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BMC Proceedings 2013, Volume 7 Suppl 6<br />
http://www.biomedcentral.com/bmcproc/supplements/7/S6<br />
MEETING ABSTRACTS<br />
23rd European Society for Animal Cell<br />
Technology (ESACT) Meeting: Better Cells for<br />
Better Health<br />
Lille, France. 23-26 June 2013<br />
Edited by Hansjörg Hauser<br />
Published: 4 December 2013<br />
These <strong>abstracts</strong> are available online at http://www.biomedcentral.com/bmcproc/supplements/7/S6<br />
Open Access<br />
INTRODUCTION<br />
I1<br />
Better Cells for Better Health: Abstracts of the 23 rd ESACT Meeting<br />
2013 in Lille<br />
Hansjörg Hauser<br />
Helmholtz-Zentrum für Infektionsforschung GmbH, Department of Gene<br />
Regulation and Differentiation, 38124 Braunschweig, Germany<br />
E-mail: hansjoerg.hauser@helmholtz-hzi.de<br />
BMC Proceedings 2013, 7(Suppl 6):I1<br />
The European Society of Animal Cell Technology (ESACT) is a society that<br />
brings together scientists, engineers and other specialists working with<br />
animal cells in order to promote communication of experiences between<br />
European and international investigators and progress development of<br />
cell systems in productions derived from them.<br />
Animal cells are being used as substrates in basic research and also for<br />
the production of proteins. Tissue engineering, gene and cell therapies,<br />
organ replacement technologies and cell-based biosensors contribute to<br />
a considerable widening of interest and research activity based on animal<br />
cell technology.<br />
Since its foundation 35 years ago, the ESACT Meeting has developed into<br />
the international reference event in animal cell technology, building on a<br />
tradition of combining both basic science and its application into<br />
industrial biotechnology.<br />
The <strong>abstracts</strong> of this supplement are from the 23 rd ESACT meeting that<br />
was held in Lille, France, June 23 - 26, 2013. The <strong>abstracts</strong> review the<br />
presentations from this meeting and should be a useful resource for the<br />
state-of-the-art in animal cell technology.<br />
ORAL PRESENTATIONS<br />
O1<br />
A novel genotype of MVA that efficiently replicates in single cell<br />
suspensions<br />
Ingo Jordan * , Volker Sandig<br />
ProBioGen AG, 13086 Berlin, Germany<br />
E-mail: ingo.jordan@probiogen.de<br />
BMC Proceedings 2013, 7(Suppl 6):O1<br />
Background: Vectored vaccines based on modified vaccinia Ankara<br />
(MVA) may lead to new treatment options against infectious diseases and<br />
certain cancers. MVA is highly attenuated and requires avian cells for<br />
production. We established avian continuous cell lines (including CR and<br />
related CR.pIX) and adapted these cells to proliferation in single-cell<br />
suspension in a chemic<strong>all</strong>y defined medium [1,2]. Replication of several<br />
viruses was efficient in CR suspension cultures [3,4] but yields for MVA<br />
were low. We suspected that cell-to-cell spread may be an important<br />
mechanism for MVA replication in agitated suspension cultures and<br />
developed a production medium that is added at the time of infection to<br />
induce cell aggregates [2]. MVA (and other host-restricted poxviruses)<br />
replicate to very high titers with this robust and fully scalable cultivation<br />
protocol but further improvement may facilitate production for large<br />
vaccine programs. We now describe a novel genotype of MVA that<br />
replicates with high efficiency in single-cell suspensions without<br />
aggregate induction.<br />
Materials and methods: Motivated to discover new phenotypes, we<br />
quantified replication of successive MVA passages in aggregated CR<br />
suspension cultures. Because titers increased slightly within 10 passages,<br />
viral genomic DNA of early and late passages was sequenced. Of the<br />
advanced passage, a contiguous sequence of 135 kb was recovered and<br />
revealed a genotype (which we c<strong>all</strong> MVA-CR) where the structural proteins<br />
A3L, A9L and A34R (in vaccinia virus nomenclature) each carry a single<br />
amino acid exchange (Figure 1A). The novel genotype appears to<br />
accumulate in our system but to completely remove traces of wildtype<br />
plaque purification was performed. The pure isolate (c<strong>all</strong>ed MVA-CR19) was<br />
further characterized and compared to the wildtype.<br />
Results: The aggregate-based process was developed to facilitate cell-to-cell<br />
spread, which appears to be an important mechanism for vaccinia virus<br />
replication. Surprisingly, multiplication of MVA-CR19 appears to be efficient<br />
also in single-cell avian suspension cultures (Figure 1B) with increased<br />
infectious titers in the cell-free supernatant. Because of this qualitative<br />
difference between wildtype and MVA-CR19, we hypothesized that a sm<strong>all</strong>er<br />
fraction of the MVA-CR isolate remains cell associated and that this capacity<br />
<strong>all</strong>ows viruses of the novel genotype to spread also in single cell<br />
suspensions. As one test of our proposed explanation we repeated the<br />
passaging experiments in adherent cultures. No mutations in the three<br />
genes that distinguish MVA-CR were detected, suggesting that the<br />
contribution of host cell properties to the observed changes in the virus<br />
population recovered from the suspension process may be negligible.<br />
However, the MVA-CR phenotype is evident also in adherent cells: compared<br />
to wildtype MVA, plaques formed by MVA-CR19 on CR cell monolayers in<br />
comet assays appear to be larger and to develop earlier [5]. These results<br />
are consistent with mechanisms that <strong>all</strong>ow MVA-CR19 to replicate, infect or<br />
uncoat faster, or be released with greater efficiency from host cells. For<br />
further characterization of this effect, adherent cells were infected with a<br />
high multiplicity of 10 and briefly subjected to a pH shift. This is predicted<br />
© 2013 various authors, licensee <strong>BioMed</strong> <strong>Central</strong> Ltd. All articles published in this supplement are distributed under the terms of the<br />
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and<br />
reproduction in any medium, provided the original work is properly cited.
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Figure 1(abstract O1) (A) Schematic of the genomic DNA of MVA-CR. The region covered by next generation sequencing is shown together with the<br />
mutations (in single letter code for amino acids, e.g. H639Y is His 639 ® Tyr) in the three genes. ITR (viral telomers) and deletion sites in MVA as light gray boxes<br />
are shown for orientation. (B) CR.pIX single-cell suspension cultures were infected with wildtype (wt) and MVA-CR19. Cells were immunostained for virus<br />
antigens 48 h post infection and quantified by FACS to investigate differences in the dissemination of infectious units in absence of aggregate induction.<br />
(C) Cell fusion is induced by wildtpe MVA but less so by MVA-CR19. Red immunofluorescence against MVA antigens serves as a positive control for infection.<br />
Blue fluorescence of DNA is shown for orientation. MVA-negative cells next to infected cells are shown in the panels where virus was added to a multiplicity<br />
of infection (MOI) of 0.1.<br />
to activate the viral fusion apparatus so that cell-associated viruses in a<br />
confluent cell monolayer can induce formation of syncitia [6]. As shown in<br />
Figure 1C, cell fusion appears to be less pronounced in cultures infected<br />
with MVA-CR suggesting that either fewer virions of this genotype remain<br />
cell associated or that fusion may be less important for entry of such virions.<br />
A molecular basis for the proposed improved MVA-CR19 dissemination is<br />
that <strong>all</strong> three of the observed mutations each target a different<br />
component of the complex viral particles, the core and the different<br />
membranes of the mature intracellular and extracellular virions. We are in<br />
the process of generating various combinations of recombinant MVAs to<br />
determine whether <strong>all</strong> three factors need to cooperate to produce the<br />
observed effects or whether a single gain of function mutation in any<br />
one or two factors is sufficient.<br />
Conclusions: Compared to wildtype MVA, plaques formed by MVA-CR19 on<br />
adherent CR cells appear to be larger and to develop earlier. Titers are<br />
slightly higher in complete lysates and significantly elevated in cell-free<br />
supernatants. MVA-CR19 replicates efficiently without aggregate induction<br />
also in single cell suspension cultures. We hypothesize that a greater fraction<br />
of MVA-CR19 escapes the hosts for infection of distant targets. In such a<br />
model the new genotype should not confer a significant advantage<br />
to viruses spreading in cell monolayers, and indeed we could not generate<br />
the MVA-CR genotype by passaging in adherent cultures. Attenuation has<br />
yet to be confirmed for MVA-CR but host cell-restriction appears to have<br />
been fully maintained for Vero and HEK 293 cells.<br />
Supply of an injectable vaccine preparation may be facilitated with this<br />
strain as production in single cell suspension using only a cell proliferation<br />
medium is less complex compared to the current protocol that requires<br />
cell aggregate induction by addition of a virus production medium.<br />
Furthermore, MVA-CR has a tendency to accumulate in the extracellular<br />
volume. Purification of live virus out of a cell-free suspension may <strong>all</strong>ow
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Page 3 of 151<br />
enhanced purity compared to a process that initiates with a complete<br />
lysate containing the full burden of unwanted host cell-derived<br />
components.<br />
References<br />
1. Jordan I, Vos A, Beilfuss S, Neubert A, Breul S, Sandig V: An avian cell line<br />
designed for production of highly attenuated viruses. Vaccine 2009,<br />
27:748-756.<br />
2. Jordan I, Northoff S, Thiele M, Hartmann S, Horn D, Höwing K, Bernhardt H,<br />
Oehmke S, von Horsten H, Rebeski D, Hinrichsen L, Zelnik V, Mueller W,<br />
Sandig V: A chemic<strong>all</strong>y defined production process for highly attenuated<br />
poxviruses. Biol J Int Assoc Biol Stand 2011, 39:50-58.<br />
3. Lohr V, Rath A, Genzel Y, Jordan I, Sandig V, Reichl U: New avian<br />
suspension cell lines provide production of influenza virus and MVA in<br />
serum-free media: studies on growth, metabolism and virus<br />
propagation. Vaccine 2009, 27:4975-4982.<br />
4. Lohr V, Genzel Y, Jordan I, Katinger D, Mahr S, Sandig V, Reichl U: Live<br />
attenuated influenza viruses produced in a suspension process with<br />
avian AGE1.CR.pIX cells. Bmc Biotechnol 2012, 12:79.<br />
5. Jordan I, Horn D, John K, Sandig V: A Genotype of Modified Vaccinia<br />
Ankara (MVA) that Facilitates Replication in Suspension Cultures in<br />
Chemic<strong>all</strong>y Defined Medium. Viruses 2013, 5:321-339.<br />
6. Ward BM: Visualization and characterization of the intracellular<br />
movement of vaccinia virus intracellular mature virions. J Virol 2005,<br />
79:4755-4763.<br />
O2<br />
Electric<strong>all</strong>y modulated attachment and detachment of animal cells<br />
cultured on an ITO patterning electrode surface<br />
Sumihiro Koyama<br />
Institute of Biogeosciences, Japan Agency for Marine-Earth Science and<br />
Technology, 2-15 Natsushima-cho, Yokosuka, 237-0061, Japan<br />
E-mail: skoyama@jamstec.go.jp<br />
BMC Proceedings 2013, 7(Suppl 6):O2<br />
Background: Micropatterning techniques of animal cells have been<br />
reported by numerous groups and f<strong>all</strong> into 6 major classifications (1).<br />
There are 1) photolithography, 2) soft lithography, 3) ink jet printing, 4)<br />
electron beam writing, 5) electrochemical desorption of self-assembled<br />
monolayers, and 6) dielectrophoresis. These six cell micropatterning<br />
techniques cannot modulate both the attachment and detachment of<br />
animal cells iteratively at the same positions, however. The present work<br />
has demonstrated that a weak electrical potential can modulate the<br />
attachment and detachment of specific<strong>all</strong>y positioned adhesive animal<br />
cells using a patterned indium tin oxide (ITO)/glass electrode culture<br />
system [1], (Figure 1).<br />
Materials and methods: A patterned indium tin oxide (ITO) optic<strong>all</strong>y<br />
transparent working electrode was placed on the bottom of a chamber<br />
slide with a counter- (Pt) and reference (Ag/AgCl) electrode. The ITO<br />
patterning was formed by a reticulate ITO region and arrayed square glass<br />
regions of varying size. Constant and rectangular potentials were applied<br />
to the working ITO/glass electrode using the Ag/AgCl reference and the Pt<br />
counterelectrode (Figure 1). The potentials were delivered via a function<br />
generator (AD-8624A, A&D Company, Tokyo, Japan) and a potentiostat<br />
(PS-14, Toho Technical Research, Tokyo, Japan).<br />
Results: Animal cells suspended in serum or sera containing medium<br />
were drawn to and attached on a reticulate ITO electrode region to<br />
which a +0.4-V vs. Ag/AgCl-positive potential was applied. Meanwhile, the<br />
cells were successfully placed on the square glass regions by -0.3-V vs. Ag/<br />
AgCl-negative potential application.<br />
Animal cells detached not only from the ITO electrode but also from the<br />
square glass regions after the application of a ± 10 mV vs. Ag/AgCl, 9-MHz<br />
triangular wave potential in PBS(-) for30-60min.Thetriangularwave<br />
potential-induced cell detachment is almost completely noncytotoxic, and<br />
no statistical differences between trypsinization and the high frequency<br />
wave potential application was observed in HeLa cell growth.<br />
Conclusions: Using the 3-electrode culture system, the author succeeded<br />
in modulation of the attachment and detachment of animal cells on the<br />
working electrode surface. The electrical modulation of specific<strong>all</strong>y<br />
positioned cell attachment and detachment techniques holds potential<br />
for novel optical microscopic cell sorting analysis in lab-on-chip devices.<br />
Reference<br />
1. Koyama S: Electric<strong>all</strong>y modulated attachment and detachment of animal<br />
cells cultured on an optic<strong>all</strong>y transparent patterning electrode. J Biosci<br />
Bioeng 2011, 111:574-583, (Erratum in: J Biosci Bioeng 2012, 114: 240-241).<br />
O3<br />
Novel strategy for a high-yielding mAb-producing CHO strain<br />
(overexpression of non-coding RNA enhanced proliferation and<br />
improved mAb yield)<br />
Hisahiro Tabuchi<br />
Chugai Pharmaceutical Co., Ltd., 5-5-1 Ukima, Kitaku, Tokyo, Japan 115-8543<br />
E-mail: tabuchihsh@chugai-pharm.co.jp<br />
BMC Proceedings 2013, 7(Suppl 6):O3<br />
Background: Innovation in mAb production is driven by strategies to<br />
increase yield. A host cell line constructed to overexpress TAUT (taurine<br />
transporter) produced a higher proportion of high-mAb-titer strains [1].<br />
From these we selected a single TAUT/mAb strain that remained viable<br />
for as long as 1 month. Its improved viability is attributed to improved<br />
metabolic properties. It was also more productive (>100 pg/cell/day) and<br />
yielded more mAb (up to 8.1 g/L/31 days) than the parent cell line [2]. These<br />
results suggested that this host cell engineering strategy has great potential<br />
for the improvement of mAb-producing CHO cells.<br />
Results: Our present ch<strong>all</strong>enge was to achieve a high yield in a shorter<br />
culture period by modulating events in the nucleus by using non-coding<br />
RNA (ncRNA). We looked for long ncRNA (lncRNA) that was abnorm<strong>all</strong>y<br />
expressed in high-titer cells. A Mouse Genome 430 2.0 array (Affymetrix)<br />
identified the lncRNA (Figure 1) as a complementary sequence of the<br />
3’ non-coding region of mouse NFKBIA (NF-kappa-B inhibitor alpha) mRNA.<br />
NFKBIA is an important regulator of the transcription factor NFKB, a<br />
positive regulator of cell growth. Since NFKBIA suppresses NFKB function,<br />
inhibition of NFKBIA by overexpression of the lncRNA might further<br />
enhance cell proliferation. We genetic<strong>all</strong>y modified the TAUT/mAb strain to<br />
overexpress part of the lncRNA. The resulting co-overexpression strains<br />
gave increased yield, and one strain increased yield in a shorter culture<br />
period (up to 6.0 g/L/14 days from 3.9 g/L/14 days). Interestingly, however,<br />
this effect might not be due to enhancement of the NFKB-dependent<br />
promoter activity of the mAb expression plasmid because mAb production<br />
under EF-1a promoter without an NFKB binding site was also enhanced by<br />
overexpression of part of the lncRNA. Since overexpression of the partial<br />
sequence still functions as an antibody production enhancing sequence in<br />
mAb-producing cell lines, many unexpected functions from ncRNAcontaining<br />
microRNA might exist.<br />
Conclusions: 1. We found a lncRNA that was abnorm<strong>all</strong>y expressed in hightiter<br />
cells. It was identified as the antisense RNA of NFKBIA. Overexpression<br />
of part of the lncRNA suppressed NFKBIA mRNA.<br />
2. Overexpression of part of the lncRNA improved CHO cell performance.<br />
The transporter/lncRNA co-overexpressing strain gave increased yield in a<br />
shorter culture period.<br />
3. This effect might not be due to enhancement of the NFKB-dependent<br />
promoter of the mAb expression plasmid.<br />
References<br />
1. Tabuchi H, Sugiyama T, Tanaka S, Tainaka S: Overexpression of taurine<br />
transporter in Chinese hamster ovary cells can enhance cell viability and<br />
product yield, while promoting glutamine consumption. Biotechnol<br />
Bioeng 2010, 107:998-1003.<br />
2. Tabuchi H, Sugiyama T: Cooverexpression of alanine aminotransferase<br />
1 in Chinese hamster ovary cells overexpressing taurine transporter<br />
further stimulates metabolism and enhances product yield. Biotechnol<br />
Bioeng 2013, 110:2208-2215.<br />
O4<br />
Improvement in a human IgE-inducing system by in vitro immunization<br />
Shuichi Hashizume 1* , Hiroharu Kawahara 2<br />
1 Idea-Creating Lab, Yokohama 236-0005, Japan;<br />
2 Kitakyushu National College<br />
of Technology, Kitakyushu 802-0985, Japan<br />
E-mail: hashizume.shu@nifty.com<br />
BMC Proceedings 2013, 7(Suppl 6):O4
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Figure 1(abstract O2) Schematic illustration of a patterned ITO/glass electrode culture system.<br />
Introduction: The immune system, which is the self-defense system of the<br />
body, occasion<strong>all</strong>y responds in a manner that is harmful to the body. The<br />
incidence and severity of <strong>all</strong>ergies caused by cedar pollen, house dust, egg<br />
protein, and many others are increasing and have recently become a<br />
serious social problem. We have previously developed an original in vitro<br />
system for inducing human IgE antibody specific to a designated antigen<br />
that can be used to study various <strong>all</strong>ergic reaction [1]. In this study, we<br />
attempted to improve this system to stimulate IgE levels in its medium to<br />
provide a highly sensitive screening method.<br />
Experimental: The original in vitro IgE-inducing system was established<br />
using lymphocytes and plasma from donors which were not natur<strong>all</strong>y<br />
immunized with <strong>all</strong>ergens. The original system contained ERDF supplemented<br />
with fetal bovine serum (final concentration, 5%) and contained human<br />
plasma (10%) as an essential component. Human peripheral blood<br />
lymphocytes and plasma were obtained by density-gradient centrifugation<br />
at 400 × g for 30 min with cell separation medium, Ficoll-Paque Plus.<br />
This system also included <strong>all</strong>ergen (100 ng/ml), interleukins (IL-) 2, 4, and<br />
6 (10 ng/ml each) and muramyl dipeptide (MDP, 10 μg/ml), as described<br />
previously [2]. Human lymphocytes were cultured in 96- or 24-well plates<br />
at a final density of 1 × 10 6 cells/ml in the medium and incubated in a CO 2<br />
incubator at 37°C for 10 days. During the 10 days, IgE was specific<strong>all</strong>y<br />
secreted into the medium.<br />
Results and discussion: Effects of human plasma and interleukins on<br />
human IgE induction: The necessity for inclusions of human plasma and<br />
interleukins was shown, when human lymphocytes and plasma from donors<br />
which were not natur<strong>all</strong>y immunized with <strong>all</strong>ergens were used. For the<br />
induction of IgE, human lymphocytes and plasma obtained from the same<br />
donor were required [2]. Addition of IL-2, 4 and 6 induced IgE. Elimination of<br />
each of these three interleukins from the medium resulted in no induction<br />
of IgE (data not shown). From these results, IL-2, 4 and 6 are considered to<br />
be essential factors to initi<strong>all</strong>y immunize lymphocytes with <strong>all</strong>ergens, when<br />
lymphocytes and plasma from donors not natur<strong>all</strong>y immunized with<br />
<strong>all</strong>ergens were used. We next attempted to improve this system to stimulate<br />
IgE levels in the medium to provide a highly sensitive screening method.
