche 

Technique (ANRT) and Genclis SAS (Vandoeuvre-lès-Nancy, France).



Figure 1(abstract P115) Western blot analysis of excreted IFN-g produced by CHO cells cultivated in various media; RPMI serum, BDM and RPMI serum 

supplemented with iron citrate. 

Table 1(abstract P115) Protease activities detected by zymography during CHO cell cultures performed in RPMI 

medium with serum and BDM medium 

Molecular Weight (kDa) RPMI medium with serum BDM medium 

Caseinase activities 220, 140, 90, 85 and 40 kDa 90 and 85 kDa 

Gelatinase activities 95, 90 and 65 kDa 90 kDa 

References 

1. Castro PML, Ison AP, Hayter PM, Bull AT: The macroheterogeneity of 

recombinant human interferon-g produced by Chinese-hamster ovary 

cells is affected by the protein and lipid content of the culture medium. 

Biotechnol Appl Biochem 1995, 21:87-100. 

2. Mols J, Peeters-Joris C, Wattiez R, Agathos SN, Schneider Y-J: Recombinant 

interferon-g secreted by Chinese hamster ovary-320 cells cultivated in 

Page 157 of 181 

suspension in protein-free media is protected against extracellular 

proteolysis by the expression of natural protease inhibitors and by the 

addition of plant protein hydrolysates to the culture medium. In Vitro 

Cell Dev- An 2005, 41:83-91. 

3. Schneider Y-J: Optimization of hybridoma cell growth and monoclonal 

antibody secretion in chemically defined serum- and protein-free 

medium. J Immunol Methods 1989, 116:67-77.



P116 

Isolation of active peptides from plant hydrolysates that promote Vero 

cells growth in stirred cultures 

Samia Rourou, Rym Hssiki, Héla Kallel * 

Viral Vaccines Research & Development Unit, Institut Pasteur de Tunis, 13, 

place Pasteur. BP.74 1002 Tunis, Tunisia 

E-mail: Hela.Kallel@pasteur.rns.tn 


Background: Vero cells are adherent cell lines commonly used for the 

production of viral vaccines. We had developed an animal component 

free medium that allows an optimal growth of this cell line in stirred 

bioreactor [1,2]. We had also showed that Vero cells grown in this 

medium (called IPT-AFM) sustained rabies virus replication, and resulted 

in an overall yield comparable to the level obtained in serumsupplemented 

medium. 

IPT-AF medium contains plant hydrolysates, namely soy (Hypep 1510) and 

wheat gluten hydrolysates (Hypeps 4601 and 4605). These peptones were 

shown to promote cell attachment and growth. However, although these 

components are of non-animal origin, their use in vaccine production 

process has several drawbacks, mainly due variability between lots. 

The aim of this work is to identify active peptides from these hydrolysates 

that show a positive effect on cell adhesion, attachment and growth. For 

this purpose, the hydrolysates were fractionated using chromatography 

and precipitation techniques. The effect of the isolated fractions on Vero 

cells growth were tested in 24 and 6-well cell culture plates using 

experimental design approach. Fractions that sustain cell growth, were 

Figure 1(abstract P116) Vero cells on 2 g/l Cytodex 1 in spinner flask, in different media. 

Page 158 of 181 

further tested in stirred culture on Cytodex1 microcarriers, to confirm 

their positive effect on Vero cells growth. 

Materials and methods: Cell line: Vero cells adapted to IPT-AF medium 

as described in Rourou et al. [2] were used in this study. 

Media: M199 medium was purchased from Invitrogen, IPT-AFM was 

prepared as detailed in Rourou et al. [2]. Hypeps were provided by 

Sheffield Bio-Science. 

Culture systems: Static cultures: Cells were grown in 24-well plates 

(Falcon) and 6-well plate experiments « Nunclon Δ». The inoculation 

density was 2x10 5 cells/ml and the working volume was equal to 1 ml for 

the 24-well plate and 3 ml when the cells were grown in 6-well plate. 

Cells were grown at 37°C in 5% CO 2 incubator. 

Stirred cultures: Cells were grown in 6-well low binding plates (Costar) 

and in spinner flasks; cultures were inoculated at a cell density of 2x10 5 

cells/ml. The Working volume was equal to 3 ml for the 6-well plate and 

200 ml when cultures were performed in spinner flask. Cells were grown 

on 2 g/l Cytodex 1 at 37°C and 30 rpm. 

Fractionnation protocols: Hypeps 1510, 4601 and 4605 were fractionated 

by either chromatography methods or sequential precipitation with 

different ethanol concentrations as described in Shen et al. [3]. 

Sephadex G-25 (GE Healthcare), BioGel P-2 fine (Biorad), Sephadex G-10 

(GE Heathcare), HiTrap Q HP (GE Healthcare) and HiTrap SP HP (GE 

Healthcare) matrixes were tested for the fractionation of the different 

solutions of Hypeps. Fractionation was monitored by measuring the 

absorbance of the collected fractions at 214 nm. Collected fractions were 

desalted, then frozen at -70°C and lyophilized. After lyophilization the 

fractions were resuspended in M199 so they would be compatible with 

cell culture.



Analytical methods: Cells grown in static cultures were first detached 

with the TrypLe Select (Invitrogen) then counted according to the Trypan 

blue method. Vero cells cultivated on Cytodex 1 microcarriers were 

counted using the Crystal Violet technique. 

Experimental design and statistical analysis: The software Modde 6.0 

(Umetrics, Sweden) was used in this study for the design of the 

experiments and the statistical analysis of the data. 

Results: Sephadex G-10, Sephadex G-25 and Biogel fine-2 were used to 

fractionate Hypeps 4605 and 4601. However, none of these matrixes was 

efficient. 

Anion and cation exchange chromatography were therefore used as an 

alternative method; two pH levels were tested: 5 and 8. Although these 

methods allowed the isolation of differentfractionsforeachpeptone, 

none of them had enhanced Vero cells when the cells were cultivated in 

24-well culture plates. In addition, most of these fractions showed a toxic 

effect on cell growth. 

Sequential precipitation with different ethanol concentration was also 

applied for the fractionation of the three peptones. The fractionation was 

conducted as described by Shen et al. [3]; 5 fractions were obtained for 

Hypep 4605 whereas for Hypep 4601 and Hypep 1510, 4 fractions were 

obtained for each. The effects of the isolated fractions on Vero cells growth 

were investigated in 24-well plates using a full factorial experimental 

design; 83 combinations were assessed in duplicate. Two combinations of 

fractions that show a cell growth comparable to that obtained in IPT-AF 

medium (positive control), were selected (combinations 1 and 2). The 

identified combinations were also tested in 6-well plates on 2 g/l Cytodex 

1 microcarriers. The highest cell density level reached under these 

conditions was similar to that achieved in IPT-AF medium. 

These combinations were further tested in spinner flask on Cytodex1 

microcarriers, to confirm their positive effect on Vero cells growth. Data 

shown in Figure 1, indicate that cell density level reached 2x10 6 cells/ml 

after 5 days of culture when Vero cells were grown in combination 1. 

Such level was slightly lower than that obtained in IPT-AF medium (2x10 6 

cells/ml versus 2.4x10 6 cells/ml). However, Vero cell growth in 

combination 2 was less efficient; the highest cell density obtained in this 

medium was equal to 1.7x10 6 cells/ml. Thus, combination 1 appears to 

be more suitable for Vero cells on Cytodex 1 microcarriers. 

Conclusions: Sephadex G-25, Sephadex G-10, Biogel-fine and ion 

exchange chromatography matrixes were not efficient for Hypeps 

fractionation. Sequential precipitation with ethanol appears to be the best 

method to isolate various fractions that promote Vero cells growth. Two 

Combinations (1 & 2) were identified as the best in terms of cell density 

and cell attachment. Spinner cultures demonstrated that these 

combinations are suitable for Vero cells growth on Cytodex 1. Further 

fractionation and characterization of these compounds are ongoing to 

identify the active components. 

Page 159 of 181 

References 

1. Rourou S, van der Ark A, Majoul S, Trabelsi K, van der Velden T, Kallel H: A 

novel animal component free medium for rabies virus production in 

Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor. Appl 

Microbiol Biotechnol 2009, 85:53-63. 

2. Rourou S, Van Der Ark A, Van Der Velden T, Kallel H: Development of an 

animal component free medium for Vero cells culture. Biotechnol Prog 

2009, 25:1752-1761. 

3. Shen CF, Kiyota T, Jardin B, Konishi Y, Kamen A: Characterization of 

yeastolate fractions that promote insect cell growth and recombinant 

protein production. Cytotechnol 2007, 54:25-34. 

P117 

Comparison of different membrane supports for monolayer culture of 

bovine oviduct epithelial cells 

Muhammad Z Tahir 1,2* , Fabien George 2 , Isabelle Donnay 2 

1 UMR-BDR 1198 INRA/ENVA, Ecole Nationale Vétérinaire d’Alfort, 94700 

Maisons-Alfort, France; 2 ISV-EMCA, Université Catholique de Louvain, 1348 

Louvain-la-Neuve, Belgium 

E-mail: mztahir@vet-alfort.fr 


Background: The oviduct epithelium consists of ciliated and secretory 

cells which play an important role in key reproductive processes such as 

sperm capacitation, fertilization and early embryonic development. Bovine 

oviduct epithelial cells have been widely used in co-culture experiments 

to condition culture media and improve early embryonic development 

[1]. However, these cells dedifferentiate during in vitro culture and 

manifest alterations like loss of cilia and secretory granules, reduction of 

cell height and flattening on the culture surface [2]. The trials for long 

term culture of oviduct cells, ensuring a better polarization and 

differentiation of the cells, include culture of oviduct cells on matrigel 

supports or collagen filter inserts. In this study, we have compared three 

different membrane supports for their potential to maintain 

ultrastructural features and monolayer integrity of bovine oviduct 

epithelial cells during in vitro culture. 

Materials and methods: Oviducts were excised from the genital tracts 

of cows slaughtered in abattoir. Following mechanical isolation, the 

oviduct epithelial cells were cultured on three different membrane 

supports i.e. polyester membrane (Thincert, Millipore Inc. USA), 

polytetrafluoroethylene membrane(Transwell, CorningInc.USA)and 

cellulose ester membrane (Millicell, Millipore Inc. USA). The culture of 

oviduct epithelial cells was done inTCM-199medium(Gibco,Grand 

Island, NY, USA) with 10% fetal bovine serum at 39°C in a humidified 

atmosphere of 5% CO 2 & 20% O 2. The development of primary cultures 

Figure 1(abstract P117) 1a) TEER (transepithelial electrical resistance) across oviduct cells monolayer (median ± extreme values) on 1. Thincert, 

2. Transwell, 3. Millicell inserts. 1b) Mannitol test (permeability to radioactive mannitol) through oviduct cells monolayer (median ± extreme values) on 

1. Thincert, 2. Transwell, 3. Millicell inserts.



was assessed daily and the medium was renewed every 48 hours. After 

3 weeks of monolayer culture, the ultrastructural features of oviduct cells 

were examined by scanning electron microscopy while the integrity of 

monolayer was tested by transepithelial electrical resistance and 

permeability to radiolabeled 14 C-Mannitol. Non-parametric statistical 

analysis was done using Kurskal-Wallis Test. 

Results: The study of ultrastructural features by scanning electron 

microscopy revealed presence of an intact monolayer of polygonal 

epithelial cells on polyester (Thincert) and cellulose ester (Millicell) 

membrane supports while no such monolayer was seen in 

polytetrafluoroethylene (Transwell) membrane support. The test of 

transepithelial electrical resistance across monolayer of oviduct epithelial 

cells showed no significant difference (P>0.25) between the three 

membrane supports (Fig. 1a). The test for permeability of radiolabeled 

14 C-Mannitol across the monolayer of oviduct epithelial cells showed a 

tendency for lowest permeability (P=0.6) in polyester (Thincert) 

membrane support (Fig. 1b). 

Conclusion: The potential of polyester membrane supports (Thincert) to 

maintain ultrastructural features (intact monolayer of polygonal epithelial 

cells) and ensure monolayer integrity (higher transepithelial electrical 

resistance and lowest permeability to mannitol) during in vitro culture 

may serve as a model to study different cellular functions such as 

transport, absorption and secretory capacity of bovine oviduct epithelial 

cells as well as embryo-maternal interactions. 

References 

1. Rottmayer R, Ulbrich S, Kölle S, Prelle K, Neumüller C, Sinowatz F, Meyer H, 

Wolf E, Hiendleder S: A bovine oviduct epithelial cell suspension culture 

system suitable for studying embryo-maternal interactions: 

morphological and functional characterization. Reproduction 2006, 

132:637-648. 

2. Reischl J, Prelle K, Schöl H, Neumüller C, Einspanier R, Sinowatz F, Wolf E: 

Factors affecting proliferation and dedifferentiation of primary bovine 

oviduct epithelial cells in vitro. Cell Tiss Res 1999, 296:371-383. 

P118 

Efficacy of an inactivated and adjuvanted “ZULVAC® 8 OVIS” vaccine 

produced using single-use bioreactors 

Lídia Garcia * , Helena Paradell, Mercedes Mouriño, Berta Alberca, Alicia Urniza, 

Ana Vila, Margarita Tarrats, Joan Plana-Durán 

Pfizer Olot S.L.U., Ctra. Camprodon s/n, La Riba, 17813 Vall de Bianya 

(Girona), Spain 

E-mail: Lidia.garcia@pfizer.com 


Introduction: Bluetongue virus (BTV) first emerged in the European 

Union (EU) in 2006, peaking at 45,000 cases in 2008. The EU spent million 

of euros in 2008 and 2009 on eradicating and monitoring programs, cofinanced 

with member states. The number of cases in 2009 was 1118, 

with only 120 reported across the EU so far last year. Vaccination has 

proven itself as the most effective tool to control and prevent the disease 

and to facilitate the safe trade of live animals. 