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Figure 1(abstract O3) The lncRNA is an antisense RNA of NFKBIA mRNA.<br />
Effects of elimination of IL-2 from the medium on human IgE<br />
production: In this study, the lymphocytes and plasma of donors natur<strong>all</strong>y<br />
immunized with various <strong>all</strong>ergens were used. Therefore, the IgE level of the<br />
control was high, i.e., more than 300 ng/ml, as shown in Table 1. Elimination<br />
of IL-2 from the medium resulted in the induction of higher IgE levels<br />
compared with medium containing IL-2 (Table 1). These data indicate that<br />
elimination of IL-2 from the medium induced higher IgE levels when human<br />
lymphocytes and plasma obtained from natur<strong>all</strong>y immunized donors were<br />
used. Furthermore, strawberry extract in the media containing Cryj1 and<br />
Derf2 decreased the secreted IgE levels by 38% and 24%, respectively. There<br />
is a possibility that strawberries may <strong>all</strong>eviate <strong>all</strong>ergies.<br />
In summary, elimination of IL-2 from the IgE-inducing system medium<br />
increased the IgE induction level when human lymphocytes and plasma<br />
obtained from donors natur<strong>all</strong>y immunized with <strong>all</strong>ergens were used. The<br />
level of about 1 μg/ml IgE reported to be secreted in this study may be<br />
the highest compared with those reported elsewhere. The original and<br />
improved systems for human IgE production are considered to be of<br />
profound use for studying <strong>all</strong>ergy mechanisms and surveying <strong>all</strong>ergy<strong>all</strong>eviating<br />
products, respectively.<br />
Table 1(abstract O4) Effects of various additives on IgE<br />
productivity<br />
Medium<br />
IgE productivity (ng/ml)<br />
Control (ERDF + hPlasma + FBS) 319 ± 19<br />
+ IL-2 + IL-4 + IL-6 + MDP + Cryj1 356 ± 85<br />
+ IL-4 + IL-6 + MDP + Cryj1 549 ± 189<br />
+ IL-4 + IL-6 + MDP + Cryj1 + 341 ± 55<br />
strawberry extract<br />
+ IL-4 + IL-6 + MDP + Derf2 660 ± 172<br />
+ IL-4 + IL-6 + MDP + Derf2 + 499 ± 167<br />
strawberry extract<br />
References<br />
1. Kawahara H, Maeda-Yamamoto M, Hakamata K: Effective induction and<br />
acquisition of human IgE antibodies reactive with house-dust mite<br />
extracts. J Immunol Methods 2000, 233:33-40.<br />
2. Hashizume S, Kawahara H: Inducing of human IgE antibodies by in vitro<br />
immunization. Proceedings of the 20th Annual Meeting of the European<br />
Society for Animal Cell Technology (ESACT) Springer Science+Business Media<br />
B.V: Noll T 2010, 833-836, Dresden, Germany, 2007.<br />
O5<br />
First CpG island microarray for genome-wide analyses of DNA<br />
methylation in Chinese hamster ovary cells: new insights into the<br />
epigenetic answer to butyrate treatment<br />
Anna Wippermann 1,2* , Sandra Klausing 1 , Oliver Rupp 2 , Thomas Noll 1,2 ,<br />
Raimund Hoffrogge 1<br />
1 Cell Culture Technology, Bielefeld University, Bielefeld, Germany;<br />
2 Center for<br />
Biotechnology, Bielefeld University, Bielefeld, Germany<br />
E-mail: anna.wippermann@uni-bielefeld.de<br />
BMC Proceedings 2013, 7(Suppl 6):O5<br />
Background: Optimizing productivity and growth of recombinant Chinese<br />
hamster ovary (CHO) cells requires insight and intervention in regulatory<br />
processes. This is to some extent accomplished by several ‘omics’<br />
approaches. However, many questions remain unanswered and bioprocess<br />
development is therefore still parti<strong>all</strong>y empirical. In this regard, the analysis<br />
of DNA methylation as one of the earliest cellular regulatory levels is<br />
increasingly gaining importance. This epigenetic process is known to<br />
influence transcriptional events when it occurs at specific genomic regions<br />
with high CpG frequencies, c<strong>all</strong>ed CpG islands (CGIs). Being methylated, CGIs<br />
attract proteins with methyl-DNA binding domains (MBD proteins) that in<br />
turn can interact with chromatin modifying complexes, thereby leading to a<br />
transcription<strong>all</strong>y inactive state of the associated gene [1]. In CHO cells, DNA<br />
methylation has yet only been investigated in gene-specific approaches, e.g.<br />
regarding the CMV promoter [2]. To analyze differential DNA methylation in<br />
CHO cultures on a genomic scale, we developed a microarray covering
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19,598 CGIs in the CHO genome. We applied it to elucidate the effect of<br />
butyrate on CHO DP-12 cultures, as this short chain fatty acid (SCFA) is<br />
known to elicit epigenetic responses by inhibiting histone-deacetylases [3].<br />
Materials and methods: Based on the genomic and transcriptomic<br />
information available for CHO cells [4,5], 21,993 promoter-associated and<br />
intragenic CGIs were identified in the CHO genome using an algorithm<br />
according to Takai and Jones [6]. We developed a customized 60 K<br />
microarray (printed by Agilent Technologies) covering 19,598 (89%) of the<br />
identified CGIs with an average probe spacing of 500 bp. Genomic DNA of<br />
each four replicate experimental and reference CHO DP-12 (clone #1934,<br />
ATCC CRL-12445) batch cultures was phenol-chloroform extracted and<br />
sheared by sonication. Methylated fragments were enriched using the<br />
methyl-CpG binding domain of MBD2 protein fused to the Fc tail of IgG1<br />
(MBD2-Fc protein) coupled to magnetic beads (New England Biolabs).<br />
Experimental samples prior to treatment with 3 mM butyrate (0 h) as well as<br />
24 hours and 48 hours after butyrate addition were directly compared to the<br />
references by two-colour co-hybridizations. Data analysis was carried out<br />
upon LOWESS normalization by Student’s t-tests with p-values ≤ 0.05 using<br />
the open source platform EMMA2 [7]. Confirmatory COBRA (combined<br />
bisulfite restriction analysis) was performed by amplifying a 541 bp fragment<br />
of the myc proto-oncogene protein-like gene (Gene ID: 100758352) following<br />
bisulfite treatment of genomic DNA using the primers myc_for 5’-atttggaagg<br />
atagtaagtatattggaag-3’ and myc_rev 5’- aaataaaactctaactcaccatatctcct-3’ and<br />
the nested primers myc_for_nested 5’- atagtaagtatattggaaggggagtg-3’ and<br />
myc_rev_nested 5’- taaaactctaactcaccatatctcctc-3’ (oligonucleotides obtained<br />
from Metabion). Purified PCR products were digested with BstUI (Fermentas)<br />
and separated in agarose gels.<br />
Results: Butyrate treated CHO DP-12 cultures stopped proliferating and<br />
decreasing viabilities could be detected 24 hours upon addition of the<br />
SCFA (Figure 1A). Simultaneously, cell specific productivities increased by<br />
nearly 100% (17 pg/cell/day 48 hours after butyrate addition compared to<br />
9 pg/cell/day in the reference cultures). Surprisingly, 228 differenti<strong>all</strong>y<br />
methylated genes could be detected in a comparison between the<br />
experimental cultures and the references even before addition of butyrate<br />
(Figure 1B), indicating substantial heterogeneity among identic<strong>all</strong>y handled<br />
par<strong>all</strong>el cultivations. 24 hours after butyrate addition we found a strongly<br />
increased number of 1221, solely at this point in time, differenti<strong>all</strong>y<br />
methylated genes. Gene ontology classification showed that, amongst<br />
others, the terms ‘stress response’, ‘chromatin modification’ or ‘sign<strong>all</strong>ing<br />
cascade’ were significantly overrepresented. Pathways such as the Ca 2+ ,<br />
MAPK and Wnt sign<strong>all</strong>ing systems were comprised within the latter group<br />
and showed a large coverage by differenti<strong>all</strong>y methylated components.<br />
48 hours upon butyrate addition the number of differential methylations<br />
decreased by about 90%. COBRA analysis of the Wnt responsive myc<br />
proto-oncogene protein-like gene showed clearly detectable cleavage<br />
products (indicating methylation of the BstUI sites in the original DNA)<br />
24 hours upon butyrate addition, that completely vanished another<br />
24 hours later (Figure 1C), confirming the results of the microarray analysis.<br />
Conclusions: Our first genome-wide screening for differential DNA<br />
methylation in CHO cells shows that the epigenetic response upon<br />
butyrate treatment seems to be highly dynamic and reversible. This was<br />
confirmed by applying the bisulfite-based single-gene method COBRA<br />
to analyze a region of the myc proto-oncogene protein-like gene.<br />
Furthermore, detection of differential methylation before butyrate addition<br />
Figure 1(abstract O5) (A) Viable cell densities, viabilities and cell specific productivities for batch CHO DP-12 reference (blue) and<br />
butyrate treated (red) cultivations. The green dashed line marks the point of butyrate addition. Error bars represent standard deviations. (B) Venn<br />
diagram showing the numbers of genes associated with differenti<strong>all</strong>y methylated CpG islands before (0 h), 24 hours and 48 hours upon butyrate addition.<br />
Gene Ontology classification was performed using DAVID [9] with an EASE score ≤ 0.01 (C) COBRA analysis of a part of the CGI (blue) of the myc<br />
proto-oncogene protein-like gene (green) differential methylation was detected for (red). Cleavage products indicate methylation of BstUI sites in the<br />
original DNA.
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indicates that heterogeneity in DNA methylation occurs even if cells<br />
originated from the same preculture and were treated identic<strong>all</strong>y. This<br />
occurrence of differenti<strong>all</strong>y methylated genes in par<strong>all</strong>el cultivations<br />
strongly fosters the hypothesis that the culture history influences final<br />
process outcomes [8]. It underlines the importance of DNA methylation<br />
analyses in CHO cells, especi<strong>all</strong>y considering the fact that DNA methylation<br />
patterns can remain stably anchored over several generations.<br />
References<br />
1. Ndlovu MN, Denis H, Fuks F: Exposing the DNA methylome iceberg.<br />
Trends Biochem Sci 2011, 36:381-387.<br />
2. Osterlehner A, Simmeth S, Göpfert U: Promoter methylation and transgene<br />
copy numbers predict unstable protein production in recombinant<br />
Chinese hamster ovary cell lines. Biotechnol Bioeng 2011, 108:2670-2681.<br />
3. Mariani MR, Carpaneto EM, Ulivi M, Allfrey VG, Boffa LC: Correlation<br />
between butyrate-induced histone hyperacetylation turn-over and<br />
c-myc expression. J Steroid Biochem Mol Biol 2003, 86:167-171.<br />
4. Xu X, Nagarajan H, Lewis NE, Pan S, Cai Z, Liu X, Chen W, Xie M, Wang W,<br />
Hammond S, Andersen MR, Neff N, Passarelli B, Koh W, Fan HC, Wang J,<br />
Gui Y, Lee KH, Betenbaugh MJ, Quake SR, Famili I, Palsson BO, Wang J: The<br />
genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line.<br />
Nat Biotechnol 2011, 29:735-741.<br />
5. Becker J, Hackl M, Rupp O, Jakobi T, Schneider J, Szczepanowski R, Bekel T,<br />
Borth N, Goesmann A, Grillari J, Kaltschmidt C, Noll T, Pühler A, Tauch A,<br />
Brinkrolf K: Unraveling the Chinese hamster ovary cell line transcriptome<br />
by next-generation sequencing. J Biotechnol 2011, 156:227-235.<br />
6. Takai D, Jones P: The CpG island searcher: a new WWW resource. In silico<br />
biology 2003, 3:235-40.<br />
7. Dondrup M, Albaum SP, Griebel T, Henckel K, Jünemann S, Kahlke T,<br />
Kleindt CK, Küster H, Linke B, Mertens D, Mittard-Runte V, Neuweger H,<br />
Runte KJ, Tauch A, Tille F, Pühler A, Goesmann A: EMMA 2–a<br />
MAGE-compliant system for the collaborative analysis and integration<br />
of microarray data. BMC Bioinformatics 2009, 10:50.<br />
8. Le H, Kabbur S, Pollastrini L, Sun Z, Mills K, Johnson K, Karypis G, Hu WS:<br />
Multivariate analysis of cell culture bioprocess data–lactate consumption<br />
as process indicator. J Biotechnol 2012, 162:210-23.<br />
9. Huang DW, Sherman BT, Zheng X, Yang J, Imamichi T, Stephens R,<br />
Lempicki RA: Extracting biological meaning from large gene lists with<br />
DAVID. Curr Protoc Bioinformatics 2009, Chapter 13, Unit 13.11.<br />
O6<br />
Aspects of vascularization in Multi-Organ-Chips<br />
Katharina Schimek 1 , Reyk Horland 1* , Sven Brincker 1 , Benjamin Groth 1 ,<br />
Ulrike Menzel 1 , Ilka Wagner 1 , Eva-Maria Materne 1 , Gerd Lindner 1 ,<br />
Alexandra Lorenz 1 , Silke Hoffmann 1 , Mathias Busek 2 , Frank Sonntag 2 ,<br />
Udo Klotzbach 2 , Roland Lauster 1 , Uwe Marx 1,3<br />
1 TU Berlin, Institute of Biotechnology, Faculty of Process Science and<br />
Engineering, 13355 Berlin, Germany;<br />
2 Fraunhofer IWS Dresden, 01277<br />
Dresden, Germany;<br />
3 TissUse GmbH, 15528 Spreenhagen, Germany<br />
E-mail: reyk.horland@tu-berlin.de<br />
BMC Proceedings 2013, 7(Suppl 6):O6<br />
Background: Enormous efforts have been made to develop circulation<br />
systems for physiological nutrient supply and waste removal of in vitro<br />
cultured tissues. These developments are aiming for in vitro generation of<br />
organ equivalents such as liver, lymph nodes and lung or even multi-organ<br />
systems for substance testing, research on organ regeneration or transplant<br />
manufacturing. Initi<strong>all</strong>y technical perfusion systems based on membranes,<br />
hollow fibers or networks of micro-channels were used for these purposes.<br />
However, none of the currently available systems ensures long-term<br />
homeostasis of the respective tissue over months. This is caused by a lack of<br />
in vivo-like vasculature which leads to continuous accumulation of protein<br />
sediments and cell debris in the systems. Here, we demonstrate a closed<br />
and self-contained circulation system emulating the natural blood perfusion<br />
environment of vertebrates at tissue level.<br />
Material and methods: The Multi-Organ-Chip (MOC) device accommodates<br />
two microvascular circuits (Figure 1a). Each circuit is operated by a separate<br />
peristaltic on-chip micropump, modified from Wu and co-workers [1].<br />
Microfluidic 3D channels were formed in PDMS by replica molding from<br />
master molds and were afterwards closed by bonding to a cover-slip by air<br />
plasma treatment. To retain PDMS hydrophilicity, channels were filled with<br />
culture medium immediately after sealing. To emulate the natural blood<br />
perfusion environment, human dermal microvascular endothelial cells<br />
(HDMEC) were used. The cells were seeded into the PDMS channels and<br />
adhered to <strong>all</strong> channel w<strong>all</strong>s after subsequent static cultivation on each<br />
channel side. Afterwards cells were cultured up to 14 days in PDMS channels<br />
under pulsatile flow conditions.<br />
Figure 1(abstract O6) HDMEC microvasculature in the MOC device. a) Exploded view of the device comprising a polycarbonate CP (blue),<br />
the PDMS-glass chip accommodating two microvascular circuits (yellow; footprint: 76 mm × 25 mm; height: 3 mm) and a heatable MOC-holder (red).<br />
b) Calcein AM assay (red) showed viable and evenly distributed HDMEC in <strong>all</strong> areas of the circulation. Scale bar = 2 mm. c) Image stack taken by<br />
two-photon laser scanning microscopy. HDMEC were able to cover <strong>all</strong> w<strong>all</strong>s of the channels forming a fluid tight layer. Functionality of the established<br />
microvascular vessel system was demonstrated by d) ac-LDL uptake of HDMEC and e) CD31 (red), vWF (green) expression throughout the entire cell<br />
population. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 100 μm.
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Results: A miniaturized circulation system has been established over a<br />
period of 14 days by fully covering <strong>all</strong> channels and surfaces of the MOC<br />
with human microvascular endothelial cells. By injecting 2 × 10 7 cells ml -1<br />
into the channels, a homogeneous distribution of cells throughout <strong>all</strong><br />
channels was achieved (Figure 1b). During the following static incubation,<br />
cells adhered well to the air plasma treated channel w<strong>all</strong>s. A peristaltic<br />
micro-pump was used to create culture medium circulation. After adaption<br />
to shear stress, HDMEC showed an elongation and alignment par<strong>all</strong>el to<br />
the flow direction. Three-dimensional reconstitutions of image stacks<br />
indicate that cells formed confluent monolayers on <strong>all</strong> w<strong>all</strong>s of the channels<br />
(Figure 1c). During the whole cultivation time they maintained adherence<br />
to the channel w<strong>all</strong>s and were positive for Calcein AM viability staining<br />
(Figure 1b). After 14 days of culture HDMEC forming the microvascular circuit<br />
were positive for ac-LDL uptake (Figure 1d) and expressed the endothelialspecific<br />
marker CD31 and von Willebrand Factor (vWF) (Figure 1e).<br />
Conclusion: A robust procedure applying pulsatile shear stress has been<br />
established to cover <strong>all</strong> fluid contact surfaces of the system with a functional,<br />
tightly closed layer of HDMEC.<br />
Long-term cultivation of elongated and flow-aligned HDMEC inside the chipbased<br />
microcirculation was demonstrated over a period of 14 days. For such<br />
endothelialized microfluidic devices to be useful for substance testing, it is<br />
essential to show long-term viability and function in the presence of<br />
physiological flow rates as shown here. These artificial vessels are an<br />
important approach for systemic substance testing in Multi-Organ-Chips.<br />
The miniaturized circulation system creates the conditions for circulation of<br />
nutrients through the organoid culture chamber, <strong>all</strong>ows for in vivo-like<br />
crosstalk between endothelial cells and tissues and prevents clumping<br />
inside the channels. Compared with conventional cell culture techniques, a<br />
microfluidic-based cell culture may mimic more accurate in vivo-like<br />
extracellular conditions, as the culture of cells and organ models in perfused<br />
microfluidic systems can improve their oxygen and nutrient supply. This<br />
makes it suitable for long-term cultivation and more efficient drug studies.<br />
In future, such endothelialized bioreactors might be used for testing<br />
vasoactive substances. Fin<strong>all</strong>y, the described system can now be used for<br />
the establishment of organ-specific capillary networks. Here, we will adhere<br />
to our recently published roadmap toward vascularized ‘’human-on-a-chip’’<br />
models to generate systemic data fully replacing the animals or human<br />
beings currently used [2].<br />
Acknowledgements: The work has been funded by the German Federal<br />
Ministry for Education and Research, GO-Bio Grant No. 0315569.<br />
References<br />
1. Wu M-H, Huang S-B, Cui Z, Cui Z, Lee G-B: A high throughput perfusionbased<br />
microbioreactor platform integrated with pneumatic micropumps<br />
for three-dimensional cell culture. Biomedical microdevices 2008,<br />
10:309-319.<br />
2. Marx U, W<strong>all</strong>es H, Hoffmann S, Lindner G, Horland R, Sonntag F,<br />
Klotzbach U, Sakharov D, Tonevitsky A, Lauster R: “Human-on-a-chip”<br />
developments: a translational cutting-edge alternative to systemic<br />
safety assessment and efficiency evaluation of substances in laboratory<br />
animals and man? Alternatives to laboratory animals: ATLA 2012,<br />
40:235-257.<br />
O7<br />
Rapid construction of transgene-amplified CHO cell lines by cell cycle<br />
checkpoint engineering<br />
Kyoungho Lee 1 , Kohsuke Honda 1 , Hisao Ohtake 1 , Takeshi Omasa 1,2*<br />
1 Department of Biotechnology, Graduate School of Engineering, Osaka<br />
University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan;<br />
2 Institute of<br />
Technology and Science, The University of Tokushima, 2-1 Minamijosanjimacho,<br />
Tokushima 770-8506, Japan<br />
E-mail: omasa@bio.tokushima-u.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):O7<br />
Introduction: Dihydrofolate reductase (DHFR)-mediated gene amplification<br />
has been widely used to establish high-producing mammalian cell lines<br />
[1-3]. However, since gene amplification is an infrequent event, in that many<br />
rounds of methotrexate (MTX) selection to amplify the transgene and<br />
screening of over several hundred individual clones are required to obtain<br />
cells with high gene copy numbers [4]. Consequently, the process for DHFRmediated<br />
gene amplification is a time-consuming and laborious step for cell<br />
line construction. Here, we present a novel concept to accelerate gene<br />
amplification through cell cycle checkpoint engineering. In our knowledge,<br />
there is no previous report which focused on controlling cell cycle<br />
checkpoint to enhance the efficiency of DHFR gene amplification system.<br />
Materials and methods: A sm<strong>all</strong> interfering RNA (siRNA) expression<br />
vector against Ataxia-Telangiectasia and Rad3-Related (ATR), a cell cycle<br />
checkpoint kinase, was transfected into Chinese hamster ovary (CHO) cells.<br />
The effects of ATR down-regulation on gene amplification and productivity<br />
in CHO cells producing green fluorescent protein (GFP) and monoclonal<br />
antibody (mAb) were investigated.<br />
Results and discussion: Analysis of GFP expression level during gene<br />
amplification process: The ratio of GFP-expressing cells was evaluated<br />
by flow cytometry analysis during the gene amplification process at 100-,<br />
250-, and 500-nM MTX concentrations. In the process of gene amplification<br />
at <strong>all</strong> MTX concentrations, the pools of ATR-downregulated cells showed a<br />
much higher percentage of GFP-positive cells as compared with the pools<br />
of mock cells. At 100-nM MTX concentration, the percentage of GFPpositive<br />
cells in the CHO-siATR cell pool was 18.7% of total cells, which<br />
was approximately twice of the 8.4% in the mock cells. At 250- and<br />
500-nM MTX concentrations, CHO-siATR cell pools had 28.6 and 39.2%<br />
GFP-positive cells, respectively, which were up to six times higher than the<br />
4.6 and 6.8% of the pools of mock cells.<br />
Comparison of IgG productivity: IgG-producing cell lines were generated<br />
to confirm the previous results obtained in GFP-producing cell lines. The<br />
ATR-downregulated cells showed a significant increase in specific production<br />
rate of an average of 0.08 pg cell −1 day −1 , which was approximately four<br />
times higher than the average of 0.02 pg cell −1 day −1 in the mock cells.<br />
The volumetric productivity of each cell line was also investigated to<br />
evaluate the influence of ATR downregulation. The volumetric productivity<br />
of ATR knockdown cells was an average of 0.035 mg L −1 day −1 , which was<br />
approximately three times higher than the average of 0.013 mg L −1 day −1<br />
of the mock cells, suggesting that ATR knockdown generated the pool of<br />
higher-producing cells during the gene amplification process.<br />
Estimation of amplified transgene copy number: Quantitative real-time<br />
PCR was used to estimate the amplified transgene copy number of GFPproducing<br />
cell lines during the gene amplification process. The average<br />
copy number of ATR-downregulated cells was 15.4 ± 0.8, 27.6 ± 0.3,<br />
and 62.0 ± 2.9 at 100-, 250-, and 500-nM MTX concentrations, respectively.<br />
These numbers were up to 24 times higher than 3.98 ± 0.09, 2.20 ± 0.03,<br />
and 2.59 ± 0.07 of the mock cells. Interestingly, the amplified transgene<br />
copy numbers in the pools of ATR-downregulated cells were increased<br />
proportion<strong>all</strong>y with the MTX concentration. The amplified transgene copy<br />
numbers in the IgG-producing cells were also investigated during the gene<br />
amplification process at 100-nM MTX concentration. The amplified light- and<br />
heavy-chain copy numbers of the pool of ATR knockdown cells were 13.2 ±<br />
3.8 and 11.8 ± 1.8, respectively, which were up to seven times higher than<br />
6.95 ± 0.07 and 1.68 ± 0.04 of the mock cells. The results from both the<br />
GFP- and IgG-producing cells showed that the pools of ATR-downregulated<br />
cells had much higher amplified transgene copy numbers as compared with<br />
the pools of mock cells during the gene amplification process.<br />
Conclusions: In conclusion, we have demonstrated that gene amplification<br />
can be accelerated by the downregulation of a cell cycle checkpoint kinase,<br />
ATR, and a pool of high-producing cells can be rapidly derived in a short<br />
time after MTX treatment. This novel method focuses on generating more<br />
high-producing cells in a heterogeneous pool as compared with the<br />
conventional method and would thus contribute to reducing the time and<br />
labor required for cell line establishment by increasing the possibility of<br />
selecting high-producing clones.<br />
Acknowledgements: This work is parti<strong>all</strong>y supported by grants from the<br />
Program for the Promotion of Fundamental Studies in Health Sciences of<br />
NIBIO and a Grant-in-Aid for Scientific Research of JSPS. We thank Prof.<br />
Yoshikazu Kurosawa at Fujita Health University for kindly providing heavyand<br />
light-chain genes of humanized IgG.<br />
References<br />
1. Gandor C, Leist C, Fiechter A, Asselbergs FA: Amplification and expression<br />
of recombinant genes in serum-independent Chinese hamster ovary<br />
cells. FEBS Lett 1995, 377:290-294.<br />
2. Kim JY, Kim YG, Lee GM: CHO cells in biotechnology for production of<br />
recombinant proteins: current state and further potential. Appl Microbiol<br />
Biotechnol 2012, 93:917-930.<br />
3. Wurm FM: Production of recombinant protein therapeutics in cultivated<br />
mammalian cells. Nat Biotechnol 2004, 22:1393-1398.
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4. Cacciatore JJ, Chasin LA, Leonard EF: Gene amplification and vector<br />
engineering to achieve rapid and high-level therapeutic protein<br />
production using the Dhfr-based CHO cell selection system. Biotechnol<br />
Adv 2010, 28:673-681.<br />
O8<br />
1 H-NMR spectroscopy for human 3D neural stem cell cultures metabolic<br />
profiling<br />
Daniel Simão 1,2 , Catarina Pinto 1,2 , Ana P Teixeira 1,2 , Paula M Alves 1,2 ,<br />
Catarina Brito 1,2*<br />
1 iBET, Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras,<br />
Portugal;<br />
2 Instituto de Tecnologia Química e Biológica, Universidade Nova de<br />
Lisboa, 2780-157 Oeiras, Portugal<br />
E-mail: anabrito@itqb.unl.pt<br />
BMC Proceedings 2013, 7(Suppl 6):O8<br />
Background: The current lack of predictable central nervous system (CNS)<br />
models in pharmaceutical industry early stage development strongly<br />
contributes for the high attrition rates registered for new therapeutics [1].<br />
Thus, there is an increasing need for a paradigm shift towards more human<br />
relevant cell models, which can closely recapitulate the in vivo cell-cell<br />
interactions, presenting higher physiological relevance by bridging the gap<br />
between animal models and human clinical trials. In this context, human 3D<br />
in vitro models are promising tools with great potential for pre-clinical<br />
research, as they can mimic some of the main features of tissues, such as<br />
cell-cell and cell-extracellular matrix (ECM) interactions [2,3]. Moreover these<br />
complex cell models are suitable for high-throughput screening (HTS)<br />
platforms, essential in drug discovery pipelines by reducing both costs and<br />
time in clinical trials [2,4]. However, despite important advances in the<br />
last years and the increasing clinical and biological relevance, the full<br />
establishment of human 3D in vitro models in pre-clinical research requires a<br />
significant increase in the power of the available analytical methodologies<br />
towards more robust and comprehensive readouts [4]. With the emergence<br />
of systems biology field and several “-omics” technologies, such as<br />
metabolomics, it became possible to have a more mechanistic approach in<br />
the understanding of cellular programs. 1 H-nuclear magnetic resonance<br />
( 1 H-NMR) spectroscopy is a powerful and widely accepted high resolution<br />
methodology for a number of applications, including metabolic profiling [5].<br />
Despite the low sensitivity when compared with mass spectrometry (MS),<br />
1 H-NMR profiling presents several advantages, enabling a non-invasive and<br />
non-destructive quantitative analysis requiring only minimal sample<br />
preparation [5].<br />
In this work we present the development of a robust and optimized<br />
workflow for the exometabolome profiling of 3D in vitro cultures of human<br />
midbrain-derived neural progenitor cells (hmNPC).<br />
Materials and methods: Cell culture: hmNPC were isolated and routinely<br />
propagated in static conditions, on poly-L-ornithine-fibronectin (PLOF)<br />
coated plates, in serum-free expansion medium, containing basic fibroblast<br />
growth factor and epidermal growth factor, as previously reported [6].<br />
hmNSC were cultured in stirred systems as neurospheres for 7 days, with a<br />
50% media changes every at day 3 [7]. All experiments were performed<br />
in 500 mL shake flasks (80 mL working volume), with orbital shaking at<br />
100 rpm. Cultures were maintained at 37°C, in 3% O 2 and 5% CO 2 .<br />
Sample Preparation: Neurospheres harvested at day 7 were plated on<br />
PLOF-coated plates. A washing step with PBS was performed before adding<br />
fresh medium (Neurobasal medium (Invitrogen) supplemented with 2% of<br />
B27, 2 mM of Glutamax (Invitrogen), 100 μM dibutyryl c-AMP (Sigma-<br />
Aldrich), and 10 μg/mL gentamycin (Invitrogen)) to the culture. Samples of<br />
supernatant were then collected at 6, 12, 24 and 48 hours after media<br />
exchange and stored at -20°C. Neurospheres were harvested and total<br />
protein was quantified with Micro BCA Protein Assay Kit (Pierce), according<br />
to manufacturer’s instructions. Prior to NMR analysis, samples were thawed<br />
and filtered using Vivaspin 500 columns (Sigma-Aldrich) at 14,000xg, in<br />
order to remove high molecular weight proteins and lipids that induce<br />
baseline distortions and peak broadening due to protein binding.<br />
To minimize variations in pH, 400 μL of filtered samples were mixed with<br />
200 μL of phosphate buffer (50 mM, pH 7.4) with 5 mM DSS-d 6 [8].<br />
1 H-NMR spectra acquisition and profiling: For NMR analysis, 500 μL of<br />
the resulting supernatants were placed into 5 mm NMR tubes. All 1 H-NMR<br />
spectra were recorded at 25°C on a Bruker Avance II+ 500 MHz NMR<br />
spectrometer. One-dimensional (1D) spectra were recorded using a NOESYbased<br />
pulse sequence (4 s acquisition time, 1 s relaxation time and 100 ms<br />
mixing time). Typic<strong>all</strong>y, 256 scans were collected for each spectrum.<br />
All spectra were phase and baseline corrected automatic<strong>all</strong>y, with fine<br />
adjustments performed manu<strong>all</strong>y. Spectra analysis was performed using<br />
Chenomx NMR Suite 7.1, using DSS-d 6 as internal standard for quantification<br />
of metabolites.<br />
Results: The approach applied in this study for metabolic profiling of the<br />
hmNPC cultures using 1 H-NMR enables an accurate screening of a wide<br />
range of metabolites in the extracellular environment (Figure 1A),<br />
including amino acids, glucose, lactate, among other substrates and<br />
by-products.<br />
Metabolism plasticity has been widely described as closely related with cell<br />
pluri/multipotency and cell fate. Stemness programs and cell identity<br />
determination are driven mainly by genetic and epigenetic switches, which<br />
can modulate cell metabolism, among other cell fate pathways [9]. Thus,<br />
the transition from pluri/multipotency towards somatic cell lineages is<br />
accompanied by significant metabolic shifts, mainly at energy metabolism<br />
levels. In this context, the metabolic study of in vitro cultures of stem cells<br />
may contribute with valuable knowledge for the mechanistic understanding<br />
of stemness and differentiation pathways.<br />
Our results showed that the hmNPC in an undifferentiated state presented<br />
a highly glycolytic metabolism, with high glucose consumption and lactate<br />
production rates (Figure 1B), in agreement with previous reports for<br />
murine NPC [10]. The profiles observed for glucose consumption and<br />
lactate synthesis suggest an almost complete conversion of pyruvate,<br />
generated as the final product of glycolysis, to lactate. One key culture<br />
parameter that can greatly contribute for a low oxidative metabolism is<br />
the fact that neural stem/progenitor cells are typic<strong>all</strong>y cultured under<br />
physiological low oxygen tension environments. Hypoxic conditions have<br />
been widely described as critical for maintaining cell viability and selfrenewal,<br />
while promoting proliferation and influencing cell fate during<br />
differentiation [11]. Moreover, the consumption and depletion of pyruvate<br />
present in culture media may suggest not only its conversion to lactate,<br />
but may also contribute for the observed alanine synthesis.<br />
Interestingly, even though glutamate could not be detected at significant<br />
levels, an accumulation of pyroglutamate was observed, which can be<br />
found as N-terminal modification in many neuronal peptides, including<br />
pathological accumulating peptides as b-amyloid in Alzheimer’s disease.<br />
As a free metabolite pyroglutamate can derive both from degradation of<br />
proteins containing N-terminal residues or from glutamate/glutamine<br />
cyclization. Although it is still a matter of debate, pyroglutamate<br />
may act as a reservoir of neural glutamate, which is the main excitatory<br />
neurotransmitter in CNS and in high levels becomes a major<br />
neurotoxicant [12].<br />
Concerning branched-chain amino acids (BCAA) metabolism it was possible<br />
to observe the extracellular accumulation of 2-oxoisocaproate and<br />
methylsuccinate as main by-products, although in low rates. In brain<br />
metabolism the balance between leucine and 2-oxisocaproate has particular<br />
relevance through the establishment of a nitrogen turnover cycle where<br />
astroglia cells catabolize leucine into 2-oxoisocaproate, which is then taken<br />
up by neurons and converted back into leucine [13,14].<br />
Conclusions: The methodology presented in this work, enables a<br />
straightforward approach for an accurate and reproducible metabolic<br />
profiling of multipotent hmNPC 3D cultures. This methodology provides a<br />
robust alternative to an array of laborious analytical methods, by taking<br />
advantage of the fast and simple sample preparation for NMR spectroscopy<br />
and the ease of user-friendly software for spectra profiling, which is often a<br />
ch<strong>all</strong>enging and time-consuming process due to peak overlapping in<br />
complex mixtures such as the mammalian cell culture media. Moreover, this<br />
approach can be applied to other multi/pluripotent cell sources, not only for<br />
metabolic profiling of in vitro cultures but also to study the impact of new<br />
therapeutics or toxicants, contributing to generate invaluable data in drug<br />
development cascades.<br />
Acknowledgements: The authors acknowledge Dr J. Schwarz (Technical<br />
University of Munich, Germany) for the supply of hmNPC, within the<br />
scope of the EU project BrainCAV (FP7-222992); this work was supported<br />
by PTDC/EBB-BIO/112786/2009 and PTDC/EBB-BIO/119243/2010, FCT,<br />
Portugal; BrainCAV (FP7-222992), EU. The NMR spectrometers are part of<br />
The National NMR Facility, supported by Fundação para a Ciência e a<br />
Tecnologia (RECI/BBB-BQB/0230/2012). Daniel Simão acknowledges the PhD<br />
fellowship (SFRH/BD/78308/2011, FCT).