Mammalian cells are commonly used as a substrate for production of 

most of the viral vaccines. BHK-21 cells are used for the production of 

bluetongue vaccines. Most companies use roller bottles or conventional 

bioreactors, but taking into account that the manufacturing cost is very 

important, the possibility of using Single-Use Bioreactor (SUB) technology 

as an alternative to roller bottles or conventional bioreactors was 

explored. Advantages like yields of production, time reduction 

Table 1(abstract P118) NA titers against BTV serotype 8 after vaccination 

(elimination of cleaning and sterilization steps needed for bioreactors, no 

validation process, etc) and quality of antigen production were studied. 

Materials and methods: Cell line: BHK-21 cell line was used due to the 

good yields. Cells were cultured at 37°C in Minimum Essential Medium 

Glasgow supplemented with serum. 

Cultivation system: The growth of the BHK-21 cells and virus production 

was conducted in roller bottles (RB), 10-L bioreactor (Biostat B-plus) and 

in 250-L SUB (Hyclone). 

Cells on bioreactors and SUB were cultivated using microcarriers 

(Cytodex-3) at a density of 3g/L. The crystal violet dye nucleus staining 

method was used to estimate cell density. 

Dissolved oxygen (DO) and pH were automatically adjusted by addition 

gases (CO 2, and air). 

Virus strain: BTV serotype 8 (BTV-8), strain BEL2006/02 was used in all 

experiments. The strain was supplied by “Veterinary and Agrochemical 

Research Centre” (VAR-CODA-CERVA), Ukkel, Belgium. 

Once the cells were 80-100% confluent, RB, 10-L bioreactor and 250-L 

SUB were infected under identical conditions and with a constant 

multiplicity of infection (MOI). Harvesting of virus was done when 

cytopathic effect (CPE) was about 90- 100%. 

Virus production was evaluated by TCID 50/ml. 

Vaccine: The antigen obtained on SUB was inactivated by Binary 

Ethylenimine (BEI) and adjuvanted with aluminum hydroxide and 

saponin. The efficacy of the vaccine was tested in lambs. 

Animals and experimental design: Forty, 1.5 month-old, lambs were 

included in the study. 

Thirty lambs were vaccinated and revaccinated 3 weeks later by 

subcutaneous route and ten lambs were left as unvaccinated controls. 

Forty-four days after revaccination all lambs were challenged with BTV-8. 

The antibody response in vaccinated lambs was evaluated from 

vaccination until the moment of challenge by a seroneutralization test 

(see table 1). 

Viremia (presence of BTV genome in blood samples) was evaluated by 

real time qRT-PCR [1] in blood samples obtained before challenge and 

during four weeks after challenge. 

Results and discussion: Some experiments to scale-up were done. An 

optimized and transferable process was developed in 10L glass vessels by 

monitoring the pH, temperature, and DO. The final process was 

transferred to 250-L stirred-tank SUB integrated with an Applikon 

controller. 

Cell growth and virus production in SUB was conducted at the optimal 

conditions determined previously on conventional bioreactors. Three 

critical parameters were taken into account: Cell concentration, growth 

rate at start virus inoculation and harvesting time. 

Cell growth: Several studies were conducted to compare the growth of 

cells in RB, 10-L bioreactor and 250-L SUB. During culture in bioreactors 

and SUB, DO was regulated by pulse of oxygen and pH was controlled. 

Figure 2, shows that cell concentration at 48 hours (when confluency is 

reached) was higher in bioreactors than in roller bottles. That 

corresponds to an increase of cell biomass by a factor of 1.5. 

Virus production: Figure 3, shows the results of the virus titers in 250-L 

SUB compared with those obtained in RB and 10-L bioreactor. 

The virus titer reached in a 10L bioreactor was comparable with the levels 

obtained in SUB. Preliminary results prove that by using SUB, the yields 

obtained in roller bottles can increase more than two times. 

Efficacy study: The potency of antigens produced using 250-L SUB 

technology was verified in target species. 

Serological study: Determination of neutralizing antibodies (NA) titer 

against BTV serotype 8 in the samples taken after vaccination and 

Mean NA Titers 

Group D



Figure 1(abstract P118) Scale-up from roller bottles to 250-L SUB. 

Figure 2(abstract P118) BHK-21 concentration at 48 hours post cultivation. 

Figure 3(abstract P118) Virus titers obtained at 72h p.i. 

Page 161 of 181



revaccination was done by means of seroneutralization test. Table 1 show 

the geometric mean NA titers. 

Evaluation of viremia after challenge: In any of the lambs of 

the vaccinated and challenged groups, viral gemone could be 

detected by RT-PCR during 27 days after challenge. Whereas in all 

unvaccinated and challenged lambs, the viral genome was detected 

from day 3-5 p.i. 

Conclusions: The results obtained using a 250-L SUB concerning the 

cell concentration and virus titer, were similar to those reached in a 10L 

bioreactor and higher than in roller bottles. 

Efficacy results on lambs confirm that the quality of antigen produced in 

SUB are similar of the antigen produced both in roller bottles and on 

conventional bioreactor. 

We can think on the possibility of using disposable systems in vaccines 

production in order to reduce the production costs. 

SUB can be an alternative to conventional production methods: 

Reduced facility complexity, reduction of the cost of building, and 

possibility of the rapid expansion of the capacity of the production. 

Reduction of the capital of equipment and equipment validation, etc. 

Avoid the cleaning process and reduction of the risk of crosscontamination. 

Reference 

1. Toussaint JF, Sailleau C, Breard E, Zientara S, De Clercq KJ: Bluetongue virus 

detection by two real-time RT-qPCRs targeting two different genomic 

segments. Virol Methods 2007, 140(1-2):115-123. 

P119 

Engineering CHO cell metabolism for growth in galactose 

Natalia E Jiménez 1,2 , Camila A Wilkens 1,2 , Ziomara P Gerdtzen 1,2* 

1 Centre for Biochemical Engineering and Biotechnology, Department of 

Chemical Engineering and Biotechnology, University of Chile, Santiago, 

8370448, Chile; 2 Millennium Institute for Cell Dynamics and Biotechnology: a 

Centre for Systems Biology, University of Chile, Santiago, 8370448, Chile 

E-mail: zgerdtze@ing.uchile.cl 


Background: Chinese hamster ovary (CHO) cells are one of the main 

hosts for industrial production of therapeutic proteins, owing to wellcharacterized 

technologies for gene transfection, amplification, and 

selection of high-producer clones. This has motivated the search for 

different strategies for the improvement of their specific productivity 

being one of the key points for this approaches the reduction of 

metabolic end-products like lactate and ammonia. 

Page 162 of 181 

The use of different carbon sources has been an alternative solution for 

this problem, as they are metabolized more slowly than glucose leading to 

lower production of metabolic end-products [1]. Particularly, it has been 

observed that cultures in presence of glucose and galactose undergo a 

metabolic shift in which they are capable of remetabolize lactate. However, 

the specific growth rate is diminished due to a slower metabolism 

associated to the incorporation of galactose [2]. In addition, cells are 

unable to survive with galactose as their unique carbon source [3]. 

In this work we aim at identifying culture conditions that extend the 

culture’s viability for tPA producing CHO cells in media with combined 

glucose and galactose as carbon sources. Furthermore, we propose 

reducing the production of secondary metabolites by over-expressing 

galactokinase (GALK1), a bottleneck point in the galactose metabolism. 

Methodology: Two sets of experiments were carried out. In the first set, 

t-PA producing CHO TF 70R cells were grown in protein and serum free 

media without glutamine and supplemented with glucose, galactose and 

glutamate to define two different culture conditions. A high glucose 

control experiment was performed with 20 mM glucose (G20), and a 

combined carbon condition with a final concentration of 20 mM of 

hexose with 6 mM glucose and 14 mM galactose (GG6/14). 

Based on these results, transfection of genes involved in galactose 

metabolism are performed to enhance cell growth by improving 

galactose metabolism. Cells were transfected with the Galactokinase 

(GALK1) gene from Mus musculus, using Lipofectamine. Experiments were 

performed to analyze cell proliferation, carbon source consumption 

lactate production and metabolic flux distribution for the obtained 

pooled clones, as a preliminary tool to determine if the over-expression 

of this protein has a positive effect. 

In the second culture set, transfected cells were grown in protein free 

media with 2% Fetal Bovine Serum, supplemented with glucose and 

galactose to define two different culture conditions with a final 

concentration of 20 mM of hexose: 6 mM glucose and 14 mM galactose 

(GalKGG6/14); and 20 mM galactose (Gal20). Extracellular metabolites 

were measured. 

Results: In combined carbon source experiments, two phases can be 

distinguished. During the glucose consumption phase cells produce 

biomass, tPA, lactate and alanine. When glucose is depleted GG6/14 

culture enters a second metabolic stage where no significant growth is 

observed and galactose is consumed along with extracellular lactate and 

alanine. This is consistent with lactate and alanine being used as a 

supplementary pyruvate source to support energy metabolism associated 

with cellular maintenance. 

Metabolic flux analysis (MFA) was performed, considering the main 

reactions of the central glucose and galactose metabolism. Figure 1(a) 

Figure 1(abstract P119) Experimental results and metabolic flux distribution. (a) Metabolic Flux distribution between G20 (red) and GG6/14 (blue) at 50 h 

and 100 h in culture. Cell density, glucose and lactate concentration in GG6/14 (b) and Gal20 media (c). (▪) CHO tPA,(●) CHO tPA-pcDNA3.1(+), (♦) CHO 

tPA-GALK1, glucose (▪,♦), lactate (▲,●).



shows results for two time-points for each culture, an early (50 h) and a 

late time-point (100 h). For GG6/14, the columns represent glucose and 

galactose consumption stages, respectively. In the pyruvate node carbon 

molecules are channeled either towards the TCA cycle, or in the direction 

of secondary metabolite synthesis such as lactate and alanine. Fluxes 

from the central carbon metabolism are reduced several times in the late 

time point for both cultures, except for the Pyr-AcCoA reaction, which 

plays a central role in the regulation of mammalian metabolism by 

connecting glycolysis with the TCA cycle. In late culture stages where 

hexose uptake is low, cell metabolism is directed towards maintaining 

TCA cycle fluxes in order to obtain energy. To achieve this in GG6/14, 

galactose and lactate are used as an additional carbon source. 

SincegalactoseconsumptionappearstobelimitingcellgrowthinGG6/ 

14, there is an open possibility to improve cell growth during galactose 

consumption while maintaining low lactate production by overexpressing 

genes involved in galactose metabolism. Transport of 

galactose has been proposed to explain the low uptake of galactose and 

the GalK reaction is identified as the limiting step of the galactose 

metabolism. To achieve this cells are transfected with stable expression 

vectors to insert the galactokinase GalK1 gene and the galactose 

transporter Slc2a8. 

Obtained clones exhibit a higher growth rate in galactose than control 

cells due to their ability of metabolize galactose at a increased rate. 

Transfected cells in GalKGG6/14 consume all available glucose before 

starting to consume galactose. They also exhibit lower lactate 

accumulation, indicating that the introduction of these gene does not 

increase galactose uptake in a manner that would generate secondary 

metabolites. Control cells are unable to grow on media with galactose as 

the only carbon source. Transfected cells were able to maintain high 

viability during 100 hours in this media. 

The over-expression of the Slc2a8 (GLUT8) Galactose transporter could 

enhance the positive effects of the introduction of the galactokinase 

gene by increasing the availability of substrate at an intracelullar level. 

Before transfection, mutation of a dileucine motif to alanine is required to 

allow the expression of this transporter in the plasmatic membrane. For 

that purpose an strategy using PCR with primers that include this 

substitutions is currently undergoing. 

Conclusions: When CHO cells are cultured with a combination of glucose 

and galactose it is possible to achieve extended viability along with a 

metabolic shift towards lactate consumption, which is triggered when the 

second hexose is consumed. 

Metabolism can be modified through cell engineering to increase cell 

growth while maintaining low lactate production rates, enabling cells to 

utilize alternative carbon sources. Specifically this can be achieved with 

galactose. 

Acknowledgements: This work was supported by FONDECYT Initiation 

Grant 11090268. 

References 

1. Wlaschin K, Hu WS: Engineering cell metabolism for high density cell 

culture via manipulation of sugar transport. J of Biotechnol 2007, 

13:1023-1027. 

2. Altamirano C, Illanes A, Becerra S, Cairó J, Gòdia F: Considerations on the 

lactate consumption by CHO cells in the presence of galactose. Jof 

Biotechnol 2006, 125:547-556. 

3. Neerman J, Wagner R: Comparative analysis of glucose and glutamine 

metabolism in transformed mammalian cell lines, insect and primary 

liver cells. J Cell Physiol 1996, 166:152-169. 