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Page 10 of 151<br />
Figure 1(abstract O8) Typical 1 H-NMR spectra for hmNPC culture at different time points (A). Concentration profiles of the main metabolites quantified<br />
in the exometabolome of hmNPC cultures that have significantly changed during 48 h of culture (B).<br />
References<br />
1. Miller G: Is pharma running out of brainy ideas? Science 2010,<br />
329:502-504.<br />
2. Pampaloni F, Reynaud EG, Stelzer EHK: The third dimension bridges the<br />
gap between cell culture and live tissue. Nat Rev Mol Cell Biol 2007,<br />
8:839-845.<br />
3. Griffith LG, Swartz M: Capturing complex 3D tissue physiology in vitro.<br />
Nat Rev Mol Cell Biol 2006, 7:211-224.<br />
4. Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J: Spheroid<br />
culture as a tool for creating 3D complex tissues. Trends Biotechnol 2013,<br />
31:108-115.<br />
5. Mountford CE, Stanwell P, Lin A, Ramadan S, Ross B: Neurospectroscopy:<br />
the past, present and future. Chem Rev 2010, 110:3060-3086.<br />
6. Storch A, Paul G, Csete M, Boehm BO, Carvey PM, Kupsch A, Schwarz J:<br />
Long-term proliferation and dopaminergic differentiation of human<br />
mesencephalic neural precursor cells. Exp Neurol 2001, 170:317-325.<br />
7. Brito C, Simão D, Costa I, Malpique R, Pereira CI, Fernandes P, Serra M,<br />
Schwarz SC, Schwarz J, Kremer EJ, Alves PM: 3D cultures of human neural<br />
progenitor cells: dopaminergic differentiation and genetic modification.<br />
Methods 2012, 56:452-460.<br />
8. Duarte T, Carinhas N, Silva AC, Alves PM, Teixeira AP: 1H-NMR protocol for<br />
exometabolome analysis of cultured mammalian cells. Animal Cell<br />
Biotechnology-Methods and Protocols Springer: Pörtner R , 3 2013 in press.<br />
9. Folmes CDL, Nelson TJ, Dzeja PP, Terzic A: Energy metabolism plasticity<br />
enables stemness programs. Ann N Y Acad Sci 2012, 1254:82-89.<br />
10. Candelario KM, Shuttleworth CW, Cunningham LA: Neural stem/progenitor<br />
cells display a low requirement for oxidative metabolism independent<br />
of hypoxia inducible factor-1alpha expression. J Neurochem 2013,<br />
125:420-429.<br />
11. Milosevic J, Schwarz SC, Krohn K, Poppe M, Storch A, Schwarz J: Low<br />
atmospheric oxygen avoids maturation, senescence and cell death of<br />
murine mesencephalic neural precursors. J Neurochem 2005, 92:718-729.
BMC Proceedings 2013, Volume 7 Suppl 6<br />
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Page 11 of 151<br />
12. Kumar A, Bachhawat AK: Pyroglutamic acid: throwing light on a lightly<br />
studied metabolite. Curr Sci 2012, 102:288-297.<br />
13. Bixel MG, Engelmann J, Willker W, Hamprecht B, Leibfritz D: Metabolism of<br />
[U-(13)C]leucine in cultured astroglial cells. Neurochem Res 2004,<br />
29:2057-2067.<br />
14. Yudkoff M, Daikhin Y, Nelson D, Nissim I, Erecińska M: Neuronal<br />
metabolism of branched-chain amino acids: flux through the<br />
aminotransferase pathway in synaptosomes. J Neurochem 1996,<br />
66:2136-2145.<br />
O9<br />
BEAT® the bispecific ch<strong>all</strong>enge: a novel and efficient platform for the<br />
expression of bispecific IgGs<br />
Pierre Moretti 1* , Darko Skegro 2 , Romain Ollier 2 , Paul Wassmann 2 ,<br />
Christel Aebischer 1 , Thibault Laurent 1 , Miriam Schmid-Printz 3 ,<br />
Roberto Giovannini 3 , Stanislas Blein 2 , Martin Bertschinger 1<br />
1 Cell Line Development and Protein Expression group, Glenmark<br />
Pharmaceuticals SA, La Chaux-de-Fonds, 2300, Switzerland;<br />
2 Antibody<br />
Engineering group, Glenmark Pharmaceuticals SA, La Chaux-de-Fonds, 2300,<br />
Switzerland;<br />
3 Downstream Processing group, Glenmark Pharmaceuticals SA,<br />
La Chaux-de-Fonds, 2300, Switzerland<br />
E-mail: pierrem@glenmarkpharma.com<br />
BMC Proceedings 2013, 7(Suppl 6):O9<br />
Background: The binding of two biological targets with a single IgGbased<br />
molecule is thought to be beneficial for clinical efficacy. However<br />
the technological ch<strong>all</strong>enges for the development of a bispecific platform<br />
are numerous. While correct pairing of heterologous heavy and light<br />
chains (Hc and Lc) can be achieved by engineering native IgG scaffolds,<br />
crucial properties such as thermostability, effector function and low<br />
immunogenicity should be maintained [1]. The molecule has to be<br />
expressed at industri<strong>all</strong>y relevant levels with a minimum fraction of<br />
contaminants and a scalable purification approach is needed to isolate<br />
the product from potenti<strong>all</strong>y complex mixtures. This article introduces a<br />
novel bispecific platform based on the proprietary BEAT® technology<br />
(Bispecific Engagement by Antibodies based on the T cell receptor)<br />
developed by Glenmark.<br />
Materials and methods: Stable cell lines were generated by co-transfection<br />
of three proprietary expression vectors pGLEX41_GA/GB coding for the Hc, Lc<br />
and Fc-scFv under optimized stoichiometric conditions in CHO-S cells. Cell<br />
lines were selected according to expression and heterodimerization during<br />
sm<strong>all</strong> scale fed-batch cultures performed in TubeSpin bioreactors (TPP,<br />
Trasadingen, Switzerland). For high throughput (HT) screening, the fraction of<br />
BEAT® molecule was evaluated using the Caliper LabChip GXII Protein Assay<br />
(PerkinElmer, Waltham, Ma, USA). Titers were measured by HPLC-PA after<br />
14 days of culture. The fraction of heterodimer in CHO supernatants was<br />
measured by CE-CGE on Protein A (ProtA) purified supernatants harvested on<br />
day 14. The actual BEAT® titer was obtained by multiplying the concentration<br />
measured by HPLC-PA by the fraction of heterodimer measured by CE-CGE in<br />
ProtA purified supernatants. The BEAT® was produced in 3 L STR bioreactors<br />
(Mobius CellReady Bioreactor, Millipore) in fed-batch. Supernatants were<br />
typic<strong>all</strong>y harvested on day 14 by centrifugation and dead-end filtration.<br />
A single Protein A step was performed for purification, where two<br />
isocratic steps <strong>all</strong>owed the selective elution of the bispecific product. The<br />
thermostability of the BEAT® molecule was measured by differential scanning<br />
calorimetry (DSC) in PBS.<br />
Results: The BEAT® bispecific molecule consists of three chains: a heavy<br />
chain (Hc), a light chain (Lc) and a Fc-scFv (see Figure 1 A). The molecule has<br />
a fully functional Fc and engages two biological targets by a Fab arm on one<br />
side and by a scFv on the other. Heterodimerization is achieved by<br />
a proprietary CH3 interface, mimicking the natural association of the T-cell<br />
surface receptors a and b between the two CH3 domains of IgG. Lc<br />
mispairing is avoided by the replacement of one Fab arm of the bispecific<br />
IgG by a scFv. In addition, the Protein A binding site in the Hc of the<br />
Figure 1(abstract O9) The BEAT®bispecific platform. In A: secretion profile of a BEAT® secreting CHO clone obtained by Caliper analysis of a<br />
non-purified supernatant. B: distribution of the heterodimerization level of stable clones at cell line development level. C: BEAT® expression level of 10<br />
selected stable clones. D: BEAT® purification strategy.
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molecule is abrogated to facilitate the isolation of the BEAT®-antibody by<br />
affinity chromatography (discussed in the following). The DSC analysis of the<br />
BEAT® indicated a good thermostability within the range of natur<strong>all</strong>y<br />
occurring antibodies. The BEAT® molecule is expressed in CHO cells. Figure 1<br />
A shows a typical secretion profile obtained by Caliper Protein Analysis of a<br />
non-purified CHO supernatant after 14 days in fed-batch culture. It can be<br />
seen that the asymmetry of the BEAT® format <strong>all</strong>ows an easy characterization<br />
of the secretion profile of generated clones using HT analytics solely based<br />
on molecular weight. The example illustrates that a very low level of<br />
monospecific IgG is secreted and that the main secreted species is the BEAT®<br />
molecule, the main monospecific contaminant being the scFv-Fc homodimer.<br />
Figure 1 B shows the distribution of the heterodimerization level of the CHO<br />
clones screened during cell line development. The median of the distribution<br />
is approx. 80% indicating that half of the generated clones secreted > 80% of<br />
heterodimer. The expression level of the best 10 clones selected in sm<strong>all</strong><br />
scale fed-batches after cell line development can be seen in Figure 1 C.<br />
Clones secreting 1-2 g/L of BEAT® could be obtained under non-optimized<br />
fed-batch conditions. Stability studies demonstrated that selected CHO<br />
clones have a stable level of heterodimerization over long term cultivation<br />
(75 population doubling level (PDL), data not shown).<br />
At 3 L bioreactor scale, titers of 3 g/L with 90% of secreted heterodimer<br />
could be obtained in fed-batch with minimal feeding optimization. After<br />
harvest the molecule is purified by Protein A (ProtA). For purification<br />
purposes the BEAT® was designed with a missing ProtA binding site on the<br />
Hc of the molecule. Consequently, residual monospecific IgG contaminants<br />
(harboring 2 Hc) do not bind to the ProtA column and are thus easily<br />
separated from the products of interest. In addition, the BEAT® molecule and<br />
the homodimeric Fc-scFv contaminant exhibit a different affinity for Protein<br />
A as the molecules harbor one and two binding sites for ProtA, respectively.<br />
Thus, the BEAT® molecule can be separated by ProtA via a two-step isocratic<br />
elution as illustrated in Figure 1 D. Applying this purification strategy for<br />
harvested bioreactor material, a level of purity of 97% could be obtained<br />
post ProtA.<br />
Conclusions: This work introduces a new bispecific IgG format c<strong>all</strong>ed the<br />
BEAT®. Glenmark’s BEAT® platform <strong>all</strong>ows the generation of stable clones<br />
with volumetric productivity of several g/L and a high heterodimerization<br />
level (> 90% secreted BEAT® in CHO supernatants). Generated clones harbor<br />
stable product quality profiles, e.g. level of heterodimerization, over at least<br />
75 PDL. The developed purification strategy <strong>all</strong>ows a purity reaching 97%<br />
post ProtA. The BEAT® platform combines a unique CH3 interface for<br />
heterodimerization, an efficient cell line selection strategy and an industrial<br />
relevant purification process for the production of pure bispecific antibody<br />
at several g/L.<br />
Acknowledgements: The authors would like to thank Emilie Vaxelaire<br />
and Farid Mosbaoui for their contribution to this work.<br />
Reference<br />
1. Klein C, Sustmann C, Thomas M, Stubenrauch K, Croasdale R, Schanzer J,<br />
Brinkmann U, Kettenberger H, Regula J T, Schaefer W: Progress in<br />
overcoming the chain association issue in bispecific heterodimeric IgG<br />
antibodies. MAbs 2012, 4:653-663.<br />
O10<br />
A quantitative and mechanistic model for monoclonal antibody<br />
glycosylation as a function of nutrient availability during cell culture<br />
Ioscani Jiménez del Val 1 , Antony Constantinou 2,3 , Anne Dell 2 , Stuart Haslam 2 ,<br />
Karen M Polizzi 2,3 , Cleo Kontoravdi 1*<br />
1 Centre for Process Systems Engineering, Department of Chemical<br />
Engineering, Imperial College London, South Kensington Campus, London,<br />
SW7 2AZ, UK;<br />
2 Department of Life Sciences, Imperial College London, South<br />
Kensington Campus, London, SW7 2AZ, UK; 3 Centre for Synthetic Biology<br />
and Innovation, Imperial College London, South Kensington Campus,<br />
London, SW7 2AZ, UK<br />
E-mail: cleo.kontoravdi@imperial.ac.uk<br />
BMC Proceedings 2013, 7(Suppl 6):O10<br />
Introduction: Monoclonal antibodies (mAbs) are currently the highestselling<br />
products of the biopharmaceutical industry, having had global sales<br />
of over $45 billion in 2012 [1]. All commerci<strong>all</strong>y-available mAbs contain a<br />
consensus N-linked glycosylation site on each of the Cg2 domains of their<br />
constant fragment (Fc). The monosaccharide composition and distribution of<br />
these N-linked carbohydrates (glycans) has been widely reported to directly<br />
impact the safety and efficacy of mAbs when administered to patients.<br />
Many studies have also shown that manufacturing bioprocess conditions<br />
(e.g. nutrient availability, metabolite accumulation, dissolved oxygen, pH,<br />
temperature and stirring speed) directly influence the composition and<br />
distribution of N-linked glycans bound to mAbs and other recombinant<br />
proteins. Given this tight interconnection between manufacturing process<br />
conditions, product quality and ensuing safety and therapeutic efficacy,<br />
mAbs and their glycosylation present a clear opportunity where process<br />
development can be guided by quality by design (QbD) principles.<br />
QbD is a conceptual framework that aims to build quality into drug products<br />
at every stage of process development. Specific<strong>all</strong>y, implementation of QbD<br />
to pharmaceutical process development requires identifying critical quality<br />
attributes (CQAs) that define the drug’s safety and therapeutic efficacy. QbD<br />
then uses <strong>all</strong> available information on the mechanisms that quantitatively<br />
relate process inputs with product quality to control the manufacturing<br />
process so that product CQAs are maintained and end-product quality is<br />
ensured. Within the QbD context, composition and distribution of the<br />
glycans present on the Fc of mAbs is defined as a CQA, and thus, the<br />
processes employed in their manufacture must be controlled so that their<br />
glycan distribution ensures the required safety and efficacy profiles.<br />
Under this perspective, we have defined a mathematical model that<br />
mechanistic<strong>all</strong>y and quantitatively describes mAb Fc glycosylation as a<br />
function of nutrient availability during cell culture. Such a model aims to be<br />
used for bioprocess design, control and optimisation, thus facilitating the<br />
manufacture of mAbs with built-in glycosylation-associated quality under<br />
the QbD scope.<br />
Materials and methods: The mathematical model consists of three<br />
distinct modular elements which have been connected to achieve a<br />
mechanistic description of mAb glycosylation as a function of nutrient<br />
availability. The first element corresponds to cell culture dynamics and uses<br />
modified Monod kinetics to describe the growth and death of cells as a<br />
function of glucose and glutamine availability. This element also describes<br />
accumulation of metabolites (lactate and ammonia) and mAb synthesis<br />
throughout cell culture.<br />
The second element describes the intracellular dynamics of nucleotide sugar<br />
(NS) metabolism. NSs are the substrates required for protein glycosylation<br />
and are synthesised via the amino sugar and nucleotide sugar metabolic<br />
pathway using glucose and glutamine as primary substrates [2]. The full<br />
metabolic pathway has been heuristic<strong>all</strong>y reduced to 8 reactions by<br />
collapsing sequential reactions along each distinct branch of the pathway<br />
into a single one, as shown with the coloured arrows in Figure 1. This<br />
module is linked with the cell culture dynamics one by equations that<br />
define intracellular glucose and glutamine accumulation as a function of<br />
their availability in the extracellular environment.<br />
The pathway shows the synthesis of uridine diphosphate N-acetylglucosamine<br />
(UDP-GlcNAc), uridine diphosphate N-acetylgalactosamine (UDP-GalNAc),<br />
uridine diphosphate glucose (UDP-Glc), uridine diphosphate galactose (UDP-<br />
Gal), guanosine diphosphate mannose (GDP-Man), guanosine diphosphate<br />
fucose (GDP-Fuc), cytosine monophosphate N-acetylneuraminic acid<br />
(CMP-Neu5Ac) and uridine diphosphate glucoronic acid (UDP-GlcA) using<br />
glucose (Glc) and glutamine as substrates. The coloured arrows represent the<br />
reduced scheme where sequential reactions have been collapsed into a single<br />
one (e.g. the blue arrow describes a single reaction that produces UDP-GlcNAc<br />
using glucose and glutamine as substrates). The remaining arrows represent<br />
the synthesis of the other NSs using glucose and glutamine or other NSs<br />
as substrates.<br />
The third element describes mAb Fc glycosylation as a function of mAb<br />
specific productivity and NS availability. This element approximates the<br />
Golgi apparatus to a plug-flow reactor and considers the transport of NSs<br />
from the cytosol, where they are synthesised, into the Golgi, where they are<br />
consumed in glycosylation reactions [3]. As inputs, this element requires<br />
intracellular NS availability and mAb specific productivity, and is thus<br />
coupled to the other two modules. All model simulation was performed<br />
with gPROMS v. 3.4.0 [4].<br />
Experiment<strong>all</strong>y, murine hybridoma cells (CRL-1606, ATCC) were cultured and<br />
typical data was collected (viable cell density, extracellular glucose,<br />
glutamine, lactate, ammonia and mAb titre). In addition, the intracellular<br />
pools of NSs were extracted using perchloric acid and quantified using a<br />
high performance anion exchange chromatographic method that <strong>all</strong>ows for<br />
quantification of 8 NSs and 8 nucleotides in under 30 minutes [5]. Fin<strong>all</strong>y,<br />
the mAb glycan profiles were obtained using MALDI mass spectrometry.