P120 

Engineering CHO cells for improved central carbon and energy 

metabolism 

Camila A Wilkens 1,2 , Ziomara P Gerdtzen 1,2* 

1 Centre for Biochemical Engineering and Biotechnology, Department of 

Chemical Engineering and Biotechnology, University of Chile, Santiago, 

8370448, Chile; 2 Millennium Institute for Cell Dynamics and Biotechnology: a 

Centre for Systems Biology, University of Chile, Santiago, 8370448, Chile 

E-mail: zgerdtze@ing.uchile.cl 


Background: Investigations have shown animal cell cultures’ 

performance, in terms of cell proliferation and production of recombinant 

Page 163 of 181 

protein, are negatively affected by both lactate’s concentration and its 

specific production rate. In a previous work, we determined that lactate 

production was caused by pyruvate accumulation due to its high 

synthesis rate in the glycolitic pathway and limited consumption in the 

TCA cycle, which leads to lactate production [1]. In this work, we use the 

ΔL/ΔHexose ratio in order to characterize the cells metabolic state. This 

ratio describes the lactate production rate vs. hexose consumption. Low 

ΔL/ΔHexose ratios indicate efficient metabolic states where carbons 

consumed are mainly used to support cell growth, protein synthesis or 

energy metabolism. 

Cell engineering has been previously used to improve cultures’ 

performance by changing the expression of genes involved in 

metabolism and apoptosis, focusing on the modification of only one 

gene at the time. These works showed that after overexpression of genes 

such as fructose transporter (Slc2a5) and yeast’s pyruvate carboxylase 

(PYC) cells are able to achieve higher cell densities and lower lactate 

production than wild-type cells under the same culture conditions [2-4]. 

In this work we aim at introducing multiple changes in the cells’ genome 

in order to obtain an engineered cell line with reduced lactate 

production and enhanced energy metabolism, which is capable of 

achieving higher cell densities and with a longer lifespan. We propose to 

control both, carbon uptake and itsusebytheTCAcycle.Cellswere 

transfected with the fructose transporter gene (Slc2a5) and pyruvate 

carboxylase gene (PYC). Metabolic flux redistribution was studied through 

metabolic flux analysis, comparing engineered cells and wild-type under 

normal culture conditions. 

Materials and methods: CHO cells were transfected with the pcDNA3.1 

(+) zeo-Slc2a5 and/or PCMVSHE-PYC2 + Hygromycine resistance vectors 

using lipofectamine. After selection, five experiments were designed to 

study cell proliferation, carbon source consumption, lactate production 

and metabolic fluxes. CHO cells overexpressing PYC (CHO-PYC) were 

cultured with glucose 17.5 mM and cells transfected with Slc2a5 (CHO- 

Slc2a5) and both PYC and Slc2a5 (CHO-PYC-Slc2a5) were grown in media 

containing fructose 17.5 mM. Two control cultures were performed with 

wild-type CHO cells in 17.5 mM glucose (GC) orfructose(FC). Results are 

shown in Figure 1. 

Results: Cultures’ performance: As seen in Figure 1.(a) and Table 1 

respectively, GC, CHO-PYC and CHO-PYC-Slc2a5 were able to reach higher 

cell densities and maximum growth rates (µ max) than FC and CHO-Slc2a5. 

Cultures with glucose have almost no lag phase while experiments with 

media supplemented with fructose have long lag phases, probably due 

tothesloweruptakeoffructose,which would delay the exponential 

growth phase. In addition, engineered cells exhibit an extended lifespan 

in comparison to wild-type cells. 

ΔL/ΔHexose values reached by the cultures are given in Table 1. CHO cells 

grown in high glucose show an inefficient metabolic state where most 

carbons consumed go towards lactate production. Engineered cells 

grown in glucose have lower lactate production per carbon consumed 

than wild-type cells (Figure 1.(b)). 

Engineered cells show a better use of glucose, producing less lactate per 

glucose consumed, as reflected in their lower ΔL/ΔHexose. In addition, 

CHO-PYCcellsareabletoproduceless lactate and achieve a longer 

lifespan than wild-type cells. CHO-Slc2a5 cells have higher fructose 

uptake rates than FC and are able to achieve longer lifespans and higher 

cell densities. 

CHO-PYC-Slc2a5 cells have the highest µmax among all experiments yet 

they produce more lactate than FC. Most of the lactate is produced in 

the lag phase. The fact that cells are capable of growing in fructose as 

well as in glucose and have a better ΔL/ΔHexose than GC, indicates that 

there is room for further improvement of this system. 

Metabolic flux analysis: Figure 1.(c) shows the flux distribution of the 

different cultures for central carbon metabolism during mid exponential 

growth phase. CHO cells grown in fructose have lower amounts of 

carbon directed towards energy metabolism. Both CHO-Slc2a5 and CHO- 

PYC-Slc2a5 have higher fluxes in glycolysis and TCA cycle than FC, 

consistent with higher cell density. CHO-PYC cells consume lower 

amounts of glucose than GC, and most of it is directed towards the TCA 

cycle. CHO-Slc2a5 cells consume higher amounts of fructose than FC and 

most of it is directed towards the TCA cycle. CHO-PYC-Slc2a5 cells show a 

more active metabolism than FC, consuming more fructose, with higher 

TCA cycle fluxes and lactate production, while reaching higher cell 

densities than the control.



Figure 1(abstract P120) Experimental and MFA results. Pink circles: GC, orangesquares:FC, purple upwards triangle: CHO-PYC , green downwards 

triangle: CHO-Slc2a5 , blue rhombus: CHO-PYC-Slc2a5. (a) Cell density, (b) Lactate concentration (c) Comparison of metabolic flux distribution in carbon 

mmol/10 9. cells/hr for the different experiments during mid exponential growth. Scale is the same in all graphs. 

Conclusions: It is possible to modify cells for a more efficient metabolism 

in media supplemented with glucose and fructose using cell engineering. 

Engineered cells show enhanced viability and more efficient metabolic 

states under high glucose or fructose concentrations than the controls. 


We would like to thank Dr. Roland Wagner for the PCMVSHE-PYC2 vector, 

Dr. Mariella Bollati for the pcDNA3.1(+)zeo vector and the Genetically 

EngineeredMouseFacilityatM.D.AndersonCancerCenterforthe 

Hygromycine resistance cassette. This work was supported by FONDECYT 

Initiation Grant 11090268. 

References 

1. Wilkens CA, Altamirano C, Gerdtzen ZP: Comparative metabolic analysis of 

lactate for CHO cells in glucose and galactose. Biotechnology and 

Bioprocess Engineering Journal 2011 in press. 

2. Elias CB, Carpentier E, Durocher Y, Bisson L, Wagner R, Kamen A: Improving 

glucose and glutamine metabolism of human HEK 293 and Trichoplusia 

Table 1(abstract P120) Parameters for cell growth and 

ΔL/ΔHexose 

Experiment µ max [10 -2 hrs -1 ] ΔL/ΔHexose 

GC 1.63 1.7 

FC 0.86 0.81 

CHO-PYC 2.1 0.81 

CHO-Slc2a5 0.65 0.88 

CHO-PYC-Slc2a5 3.68 1.1 

Page 164 of 181 

ni insect cells engineered to express a cytosolic pyruvate carboxylase 

enzyme. Biotechnol Prog 2003, 19(1):90-97. 

3. Irani N, Wirth M, van Den Heuvel J, Wagner R: Improvement of the 

primary metabolism of cell cultures by introducing a new cytoplasmic 

pyruvate carboxylase reaction. Biotechnol Bioeng 1999, 66(4):238-246. 

4. Wlaschin KF, Hu WS: Engineering cell metabolism for high-density cell 

culture via manipulation of sugar transport. J Biotechnol 2007, 

131(2):168-176. 

P121 

Mitogenic effect of sericin on mammalian cells 

Wataru Sato 1* , Ken Fukumoto 1 , Kana Yanagihara 1 , Masahiro Sasaki 2 , 

Yoshihiro Kunitomi 2 , Satoshi Terada 1 

1 Department of Applied Chemistry and Biotechnology, University of Fukui, 

Fukui, 910-8507, Japan; 2 Development Department, SEIREN Co., Ltd., Fukui, 

913-0038, Japan 


Fetal bovine serum (FBS) or other mammal-derived factors are extensively 

supplemented as growth factor into culture medium. Since mammalderived 

factors arouses the concern about the risk of zoonosis, serumand 

mammal-free culture is strongly required. We focused on sericin 

hydrolysates, originating from silkworm, and reported that sericin is 

effective as growth factor to various cells. But it is not elucidated how 

sericin induces the proliferation and inhibits apoptosis of the cells. In this 

study, inhibitor assay was done in order to identify signaling factors



Figure 1(abstract P121) The pathway deduced from our previous study. cDNA microarray analysis detected up-regulation of myc, tbp, fos, and Bcl-xL, 

and down-regulation of atf3 after sericin treatment. Inhibition assay confirmed the involvement of Ras and MEK1/2 in the mitogenic effect of sericin. 

Figure 2(abstract P121) Different pathways depending on cell line. 

Page 165 of 181



involved in sericin effect. In hybridoma cells, the involvement of Src was 

suggested, while ERK1/2, PP2A and p38 were not involved. In HepG2 cells, 

the involvement of Src and ERK1/2 was suggested. From these results, it 

was implied that the mitogenic effect of sericin might be transduced 

through different pathways depending on cell line. 

Background: In in vitro cell culture, mammal-derived factors including 

FBS are usually used as growth factors and supplemented into the media. 

However, supplementing mammal-derived factors causes the concern 

about the risk of zoonosis such as abnormal prions and various viruses. 

Therefore, mammal-free culture is strongly required in the industry of 

antibody therapeutics production and in regenerative medicine including 

cell therapy. As an alternative to mammal-derived factors, we focused on 

hydrolysate of sericin, which is glue protein included in silk fiber. Our 

previous studies revealed that sericin has mitogenic and anti-apoptotic 

effect on various cell lines [1-3]. Additionally, sericin hydrolysates treated 

with autoclave sterilization maintained its original mitogeneic activity and 

so we successfully developed a mammal-free medium, Sericin-GIT (Wako 

Pure Chemical Industries, Ltd., Japan). Although sericin is effective, it is 

not revealed how sericin up-regulates the proliferation and downregulates 

apoptosis. 

In the previous study, the following results were obtained. One, cDNA 

microarray analysis detected up-regulation of myc, tbp, fos and Bcl-xL, and 

down-regulation of atf3 after sericin treatment. Two, Inhibition assay 

confirmed the involvement of Ras and MEK1/2 in the mitogenic effect of 

sericin. From these results, we suppose a following pathway (Figure 1). In 

this study, inhibitor assays against the factors shown in the figure were 

done in order to identify signaling factors involved in sericin effect. 

Materials and methods: Cells. Mouse hybridoma 2E3-O cells and human 

hepatoblastoma HepG2 cells were cultured in ASF104 (Ajinomoto Japan) 

serum-free medium and DME medium (Nissui, Japan) without FBS, 

respectively. 

Inhibitors. PP2 (BioMoL, USA), ERK Activation Inhibitor 1, Cell-Permeable 

(Calbiochem, USA), Okadaic acid (Calbiochem), SB239063 (SIGMA, USA) 

were used at inhibition assay. 

Inhibition Assays. Mouse hybridoma 2E3-O cells and human 

hepatoblastoma HepG2 cells were cultivated in addition of each of 

inhibitors. After two days, the number of cells and the viability were 

determined by trypan blue-exclusion test with hemocytometer. 

Results: Inhibition assays were done in order to identify signaling factors 

involved in sericin effect. 

Since Ras and MEK1/2 are involved in Map kinase pathway, PP2, a specific 

inhibitor against Src, was used. PP2 successfully neutralized the mitogenic 

effect of sericin, indicating that Src would be involved. 

Involvement of ERK1/2 was tested by using ERK Activation Inhibitor 1. The 

inhibitor neutralized the mitogenic effect in HepG2 cells, but did not in 

hybridoma cells, suggesting that ERK1/2 would be involved in HepG2 

cells but not in hybridoma cells. In order to confirm that ERK1/2 is not 

involved in hybridoma cells, inhibition assay against PP2A was done; the 

mitogenic effect could be enhanced by inhibition of PP2A if MAPK 

pathway would be involved in the mitogenic effect of sericin. Inhibition 

against PP2A failed to improve the proliferation of the hybridoma cells 

treated with sericin, implying that PP2A would not be involved. 

Further cDNA microarray analysis implied that several other genes might 

be affected by sericin treatment. Among the genes, myc and stat1 are the 

lower signaling factors of p38 and so SB239063, a specific inhibitor 

against p38, was tested. It failed to neutralize, indicating that p38 would 

not be involved in the mitogenic effect of sericin. 

Conclusions: In hybridoma cells, Src was involved in the mitogenic effect 

of sericin, while ERK1/2, PP2A and p38 were not, suggesting that Src could 

activate other factors to transduce signal of sericin. In HepG2 cells, the 

involvement of Src and ERK1/2 was confirmed, suggesting that MAPK 

pathway could be involved. From these results, the mitogenic effect of 

sericin is transduced through different pathways depending on cell lines 

as shown in figure 2. Further study should be done to reveal the 

involvement of these factors. 

References 

1. Morikawa M, Kimura T, Murakami M, Katayama K, Terada S, Yamaguchi A: 

Rat islet culture in serum-free medium containing silk protein sericin. 

J Hepatobiliary Pancreat Surg 2009, 16:223-228. 

2. Yanagihara K, Terada S, Miki M, Sasaki M, Yamada H: Effect of the silk 

protein sericin on the production of adenovirus-based gene-therapy 

vectors. Biotechnol Appl Biochem 2006, 45:59-64. 

Page 166 of 181 

3. Terada S, Sasaki M, Yanagihara K, Yamada H: Preparation of silk protein 

sericin as mitogenic factor for better mammalian cell culture. J Biosci 

Bioeng 2005, 100:667-671. 