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Figure 1(abstract O10) Nucleotide sugar metabolic network.<br />
The obtained experimental data was then used to estimate the unknown<br />
parameters of the model. Estimation was performed with the maximum<br />
likelihood formulation available in gPROMS v. 3.4.0, where the values for<br />
uncertain physical parameters are obtained to maximise the probability that<br />
the model will predict values from experimental measurements [4].<br />
Results: Time-courses for <strong>all</strong> data were produced, including intracellular<br />
profiles for six NSs (GDP-Man, GDP-Fuc, UDP-Glc, UDP-Gal, UDP-GlcNAc and<br />
CMP-Neu5Ac). This, along with data on cell culture dynamics and mAb Fc<br />
glycosylation were used to estimate the unknown parameters of the model<br />
as described previously. With the estimated parameters, the mathematical<br />
model was found to reproduce cell culture dynamics, intracellular NS pools<br />
and terminal mAb Fc glycan distributions accurately.<br />
With the obtained parameters, a case study for glutamine depletion<br />
was simulated. This study showed that under glutamine deprivation,<br />
intracellular availability of UDP-GlcNAc decreases to a point where mAbs<br />
with high-mannose (Man5) glycan structures begin accumulating in the<br />
extracellular environment, a phenomenon that is consistent with previous<br />
observations [6].<br />
Conclusions: We have shown the construction of a mathematical model<br />
which mechanistic<strong>all</strong>y and quantitatively describes mAb Fc glycosylation<br />
as a function of nutrient availability during cell culture. In addition,<br />
experimental methods have been developed to generate data which was<br />
used to estimate the unknown parameters of the model. Fin<strong>all</strong>y, the<br />
model and obtained parameters were found to be capable of reproducing<br />
previously observed effects of glutamine depletion on protein glycosylation.<br />
With further validation, this quantitative and mechanistic model could prove<br />
useful in aiding process development, control and optimisation for the<br />
manufacture of mAbs with desired glycosylation-associated quality.<br />
References<br />
1. World Preview 2013, Outlook to 2018: Returning to Growth.<br />
EvaluatePharma Report 2013.<br />
2. Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M: KEGG for integration<br />
and interpretation of large-scale molecular data sets. Nucl Acids Res 2012,<br />
40(D1):D109-D114.<br />
3. del Val IJ, Nagy JM, Kontoravdi C: A dynamic mathematical model for<br />
monoclonal antibody N-linked glycosylation and nucleotide sugar donor<br />
transport within a maturing Golgi apparatus. Biotechnol Progr 2011,<br />
27:1730-1743.<br />
4. Process Systems Enterprise: gPROMS Introductory User Guide. 2009.<br />
5. Jimenez del Val I, Kyriakopoulos S, Polizzi KM, Kontoravdi C: An optimised<br />
method for extraction and quantification of nucleotides and nucleotide<br />
sugars from mammalian cells. Analytical Biochemistry 2013, under review.<br />
6. Wong DCF, Wong KTK, Goh LT, Heng CK, Yap MGS: Impact of dynamic online<br />
fed-batch strategies on metabolism, productivity and N-glycosylation<br />
quality in CHO cell cultures. Biotechnol Bioeng 2005, 89:164-177.<br />
POSTER PRESENTATIONS<br />
P1<br />
Generation of genetic<strong>all</strong>y engineered CHO cell lines to support the<br />
production of a difficult to express therapeutic protein<br />
Holger Laux 1* , Sandrine Romand 1 , Anett Ritter 1 , Mevion Oertli 1 , Mara Fornaro 2 ,<br />
Thomas Jostock 1 , Burkhard Wilms 1<br />
1 Novartis Development Integrated Biologic Profiling, 4002 Basel, Switzerland;<br />
2 Novartis Institutes for Biomedical Research, 4056 Basel, Switzerland<br />
E-mail: holger.laux@novartis.com<br />
BMC Proceedings 2013, 7(Suppl 6):P1<br />
Introduction: Chinese Hamster Ovary (CHO) cells are widely used for the<br />
large scale production of recombinant biopharmaceuticals. These cells<br />
have been extensively characterised and approved by regulatory<br />
authorities for production of biopharmaceuticals. During the last years<br />
more and more cell-line engineering strategies have been developed to<br />
enhance productivity and quality. CHO cell line engineering work has<br />
made remarkable progress in optimizing products or titers by focusing on<br />
manipulating single genes and selecting clones with desirable traits. In<br />
this work it is shown how cell line engineering approaches enable the
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expression of a ch<strong>all</strong>enging to express “novel therapeutic protein”. The<br />
expression of the “novel therapeutic protein” in CHO cells resulted in<br />
significant reduced cell growth as well as low productivity.<br />
Results: Transcriptomics analysis: Using customised CHO specific<br />
microarrays the gene expression profile of CHO cells expressing the “novel<br />
therapeutic protein” was analysed. The expression of the “novel therapeutic<br />
protein” resulted in a significant downregulation of <strong>all</strong> mitochondria encoded<br />
genes. The downregulation was more than 40 fold for some of these genes<br />
(Figure 1A). This massive reduced transcription of mitochondrial encoded<br />
genes was very likely causing the reduced cell growth and reduced<br />
expression of the “novel therapeutic protein”. A decrease in mitochondrial<br />
function reduces over<strong>all</strong> metabolic efficiency and a change of metabolic<br />
pathways could also be detected on gene expression level. Addition<strong>all</strong>y the<br />
expression of “gene A” was detected in the applied CHO cell line, which<br />
might have the potential to trigger the down regulation of the mitochondrial<br />
encoded genes in the presence of the “novel therapeutic protein”.<br />
Gene knockdown using shRNA (short hairpin RNA) technique: A variety<br />
of cell line engineering approaches were performed to circumvent cell<br />
growth inhibition caused by down regulation of mitochondrial encoded<br />
genes with the aim to improve expression of the “novel therapeutic<br />
protein”. In the first approach the expression of “gene A”, whichwas<br />
assumed to trigger the down regulation of the mitochondrial encoded<br />
genes, was repressed more than 10 fold using shRNA technique. shRNA is<br />
a sequence of RNA that makes a tight hairpin turn that can be used to<br />
silence target gene expression via RNA interference. Expression of shRNA<br />
was accomplished by delivery of stable integrated plasmids. Cells with<br />
reduced expression of “gene A” showed an improved cell growth and<br />
higher expression of the “novel therapeutic protein” (Figure 1 B). However<br />
cell growth was still repressed, although to a lower extent, and titers were<br />
still lower in comparison to other therapeutic protein formats. Despite the<br />
significant decrease in the expression of “gene A”, the remaining “protein A”<br />
seemed to be sufficient to trigger these effects although to a lower<br />
magnitude.<br />
Gene knockout using zinc finger nucleases (ZFN): To completely<br />
eliminate the cell growth inhibition a knockout of “gene A” was<br />
performed using ZFN technique. ZFNs are artificial restriction enzymes<br />
generated by fusing a zinc finger DNA-binding domain to a DNAcleavage<br />
domain. Plasmids encoding ZFNs (specific<strong>all</strong>y designed to detect<br />
and cleave “gene A”) were transiently transfected in the parental CHO cell<br />
line. ZFN cleaves “gene A” which is then repaired by non-homologous<br />
end joining. This is often error prone and resulted in the generation of<br />
mutant <strong>all</strong>eles. Three clones were identified with mutation in both <strong>all</strong>eles<br />
of “gene A” resulting in shifts of the reading frame and therefore only<br />
nonfunctional premature termination products are encoded.<br />
Knockout of “gene A” resulted in complete elimination of cell growth<br />
inhibition and the expression of mitochondria encoded genes (Figure 1D)<br />
was restored to levels comparable to parental CHO cells. In addition there<br />
was no change in the expression of genes that are involved in metabolic<br />
pathways. Most striking is the significant improved cell growth and<br />
productivity resulting in a 6-7 fold titer increase using this genetic<strong>all</strong>y<br />
engineered knockout cell line (Figure 1B and 1C).<br />
Conclusion: This example illustrates that transcriptomic analysis can<br />
support and facilitate the understanding and solving of specific issues<br />
during the expression of therapeutic proteins. Novel cell line engineering<br />
methods as ZFN technique are powerful tools to solve definite issues in<br />
production of therapeutic proteins in biopharmaceutical industry.<br />
P2<br />
Expansion of mesenchymal adipose-tissue derived stem cells in a<br />
stirred single-use bioreactor under low-serum conditions<br />
Carmen Schirmaier 1* , Stephan C Kaiser 1 , Valentin Jossen 1 , Silke Brill 2 ,<br />
Frank Jüngerkes 2 , Christian van den Bos 2 , Dieter Eibl 1 , Regine Eibl 1<br />
1 Zurich University of Applied Sciences, Institute of Biotechnology,<br />
Biochemical Engineering and Cell Cultivation Technique, 8820 Wädenswil,<br />
Switzerland;<br />
2 Lonza Cologne GmbH, 50829 Cologne, Germany<br />
E-mail: *carmen.schirmaier@zhaw.ch<br />
BMC Proceedings 2013, 7(Suppl 6):P2<br />
Background: The need for human mesenchymal stem cells (hMSCs) has<br />
increased enormously in recent years due to their important therapeutic<br />
potential. Efficient cell expansion is essential to providing clinic<strong>all</strong>y relevant<br />
cell numbers. Such cell quantities can be manufactured by means of<br />
scalable microcarrier (MC)-supported cultivations in stirred single-use<br />
bioreactors.<br />
Materials and methods: Preliminary tests in disposable-spinners (100 mL<br />
culture volume, Corning) were used to determine two suitable media and<br />
MC-types for serum reduced expansions (< 10%) of human adipose tissuederived<br />
stem cells (hADSCs; passage 2, Lonza). Using such optimized<br />
media-MC-combinations, hADSCs expanded 30 to 40-fold, which compares<br />
well with expansion rates in planar culture. Based on computational fluid<br />
dynamics simulations and suspension analyses in spinners [1], optimal<br />
operating parameters were determined in a BIOSTAT® UniVessel® SU 2 L<br />
(2 L culture volume, Sartorius Stedim Biotech).<br />
Results: In subsequent batch tests with the BIOSTAT UniVessel® SU 2 L,<br />
expansion rates of between 30 and 40-fold were reached and up to 4.4·10 8<br />
cells with a cell viability exceeding 98% were harvested. Flow cytometry<br />
tests demonstrated typical marker profiles following cell expansion and<br />
harvest. A 40-fold expansion rate delivered a total of 1·10 10 cells in a first<br />
cultivation with the BIOSTAT® CultiBag STR 50 L (35 L culture volume,<br />
Sartorius Stedim Biotech).<br />
Conclusions: In summary, the foundations for successfully expanding<br />
therapeutic stem cells in truly scalable systems have been laid. Strategies<br />
ensuring expansion rates between 60 and 70-fold and, thus, generating<br />
cell amounts over 10 10 are now in preparation.<br />
Acknowledgements: This work is part of the project “Development of a<br />
technology platform for a scalable production of therapeutic<strong>all</strong>y relevant<br />
stem cells” (No. 12893.1 VOUCH-LS). It is supported by the Commission for<br />
Technology and Innovation (CTI, Switzerland). The authors would like to<br />
thank the CTI for parti<strong>all</strong>y financing the investigations presented.<br />
Reference<br />
1. Kaiser S C, Jossen V, Schirmaier C, Eibl D, Brill S, van den Bos C, Eibl R:<br />
Investigations of fluid flow and cell proliferation of mesenchymal<br />
adipose-derived stem cells in sm<strong>all</strong>-scale, stirred, single-use bioreactors.<br />
Chem Ing Tech 2013, 85:95-102.<br />
P3<br />
Evaluating the effect of chromosomal context on zinc finger nuclease<br />
efficiency<br />
Scott Bahr * , Laura Cortner, Sara Ladley, Trissa Borgschulte<br />
CHOZN® Platform Development Team, SAFC/Sigma-Aldrich, St Louis, MO<br />
63103, USA<br />
E-mail: scott.bahr@sial.com<br />
BMC Proceedings 2013, 7(Suppl 6):P3<br />
Introduction: Zinc Finger Nuclease (ZFN) technology has provided<br />
researchers with a tool for integrating exogenous sequences into most cell<br />
lines or genomes in a precise manner. Using current methods, the<br />
efficiency of targeted integration (TI) into the host genome is gener<strong>all</strong>y low<br />
and is highly dependent on the ZFN activity at the genomic locus of<br />
interest. It is unknown if the ZFN binding and cutting efficiency is more<br />
dependent on the nucleotide recognition sequence or the chromosomal<br />
context in which the sequence is located.<br />
We have taken a highly efficient ZFN pair (hAAVS1) from human studies<br />
and introduced the exogenous DNA sequence into the Chinese Hamster<br />
Ovary (CHO) genome in an attempt to improve the efficiency of targeted<br />
integration. A “Landing Pad” comprised of human AAVS1 sequence has<br />
been integrated into the CHO genome at 3 separate loci to determine if<br />
the ZFN’s will work across species and if the cutting efficiency is affected<br />
by chromosomal context. The results of this study will help us to improve<br />
the over<strong>all</strong> efficiency of TI by using Landing Pads, particularly for<br />
genomic targets in which suitable ZFN’s may not be available.<br />
Methods: 3 CHO Loci were chosen for this study based on previous gene<br />
expression studies. Rosa26 and Neu3 show consistent but low levels of<br />
expression while Site #1 appears to have no known coding sequence.<br />
Addition<strong>all</strong>y, Rosa26 and Site#1 were chosen as potential safe harbor sites<br />
in CHO. The ZFN cutting efficiency at the endogenous CHO loci Rosa26,<br />
Site #1 and Neu3 are approximately 15%, 30% and 40% respectively. Based<br />
on other studies the cutting efficiency of human AAVS1 ZFN’s was as high<br />
as 50% depending on the human cell line used. A plasmid donor carrying<br />
the hAAVS1 ZFN recognition sequence Landing Pad was introduced into<br />
CHO Rosa26, Site #1, and Neu3 via targeted integration (Figure 1).
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Figure 1(abstract P1) A highlights the reduced expression of mitochondria encoded genes in CHO cells expressing the “novel therapeutic protein” in<br />
comparison to parental CHO cells. The y-axis shows the gene expression values in signal intensities. B: Batch culture titers of the “novel therapeutic<br />
protein” in shake flask are shown. Titer for CHO WT cells are labeled in red, titer for CHO cells with reduced expression of gene A (shRNA approach) are<br />
labeled in yellow and titer for CHO cells with non-functional gene A (knockout) are labeled in green. C: Cell growth of CHO WT cells and CHO knockout<br />
(KO) cells with and without “novel therapeutic protein” in shake flasks. Y-axis shows viable cell density and x-axis cultivation time. D: Gene expression of<br />
mitochondrial encoded genes is not reduced in <strong>all</strong> three generated CHO KO cell lines with the “novel therapeutic protein”. Hierarchical clustering reveals<br />
that the “novel therapeutic protein” does not affect the gene expression profile of the KO cell lines. In contrast the “novel therapeutic protein” has a clear<br />
effect on the WT cell line.
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Figure 1(abstract P3) Schematic of ZFN mediated Integration of the hAAVS1 Landing Pad into CHO.<br />
Results: Clones carrying the exogenous hAAVS1 Landing Pads at Rosa26, Site<br />
#1 and Neu3 were transfected with hAAVS1 ZFN’s and the cutting efficiency<br />
was measured. We found that the human AAVS1 ZFN’s wereableto<br />
successfully cut at their recognition sequence in the Landing Pad at <strong>all</strong> 3 CHO<br />
loci to varying degrees (Table 1). ZFN efficiency at each loci was measured by<br />
Cel1 Assay or direct sequencing of Indels in PCR amplicons. We see<br />
successful ZFN activity at <strong>all</strong> 3 loci but with varying efficiency. **The Landing<br />
Pad integration at Neu3 locus caused phenotypic changes in the cell growth<br />
and viability following transfection which may explain low ZFN activity.<br />
Conclusions: These results indicate that the chromosomal context of the<br />
ZFN recognition sequence has an effect on cutting efficiency. This study<br />
shows that TI can be performed with Landing Pads across species with high<br />
efficiency and provide researchers with additional tools for cell line<br />
engineering. Further development of Landing Pads could create highly<br />
engineered and multi-functional platforms that would facilitate more<br />
efficient and more tailored CHO cell modifications.<br />
P4<br />
Insights into monitoring changes in the viable cell density and cell<br />
physiology using scanning, multi-frequency dielectric spectroscopy<br />
John Carvell 1* , Lisa Graham 2 , Brandon Downey 2<br />
1 Aber Instruments Ltd, Abersytwyth, UK;<br />
2 Bend Research Inc., Oregon, USA<br />
E-mail: johnc@aberinstruments.com<br />
BMC Proceedings 2013, 7(Suppl 6):P4<br />
Table 1(abstract P3) Comparing ZFN activity in CHO<br />
before and after Landing Pad Integration<br />
CHO<br />
Site<br />
Rosa<br />
26<br />
Site<br />
#1<br />
ZFN Activity at Endogenous<br />
CHO Locus<br />
ZFN Activity at Integrated<br />
Landing Pad<br />
16% 18%<br />
31% 51%<br />
Neu3 41% 16%**<br />
Background: Real-time bioprocess monitoring is fundamental for<br />
maximizing yield, improving efficiency and process reproducibility,<br />
minimizing costs, optimizing product quality, and full understanding of how<br />
asystemworks.TheFDA’s Process Analytical Technology initiative (PAT)<br />
encourages bioprocess workflows to operate under systems that provide<br />
timely, in-process results. At the same time the demand for ever increasing<br />
supplies of biological pharmaceuticals, such as antibodies and recombinant<br />
proteins, has fueled interest in streamlined manufacturing solutions.<br />
Bioreactors that are monitored continuously and in real-time offer the<br />
advantage of meeting current and future supply demands with biological<br />
product of the utmost quality and safety, achieved at the lowest over<strong>all</strong> cost<br />
and with least risk. This paper will focus on how one research groups in has<br />
used scanning multi-frequency dielectric spectroscopy to comparatively<br />
profile multiple bioreactor runs and elucidatefinedetailsconcerningcell<br />
viability and mechanism of cell death. The cellular information observed has<br />
not been available through other technologies. The presentation will also<br />
focus on how the technology can also be applied to Single use Bioreactors<br />
in a cGMP environment and on samples down to 1 ml volume.<br />
Introduction: • Dielectric spectroscopy (DS) is now the most common<br />
method for estimating the in situ live cell concentration in animal cell<br />
culture.<br />
• DS and traditional offline methods for cell counting based on<br />
Trypan Blue correlate well during the growth phases but with some<br />
cell lines, deviations are observed during the late growth phase.<br />
• Scanning multi-frequency DS can detect the physiological changes<br />
of the cells during the death phase of the culture including changes<br />
in cell size, membrane capacitance and internal conductivity [1-3].<br />
• The concept of using the Area Ratio Algorithm (ARA) looks to be a<br />
relatively simple and promising method for providing on-line cell<br />
counts that correlate well with traditional methods for the complete<br />
cell growth cycle.<br />
Background of DS and the Futura Biomass Monitor: • DS measures<br />
the passive electrical properties of cells in suspension through the<br />
cells’ interaction with RF excitations.<br />
• Viable cells are composed of a conducting cytoplasm surrounded by a<br />
non-conducting membrane suspended in a conducting medium. When<br />
an alternating current is applied to the suspension, each cell becomes<br />
polarised and behaves electric<strong>all</strong>y as a tiny spherical capacitor.
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Page 17 of 151<br />
• The suspensions reaction to the current is expressed as its permittivity<br />
can be measured by its capacitance and conductivity as a function of<br />
frequency. Viable cells possess intact membranes which prevent the<br />
free flow of ions and <strong>all</strong>ow the cells to polarise. Dead, porous cells and<br />
debris lack an enclosing membrane and are unable to build up charge<br />
separation. Hence, DS measures only viable cells.<br />
• The Futura Biomass Monitor (Aber Instruments Ltd, UK) measures the<br />
capacitance created directly from the cells. The capacitance signature<br />
of cells is measured between 50 KHz and 20 MHz with readings every<br />
30 seconds.<br />
• At low excitation frequencies the cells can fully polarise and the<br />
capacitance of the suspension is maximised. As the excitation<br />
increases, the cells lose their ability to fully polarize and the measured<br />
capacitance drops eventu<strong>all</strong>y measuring no polarisation at high<br />
frequencies.<br />
Concept of the Area Ratio algorithm: • A novel method for obtaining an<br />
enhanced prediction of viable cell volume fraction (VCV) compared to<br />
currently employed methods has been developed, wherein changes in<br />
cell health are quantified using frequency scanning data. In the novel<br />
method, cell health is measured by using an area ratio (AR) to quantify<br />
the shape of the measured dielectric spectrum using the following<br />
algorithm: AR = ∫ fQfHC(f )df ∫ fLfHC(f )df fH < fQ < fL<br />
Where:<br />
AR = area ratio for a given scan<br />
f H = highest frequency of the scan<br />
f L = lowest frequency of the scan<br />
f Q = semi arbitrary chosen frequency between f H and f L<br />
C(f) = capacitance as a function of frequency<br />
• The AR is used as a correction factor to correct for the death phase<br />
divergence in the following manner: VCV(t) = A × (C(t) - B × AR(t) -<br />
k2) + k1<br />
Where:<br />
VCV = predicted viable cell volume fraction<br />
A and B = fit constants of proportionality relating dielectric<br />
measurements to offline cell measurements<br />
k 1 and k 2 = constant offset values<br />
• Changes in cell health are quantified using frequency scanning data.<br />
When the ARA is applied to the uncorrected VCV derived from the<br />
capacitance data, there is a good match with the off-line derived VCV<br />
(Figure 1).<br />
Applying multi-frequency scanning DS to single use bioreactors and<br />
samples off-line: • A single use sensor has been developed by Aber<br />
Instruments and the early versions utilized stainless steel electrodes.<br />
This sensor was suitable for single or dual frequency DS and the<br />
performance has been compared with traditional probes that are used<br />
on reusable bioreactors [4].<br />
• Samples as low as 100 microlitre can be withdrawn from a bioreactor<br />
and scanning DS can be applied using existing DS probes. An example<br />
of this is shown in the full version of the poster with distinctly<br />
different frequency scans for healthy and unhealthy cells. The<br />
unhealthy cells were generated by treatment with 1 uM staurosporine<br />
to induce apoptosis.<br />
Discussions and conclusions: The work presented here shows the utility<br />
of frequency scanning data to obtain enhanced measurement of VCV using<br />
non-invasive capacitance sensors in reusable and single use bioreactors. The<br />
information-rich nature of dielectric frequency scanning <strong>all</strong>ows interrogation<br />
of biophysical properties of cells. The concept can be extended to samples<br />
off-line.<br />
References<br />
1. Asami K: Characterization of heterogeneous systems by dielectric<br />
spectroscopy. Prog Polymer Sci 2002, 27:1617-1659.<br />
2. Cannizzaro C, Gügerli R, Marison I, Von Stockar U: On-line biomass<br />
monitoring of CHO perfusion culture with scanning dielectric<br />
spectroscopy. Biotechnol Bioeng 2003, 84:597-610.<br />
3. Ron A, Singh RR, Fishelson N, Shur I, Socher R, Benayahu D, Shacham-<br />
Diamand Y: Cell-based screening for membranal and cytoplasmatic<br />
markers using dielectric spectroscopy. Biophys Chem 2008, 135:59-68.<br />
4. Carvell JP, Williams J, Lee M, Logan D: On-Line Monitoring of the Live Cell<br />
Concentration in Disposable Bioreactors (poster). European Society for<br />
Animal Cell Technology biennial conference, Dublin, Ireland 2009.<br />
P5<br />
Multidimension cultivation analysis by standard and omics methods for<br />
optimization of therapeutics production<br />
Julia Gettmann 1† , Christina Timmermann 1† , Jennifer Becker 1 , Tobias Thüte 1 ,<br />
Oliver Rupp 2 , Heino Büntemeyer 1 , Anica Lohmeier 1 , Alexander Goesmann 2 ,<br />
Thomas Noll 1,3*<br />
1 Institute of Cell Culture Technology, Bielefeld University, 33615 Bielefeld,<br />
Germany;<br />
2 Bioinformatics Resource Facility, Center for Biotechnology<br />
(CeBiTec), Bielefeld University, 33615 Bielefeld, Germany; 3 Center for<br />
Biotechnology (CeBiTec), Bielefeld University, 33615 Bielefeld, Germany<br />
E-mail: thomas.noll@uni-bielefeld.de<br />
BMC Proceedings 2013, 7(Suppl 6):P5<br />
Background: During the last decades Chinese Hamster Ovary (CHO) cells<br />
have been extensively used for research and biotechnological applications.<br />
About 40% of newly approved glycosylated protein pharmaceuticals are<br />
produced in CHO cells today [1]. Despite the increasing relevance of these<br />
Figure 1(abstract P4) Implementation of the Area Ratio Algorithm (ARA) yields enhanced prediction of viable cell volume fraction compared<br />
with uncorrected methods.
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Page 18 of 151<br />
cells for biopharmaceutical production little is known about effects of<br />
intracellular processes on productivity and product quality.<br />
In the last years supplementation of serum-free media with insulin - more<br />
and more replaced by IGF-1 and its analogue LongR 3 - was utilized to<br />
enhance product titer and quality. To compare the intracellular effects of<br />
these two supplements an antibody producing CHO cell line was cultivated<br />
in batch mode using insulin, LongR 3 or no growth factor as reference.<br />
Subsequently, different omics-techniques were applied to analyze medium<br />
and cell samples.<br />
Materials and methods: CHO cells producing an antibody were cultured<br />
in chemic<strong>all</strong>y defined serum-free medium TC-BN.CHO (Teutocell AG) with<br />
addition of 6 mM glutamine. Three cultivations (37°C, pH 7.1, 40% DO,<br />
120 rpm) were performed in 2l-bioreactor systems with supplementation<br />
of 10 mg/l insulin or 0.1 mg/l LongR 3 . The third culture was untreated and<br />
served as reference. Samples were taken every 24 h.<br />
Viable cell density and cell viability were measured using Cedex (Roche).<br />
Glucose and lactate were determined via YSI 2300 STAT Plus Glucose &<br />
Lactate Analyzer (YSI Life Science). Quantitation of antibody production was<br />
determined using POROS® A columns (Invitrogen). N-Glycan abundance was<br />
analyzed by HPAEC-PAD method [2].<br />
For RNA samples ‘Total RNA NucleoSpin Kit’ (Macherey-Nagel) was used.<br />
Quality and quantity of RNA were determined using Nano Drop 1000<br />
(Peqlab) and Bioanalyzer (Agilent).<br />
An in-house developed customized cDNA microarray with 41,304 probes<br />
was applied for transcriptome analysis. RNA was labeled using Agilent<br />
LIQUA Kit, one-color. Processing of microarray data was performed in<br />
ArrayLims and EMMA2 [3]. Raw data were standardized using Feature<br />
Extractor (Agilent) and LOWESS normalization.<br />
Results: Cultivation data illustrated that maximal cell density was higher in<br />
cultivations with insulin and LongR 3 compared to that without growth<br />
factor. Addition<strong>all</strong>y, glucose consumption and lactate production was<br />
slightly higher in cultivations with these supplements but time point of<br />
glutamine depletion was similar in <strong>all</strong> reactors after similar cultivation time<br />
(Figure 1A). Furthermore, product quantity and product quality was not<br />
influenced by growth factor addition. The most abundant glycoforms after<br />
7 days of cultivation were G0F with about 50% and G1F with about 40% in<br />
<strong>all</strong> cultivation set-ups (Table 1).<br />
For transcriptome analysis samples on day 5 were compared with those on<br />
day 3. Therefore, the following settings were used in statistical tests: a twosample<br />
t-test with a p-value ≤ 0.01, signal intensity ≥ 6(forA1orA2)and<br />
intensity ratio ≥ 0.6 or ≤ -0.6 (for M1 or M2). Transcriptome data showed<br />
that LongR 3 supplementation resulted in the highest transcription change<br />
(1259 up- and 1689 down-regulated). Insulin supplementation resulted in<br />
second highest transcriptomic change (1026 up- and 1404 downregulated)<br />
and reference cultivation led to lowest changes (344 up- and<br />
301down-regulated). Supplemented cultures showed a higher transcription<br />
change in the selected pathways, like pentose phosphate pathway, TCA<br />
and glycolysis, than the reference culture, too. In LongR 3 containing<br />
cultures even more genes from these pathways were higher changed<br />
(Figure 1B).<br />
Conclusions: Data on cell growth and productivity as well as omics results<br />
were brought together to achieve a deeper insight into cellular processes<br />
and their influence on productivity and product quality.<br />
Cultivation data showed faster growth, glucose consumption and lactate<br />
formation for cultivations with insulin and LongR 3 compared to reference<br />
culture. However, antibody titer and glycan profiles were almost similar in <strong>all</strong><br />
cultures. This indicates that supplementation with insulin or LongR 3 does<br />
not have an enhancing effect on product quality and quantity in antibody<br />
production with our CHO-K1 cells.<br />
Addition<strong>all</strong>y, transcriptome data showed that growth factor supplementation<br />
resulted in a higher transcription change than in reference cultivation.<br />
Thus, for more understanding of the influence of insulin or LongR 3<br />
supplementation on cultured CHO cells, further analysis of pathway<br />
regulation with full details is required.<br />
Acknowledgements: The project is co-funded by the European Union<br />
(European Regional Development Fund - Investing in your future) and the<br />
German federal state North Rhine-Westphalia (NRW).<br />
References<br />
1. Higgins E: Carbohydrate analysis throughout the development of a<br />
protein therapeutic. Glycoconj J 2010, 2:211-225.<br />
2. Behan JL, Smith KD: The analysis of glycosylation: a continued need for high<br />
pH anion exchange chromatography. Biomed Chromatogr 2011, 25:39-46.<br />
3. Dondrup M, Albaum SP, Griebel T, Henckel K, Junemann S, Kahlke T,<br />
Kleindt CK, Kuster H, Linke B, Mertens D, Mittard-Runte V, Neuweger H,<br />
Runte KJ, Tauch A, Tille F, Puhler A, Goesmann A: EMMA 2–a MAGEcompliant<br />
system for the collaborative analysis and integration of<br />
microarray data. BMC Bioinformatics 2009, 10:50.<br />
P6<br />
Toward a serum-free, xeno-free culture system for optimal growth and<br />
expansion of hMSC suited to therapeutic applications<br />
Mira Genser-Nir * , Sharon Daniliuc, Marina Tevrovsky, David Fiorentini<br />
Biological Industries, Kibbutz Beit Haemek, Israel<br />
E-mail: mira@bioind.com<br />
BMC Proceedings 2013, 7(Suppl 6):P6<br />
Background: Human mesenchymal stem cells (hMSC) hold great promise<br />
as a tool in regenerative medicine and cell therapy. Application of hMSC in<br />
cell therapy requires the elaboration of an appropriate serum-free (SF),<br />
xeno-free (XF) culture system in order to minimize the health risk of using<br />
xenogenic compounds, and to limit the immunological reactions in-vivo.<br />
Besides the well-known disadvantages of serum, in comparison to a SF, XF<br />
culture system, serum also exhibits poor performance in the context of<br />
hMSC proliferation. In the present study, a novel SF, XF culture system for<br />
hMSC suitable for therapeutic applications was developed and evaluated.<br />
Figure 1(abstract P5) (A) Time chart of viable cell density (VCD), cell viability (CV) and extracellular metabolites [glucose (Glc), lactate (Lac), glutamine<br />
(Gln)]. (B) Number of significantly up- and down-regulated genes on day 5 in selected pathways (compared to day 3).