P122 

Challenges in scaling up a perfusion process 

Vana Raja, Saravanan Desan, Ankur Bhatnagar * , Anuj Goel, Harish Iyer 

Cell Culture Lab, Biocon Limited, Bangalore, India 

E-mail: ankur.bhatnagar@biocon.com 


Introduction: Perfusion process involves retention of the cells inside the 

bioreactor while simultaneously removing spent medium and adding 

fresh medium continuously. The flow rate of addition of fresh and 

removal of spent medium are generally kept the same (perfusion rate) to 

maintain the culture volume inside the bioreactor. Cell retention is 

possible with many devices but the reliability and consistency in 

performance of these devices remains a major challenge for design, 

operation and scale-up of perfusion processes. For a filtration based 

retention device, successful scale up depends on cell retention efficiency, 

prevention of filter fouling and the similarity of perfusion equipment 

between small and large scale. We present a case study where a 

perfusion process from a 2L (Liter) bioreactor is scaled up to 

manufacturing scale of 1KL (Kilo Litre). 

Materials and methods: A NS0 host cell line cultured in protein free 

medium was used. At lab scale 2L stirred tank bioreactors with internal 

spin filters as the retention device were used. The 1KL production 

bioreactor used closed external rotating filters for cell retention. Cell 

count and viability were determined using Hemocytometer and Trypan 

Blue dye exclusion. Glucose and lactate were measured using YSI 2700 

analyzer and product concentration by Affinity chromatography. 

Results and discussions: Development of perfusion process and 

scale up: A perfusion process was developed which consisted of a batch 

phase for cell growth followed by perfusion phase once the cell density 

reaches the desired level. This process was scaled up by scaling up the 

scale dependent parameters linearly same while maintaining the scale 

independent factors within the acceptable range. The filter parameters 

such as mesh type and filter area per unit bioreactor volume were 

however not comparable between the scales. 

PB-1 (Production Batch no. 1): The growth rate during the batch phase 

of the run was comparable to the lab scale. However, during the 

perfusion phase, the maximum cell densitywasobservedtobeonly 

about 25% of the lab scale. Due to the lower cell counts, the perfusion 

rates were also proportionally reduced. Together this resulted in 

obtaining only ~25% of the expected product yield. 

As part of investigation, the following factors were analyzed: 

a) Inoculum and “medium lot” used in PB-1 – A control batch was run in 

the lab with the same inoculum and medium as used in the PB-1 run. 

This batch showed normal lab batch profiles ruling out these factors as 

possible cause for the underperformance of the production batch. 

b) Pumps used for perfusion – The lab scale used peristaltic pumps while 

in manufacturing pulsating pumps were used. These pulsating pumps 

could influence cell retention and hence were replaced with peristaltic 

pumps for future production batches. 

PB-2 (Production Batch no. 2): Changing the pump type resulted in 

better cell retention. However, soon the retention started dropping, 

indicating cell loss from the bioreactor. Visual inspection of the filter 

mesh (mesh Type A) showed significant mesh deformation. This could 

have resulted due to the fragile nature of the mesh which could not 

withstand the negative pressure generated inside the closed filter 

housing due to suction forces and filter fouling. The mesh Type A was 

also found to be fouling very fast due to it mesh weave design. Two 

other mesh types (B & C) with different mesh weave patterns and 

mechanical strengths were evaluated. Type B showed poor cell retention 

due to bigger pore size. Type C showed better cell retention and also did 

not deform easily during filter fouling. Thus the filter mesh was changed 

from Type A to Type C in the production system. 

PB-3 (Production Batch no. 3): The filter with mesh C showed cell 

retentionbetterthaneventhelabscale.Thisresultedinachievingcell 

densities higher than the range of the lab batches (Figure 1). The 

perfusion rates were increased by about 20% from the lab scale to



Figure 1(abstract P122) Cell count profiles from the lab batches and the production batches with different mesh types. 

address the nutritional requirements of the higher cell numbers. The 

product yield was also proportionally higher by ~20%. 

Note: N. VCC – normalized viable cell concentration 

Product quality analysis: Analysis showed that the product obtained 

from batch PB-3 was significantly different in quality (charge distribution) 

compared to product from PB-1,2 and lab batches. The following were 

evaluated as possible reasons for the observed differences: 

a) High perfusion rates compared to lab and PB-1,2 batches resulting in 

shorter product residence time inside the bioreactor. 

b) Different lactate and pCO2 levels maintained (due to higher perfusion 

rates) which also resulted in different pH profiles. 

PB-4 (Production Batch no. 4): Batch PB-4 was run with perfusion flow 

rates comparable to the lab batches. The additional nutritional 

requirement for the higher cell concentration was addressed by making 

the fresh medium more concentrated. pH was also controlled using the 

CO 2 and base combination. The cell concentration obtained was similar 

to PB-3. Lowering the perfusion flow rates also helped in delaying the 

clogging of the filter. 

Product analysis showed that the changes done in the batch helped in 

bringing the product quality closer to (within acceptable range) the lab 

batches. The results are shown in the Table 1. Two differently charged 

species Type-1 and Type-2 are used for comparison. 

Table 1(abstract P122) Product quality comparison of PB-3 

and PB-4 batches 

Perfusion lots PB-3 PB-4 

Type-1* Type-2* Type-1* Type-2* 

1 112 74 59 139 

2 47 184 66 126 

3 43 195 73 116 

4 41 198 73 120 

5 39 208 82 109 

6 39 209 82 118 

* % of Lab batch profiles. 

Page 167 of 181 

Summary: Development and scale-up of a perfusion process has challenges 

due to the complex nature of the process and unavailability of direct scale-up 

of the perfusion equipment. The initial scale-up to production scale resulted 

in poor cell growth profile. Upon investigation, the reason for low cell 

concentration was attributed to poor cell retention by the perfusion device. 

Changes were introduced in the type of mesh used for filter construction and 

perfusion pumps to improve retention. These modifications helped in better 

cell culture profiles and yields. However, the product quality was impacted 

because of these changes. Further changes in the perfusion flow rates were 

done to address the product quality differences. 

P123 

High cell density growth of High Five suspension cells in DO-controlled 

wave-mixed bioreactors 

Teddy Beltrametti 1 , Nicole C Bögli 1* , Gerhard Greller 2 , Regine Eibl 1 , Dieter Eibl 1 

1 Institute of Biotechnology, Zurich University of Applied Sciences and Facility 

Management (ZHAW), Wädenswil CH-8820, Switzerland; 2 Sartorius Stedim 

Biotech, Göttingen, D-37075, Germany 


Insect cells such as High Five cells used in the manufacture of 

biopharmaceuticals are best grown in wave-mixed bioreactors (1). This is 

due to the continual blending of foam with the culture medium which 

results from the wave-induced mixing and permanent renewal of the 

medium surface. Even the addition of an antifoam agent is not required. 

Process conditions which ensure maximum High Five cell densities and 

whichhavebeenreportedtobeabout8x10 6 cells x mL -1 (2) were 

determined in Biostat CultiBag RM50 optical experiments for batch mode 

and 1 L culture volume. Seed inoculum for these experiments was generated 

in single-use shake flasks (Corning) incubated in an Infors`Multitron shaker 

(27°C, 100 rpm, 25 mm shaking diameter). Biostat CultiBag RM50 optical was 

controlled in four different modes: non-pH- and non-DO-controlled, DOcontrolled, 

pH-controlled, DO- as well as pH-controlled. The DO level was 

guaranteed by increasing the rocking rate up to 28 rpm and, if required by 

addition of pure oxygen. In process control was supplemented with off-line 

analyses of cell density, viability, metabolites (glucose, glutamine, glutamate, 

lactate, ammonium) and pH.



While the influence of the type of bioreactor`s control on the maximum 

growth rate (0.041-0.044 h -1 ) and doubling time (15.6-17.7 h) was 

negligible, maximum cell densities were achieved with DO regulation (set 

point 50%). Maximum cell densities ranged between 8.2 and 9.4 x 10 6 

cells x mL -1 and represent the highest values described for High Five cells 

so far in the literature. They are 35% higher compared to those seen in 

pH-controlled and non-controlled experiments. Controlling both DO and 

pH level did not lead to any further improvement of cell growth i.e. the 

range of growth parameter values was the same as that observed in the 

previous experiments. For High Five cell-based biopharmaceuticals this 

knowledge enables optimized seed inoculum/seed train production in 

wave-mixed bag bioreactors. 

References 

1. Werner S, et al: Innovative, Non-stirred Bioreactors in Scales from Millilitres 

up to 1000 Liters for Suspension Cultures of Cells using Disposable Bags 

and Containers – A Swiss Contribution. CHIMIA 2010, 64:819-823. 

2. Rhiel M, Mitchell-Logean CM, Murhammer DW: Comparison of Trichoplusia 

ni BTI-Tn-5B1-4 (High FiveTM) and Spodoptera frugiperda Sf-9 Insect 

Cell Line Metabolism in Suspension Cultures. Biotechnology and 

Bioengineering 1997, 55:909-920. 

P124 

Bag-based rapid and safe seed-train expansion method for Trichoplusia 

ni suspension cells 

Nicole C Bögli 1* , Christoph Ries 1 , Irina Bauer 1 , Thorsten Adams 2 , 

Gerhard Greller 2 , Regine Eibl 1 , Dieter Eibl 1 

1 Institute of Biotechnology, Zurich University of Applied Sciences and Facility 

Management (ZHAW), Wädenswil CH-8820, Switzerland; 2 Sartorius Stedim 

Biotech, Göttingen, D-37075, Germany 


Trichoplusia ni suspension cells (High Five) used in conjunction with 

the baculovirus vector expression system (BEVS) are regarded as 

potential product system of new, recombinant virus-like particle (VLP) 

vaccines. In order to push vaccine development and production, 

biomanufacturers use single-use technology when- and wherever 

possible. This applies to upstream processing and in particular seedtrain 

expansion ranging from cryopreserved vials via t-flasks, spinners 

(respectively shake flasks) to stirred stainless steel bioreactors. The 

stainless steel bioreactors deliver inoculum for seed bioreactors and 

have been increasingly replaced by wave-mixed single-use bag 

bioreactors during the last 5 years [1]. 

Page 168 of 181 

The approach presented for seed-train cell expansion of High Five 

suspension cells is based on the Biostat CultiBag RM50 optical (Sartorius 

Stedim Biotech). It was used for the production of cells for long-term 

storage and for the expansion of cells for subsequent production 

experiments. For long-term storage the cells were frozen at high cell 

concentrations (20 - 40 x 10 6 cells x mL -1 ) in 60 mL Cryobags and stored 

in nitrogen at -196 °C in vapour phase. 

Initial experiments were aimed at the growth characterization of High Five 

suspension cells from a vial working cell bank (WCB). The High Five cells 

were grown in batch mode and in 250 mL single-use shake flasks (Corning 

and Sartorius Stedim Biotech) on a Certomat® CT Plus shaker (Sartorius 

Stedim Biotech) during six days (triplicates, 27 °C, 100 rpm, 25 mm shaking 

diameter). Afterwards a procedure was developed in which thawed cells 

from a Cryobag were directly transferred into and expanded in a Biostat 

CultiBag RM. Under optimal process conditions (500 mL starting volume, a 

starting cell density of 1 x 10 6 cells x mL -1 , 27 °C, rocking angle of 6 °, 20 - 

30 rpm, 0.2 vvm, DO set point 50%) growth rate (0.039 - 0.042 h -1 ), 

doubling time (18 - 20 h) and maximal cell density (7.8 - 8.9 x 10 6 cells x 

mL -1 ) showed good correlation with results arising from CultiBags which 

were inoculated with cells from shake flasks. This bag-based seed-train 

expansion allows time saving of about one week and reduces crosscontamination, 

both advantages being due to omitted intermediate 

cultivation steps in shake flasks. 

Reference 

1. Eibl R, Löffelholz C, Eibl D: Single-Use Bioreactors-An Overview. Single-Use 

Technology in Biopharmaceutical Manufacture Eibl R, Eibl D. Wiley , 1 2010, 

1:33-51. 

P125 

On-line monitoring of the live cell concentration in bioreactors based 

on a rocking platform 

John Carvell * , Matt Lee 

Aber Instruments Ltd., Aberystwyth, SY23 3AH, UK 

E-mail: johnc@aberinstruments.com 


Background: Aber Instruments first introduced the concept of using 

disposable live-biomass probes in 2009 [1]. The probe has been carefully 

designed to be welded into most single use bioreactors and is suitable 

for bags with agitators eg the Hyclone SUB and the Sartorius Stedim 

Biostat Cultibag STR, or those using the rocker type platform eg Sartorius 

Stedim Biostat Cultibag RM and GE Wave bioreactors . The disposable 

Figure 1(abstract P125) Capacitance and conductivity measurements in a 50-L Sartorius (25 L working volume) rocking-motion CultiBag system 

(R. Tanner, GlaxoSmithKline, UK)



biomass probe has four electrodes with the same dimensions as the 

existing reusable production biomass probes with flush platinum 

electrodes that are used in cGMP manufacture worldwide. The electrode 

support material is HDPE that meets FDA and USP Class VI requirements 

and the probe can withstand gamma sterilisation and be stored for 

prolonged periods before use. The disposable probe is easily connected 

to a mini-lightweight Futura pre-amplifier so that the weight load or 

torque on the bag is minimal and the bulk of the electronics is then 

located well away from the bag. The disposable probe has already been 

welded into different bags including Sartorius Stedim Biotech CultiBag 

RM bioreactors. 