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Table 1(abstract P5) N-Glycan abundance [%] after<br />
7 days of cultivation<br />
Culture G0F G0 G1F G1 G2F G2<br />
Reference 51,8 4,1 35,6 0,9 7,4 0,2<br />
Insulin 50,6 3,4 38,6 0,9 6,3 0,2<br />
LongR 3 52,5 1,4 38,5 0,5 6,9 0,3<br />
The SF, XF culture system includes speci<strong>all</strong>y developed solutions for<br />
attachment, dissociation, and freezing, as well as a culture medium, MSC<br />
NutriStem® XF, that enables long-term growth of multipotent hMSC.<br />
Development of the SF, XF culture system was conducted on hMSC from a<br />
variety of sources: bone marrow (BM), adipose tissue (AT) and Wharton’s<br />
jelly (WJ).<br />
Materials and methods: MSC NutriStem® XF culture medium was<br />
examined in combination with MSC Attachment Solution (BI, 05-752-1) and<br />
either Recombinant Trypsin Solution (BI, 03-078-1) or MSC Dissociation<br />
Solution (BI, 03-075-1). The performance of MSC NutriStem® XF was<br />
evaluated based on the following parameters: proliferation rate, viability,<br />
morphology, stemness (estimated from CFU-F), multilineage differentiation<br />
capability, and phenotypic surface marker profile [1].<br />
Cells: hMSC (passage 1-5) from a variety of sources: BM (Lonza, Promocell),<br />
AT (Promocell, ATCC), and WJ (ATCC, Prof. Mark Weiss - self isolation) were<br />
used in this study.<br />
Culture System: hMSC were cultured in a SF, XF expansion medium (MSC<br />
NutriStem® XF, BI) on pre-coated dishes (MSC Attachment Solution, BI) or<br />
other media; commercial SF media (Invitrogen; SCT, Promocell), in-house<br />
serum-containing formulation (Prof. Mark Weiss). Cells were seeded at<br />
5000-6000 viable cells/cm 2 , and harvested using either MSC Dissociation<br />
Solution (BI) or recombinant Trypsin Dissociation Solution (BI).<br />
Medium Performance Evaluation: Medium performance was evaluated<br />
by conducting a comparison of proliferation rate, cell morphology,<br />
multilinage differentiation potential into adipocytes, osteocytes, and<br />
chondrocytes, self-renewal potential and cell immunophenotype.<br />
Cell Expansion: Cell proliferation was assessed by cell count using a<br />
trypan blue exclusion assay at each time point.<br />
Differentiation: hMSC expanded for 3-5 passages in MSC NutriStem® XF<br />
were tested for maintenance of multilineage differentiation potential (into<br />
adipocytes, osteocytes, and chondrocytes) using in-house differentiation<br />
formulations. Undifferentiated control cells were cultured in MSC<br />
NutriStem® XF. Cells were fixed and stained with Oil Red O, Alizarin Red/<br />
von Kossa, and Alcian blue/Masson’s trichrome, respectively.<br />
CFU-F Assay: hMSC were seeded at low densities (10, 50, and 100 cells/<br />
cm 2 ) in MSC NutriStem® XF, cultured for 14 days, and stained with 0.5%<br />
crystal violet.<br />
Flow Cytometry: WJ-derived hMSC were cultured for five passages in<br />
MSC NutriStem® XF, followed by immunophenotype evaluation by flow<br />
cytometry expression of CD73, CD90, CD105, HLA-ABC (positive), HLA-DR,<br />
and CD45 (negative).<br />
Results: An optimized SF, XF culture system for hMSC was developed,<br />
composed of growth medium, MSC NutriStem® XF, and <strong>all</strong> the required<br />
auxiliary solutions for the attachment, dissociation, and freezing of the cells.<br />
This SF, XF culture system for hMSC, supported optimal expansion of hMSC<br />
from a variety of sources, and exhibited superior proliferation compared<br />
with serum-containing media and commerci<strong>all</strong>y available SF media. hMSC<br />
expanded in the SF, XF culture system maintain their typical fibroblast-like<br />
cell morphology and phenotypic surface marker profile of CD73, CD90,<br />
CD105, HLA-ABC (<strong>all</strong> positive), or CD34, CD45, HLA-DR (<strong>all</strong> negative). hMSC<br />
differentiated efficiently after expansion in the developed SF, XF culture<br />
system into osteocytes, chondrocytes, and adipocytes. The self-renewal<br />
potential was maintained as well, demonstrated by a colony-forming unit<br />
fibroblast (CFU-F) assay (Figure 1).<br />
Conclusions: The use of serum is not an option from a regulatory point of<br />
view. A SF, XF culture system for hMSC was developed and enables longterm<br />
growth of multipotent hMSC suitable for therapeutic applications.<br />
The performance of MSC NutriStem® XF medium was proved to be<br />
superior to serum-containing medium and commerci<strong>all</strong>y available SF<br />
media. MSC NutriStem® XF medium supports long-term culture of hMSC<br />
from a variety of sources, while retaining the essential hMSC characteristics<br />
(fibroblast-like morphology, surface markers phenotype, multilineage<br />
differentiation, and self-renewal potential).The developed SF, XF culture<br />
system (MSC NutriStem® XF medium, MSC Attachment Solution, either<br />
MSC Dissociation Solution or Recombinant Trypsin solution, and MSC<br />
Freezing Solution) supports the expansion of hMSC suitable for clinical<br />
applications.<br />
Acknowledgements: We would like to thank Professor Mark L. Weiss,<br />
Kansas State University, Department of Anatomy and Physiology,<br />
Manhattan, KS, for his invaluable contribution to this study.<br />
Reference<br />
1. Poster: ISCT 2012 Seattle, USA. Identification of optimal conditions for<br />
generating MSCs for preclinical testing: Comparison of three commercial<br />
serum-free media and low-serum growth medium. Weiss, Kansas State<br />
University, Department of Anatomy and Physiology, Manhattan, KS: Yelica<br />
López, Elizabeth Trevino, Mark L .<br />
P7<br />
Highly efficient inoculum propagation in perfusion culture using WAVE<br />
Bioreactor systems<br />
Christian Kaisermayer 1* , Jianjun Yang 2<br />
1 GE Healthcare Life Sciences, Björkgatan 30, 751 84 Uppsala, Sweden;<br />
2 GE<br />
China Research and Development Center Co. Ltd. Shanghai, China<br />
E-mail: Christian.Kaisermayer@ge.com<br />
BMC Proceedings 2013, 7(Suppl 6):P7<br />
Introduction: A perfusion-based process was developed to increase the<br />
split ratio during the scale-up of CHO-S cell cultures. Fedbatch cultures<br />
were inoculated with cells propagated in either batch or perfusion<br />
cultures. All cultures were grown in disposable Cellbag bioreactors using<br />
the WAVE Bioreactor system. Cell concentrations of 4.8 × 10 7 cells/mL were<br />
achieved in the perfusion culture, whereas the final cell concentration in<br />
the batch culture was 5.1 × 10 6 cells/mL. The higher cell concentration of<br />
the perfusion culture <strong>all</strong>owed for a more than six-fold increase of the split<br />
ratio to about 1:30. The method described here, can reduce the number of<br />
required expansion steps and eliminate the need for one or two<br />
bioreactors in the seed train. Single-use bioreactors at benchtop scale can<br />
be used for direct inoculation of production bioreactors. Alternatively, high<br />
biomass concentrations accumulated in perfusion culture can be used to<br />
seed production vessels at increased cell concentrations. Thus, the process<br />
time in these bioreactors, which often is the bottleneck in plant<br />
throughput, can be shortened.<br />
Materials and methods: • CHO-S cells (Life Technlologies)<br />
• Cultivation medium and feed concentrate: T13 and T13-F (Shanghai<br />
Hankang Biotech Co.)<br />
• WAVE Biorereactor 20/50 system (GE Healthcare)<br />
• Cellbag bioreactors (GE Healthcare)<br />
Batch and fed-batch cultivations were run in Cellbag 10 L bioreactors,<br />
perfusion cultures in Cellbag 2 L bioreactors. Cultivation conditions: T 37°C,<br />
pH 7.10, DO > 40%, agitation for <strong>all</strong> cultures 25 rpm/6°.<br />
Analytics: Cell concentration and viability, glucose and lactate concentration.<br />
Perfusion and feed rates were adjusted to maintain the residual glucose<br />
concentration above 0.5 g/L.<br />
Results and discussion: CHO cells are the production system of choice for<br />
complex recombinant proteins. The prevalent mode of production is<br />
fedbatch cultivation because of the generated titers achieved with limited<br />
process complexity [1]. Perfusion processes have been reported as an<br />
alternative strategy that substanti<strong>all</strong>y increases volumetric productivity but<br />
because of the higher process complexity, they are less frequently used in<br />
manufacturing [2,3]. An alternative strategy is to use perfusion technology in<br />
the seed train to improve process flexibility and maximize equipment<br />
utilization [3]. In this comparative study, CHO-S cells were grown in either<br />
batch or perfusion (Figure 1) culture to generate inocula for subsequent fedbatch<br />
cultivations. During the initial phase, cell growth in both cultures was<br />
similar (Figure 1). However, despite high cell viability, the growth rate in the<br />
batch culture decreased from 0.8 d -1 during the first two days to about 0.3 d -1<br />
between day 2 and 6 (data not shown). In contrast, the nutrient supply in<br />
the perfusion culture supported an average growth rate of 0.8 d -1 and an<br />
exponential growth until day 5 (Figure 1). Inoculum was removed from each<br />
seed culture while the cells were still growing at their maximum rate and<br />
while viability was above 95%. The higher cell concentration achieved in the<br />
perfusion culture was used to seed a subsequent fedbatch culture at an
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Figure 1(abstract P6) hMSC Features after culturing in MSC NutriStem® XF. Characterization of hMSC-WJ expanded for 5 passages in MSC<br />
NutriStem® XF and in-house serum-containing formulation. Immunophenotype using FACS analysis (A), multilineage differentiation into adipocytes (Oil<br />
Red O), osteocytes (von Kossa), and chondrocytes (Masson’s trichrome) (B), CFU-F assay (C). hMSC cultured in MSC NutriStem® XF maintains the essential<br />
MSC characteristics; classical profile of MSC markers, multilineage differentiation, and self-renewal potential [1].<br />
increased split ratio of 1:30, compared with 1:5 used for the inoculum from<br />
the batch culture. Cell growth in the two subsequent fed-batch cultures is<br />
shown in Figure 1. The cultures inoculated from either batch or perfusion<br />
culture showed comparable growth and no lag phase was observed after<br />
inoculation. A comparison of the individual culture parameters is presented in<br />
Table 1. The higher split ratio in the perfusion culture saves at least one step<br />
in the inoculum propagation as compared with cultivation in batch mode, for<br />
which two subsequent cultures with a split ratio of 1:5 would be required to<br />
obtain a similar ratio. Even higher split ratios could be achieved in perfusion<br />
cultures. On day 6, the cell concentration was 4.06 × 10 7 cells/mL with a<br />
viability of 96%. (Figure 1). Although the cells were already at the end of the<br />
exponential growth phase, a split ratio of 1:100 could be achieved at this<br />
timepoint. The fed-batch culture inoculated from the perfusion culture was<br />
started at a substanti<strong>all</strong>y higher cell concentration than the one inoculated<br />
from the batch culture and, thus, reached its maximum cell concentration<br />
about two days earlier (Figure 1). Addition<strong>all</strong>y the viable cell integral was<br />
increased by about 20% (data not shown). Assuming constant product<br />
formation during cell growth, this would <strong>all</strong>ow to reach the same amount of<br />
product two days earlier and, thus, shorten process time in the main<br />
bioreactor. The use of perfusion cultures for seeding the production<br />
bioreactor at high cell concentrations has also been reported for an industry<br />
process at 13,500 L working volume where it resulted in a 20% decrease in<br />
the occupation of the production vessel [3].<br />
Conclusions: • Perfusion culture maintained cells in exponential growth<br />
phase for an extended period of time compared with batch culture.<br />
• The high cell concentrations obtained in perfusion culture can<br />
substanti<strong>all</strong>y increase the split ratio, thus, minimizing the number of<br />
vessels needed in the seed train.<br />
• Alternatively, the production bioreactor can be inoculated at high cell<br />
concentration, which can help shortening process time in the<br />
production vessel and improving facility utilization.<br />
• One WAVE Bioreactor 20/50 system, run in perfusion mode at the<br />
maximum operating volume of 25 L, could provide inoculum for a<br />
2000 L bioreactor.<br />
References<br />
1. Shukla A, Thömmes J: Recent advances in large-scale production of<br />
monoclonal antibodies and related proteins. Trends Biotechnol 2010,<br />
28:253-261.<br />
2. Wang L, Hu H, Yang J, Wang F, Kaisermayer C, Zhou P: High yield of<br />
human monoclonal antibody produced by stably transfected drosophila<br />
schneider 2 cells in perfusion culture using wave bioreactor. Mol<br />
Biotechnol 2012, 52:170-179.<br />
3. Pohlscheidt M, Jacobs M, Wolf S, Thiele J, Jockwer A, Gabelsberger J,<br />
Jenzsch M, Tebbe H, Burg J: Optimizing capacity utilization by large scale<br />
3000 L perfusion in seed train bioreactors. Biotechnol Prog 2013,<br />
29:222-229.
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Figure 1(abstract P7) CHO-S cells grown in batch and perfusion. Arrow indicates seed removal for subsequent fedbatch cultures (upper panel).<br />
Comparison of CHO-S fed-batch cultures inoculated from either batch or perfusion (lower panel).<br />
P8<br />
Intact cell MALDI mass spectrometry biotyping for “at-line” monitoring<br />
of apoptosis progression in CHO cell cultures<br />
Sebastian Schwamb 1* , Bogdan Munteanu 1 , Björn Meyer 1 , Carsten Hopf 1,2 ,<br />
Mathias Hafner 1,2,3 , Philipp Wiedemann 1,2<br />
1 Center for Applied Biomedical Mass Spectrometry (ABIMAS), Mannheim,<br />
Baden-Württemberg, 68163, Germany;<br />
2 Mannheim University of Applied<br />
Sciences, Mannheim, Baden-Württemberg, 68163, Germany;<br />
3 Heidelberg<br />
University, Institute for Medical Technology, Mannheim, Baden-Württemberg,<br />
68163, Germany<br />
E-mail: s.schwamb@hs-mannheim.de<br />
BMC Proceedings 2013, 7(Suppl 6):P8<br />
Background: Mammalian cell cultures, especi<strong>all</strong>y Chinese Hamster Ovary<br />
(CHO), are the predominant host for the production of biologics. Despite<br />
considerable progress in industry and academia alike (also enforced e.g.<br />
by the Process Analytical Technology Initiative of the FDA), particularly in<br />
Table 1(abstract P7) Comparison of fed-batch cultures<br />
FB seeded from batch<br />
FB seeded from perfusion<br />
Cell conc. at cell removal [c/mL] 2.2 × 10 6 2.3 × 10 7<br />
Split ratio 1:5 1:30<br />
Inoculum conc. [c/mL] 4.1 × 10 5 7.4 × 10 5<br />
Process time [d] 14 14<br />
Peak cell conc. [c/mL] 1.4 × 10 7 1.7 × 10 7<br />
Av. μ during growth phase [d -1 ] 0.44 0.52<br />
Inoculum propagated either in batch or perfusion culture
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the field of process monitoring there is still a need for innovative<br />
methods enabling improvement of process monitoring. For optimized<br />
process control it would be imperative to know as early as possible<br />
“when a cell needs what”, when it is stressed, running into substrate<br />
limitations etc., at best in an online or robust at line format.<br />
Intact cell MALDI mass spectrometry (ICM-MS) biotyping, a method used<br />
successfully in the field of clinical and environmental microbiology, is<br />
getting more attention in the context of mammalian cell cultivation. Here<br />
we report preliminary results of an assessment of a fast and high<br />
throughput at line capable ICM MS method for cell culture monitoring. As<br />
a first example, we choose apoptosis monitoring.<br />
The identification of specific mass spectrometric signatures related to<br />
early stages of apoptosis using ICM-MS biotyping as reported here could<br />
be a promising tool for CHO culture.<br />
Material and methods: An exponenti<strong>all</strong>y growing CHO suspension cell<br />
line was inoculated at a seeding density of 2 × 10 5 cells/ml and an initial<br />
volume of 30 ml in 125 ml Erlenmeyer flasks. Samples for assessing viabilityand<br />
apoptosis-progression and for ICM MS biotyping were taken at 48, 72,<br />
96,120,144,192and240h.Experimentswerecarriedoutasbiological<br />
triplicates.<br />
Viability was determined by trypan blue dye exclusion using a ViCell<br />
(Beckman Coulter, Krefeld, Germany) for automated processing. Apoptosis<br />
was measured in triplicate for each biological sample by means of caspase-9<br />
activity (Caspase-Glo®9 assay kit; Promega, Mannheim, Germany) using a<br />
microplate format (plate reader POLARstar Omega, BMG Labtech, Ortenberg,<br />
Germany).<br />
ICM MS biotyping (using a Bruker Autoflex III MALDI-TOF/TOF MS) analysis<br />
samples were prepared from as little as 2500 cells. The method is<br />
described in detail by Munteanu et al. (2012) [1].<br />
Results: To evaluate the power of ICM MS as an at-line analytical method<br />
for apoptosis monitoring, batch cultivations of CHO suspension cells were<br />
analyzed by standard analytical methods and ICM MS in comparison.<br />
Cell viabilities as assessed by trypan blue remained constant over 120 h of<br />
batch cultures. A first drop in cell viability was noticed between 120 and<br />
144 h (Figure 1 a).<br />
In ICM MS analysis, a total of approx. 160 m/z values was monitored in a<br />
mass to charge (m/z) range of 4,000 to 30,000. Principle component analysis<br />
(PCA; Figure 1 c) of ICM MS results showed no clear group discrimination<br />
during the first 96 h of cultivation. Interestingly, cell samples obtained from<br />
120 h of cultivation onwards appear as distinct groups in PCA analysis.<br />
Figure 1(abstract P8) Viability (a), caspase-9 activity (b) and ICM MS biotyping (c) during batch cultivation. FC RLU: Fold change of relative<br />
luminescence units; PC: Principal component of the respective analysis. (a) and (b): given are means of measurements of three experiments (i.e. n = 3) ±SD;<br />
(3): each dot represents one ICM MS measurement. Dashed lines illustrate at which point culture alteration is detectable with the respective method.