Materials and methods: Experiments were performed with a Futura MRF 

Mini-Remote Futura (Aber Instruments Ltd) and 58mm diameter 

disposable “Coupon” probes. The probes were welded into the RM 

Cultibags by Sartorius Stedim. The tests performed with SF9 cells at GSK 

used a combination of the Biomass Monitor 220 (Aber Instruments, UK) 

and a Futura MRF Mini-Remote Futura pre-amplifier. The media used was 

Excell 420 from SAFC Biosciences. 

Tests with Rocking Motion bags: One of the challenges of installing 

any on-line probe in a rocking motion bioreactor is that the sensor will 

be exposed to varying levels of fluid. When the bag is used at the 

minimum recommended volume, the probe will also be exposed 

momentarily to the gaseous headspace of the bioreactor. In the very first 

tests with the disposable probe installed in a 5L Sartorius Stedim Biotech 

CultiBag RM filled with just media, the unfiltered capacitance and 

conductivity profiles were shown to become increasingly noisier as the 

speed of the rocking action was increased. 

An advanced rocker filter algorithm, including an “anti-beat” mechanism, 

was developed to work automatically at varying rocker speeds such that 

the Futura does not have to monitor the rocking speed or synchronise 

the readings to the angle of the rocking table. Further experiments were 

then performed with disposable probes mounted within a simulated bag 

platform on a rocker set at a higher than average speed for cell culture 

applications (47rpm). The volume of media (500ml) was set to a 

maximum depth of 20mm at full tilt and the minimum depth over the 

electrodes was 1mm during the rocking process. 

The resulting capacitance and conductivity traces showed that a normal 

average filter flattened out the noise, however, if only the averaging filter is 

used, the output filtered value was markedly lower than the steady state 

value. The new rocker algorithm successfully filtered out the noise caused by 

the rocking motion and was far better at maintaining the steady state value. 

The disposable probe also has been assessed with a prototype 50-L 

Sartorius Stedim Biotech CultiBag RM (25L working volume) in monitoring 

thegrowthofSf9insectcells(Figure1).Theprobetrackedviablecell 

density before addition of a baculovirus for transient recombinant protein 

expression. The biomass probe successfully detected a valid infection by 

showing a rapid increase in signal caused by increasing volumes of the 

infected cells. 

Conclusions: Use of RF impedance to monitor cell culture processes is 

well established in biopharmaceutical applications. Introduction of the 

Futura biomass monitor allows this technology to be used with 

confidence on rocking motion disposable bioreactors, from process 

development through cGMP production. 


Robert Tanner of GSK, UK, and Henry Weichert of Sartorius Stedim, 

Germany, are gratefully acknowledged for providing data and 

photographs for this poster. 

Reference 

1. Carvell JP, Williams J, Lee M, Logan D: On-Line Monitoring of the Live Cell 

Concentration in Disposable Bioreactors (poster). European Society for 

Animal Cell Technology biennial conference, Dublin, Ireland 2009. 

P126 

Optimization of HEK 293 cell growth by addition of non-animal derived 

components using design of experiments 

Laura Cervera, Sonia Gutiérrez, Francesc Gòdia, María M Segura * 

Departament d’Enginyeria Química, Universitat Autònoma de Barcelona, 

Bellaterra, Barcelona, 08193, Spain 

E-mail: mersegura@gmail.com 


Page 169 of 181 

Background: Mammalian cells are a widely used expression platform for 

the production of recombinant therapeutic proteins or viral particle-based 

vaccines since they typically perform appropriate protein posttranslational 

modifications and authentic viral particle assembly. Of the 

available mammalian cells, HEK 293 is one of the most industrially 

relevant cell lines because it is cGMP compliant and is able to grow in 

suspension in a variety of commercial serum-free media. Of note, 

production of human therapeutics in mammalian cell culture has become 

more and more stringent in past recent years and not only demands 

serum-free but also animal-component free production conditions to 

ensure safety. This work is part of a project aimed to optimize HEK 293 

cell growth by addition of non-animal derived components to serum-free 

and protein-free media through design of experiments (DoE) in order to 

maximize productivity of a recombinant VLP vaccine by PEI-mediated 

transient transfection. 

Materials and methods: Thecelllineusedinthisworkisaserum-free 

suspension-adapted HEK 293 cell line from a cGMP master cell bank from 

the Biotechnology Institute of the National Research Council in Montreal, 

Canada. Cells were maintained in exponential growth in 125-mL 

disposable polycarbonate Erlenmeyer flasks shaken at 110 rpm using an 

orbitalshakerplacedinanincubator with a 37°C, humidified, 5% CO 2 

atmosphere. Cell count and viability were determined using trypan blue 

and a microscope counting chamber. 

Results: We have analyzed the kinetics of HEK 293 cell growth in HyQ 

SFM4 Transfx293 from HyClone Thermo Scientific (Logan, UT, USA). 

The cells grow to a maximum concentration of ~ 3×10 6 cells/ml with 

over 90% viability and show an average doubling time of 24 h. In 

addition, HEK 293 cell growth was assessed in two other commercial 

serum-free culture media, namely ExCell 293 from SAFC Biosciences 

(Hampshire, UK) and Freestyle 293 from Invitrogen (Carlsbad, CA, 

USA) both compatible with PEI-mediated transient transfection , 

showing similar results (Figure 1a). The effect of foetal bovine sera 

(FBS) in these serum-free culture media was also evaluated. Cells can 

triplicate their maximum cell densities in the presence of FBS (i.e. 

they reach 9,3×10 6 cells/ml in HyQ SFM4 Transfx293 medium + 10% 

FBS). 

Due to the important effect of serum on HEK 293 cell growth, we decided 

to evaluate the effect of non-animal derived serum components on cell 

growth in attempt to improve cell densities while keeping animal-origin 

free production conditions. For these studies, a pre-defined mixture of 

supplements composed of r-albumin (1 g/L), r-insulin (10 mg/L), rtransferrin 

(10 mg/L) from Merck Millipore (Kankakee, IL, USA), and an inhouse 

developed animal-component free lipid mix (1X) composed of 

synthetic cholesterol (SyntheChol®, Sigma-Aldrich, Steinheim, Germany), 

fatty acids (F7050, SAFC Biosciences), tocopherol (T1157, Sigma) and 

emulsifying agents (PS80, Sigma) at concentrations recommended in the 

literature [1] were used. HEK 293 cell density is improved in the presence 

of the mix, but only in Freestyle medium a significant difference is 

observed (Figure 1b). This medium was selected for further optimization 

by DoE. 

Screening of supplements with significant effect on HEK 293 cell growth 

was performed using a Placket-Burman experimental design [2]. Two 

levels of concentrations were assigned to each variable: a low one with 

no additives and a high one based on the typically recommended values 

mentioned above. Using this strategy, we were able to determine in 12 

experimental runs (performed in duplicate) that r-insulin, r-transferrin and 

an in-house developed lipid mix positively affect HEK 293 cell growth in 

serum-free media formulations, whereas r-albumin showed no significant 

effect (Figure 1c). 

Optimal concentrations for each supplement showing a significant effect 

on HEK 293 cell growth were defined based on a Box-Behnken 

experimental design [3]. Three levels of concentrations for each variable 

were selected (Table 1). Using this experimental design, we were able to 

define in 15 experimental runs (performed in duplicate) a model that 

accurately predicts HEK 293 cell concentrations in the presence of 

different concentrations of r-insulin (r-Ins), r-transferrin (r-Trans) and lipid 

mix (LipMix) (Table 1). 

The Box-Behnken model equation was: 

Cell density (10 6 cells/mL) = 4,53 + 0,63 × r-Ins - 0,17 × r-Trans - 0,41 × 

LipMix - 0,68 × r-Ins × r-Trans + 0,02 × r-Ins × LipMix - 0,07 × rTrans × 

LipMix - 0,18 × r-Ins 2 - 0,45 × r-Trans 2 - 1,02 × LipMix 2



Page 170 of 181 

Figure 1(abstract P126) Optimization of HEK 293 cell growth a) The effect of 10% FBS supplementation in commercially av ailable serum-free media, 

b) Growth kinetics of HEK 293 cells in 3 different serum-free protein-free formulations (bI, bII and bIII) in the presence or absence of a pre-defined mixture of 

supplements, c) Effect of recombinant proteins or synthetic lipid mix on HEK 293 cell growth, d) Response surface graphs. HEK 293 peak cell density as a 

function of the concentrations of Lipid Mix (X) vs. r-Transferrin (mg/L) (dI), r-Transferrin (mg/L) vs. r-Insulin (mg/L) (dII) and Lipid Mix (X) vs. r-Insulin (mg/L) 

(dIII) based on Box-Behnken experimental results. Cell density values presented are in millions of cells/mL and represent mean ± SD (n=3).



Table 1(abstract P126) Box-Behnken results. a Three levels of concentrations for each variable including a maximum 

(1), a minimum (-1) and a center point (0) were used. Values shown in parenthesis are concentrations employed for 

r-transferrin and r-insulin (mg/mL) and lipid mix (based on Synthechol® concentrations provided in X). b Cell density 

values presented are mean of duplicate runs in million cells/mL 

EXP N° r-Insulin a 

r-Transferrin a 

Lipid Mix a 

Max Cell density b 

Experimental According to model 

1 -1 (1) -1 (1) 0 (1) 3,1 2,8 

2 1 (20) -1 (1) 0 (1) 5,5 5,4 

3 -1(1) 1 (20) 0 (1) 3,7 3,8 

4 1 (20) 1 (20) 0 (1) 3,3 3,7 

5 -1 (1) 0 (10) -1 (0.1) 3,1 3,1 

6 1 (20) 0 (10) -1 (0.1) 4,6 4,4 

7 -1 (1) 0 (10) 1 (2) 2,0 2,3 

8 1 (20) 0 (10) 1 (2) 3,6 3,6 

9 0 (10) -1 (1) -1 (0.1) 3,2 3,6 

10 0 (10) 1 (20) -1 (0.1) 3,5 3,4 

11 0 (10) -1 (1) 1 (2) 2,8 2,9 

12 0 (10) 1 (20) 1 (2) 2,8 2,4 

13 0 (10) 0 (10) 0 (1) 4,9 4,5 

13 0 (10) 0 (10) 0 (1) 4,9 4,5 

13 0 (10) 0 (10) 0 (1) 3,8 4,5 

Optimal concentrations for each supplement in HEK 293 cell culture 

medium were defined based on this model to be 19,8 mg/L of r-insulin, 

1,6 mg/L of r-transferrin and 0,9X for the lipid mix. These results can be 

inferred from response surface graphs (Figure 1d). Finally, the model was 

validated experimentally. For this purpose, the growth kinetics of HEK 293 

cells in the optimized cell culture medium was analyzed. In the presence 

of the suitable combination/concentrations of supplements, HEK 293 cells 

reached a maximum cell density of 5,4x10 6 cells/mL (n=3), same value as 

predicted using the Box-Behnken model, as opposed to the 

unsupplemented Freestyle medium that supported cell growth up 3x10 6 

cells/mL (n=3) (Figure 1a & 1bIII). 

Conclusions: Results have shown that by adding a mixture of animal-free 

supplements to serum-free culture medium, it is possible to reach high 

cell densities comparable to those attained in the presence of FBS while 

avoiding the problems derived from its use. 


We would like to thank Dr. Amine Kamen (BRI-NRC, Canada) for kindly 

providing the HEK 293 cell line. The recombinant albumin was a 

generous gift from Merck Millipore. 

References 

1. Keenan J, Pearson D, Clynes M: The role of recombinant proteins in the 

development of serum-free media. Cytotechnology 2006, 50:49-56. 

2. Plackett RL, Burman JP: The design of optimum multifactorial 

experiments. Biometrika 1946, 33:305-325. 

3. Box GEP, Behnken DW: Some new three level designs for the study of 

quantitative variables. Technometrics 1960, 2:455-475. 

P127 

Evaluation of three commercial kits for mycoplasma NAT assays: 

selection and quality improvement 

Fabien Dorange * , Frédérick Le Goff, Nicolas Dumey 

Texcell, Evry, France, 91058 

E-mail: dorange@texcell.fr 


Background: Mycoplasma testing on cell lines or biological products 

used to be performed based on classical methods such as agar and broth 

medium and/or indicator cell culture. However, these methods require a 

long incubation period and are not adapted for samples, like liveattenuated 

vaccine viruses (which can not completely be neutralized) or 

Page 171 of 181 

cell therapy products (with short shelf life). NAT assays have several 

advantages including rapid-time to results, robustness and sensitivity. The 

European Pharmacopoeia updated the 2.6.7 section by adding the 

detection of Mycoplasma with NAT methods as an alternative to one or 

both classical methods. 

Texcell’s offers for Mycoplasma testing, which already included 

classical methods, were incremended with NAT assay. For this 

purpose, 3 commercial kits based on NAT assay were evaluated based 

of their claim to meet the European Pharmacopeia guidance for 

nucleic acid amplification techniques for Mycoplasma testing, 

including sensitivity and range of detection: CytoCheck® from Greiner, 

MycoTOOL® from Roche, MycoSEQ® Mycoplasma detection kit from 

Life Technologies. 

Material and methods: 5 mycoplasma species were chosen among the 

9 strains listed in the European Pharmacopeia. 

Mycoplasma Pneumoniae: CIP 103766T 

Mycoplasma Hyorhinis: ATCC 179891 

Acholeplasma Laidlawii: ATCC 23206 

Mycoplasma Orale: provided by Greiner Bio-One 

Mycoplasma Synoviae: provided by Greiner Bio-One 

1 ml of different concentrations of mycoplasma were tested according to 

the supplier’s instructions. 

Results: For the mycotool results, none of the mycoplasma species tested 

were detected above the required limit of detection of 10 cfu/ml. 

Investigations showed that quantity of nucleic acids in our experimental 

design was too small to be efficiently recovered with the precipitation 

based extraction procedure. Indeed, the mycoTOOL® kit was designed to 

be used in conjonction with CHO cells (5x10 6 cells/ml), acting like carrier 

DNA for the precipitation step. Since this study, the kit’s supplier has 

included a carrier DNA for samples containing low level of nucleic acid. 

Both remaining selected kits (MycoSEQ® and CytoCheck®) met the 

sensitivity parameters and were compared (Table 1). 

Although these kits use a different technology, similar results (sensitivity, 

range of detection) were obtained. The lower sensitivity observed with 

the MycoSEQ® kit for M. Pneumoniae is explained by the presence of nonviable 

mycoplasmas induced during thawing/freezing cycle for stocks 

preparation. 

According to its safer use (no post-amplification handling), its lower cost 

and quantitation possibilities, the MycoSEQ® kit was preferred. 

Performance validation was conducted in Texcell facilities using the 

MycoSEQ® Mycoplasma detection kit.



Table 1(abstract P127) Comparison of the MycoSEQ® Mycoplasma detection kit and CytoCheck® kit 

KIT supplier 

Parameters MycoSEQ® CytoCheck® Priority 

Applied Biosystems Greiner 

tested sensitivity 40% saturation by 

pulsing or constant aeration with sterile air. A biphasic process scheme 

was applied; cell growth phase (0 – 120hcultivationtime)andvirus 

propagation phase (120 – 192 h cultivation time). For infection a media 

change was performed: cells attached to the microcarriers were allowed 

to settle down by stopping agitation for 2 h, after two rinsing steps with 

PBS the cell culture medium was completely exchanged against the same 

MEM with 1% L-Glutamin but without FBS (infection medium). 

Afterwards, a small amount of infection medium containing porcine 

Influenza A virus/H1N1/strain 2617 originating from WSV (IDT Biologika 

GmbH) with a moi of 10 -4 and a trypsine concentration of 40 units/ml 

was given into the bioreactor. Samples were taken in distinct intervals 

and aliquoted for offline analytics including cell count with CASY model 

TT (Roche Diagnostics), further cell analytics for apoptosis and necrosis 

with a flow cytometer Guava easycyte and respective commercial assays 

(Millipore GmbH), automated enzymatic assays for quantification of 

glucose, lactate, glutamine and ammonia levels (YSI 7100 MBS, Yellow 

Springs Instruments) and quantification of porcine Influenza virus with 

hemagglutination (HA) and virus titration assay (TCID 50) according to IDT 

internal protocols. Online data, especially for temperature, stirrer speed, 

pH-value, pO2 and gassing rate were recorded by the software MFCS/DA 

(Sartorius Stedim Biotech GmbH). 

Results and discussion: After seeding the cells attached rapidly onto the 

microcarriers; at first analytics after 24 h cells showed a good distribution 

on the microcarriers with typical morphology. Cell number increased up 

to a maximum of 2.1 x 10 6 cells/ml with a viability of 92% after 120 h, 

which was quite in accordance with expectations when using a carrier 

concentrationof2g/linabatchmodeprocess(figure1).Thegrowth 

rate µ max in the interval from 24-120 h was 0.037 h -1 which corresponds 

to a doubling time of about 19 h. The handling of medium change at the 

beginning of the virus propagation phase was tricky but can be easily 

improved by using customized bags for microcarrier cultivations with 

media changes. The cell culture during growth phase (both populations, 

cells on microcarriers and in suspension) was also monitored via nexin 

assay determining the part of apoptotic (early and late phase) and 

necrotic cells. The obtained data confirm the cell count and viability data 

measured with the CASY TT system and microscopic observations. 

The viral infection proceeded very rapidly with first signs of Influenza 

related cytopathic effect 24 h post infection, leading to virus yields of 

maximum log 3.0 HA units and 10 8.00 TCID50/ml after 48 h post infection. 

Afterwards the infectious titer dropped slightly while HA remained stable. 

The virus yields achieved were 2-4-fold higher compared to standard



Figure 1(abstract P128) MDBK cell growth and viability for cell populations on microcarriers and in suspension during cultivation in a 50 l scale 

disposable stirred-tank bioreactor run for propagation of porcine Influenza A/H1N1 (black line indicates virus infection). 

roller bottle cultivations. For a non optimized process, these virus yields 

are very promising. Very likely, by finetuning of infection parameters 

(moi, trypsine concentration), harvest time point and increase of cell 

numbers before infection even higher titers are achievable. 

One of the most important advantages of a bioreactor system is its 

controlled surrounding in terms of pH and oxygen level. In this study that 

fact was proven very well by the good performance of the control system 

(figure 2 and 3). The profile for pO2 and air flow rate corresponds well to 

the cultivation curve; in the first hours of cultivation the pulsing aeration 

can be seen as only a few cells were in the reactor system. With 

increasing cell numbers a constant air flow is applied starting approx. 

after 48 h cultivation time leading to a maximal airflow of 3.5 l/min 

keeping the oxygen level constant also when higher cell concentrations 

are present. The virus infection led to a rapid decrease in oxygen uptake; 

Page 173 of 181 

at the end of cultivation oxygen consumption tended to be zero (figure 

2). Temperature and stirrer speed control was not a problem throughout 

cultivation (figure 3); also control of pH value worked very well (7.40 ± 

0.15). 

Conclusions: The results of this study prove a successful tech transfer for 

a porcine Influenza virus production process from roller bottles into a 

novel disposable STR bioreactor system with advances in both cell and 

virus yields and process control possibilities. These are important factors 

in near future, keeping upstream processing competitive in terms of 

prices, productivity and surely also from a regulatory point of view. It is a 

further step in order to be prepared for pandemic situations as it was 

seen for 2009 H1N1 occurrence. There should be no obstacles for 

implementing such systems into GMP surrounding. As result of the study 

also a lot of valuable data were generated which can be used for process 

Figure 2(abstract P128) Online data from cultivation in a 50 l scale disposable stirred-tank bioreactor run for propagation of porcine Influenza A/H1N1 

for oxygen partial pressure and air flow rate.



Figure 3(abstract P128) Online data from cultivation in a 50 l scale disposable stirred-tank bioreactor run for propagation of porcine Influenza A/H1N1 

for temperature, stirrer speed and pH-value. 

validation and establishment of descriptive mathematical process models 

thus developing a deeper understanding of production processes for 

biologicals. 

P129 

Effect of influenza virus infection on key metabolic enzyme activities in 

MDCK cells 

Robert Janke 1* , Yvonne Genzel 1 , Maria Wetzel 2 , Udo Reichl 1,2 

1 Max Planck Institute for Dynamics of Complex Technical Systems, 

Bioprocess Engineering group, Sandtorstraße 1, 39106 Magdeburg, Germany; 

2 Chair for Bioprocess Engineering, Otto von Guericke University of 

Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany 

E-mail: janke@mpi-magdeburg.mpg.de 


Background: Influenza, or “flu”, is an upper respiratory tract infection 

caused by a virus belonging to the family of Orthomyxoviridae. Influenza 

can pose a serious risk to the health of mainly the elderly, the very 

young, and to people suffering from medical conditions (e.g. weak 

immune system). For example, seasonal influenza strains are fatal to 

more than 50,000 people annually in the United States alone [1]. The 

most effective measure for preventing influenza-related morbidity and 

mortality is annual vaccination. Seasonal influenza vaccines are almost 

exclusively produced using the traditional egg-based manufacturing 

process. However, the main limitation of egg-based technology 

(especially in the case of a pandemic) is the time-consuming production 

process (~6 months). Furthermore, people with serious egg allergy 

cannot be vaccinated when trace amounts of egg protein remain in the 

final formulation. Therefore, new production processes using continuous 

cell lines for influenza vaccine manufacturing are currently being 

established [2]. 

Influenza viruses take advantage of the host cell metabolism to replicate 

their genetic material and to synthesize viral proteins. The influenza virus 

particle consists of three major parts: the ribonucleocapsid, the matrix 

protein M1, and the envelope, which is derived from the plasma 

membrane of the host cell. The lipid bilayer contains the ion channel 

protein M2 and the immunogenic glycoproteins hemagglutinin and 

neuraminidase [3]. The replication cycle of influenza viruses including 

entry, uncoating, genome transcription and replication, assembly and 

release has been studied extensively with type A strains [4]. So far, only 

few studies have characterized the influence of influenza infection on the 

Page 174 of 181 

central carbon metabolism of host cells [5]. Madin-Darby canine kidney 

(MDCK) cells are considered a suitable substrate for cell culture-based 

influenza vaccine manufacturing [2,6]. In this study, key metabolic 

enzyme activities were analyzed in MDCK cells infected with an influenza 

A virus strain to improve our understanding of virus-host cell interaction 

and cell response. 

Materials and methods: All chemicals and enzymes were purchased 

from Sigma (Taufkirchen, Germany) or Roche (Mannheim, Germany). 

Adherent MDCK cells obtained from the ECACC (No. 84121903) were 

routinely cultured in 6-well plates containing 4 mL of GMEM-based 

medium (2 mM glutamine, 30 mM glucose, 10% (v/v) fetal calf serum, 

2 g/L peptone, 48 mM NaHCO3) inaCO2 incubator at 37 °C and 5% CO2 

to the stationary phase (~5 days of growth, 4.0-4.2 x 10 6 cells) [7]. Cell 

concentration and viability was determined for samples from 6-well 

plates as described previously [8]. MDCK cells were either mock-infected 

or infected with MDCK cell-adapted human influenza virus A/Puerto Rico/ 

8/34 (H1N1) from the Robert Koch Institute (Berlin, Germany) at a 

multiplicity of infection of 20 as described previously [5,9]. Cells were 

washed twice with ice-cold phosphate-buffered saline 9 hours post 

infection (hpi), and the complete plate was then snap-frozen in liquid 

nitrogen and stored at -80 °C until further use. After thawing, samples 

were extracted by sonification on maximum power for 30 s with 1 mL 

extraction buffer [7] and kept at 0–4 °C. To remove cell debris, samples 

were centrifuged at 16,000 x g for 5 min. The supernatant was used to 

measure the respective enzyme activities. The procedure for the enzyme 

activity analysis and the details for the specific assay mixes were as 

described previously [7]. 

Results: The maximum catalytic activities of 28 enzymes from central 

carbon metabolism were measured under substrate saturation using a 

recently developed assay platform for mammalian cells [7]. Table 1 shows 

the maximum metabolic enzyme activities in mock-infected and influenza 

A (H1N1) infected MDCK cells. The activities of different enzymes 

comprise several orders of magnitude. The overall range covers values 

from 0.24±0.10 nmol/min/10 6 cells (pyruvate dehydrogenase, PDH) to 

10348.06±1663.65 nmol/min/10 6 cells (triose-phosphate isomerase, TPI) in 

H1N1 and mock-infected cells, respectively. Highest activities (>1000 

nmol/min/10 6 cells) were found for TPI, pyruvate kinase, lactate 

dehydrogenase, and malate dehydrogenase, while the other activities 

were in the range of 1 to 500 nmol/min/10 6 cells. Very low enzyme 

activities, which indicate possible rate-limiting steps in the respective 

metabolic pathway, were found for PDH, pyruvate carboxylase (PC), 

NAD + -dependent isocitrate dehydrogenase (NAD-ICDH), and glutamine



Table 1(abstract P129) Maximum enzyme activities of glycolysis, pentose phosphate pathway, TCA cycle, and 

glutaminolysis in MDCK cells infected with influenza A (H1N1) compared to mock-infected cells 

Enzyme activities in adherent MDCK cells a (nmol/min/10 6 cells) 

Enzyme EC number Mock-infected H1N1 infected 

Glycolysis 

Hexokinase 2.7.1.1 21.80 ± 3.95 21.89 ± 1.48 

Phosphoglucose isomerase 5.3.1.9 465.12 ± 73.94 456.93 ± 111.90 

Phosphofructokinase 2.7.1.11 29.28 ± 6.14 30.56 ± 2.08 

Fructose-1,6-bisphosphate aldolase 4.1.2.13 23.62 ± 3.32 20.74 ± 8.41 

Triose-phosphate isomerase 5.3.1.1 10348.06 ± 1663.65 10148.62 ± 698.01 

Glyceraldehyde-3-phosphate dehydrogenase 1.2.1.12 412.90 ± 73.27 413.61 ± 31.61 

Pyruvate kinase 2.7.1.40 1004.26 ± 43.53 1001.92 ± 112.95 

Lactate dehydrogenase 

Pentose phosphate pathway 

1.1.1.27 1266.97 ± 134.69 1302.10 ± 107.37 

Glucose-6-phosphate dehydrogenase 1.1.1.49 51.91 ± 1.10 62.71 ± 4.59 b 

6-phosphogluconate dehydrogenase 1.1.1.44 30.61 ± 5.03 38.13 ± 1.05 b 

Transketolase 2.2.1.1 18.63 ± 6.65 19.19 ± 2.52 

Transaldolase 

Tricarboxylic acid cycle 

2.2.1.2 23.11 ± 5.84 23.23 ± 6.30 

Pyruvate dehydrogenase 1.2.4.1 0.30 ± 0.07 0.24 ± 0.10 

Pyruvate carboxylase 6.4.1.1 0.48 ± 0.11 0.82 ± 0.23 b 

Citrate synthase 2.3.3.1 21.55 ± 1.70 25.40 ± 2.84 b 

Citrate lyase 2.3.3.8 4.48 ± 0.55 6.00 ± 0.98 b 

NAD + -linked isocitrate dehydrogenase 1.1.1.41 0.27 ± 0.02 0.34 ± 0.05 b 

NADP + -linked isocitrate dehydrogenase 1.1.1.42 44.27 ± 2.73 44.57 ± 5.37 

Fumarase 4.2.1.2 74.62 ± 6.56 77.85 ± 11.75 

Malate dehydrogenase 

Glutaminolysis 

1.1.1.37 1115.42 ± 90.90 1158.66 ± 145.10 

Glutaminase 3.5.1.2 3.29 ± 0.29 3.90 ± 0.34 b 

Glutamine synthetase 6.3.1.2 0.68 ± 0.25 0.80 ± 0.25 

Glutamate dehydrogenase 1.4.1.2 2.54 ± 0.51 2.20 ± 0.31 

Alanine transaminase 2.6.1.2 3.21 ± 0.50 3.31 ± 0.03 

Aspartate transaminase 2.6.1.1 78.67 ± 5.36 79.12 ± 13.17 

Malic enzyme 1.1.1.40 7.61 ± 0.61 9.87 ± 0.62 b 

Phosphoenolpyruvate carboxykinase 

Miscellaneous 

4.1.1.32 138.64 ± 15.33 131.48 ± 11.42 

Acetate-CoA ligase 6.2.1.1 2.01 ± 0.13 2.69 ± 0.34 b 

a 

Mean values and 95 % confidence intervals for three biological replicates. 

b 

Significantly different from mock-infected cells as determined by Student’s t-test (p ≤ 0.05). 

synthetase (



Figure 1(abstract P129) Reaction network scheme of the central carbon metabolism of adherent MDCK cells. Enzymes up-regulated in influenza A 

(H1N1) infected cells are highlighted in blue. Abbreviations: 6PGDH, 6-phosphogluconate dehydrogenase; ACoAL, acetate-CoA ligase; CL, citrate lyase; CS, 

citrate synthase; G6PDH, glucose-6-phosphate dehydrogenase; GLNase, glutaminase; MDH, malate dehydrogenase; ME, malic enzyme; PDH, pyruvate 

dehydrogenase; PC, pyruvate carboxylase. 

Ltd: Mahy BWJ, ter Meulen V , 10 2010, 3 and 4:634-698[http://onlinelibrary. 

wiley.com/doi/10.1002/9780470688618.taw0238/abstract]. 

5. Ritter JB, Wahl AS, Freund S, Genzel Y, Reichl U: Metabolic effects of 

influenza virus infection in cultured animal cells: Intra- and extracellular 

metabolite profiling. Bmc Syst Biol 2010, 4. 

6. Doroshenko A, Halperin SA: Trivalent MDCK cell culture-derived influenza 

vaccine Optaflu (Novartis Vaccines). Expert Rev Vaccines 2009, 8(6):679-688. 

7. Janke R, Genzel Y, Wahl A, Reichl U: Measurement of Key Metabolic 

Enzyme Activities in Mammalian Cells Using Rapid and Sensitive 

Microplate-Based Assays. Biotechnol Bioeng 2010, 107(3):566-581. 

8. Genzel Y, Ritter JB, König S, Alt R, Reichl U: Substitution of glutamine by 

pyruvate to reduce ammonia formation and growth inhibition of 

mammalian cells. Biotechnol Progr 2005, 21(1):58-69[http://onlinelibrary. 

wiley.com/doi/10.1021/bp049827d/abstract]. 

9. Genzel Y, Behrendt I, König S, Sann H, Reichl U: Metabolism of MDCK 

cells during cell growth and influenza virus production in large-scale 

microcarrier culture. Vaccine 2004, 22(17-18):2202-2208. 

P130 

Novel human partner cell line for immortalisation of rare antigenspecific 

B cells in mAb development 

Galina Kaseko * , Marjorie Liu, Qiong Li, Tohsak Mahaworasilpa 

The Stephen Sanig Research Institute, Suite G17, National Innovation Centre, 

Australian Technology Park, 4 Cornwallis Street, Eveleigh, NSW, 2015, 

Australia 

E-mail: g.kaseko@ssri.org.au 


It is well-documented that post-translational modification (PTM) events, 

such as glycosylation, play an important role in antibody-dependent cellmediated 

cytotoxicity (ADCC) [1,2]. In current technological processes, 

monoclonal antibody (mAb) production is widely achieved using 

heterologous hybridoma systems or genetic engineering using various 

Page 176 of 181 

non-human cell lines as expression host. As a consequence, PTMs 

generated from non-human cell lines may differ from their human 

counterparts, resulting in diminished antibody efficacy, aberrant folding 

and adverse immunogenic response. Therefore, the use of human partner 

cell lines to generate “fully human” mAbs is beneficial as it circumvents 

functional complications associated with non-human cell lines. 

A human cross-lineage hybrid cell line was developed in our laboratory as 

a candidate partner for immortalisation of rare primary human antigenspecific 

B lymphocytes using binary electrical cell hybridisation technique 

which has been developed in house. This novel partner cell line is a trihybrid 

of IL-4 secreting Th2 lymphoblast derived from a patient with 

acute lymphoblastic leukaemia (T), CD20 + B lymphoblast, also derived 

from a patient with acute lymphoblastic leukaemia (W), and IL-6 secreting 

peripheral blood-derived CD14 + monocyte (M). The selection of cell 

phenotypes used to create the tri-hybrid was based on factors known to 

maintain and promote antibody production. 

The resulting tri-hybrid (WTM) displayed characteristics of mixed CD 

phenotypes with the majority of cells being CD20 + (95%) with coexpression 

of CD4 + (54%) and CD14 + (24%). It secreted IL-4, IL-6, IL-8 and 

GM-CSF but was negative for IL-1A, IL-1B, IL-2, IL-5, IL-10, IL-12, IL-13 and 

IL-17.Thecelllinedidnotexpressthe tumour suppressor protein, p53, 

and neither did it secrete immunoglobulins (Ig)/ Ig chains nor were they 

expressed on the surface. 

Table 1(abstract P130) Success rate of stable hybrid 

generation and Ig producing hybrids 

Event Success rate, 

Hybridisation 100 (%) of attempts 

Stable hybrids 48 (%) of hybridised 

Ig producing hybrids 23 (%) of stable hybrids



WTM cells were then used as a fusion partner with primary antigenexperienced 

CD19 + B cells which had been isolated from peripheral 

blood, activated in vitro, and the resultant hybrids were sorted for IgM + 

and IgG + expression. 100% hybridisation success rate was achieved using 

a binary electrical cell hybridisation technique and the number of 

resulting stable hybrids varied from 48% to 78% depending on the 

phenotype of B lymphocytes used in experiments. 23% to 68% of those 

stable hybrids secreted Ig with production ranging between 0.2 to 1.2 ìg/ 

10 6 cells (Table 1). Cytokine screening of some of the Ig producing 

hybrids revealed a cytokine profile which was inherently different to that 

of the WTM partner cell line. The Ig producing hybrids concurrently 

expressed IL-10 and GM-CSF but not IL-4, IL-6 or IL-8. These hybrids were 

also positive for TGF-â, RANTES, MIP, MCP and MDC. 

In conclusion, major advantages of our method involve the rapid 

generation of stable Ig producing hybrids from a small B lymphocyte 

population size (50 cells) and the elimination of conventional laborious 

screening methods for hybrids and Ig producing clones. Thus, when the 

number of rare antigen-specific B cells available is a limiting factor in 

generating hybridoma, EBV transfection or direct sequencing, binary 

electrical B lymphocyte hybridisation with WTM cells can provide a very 

attractive approach for the generation of stable hybrid cell lines 

producing monoclonal antibodies. 

References 

1. Raju TS: Impact of Fc Glycosylation on Monoclonal Antibody Effector 

Functions and Degradation by Proteases. In Current Trends in Monoclonal 

Antibody Development and Manufacturing Biotechnology: Pharmaceutical 

Aspects NY 3, USA: Springer Science + Business Media: Shire S 2010 1001, 

XI(6):249-269. 

2. Werner RG, Kopp K, Schlueter M: Glycosylation of therapeutic proteins in 

different production systems. Acta Paediatri 2007, 96:17-22. 

Page 177 of 181 

P131 

Application of the novel and convenient IR/MAR gene amplification 

technology to the production of recombinant protein pharmaceuticals 

Yoshio Araki 1 , Chiemi Noguchi 1 , Tetsuro Hamafuji 2 , Hiroshi Nose 2 , 

Daisuke Miki 3 , Noriaki Shimizu 1* 

1 Graduate School of Biosphere Science, Hiroshima University, Higashihiroshima, 

739-8521, Japan; 2 Transgenic Inc., Kobe, 650-0047, Japan; 3 Tosoh 

Co., Tokyo, 252-1123, Japan 

E-mail: shimizu@hiroshima-u.ac.jp 


Amplification of DHFR gene in CHO cells by selection of MTx has been 

widely applied to the establishment of stable cell lines that efficiently 

produce recombinant protein pharmaceuticals. However, the DHFR/ 

MTx technology was highly time-and labor-consuming. On the other 

hand, we had found that a plasmid bearing a mammalian replication 

initiation region (IR) and a matrix attachment region (MAR) initiates 

gene amplification in mammalian cells, and it quite efficiently 

generate the chromosomal HSR and/or the extrachromosomal DMs 

[1,2]. This is a completely original technology of gene amplification, 

and we have revealed the mechanism why and how such plasmid 

may mimic gene amplification [2,3]. Now, we aimed to adopt this 

technology to the industrial production of recombinant protein 

pharmaceuticals. 

We constructed plasmids with or without IR/MAR, with several promoters 

for drug resistant gene, antibody gene or Fc receptor gene, and with 

various orientations of these elements. The plasmid DNA with several 

physical structures were transfected to human COLO 320DM or hamster 

CHO DG44 cells, with or without another DNA. The transformed cells 

Figure 1(abstract P131) The IR/MAR plasmid can generates DMs (A), HSR (B), Ladder-HSR (C), and Fine ladder-HSR (D). Among the metaphase 

chromosome spread, we detected the plasmid sequence by FISH in green. Gene expression was generally higher from DMs than HSR. However, DMs are 

usually hard to be generated in CHO cells. The IR/MAR plasmid can efficiently generate the ladder and the fine ladder structure that is active in 

transcription.



were selected by various conditions. The polyclonal transformants or the 

cloned cells were evaluated by the protein production (ELISA), as well as 

by the structure of amplified region (FISH). 

As a result, the usage of IR/MAR technology enabled us to obtain cells, in 

which the introduced genes were amplified to a few hundreds to 

thousands copies per cells as DMs or HSR of various size and shape 

(Figure 1), which depended both on the vector constructs and the host 

cell lines. Such stable cells with amplified genes could be obtained within 

one month, and the protein production was increased more than a 

hundred-fold compared with the case without IR/MAR. A cell clone 

showed the specific production rate that reached almost the highest 

reported for antibody protein (45 pg/cell/day). Furthermore, we have 

found several novel ways that further improve the protein production 

level. For example, the combination of the IR/MAR and the DHFR/MTx 

technologies synergistically work and far more rapidly and easily generate 

the cells of higher production rate than previously. 

In conclusion, the IR/MAR technology is a novel highly-competitive 

technology for use in recombinant protein production, and it further has 

potentials for improvement. 

References 

1. Shimizu N, Miura Y, Sakamoto Y, Tsutsui K: Plasmids with a Mammalian 

Replication Origin and a Matrix Attachment Region Initiate the Event 

Similar to Gene Amplification. Cancer Res 2001, 61:6987-6990. 

2. Shimizu N, Hashizume T, Shingaki K, Kawamoto J: Amplification of 

plasmids containing a mammalian replication initiation region is 

mediated by controllable conflict between replication and transcription. 

Cancer Res 2003, 63:5281-5290. 

3. Harada S, Sekiguchi N, Shimizu N: Amplification of a plasmid bearing a 

mammalian replication initiation region in chromosomal and 

extrachromosomal contexts. Nuc Acids Res 2011, 39:958-969. 

P132 

About making a CHO production cell line “research-friendly” by genetic 

engineering 

Bernd Voedisch 1* , Agnès Patoux 1 , Jildou Sterkenburgh 2 , Mirjam Buchs 1 , 

Emily Barry 3 , Cyril Allard 1 , Sabine Geisse 1 

1 Novartis Pharma AG, Basel, Switzerland; 2 University of Cambridge, 

Cambridge, United Kingdom; 3 Cardiff University, Cardiff, United Kingdom 


The use of Chinese Hamster Ovary (CHO) cells for transient gene expression is 

gaining importance steadily, as it has been shown that both quality and 

quantity of proteins derived from CHO cells differs from and can even be 

superior to material derived from Human Embryonic Kidney (HEK293) cells 

[1-4]. In order to augment yields HEK cell lines have been genetically modified, 

by e.g. stable integration of an expression cassette coding for the Epstein-Barr 

Virus Nuclear Antigen 1 (EBNA1) in conjunction with expression plasmid 

vectors containing the EBNA1 interaction site oriP. Here we describe the 

generation of a CHO cell line stably expressing the EBNA1 gene to enhance 

yields after large scale transient transfection with polyethylenimine (PEI). 

An expression plasmid featuring the EBNA1 gene was transfected into an 

in-house available CHO wild type cell line by nucleofection and several 

stable pools were established by antibiotic selection. In a first approach, 

133 clones were then isolated by limiting dilution cloning and 

subsequently expanded for further analysis. The approach aimed at 

identifying clones showing both the presence of EBNA1 protein and 

enhanced yields after PEI-mediated transient expression of a reporter 

protein at the same time. 

Cell lysates or nuclear and cytoplasmic protein extracts from these clones 

were tested for presence of EBNA1 protein by Western Blot. An inlicensed 

cell line, CHO EBNALT85 (Icosagen AS, Tartu, Estonia) expressing 

the full length EBNA1 gene, and the HEK293-6E cell line (from the group 

of Y. Durocher, NRC, Canada) expressing a truncated EBNA1 gene served 

as positive controls. A number of commercially available anti-EBNA1 

antibodies were tested in Western blotting, but most antibodies failed to 

detect the EBNA1 protein produced by these positive control cells. Only 

one antibody (Antibody 1EB12, Santa Cruz Biotechnology) was identified 

that detected the EBNA1 proteins in both positive control cell lines. 

However, by applying this antibody on the 133 generated clones it 

became obvious that in most of them the EBNA1 protein appeared to be 

largely degraded. 

Page 178 of 181 

The functionality of the EBNA1 protein in the cells was probed by PEImediated 

transient transfection of expression plasmids encoding the 

gene for the reporter protein Secreted Alkaline Phosphatase (SeAP). SeAP 

expression levels were compared with respect to presence or absence of 

the EBNA1 interaction sequence oriP on the SeAP expression plasmid. 

Only one clone showed a twofold enhanced production level of the 

reporter protein compared to the non-engineered parental CHO cell line. 

However, in this specific clone no EBNA1 protein could be detected by 

Western Blot, and the enhanced expression level was independent from 

the presence or absence of the oriP sequence on the SeAP expression 

plasmid. We therefore conclude that this clone does not possess a 

functional EBNA1 protein and an EBNA1/oriP based enhancement of 

productivity. The mechanism underlying the enhanced yield of the 

reporter protein in this particular clone remains currently elusive. 

In an alternative approach flow cytometric cell sorting was applied to 

generate CHO clones producing a functional EBNA1 protein. Using the 

EBNA1 positive CHO cell line CHO EBNALT85 it could be shown that 

expressing eGFP from a transiently transfected expression plasmid with 

oriP sequence resulted in a 30- to 40-fold increase of highly fluorescent 

cells in comparison to an expression plasmid lacking the oriP sequence. 

Thus, the already generated CHO pools stably transfected with the EBNA1 

gene were transiently supertransfected with an eGFP expression plasmid 

containing the oriP sequence. Highly fluorescent cells were collected by 

cell sorting and expanded. Analysis by Western Blot revealed that this 

sub-poolwasenrichedforcellsexhibiting the presence of a full length 

EBNA1 protein besides a major breakdown product. Transient transfection 

of this sub-pool with SeAP expression vectors led to the conclusion that 

the pool contained cells with a functional EBNA1 protein capable of 

enhancing SeAP yields in dependence on the presence of the oriP 

sequence on the SeAP expression plasmid. Clones were again generated 

from this sub-pool by limiting dilution cloning and characterized in 

analogy to the first approach. 9 out of 12 clones that were analyzed 

further showed increased levels of reporter protein production in 

dependence on the presence of the oriP element on the expression 

plasmid. The series of clones exhibiting highest expression levels will now 

be further evaluated by expression of other protein candidates. 

References 

1. Gaudry JP, Arod C, Sauvage C, Busso S, Dupraz P, Pankiewicz R, 

Antonsson B: Purification of the extracellular domain of the membrane 

protein GlialCAM expressed in HEK and CHO cells and comparison of 

the glycosylation. Protein Expr Purif 2008, 58(1):94-102. 

2. Haack A, Schmitt C, Poller W, Oldenburg J, Hanfland P, Brackmann HH, 

Schwaab R: Analysis of expression kinetics and activity of a new Bdomain 

truncated and full-length FVIII protein in three different cell 

lines. Ann Hematol 1999, 78(3):111-116. 

3. Suen KF, Turner MS, Gao F, Liu B, Althage A, Slavin A, Ou W, Zuo E, 

Eckart M, Ogawa T, et al: Transient expression of an IL-23R extracellular 

domain Fc fusion protein in CHO versus HEK cells results in improved 

plasma exposure. Protein Expr Purif 2010, 71(1):96-102. 

4. Van den Nieuwenhof IM, Koistinen H, Easton RL, Koistinen R, Kamarainen M, 

Morris HR, Van Die I, Seppala M, Dell A, Van den Eijnden DH: Recombinant 

glycodelin carrying the same type of glycan structures as contraceptive 

glycodelin-A can be produced in human kidney 293 cells but not in 

chinese hamster ovary cells. Eur J Biochem 2000, 267(15):4753-4762. 

P133 

CAP-T cell expression system: a novel rapid and versatile human cell 

expression system for fast and high yield transient protein expression 

Jens Wölfel * , Ruth Essers, Corinna Bialek, Sabine Hertel, 

Nadine Scholz-Neumann, Gudrun Schiedner 

CEVEC Pharmaceuticals GmbH, Cologne, Germany 


Backround: CAP (CEVEC’s Amniocyte Production) cells are an 

immortalized cell line based on primary human amniocytes. They were 

generated by transfection of these primary cells with a vector containing 

the functions E1 and pIX of adenovirus 5. CAP cells allow for competitive 

stable production of recombinant proteins with excellent biologic activity 

and therapeutic efficacy as a result of authentic human posttranslational 

modification. In order to gain access to the benefits of the CAP technology 

also for early research, target evaluation or assay development, the



transient expression system CAP-T was developed. CAP-T cells are based 

on the original CAP cells and additionally express the large T antigen of 

simian virus 40 (SV40). 

Results: To characterize the CAP-T expression system, they were 

transiently transfected with an expression plasmid for the highly complex 

and glycosylated human a1-Antitrypsin (hAAT). This resulted in 

remarkably high expression levels of up to 60 mg/L. These levels could 

be even increased 2.5 fold by adding an SV40 origin of replication 

(SV40ori) to the expression vector (Figure 1). When compared to HEK293T 

cells, CAP cells showed a significantly higher expression titer than 

HEK293T cells (data not shown). In order to understand these 

phenomena, the copy number of the expression plasmid, upon 

transfection, was determined. CAP-T efficiently replicated the expression 

plasmid containing the SV40ori, resulting in increasing copy numbers per 

cell over time yielding in about 4 times higher copy numbers than in 

HEK293T cells transfected with the same plasmid and in CAP-T cells 

transfected with the plasmid lacking the SV40ori (data not shown) and 

consequently in higher expression levels. In order to establish a more 

scalable transfection method than nucleofection, the suitability of 

different transfection reagents for the transfection of CAP-T cells was 

determined. In these experiments beside nucleofection, 293fectin and 

polyethylenimin (PEI) based transfection methods showed best results in 

Page 179 of 181 

Figure 1(abstract P133) Transient expression of hAAT in CAP-T: Two plasmids containing a hAAt expression cassette were transfected by nucleofection 

(1 x 10 7 cells). The plasmid containing a SV40ori (ori) yielded about 2.5 time higher expression levels at maximum product concentration than a plasmid 

lacking this ori (w/o ori). 

small scale transfections, enabling the scalability of the transient 

transfection in CAP-T cells (data not shown). With the PEI based 

transfection method hAAT could be produced also in 300 mL shaking 

culture and 1L bioreactor with product titers of up to 180 mg/L in simple 

batch processes of up to 10 days (data not shown). Several other 

glycosylated proteins have been tested in transient transfections of CAP-T 

cells yielding comparable product titers (Table 1). 

Summary: In summary, CAP-T cells present a highly efficient transient 

expression system enabling the generation of mg amounts of the protein 

of interest for early research and development within only two weeks 

from gene to product. Furthermore, CAP-T cell produced proteins showed 

fully human posttranslational modification pattern, which was also 

observed for the original human CAP cells, the CAP-T cells were derived 

from. The CAP technology based on CAP-T cells for transient transfection 

and CAP cells for stable protein production [1] therefore provides a 

unique system in which the whole process from early research to 

production of therapeutic proteins can be run through with the same cell 

type. 

Reference 

1. Essers R, Kewes H, Schiedner S: Improving volumetric productivity of a 

stable human CAP cell line by bioprocess optimization. BMC Proceedings , 

abstract within the same supplement. 

Table 1(abstract P133) summarizes the relevant data from transient transfections of CAP-T cells in different scales 

with plasmids coding for different proteins of interest (hAAT, erythropoietin (EPO), C1-Inhibitor and IgG). Cells were 

either transfected by nucleofection (1 x 10 7 cells) or by PEI (1.7 x 10 9 cells) 

hAAT EPO C1-Inhibitor IgG 

cells transfected 1 x 10 7 

1.7 x 10 9 

1x10 7 

1x10 7 

1x10 7 

culture volume [ml] 30 1000 60 30 30 

culture time [days] 9 6 10 12 6 

viability at harvest [%] 85 70 80 87 80 

volumetric productivity [mg/L] 170 180 38 35 150



P134 

Production and purification of TGFb-1 in CHO-Cells 

Estabraq Abdulkerim 1* , Sabrina Baganz 1 , Axel Schambach 2 , Cornelia Kasper 1 , 

Thomas Scheper 1 

1 Institute of Technical Chemistry, Leibniz University of Hanover, 30167 

Hannover, Germany; 2 Department of Experimental Hematology, Hanover 

Medical School, 30625 Hannover, Germany 


Introduction: The development of chemically well defined media is a 

demanding task in order to create the optimal conditions for an in vitro 

stem cell (SC) proliferation and differentiation system. Signals that govern SC 

differentiation into multiple mature cell types are provided by growth 

factors. TGFb-1 regulates a number of biological processes, including cell 

differentiation and proliferation, embryonic development, apoptosis and 

immune responses, together with cell surface receptors and signal 

transduction molecules within the cell. The whole signal transduction 

pathway leads to the specific production of distinct proteins. In this work, 

TGFb-1 fragment (A280 – S391) is produced using a tailor-made CHO cell 

line (CHO SFS ) and purified. 

Results: Chinese Hamster Ovary cells have been subjected to lentiviral 

transduction of TGF beta 1 vector (figure 1) resulting in the expression 

of His- and HA-tagged protein from (CHO SFS ) cells. This transduction 

was accomplished at the department of Hematology, Hanover Medical 

School [1]. 

Production test for TGFb-1: The cells were verified via flow cytometry for 

the successful transduction. The selected method involves the specific 

Figure 1(abstract P134) TGFb-1 vector used for the lentiviral transduction of CHO cell line (CHO SFS ). 

Page 180 of 181 

intracellular detection of His-tag by the help of fluorescence-labelled 

antibody. 

The fixation of 2*10 5 cells was performed by 4 % paraformaldehyde solution, 

cells were permeated with 0.1 % saponin followed by an incubation of the 

cells in 100 µL primary antibody. Afterwards the antibody was coupled to a 

Phycoerythrin (PE) labelled secondry antibody with a fluorescent character. 

To control staining specifity, non trasfected CHO cells were used as 

negative control which was treated with the same procedure as 

transfected cells. The results of the staining show that about 77% of the 

cells are successfully transfected. 

For the localisation of TGFb-1 cell culture supernatant and cell pellets after 

lysis via ultrasonic, were analysed via western blot using a combination of 

mouse-anti-His-tag and goat-anti-mouse-IgG-AP-conjugate. The results 

indicate that only a part of the protein is secreted extracellulary and the rest 

is present intracellulary. 

Cultivation of CHO cell line (CHO SFS ): CHO cells were cultured in serum 

free ProCHO 5 with 1% penicillin/streptomycin (PAA) and 2% L-glutamine 

solution (4mM). A 250 ml spinner flask (rpm. 80) was used for cell growth 

starting with a cell density of 0.4 * 10 6 cells*ml -1 and a volume of 100 ml. 

The cultivation was carried out for 108 hours and medium was changed 

every 2-3 days. Samples were taken every 24 hour to determine cell 

density and viability. A maximal cell density of 1.8*10 6 cells/ml could be 

achieved with a viability of 80 %. 

Purification of TGF beta 1: The supernatant of the culture was used, to 

perform the downstream processing. 

The cells were separated by means of centrifugation and the clean 

supernatant was purified via heparin affinity chromatography (HiTrap TM 

Heparin HP columns, GE Healthcare).



The elution was performed using the binding buffer (10 mM NaH 2PO 4, 

pH 7) in addition to a linear NaCl gradient (max. 2M). 

Afterwards all protein containing fractions obtained by FPLC were 

analysed using silver stained SDS-PAGE. The results show that TGFb-1 

could be purified with a purity of 70-80 %. 

Conclusion: The production and secretion of TGFb-1 in the CHO cell line 

(CHO SFS ) was successfully performed. The purification of protein was 

achieved using heparin affinity chromatography. Further upscaling of the 

procedure will be performed for achieving higher yield of the targeted 

protein. 

Acknowledgement: This work was performed within the activities of the 

JRG “large scale cultivation” of the DFG cluster of excellence “Rebirth”. 

Page 181 of 181 

Reference 

1. Schambach A, Galla M, Modlich U, Will E, Chandra S, Reeves L, Colbert M, 

Williams DA, von Kalle C, Baum C: Lentiviral vectors pseudotyped with 

murine ecotropic envelope: Increased biosafety and convenience in 

preclinical research. Experimental Hematology 2006, 34:588-592. 

Cite abstracts in this supplement using the relevant abstract number, 

e.g.: Abdulkerim et al.: Production and purification of TGFb-1 in CHO-Cells. 

BMC Proceedings 2011, 5(Suppl 8):P134

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