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Table 1(abstract P8) Details of classifying “unknown”<br />
samples using the CPT model<br />
“unknown”<br />
sample [h]<br />
Drop of<br />
viability [Y/N]<br />
Apoptosis<br />
detection [Y/N]<br />
48 N N Viable<br />
(no<br />
94<br />
72 N N<br />
96 N N<br />
Class PPV [%]<br />
apoptosis<br />
signal)<br />
120 N Y Early apoptotic<br />
83<br />
144 Y Y Late apoptotic<br />
100<br />
192 Y Y<br />
240 Y Y<br />
The concentration of the monitored apoptosis marker (caspase-9 activity;<br />
Figure 1 b) began to increase between 96 and 120 h, i.e. concomitantly<br />
with PCA analysis (Figure 1).<br />
As a result, ICM MS as reported here <strong>all</strong>owed for rapid detection of cell<br />
viability changes approx. 24 h earlier than standard culture monitoring<br />
and concomitant with the detection of an early, not “at-line” applicable<br />
apoptosis marker.<br />
Closer data analysis <strong>all</strong>owed the identification of an apoptosis related<br />
subset of m/z values. Using the software ClinProTools (CPT; Bruker<br />
Daltonik) it was possible to develop a classification model which points<br />
toward classification of unknown samples regarding their viability/<br />
apoptosis state (Table 1). The classification power was illustrated as<br />
positive predictive value (PPV) which is the number of correctly classified<br />
samples over the total number of classified samples. All biological samples<br />
were analyzed as 6-8 technical replicates, meaning in theory a PPV > 50%<br />
is sufficient for classification.<br />
Conclusion: We introduced a fast and robust ICM MS method for<br />
predictive cell culture monitoring. Viability changes can be detected up to<br />
24 h earlier compared to standard methods (e.g. trypan blue).<br />
We identified a specific MS signature (condensed subset of original<br />
spectra) of m/z values related to cell stress and apoptosis.<br />
A model built on the basis of this signature <strong>all</strong>ows classification of unknown<br />
samples regarding their viability/apoptosis level.<br />
These results will be substantiated by assessment of further cell lines as well<br />
as monitoring attributes other than cell stress/apoptosis (e.g. product titer or<br />
metabolite progression).<br />
Reference<br />
1. Munteanu B, von Reitzenstein C, Hänsch GM, Meyer B, Hopf C: Sensitive,<br />
robust and automated protein analysis of cell differentiation and of<br />
primary human blood cells by Intact cell MALDI mass spectrometry<br />
biotyping. Anal Bioanal Chem 2012, 408:2277-2286.<br />
P9<br />
Seed train optimization for suspension cell culture<br />
Tanja Hernández Rodríguez 1 , Ralf Pörtner 2 , Björn Frahm 3*<br />
1 Department of Mathematics, Bielefeld University, Bielefeld, D-33615,<br />
Germany;<br />
2 Institute of Bioprocess and Biosystems Engineering, Hamburg<br />
University of Technology, Hamburg, D-21073, Germany;<br />
3 Biotechnology &<br />
Bioprocess Engineering, Ostwestfalen-Lippe University of Applied Sciences,<br />
Lemgo, D-32657, Germany<br />
E-mail: bjoern.frahm@hs-owl.de<br />
BMC Proceedings 2013, 7(Suppl 6):P9<br />
Fields of application: Fields of application are the production of<br />
biopharmaceuticals (antibodies, proteins for diagnostic and therapeutic<br />
purposes) based on suspension cell culture and cultivation scales and<br />
-systems of any kind.<br />
Introduction: The purpose of a seed train is the generation of an<br />
adequate number of cells for the inoculation of a production bioreactor.<br />
This is time- and cost-intensive: From volumes used for cell thawing or cell<br />
line maintenance the cell number has to be increased. The cells are usu<strong>all</strong>y<br />
run through many cultivation systems which become larger with each<br />
passage (e.g. T-flasks, roller bottles or shake flasks, sm<strong>all</strong> scale bioreactor<br />
systems and subsequently larger bioreactors. Single-use systems may be<br />
applied and systems which are inoculated at a partly filled state and<br />
culture volume is increased afterwards by medium addition). The<br />
production bioreactor is inoculated out of the largest seed train scale.<br />
Motivation: A seed train offers space for optimization, e.g. choice of<br />
optimal points in time for cell passaging from one scale into the larger<br />
one. Furthermore choice of inoculation cell density as well as culture<br />
volume at inoculation in bioreactor scales (when inoculation volume is<br />
below maximum working volume). When designing a new seed train, the<br />
volumes of the cultivation scales may also be open for optimal choice.<br />
Results: Tool strucbture: A seed train structure has been programmed in<br />
Matlab®. The implemented model calculates cell growth, cell death, uptake<br />
of substrates and production of metabolites. The tool is suitable for<br />
different cell lines via entering corresponding model parameters, medium<br />
and seed train information. Seed train optimization is possible regarding<br />
cell passaging at optimal Space-Time-Yield (STY) or other optimization<br />
criteria [1].<br />
Application example for CHO cell line: Based on three cultivations, cell<br />
line model parameters have been determined using the simplex algorithm<br />
by Nelder and Mead. The whole seed train is modeled for cell passaging at<br />
fixed time intervals (current method, reference) and cell passaging at<br />
optimal points in time (optimized method). For this, the tool calculates<br />
Space-Time-Yield-(STY)-courses for every scale and selects the optima.<br />
As examples, Figure 1 shows an input mask of the seed train starting<br />
conditions as well as the courses of STY and viability over time during<br />
growth for flask scale 2:<br />
Figure 1 indicates that the reference method passages the cells in T-flasks<br />
and roller bottles when Space-Time-Yield (STY) is already decreasing and<br />
viability dropping which is too late (beginning of stationary phase, not<br />
presented).<br />
The whole optimized seed train is calculated including optimal points in<br />
time for cell passaging and optimal inoculation volumes and -densities in<br />
reactor scales. Table 1 gives an example of an output screen.<br />
In this example, time saving until inoculation of a 5,000 L production<br />
bioreactor is 108 hours. When the averages of point in time of optimal<br />
Space-Time-Yield (STY) and point in time of growth rate decreased to 90%<br />
are taken, time saving is 114 hours. This method also offers a ‘safety’ time<br />
span between cell passaging and beginning of stationary phase.<br />
Conclusions: The tool improves seed train understanding and <strong>all</strong>ows seed<br />
train design and optimization. Time savings as well as increased viabilities<br />
for passaging are possible. The tool has also been tested using a known<br />
and manu<strong>all</strong>y optimized seed train. Without such time consuming lab<br />
work, the tool has delivered the same optimized seed train only based on<br />
data of two batches [2].<br />
References<br />
1. Frahm B: Seed train optimization for cell culture. Animal Cell<br />
Biotechnology-Methods and Protocols Springer/Humana Press, in print:<br />
Pörtner R, 3.<br />
2. Kern S: Model-based design of the first steps of a seed train for cell<br />
culture processes. BMC Proceedings 2013, 7(6):P11.<br />
P10<br />
In vitro safety assessment of nanosilver with improved cell culture<br />
systems<br />
Alina Martirosyan * , Madeleine Polet, Yves-Jacques Schneider<br />
Laboratory of Cellular, Nutritional and Toxicological Biochemistry, Institute of<br />
Life Sciences & UCLouvain, Croix du Sud, L7.07.03, Louvain-la-Neuve, B1348,<br />
Belgium<br />
E-mail: alina_mart@list.ru<br />
BMC Proceedings 2013, 7(Suppl 6):P10<br />
Background: Silver nanoparticles (Ag-NPs) become increasingly<br />
prevailing in consumer products as antibacterial agents [1] and their<br />
potential threat on human health makes the risk assessment of utmost<br />
importance. In order to elucidate the complex interactions of Ag-NPs<br />
upon digestion in the gastrointestinal tract, an improved in vitro cell<br />
culture system was used. The model contained, beside the enterocytes,
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Page 24 of 151<br />
Figure 1(abstract P9) One input mask of the seed train starting conditions as an example for the tool’s user interface and courses of Space-<br />
Time-Yield (STY) and viability over time during growth for flask scale 2.<br />
specialized microfold (M) cells, able to increase the absorption of micro- and<br />
nanoparticles [2,3].<br />
In the current study, different aspects of the toxicity of Ag-NPs on the cell of<br />
intestinal epithelium were studied, i.e. cytotoxicity, inflammatory response<br />
and barrier integrity of the epithelial monolayer.<br />
Materials and methods: The cytotoxic effect of AgNPs < 20 nm (10-90<br />
μg/ml, Mercator GmbH, DE) was assessed by MTT assay on Caco-2 cells<br />
(clone 1, from Dr. M. Rescigno, University of Milano-Bicocca, IT). The<br />
co-culture model was received by co-culturing Caco-2 cells with RajiB cells<br />
(ATCC, Manassas, VA) in Transwell permeable supports (Corning Inc., NY)<br />
[1,2]. The inflammatory mediators chemokine IL-8 and nitric oxide (NO)<br />
levels were analysed in both apical (AP) and basolateral (BL) compartments<br />
by ELISA (BD Biosciences Pharmingen, San Diego, CA) and by Nitrate/Nitrite<br />
Colorimetric Assay Kit (Cayman Chemical Company, Ann Arbor, MI),<br />
respectively, according to the manufacturer’s instructions.<br />
The expression levels of the IL-8 and iNOS (inducible Nitric Oxide Synthase)<br />
genes were evaluated by quantitative real-time PCR (qRT-PCR), where the<br />
primers used were: for IL-8 CTGGCCGTGGCTCTCTTG (sense) and GGGT<br />
GGAAAGGTTTGGAGTATG (antisense) and for iNOS - TGTGCCACCTC<br />
CAGTCCAGT (sense) CTTATGGTGAAGTGTGTCTTGGAA (antisense). Levels of<br />
individual transcripts were normalized to those of glyceraldehyde-3-<br />
phosphate dehydrogenase (GAPDH). Relative quantification (RQ) values -<br />
fold change of the target gene expression compared to the untreated<br />
sample, were calculated by 2 -ΔΔCt method [4].<br />
The barrier integrity of the cell monolayers of mono- and co-cultures under<br />
the influence of AgNPs was evaluated on 21 days fully differentiated
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Table 1(abstract P9) Output screen example displaying the whole seed train including inoculation of production bioreactor (reactor scale 4,<br />
5,000 L).<br />
cultures in bicameral inserts by measuring the transepithelial electrical<br />
resistance and the passage of Lucifer Yellow.<br />
The immunofluorescence staining of two tight junctions (TJs) proteins, i.e.<br />
occludin and ZO-1 was realized by mouse anti-occludin/anti-ZO-1 as<br />
primary and Alexa Fluor 488 goat anti-mouse as secondary antibodies<br />
(Invitrogen). Images were collected by Zeiss LSM 710 confocal microscope.<br />
Results: Ag-NPs displayed a dose-dependent cytotoxic effect on Caco-2 cells<br />
starting from 30 μg/ml. The pro-inflammatory chemokine IL-8 levels were<br />
reduced under the influence of Ag-NPs (Figure 1a) in AP compartments in<br />
both mono- and co-cultures. In contrast, practic<strong>all</strong>y no changes in IL-8 levels<br />
were observed in the BL compartments. The ELISA analysis data were<br />
confirmed by qRT-PCR analysis, where the expression levels of the IL-8 gene<br />
showed a tendency to decrease in both mono- (fold change ≈ 0.86) and<br />
co-cultures (fold change ≈ 0.7) under the influence of Ag-NPs.<br />
NO content was increased in both AP and BL compartments in both monoand<br />
co-cultures (Figure 1b), although more marked in the latter case. In BL<br />
compartments, the NO levels increase was dependent on the Ag-NPs<br />
concentration. In contrast to IL-8, there were practic<strong>all</strong>y no changes<br />
observed in the iNOS gene expression levels in Caco-2 cells, indicating that<br />
Ag-NP-induced NO generation increase is likely independent of the iNOS<br />
gene expression.<br />
Immunostaining with confocal microscopy analysis of two TJs proteins, i.e.<br />
occludin and ZO-1, revealed that, in Ag-NP-treated cells, the continuity of<br />
both occludin and ZO-1 was disrupted as compared to control and the<br />
aggregationofbothproteinswasobserved. The Ag-NP-induced dashed<br />
and degraded distributions of occludin and ZO-1 suggest the opening of<br />
TJs (not illustrated). The opening of junctions was further confirmed by<br />
decreased TEER values and increased LY passage rates in Ag-NP-treated<br />
samples. These effects were less obvious in co-cultures, a more accurate<br />
model to reflect in vivo conditions, suggesting that the presence of<br />
M-cells seemingly decreases the toxicity of AgNPs.<br />
Conclusions: These results suggest that Ag-NPs: (i) are cytotoxic<br />
for intestinal epithelial cells; (ii) possess anti-inflammatory properties; and<br />
(iii) mediate the intestinal barrier function disruption. Differences in response<br />
to Ag-NPs were observed in mono- and co-cultures, where the NPs affected<br />
less obviously the IL-8 levels and barrier function in co-cultures, while, in<br />
contrast, led to more marked increase of NO concentration in comparison<br />
with mono-cultures. These differences demonstrate the advisability of<br />
application of more complex in vitro models and further need of<br />
improvement of the model by addition of e.g. mucus producing cells and/or<br />
dendritic cells that would provide a tool to achieve even more reliable and<br />
predictive correlations between in vitro studies and in vivo outcomes.<br />
Acknowledgements: This work was supported by a mobility grant of the<br />
Belgian Federal Science Policy Office (BELSPO) co-funded by the Marie<br />
Curie Actions from the European Commission.<br />
References<br />
1. Des Rieux A, Ragnarsson EG, Gullberg E, Preat V, Schneider Y-J, Artursson P:<br />
Transport of nanoparticles across an in vitro model of the human<br />
intestinal follicle associated epithelium. Eur J of Pharm Sci 2005, 25:455-465.<br />
2. Des Rieux A, Fievez V, Theate I, Mast J, Preat V, Schneider Y-J: An improved in<br />
vitro model of human intestinal follicle-associated epithelium to study<br />
nanoparticle transport by M cells. Eur J of Pharm Sci 2007, 30:380-391.<br />
Figure 1(abstract P10) IL-8 (a) and NO (b) levels in mono- and co-cultures in AP and BL compartments upon exposure to Ag-NPs (45 μg/ml).<br />
*samples significantly different from the corresponding control. Means of 3 independent experiments ± SD are given, P < 0.001.
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Page 26 of 151<br />
3. The Project on Emerging Nanotechnologies. [http://www.nanotechproject.<br />
org].<br />
4. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using<br />
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods<br />
2001, 25:402-408.<br />
P11<br />
Model-based design of the first steps of a seed train for cell culture<br />
processes<br />
Simon Kern 1,2 , Oscar B Platas 1 , Martin Schaletzky 1 , Volker Sandig 3 ,<br />
Björn Frahm 2 , Ralf Pörtner 1*<br />
1 Institute of Bioprocess and Biosystems Engineering, Hamburg University of<br />
Technology, Hamburg, D-21073, Germany;<br />
2 Biotechnology & Bioprocess<br />
Engineering, Ostwestfalen-Lippe University of Applied Sciences, Lemgo, D-32657,<br />
Germany;<br />
3 ProBioGen AG, Berlin, D-13086, Germany<br />
E-mail: poertner@tuhh.de<br />
BMC Proceedings 2013, 7(Suppl 6):P11<br />
Concept: Production of biopharmaceuticals for diagnostic and therapeutic<br />
applications with suspension cells in bioreactors requires a seed train up to<br />
production scale [1]. For the final process steps in pilot and production<br />
scale the scale-up steps are usu<strong>all</strong>y defined (e.g. a factor of 5 - 10). More<br />
difficult in this respect are the first steps, the transitions between T-flasks,<br />
spinnertubes,rollerbottles,shakeflasks,stirredbioreactorsorsingle-use<br />
reactors, because here often scale-up steps are different. The experimental<br />
effort to lay these steps out is correspondingly high. At the same time it is<br />
known that the first cultivation steps have a significant impact on the<br />
success or failure on production scale. The concept for a model based<br />
design of the seed train consists of the following steps:<br />
➢ A simple unstructured kinetic model, where kinetic parameters<br />
can be obtained from a few experiments only.<br />
➢ A Nelder-Mead-algorithm to determine model parameters.<br />
➢ A MATLAB simulation based on this model to determine optimal<br />
points in time or viable cell concentrations respectively for harvest of<br />
seed train scales from spinner tubes over shake flasks up to a stirred<br />
bioreactor based on an optimization criterion.<br />
Verification: The concept was verified for a suspendable cell line (AGE1.<br />
HN, ProBioGen AG) grown in serum-free 42-Max-UB medium (Teutocell<br />
AG, Germany) containing 5 mM-Glutamine.<br />
Two batch experiments were performed in shake flasks for determination<br />
of kinetic parameters.<br />
The average value of time for minimal and maximal Space-Time-Yield for<br />
cells was used as optimization criterion for cell transfer.<br />
The concept was tested successfully up to a 5 L scale for 6 scale-up steps<br />
(Figure 1).<br />
Conclusions: The concept offers a simple and inexpensive strategy for<br />
design of the first scale-up steps. The results show that the tool was able<br />
to perform a seed train optimization only on the basis of two batches, the<br />
underlying model and its parameter identification. This quick optimization<br />
led to the same results as the extensive manual optimization carried out in<br />
the past.<br />
Acknowledgements: The bioreactor (Labfors 5 Cell) was kindly provided<br />
by the company Infors AG, the cell line AGE1.HN by ProBioGen AG.<br />
Reference<br />
1. Eibl R, Eibl D, Pörtner R, Catapano G, Czermak P: Cell and Tissue Reaction<br />
Engineering. Springer 2008, ISBN 978-3-540-68175-5.<br />
P12<br />
Novel approaches to render stable producer cell lines viable for the<br />
commercial manufacturing of rAAV-based gene therapy vectors<br />
Verena V Emmerling 1* , Karlheinz Holzmann 3 , Karin Lanz 3 , Stefan Kochanek 2 ,<br />
Markus Hörer 1<br />
1 Rentschler Biotechnologie GmbH, Erwin-Rentschler-Straße 21, 88471<br />
Laupheim, Germany;<br />
2 Division of Gene Therapy, University of Ulm, Helmholtz<br />
Str. 8/1, 89081 Ulm, Germany;<br />
3 Department of Internal Medicine III, University<br />
Hospital of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany<br />
E-mail: Verena.Emmerling@rentschler.de<br />
BMC Proceedings 2013, 7(Suppl 6):P12<br />
Figure 1(abstract P11) Time course of simulated and experiment<strong>all</strong>y<br />
determined viable cell density and cell number during model based<br />
seed from culture tube to lab-scale-bioreactor. 1: culture tube (0.01 L);<br />
2: shake flask (0.035 L); 3: shake flask (0.13 L), 4: Vario 1000 (medorex, 0.35 L),<br />
5: VSF 2000 (Bioengineering, 1 L); 6: Labfors 5 Cell (Infors, 2.5 L).<br />
Background: Recombinant Adeno-associated virus (rAAV) based vectors<br />
recently emerged as very promising candidates for viral gene therapy due<br />
to a large toolbox available including twelvedifferentAAVserotypes,<br />
natural isolates, designer capsids and library technologies [2]. Furthermore,<br />
rAAV vectors have favourable properties such as non-pathogenicity of<br />
AAV, low B-/T-cell immunogenicity against transgenes delivered and longterm<br />
transgene expression from a non-integrating vector [5,9]. Promising<br />
data from clinical trials using rAAV-based vectors for the treatment of e.g.<br />
haemophilia or retinal diseases as well as the recent approval of the first<br />
gene therapy drug in the European Union, Glybera® to treat lipoprotein<br />
lipase deficiency, emphasise the potential of rAAV vectors for gene therapy<br />
approaches in a wide variety of indications [8,7,15]. Thereby, the demand<br />
for robust and cost-effective manufacturing of those vectors for market<br />
supply rose steadily. Standard production systems comprise transient<br />
transfection- and/or infection-based approaches using mammalian cells [3],<br />
or insect cells [16]. However, high production costs combined with<br />
considerable regulatory effort and safety concerns gave rise to the<br />
development of producer cell lines enabling stable rAAV production [3].<br />
AAVs are parvoviruses whose productive infection is depending on the<br />
presence of helper viruses like e.g. adenovirus (AdV). Their singlestranded<br />
DNA genome carries two genes. The rep gene encodes proteins<br />
responsible for site-specific integration, viral genome replication as well<br />
as packging. The cap gene is translated into three structural proteins<br />
building the capsid shelf. Furthermore, cap encodes a protein required<br />
for capsid assembly (AAP or assembly-activating protein) that has been<br />
described recently [13]. The AAV genes are flanked by inverted terminal<br />
repeat (ITR) sequences constituting the replication, integration and<br />
packaging signal. In a stable producer cell line with integral helper<br />
functions, <strong>all</strong> required genetic elements are stably integrated into the<br />
genome of the host cell as independent expression constructs: the<br />
recombinant vector implying a transgene flanked by AAV ITRs, the AAV<br />
genes rep and cap required for replication and encapsidation, as well as<br />
adenoviral helper function delivered by sequences encoding genes E1a,<br />
E1b, E2a, E4orf6 and viral associated (VA) I/II RNA [9]. In a timely regulated<br />
fashion, viral proteins are expressed and the AAV genome is replicated<br />
and encapsidated. As some of the gene products arising during rAAV<br />
production are toxic, an inducible expression of the gene products is<br />
indispensable for generation of stable production cells.<br />
The aim of the underlying study is to provide <strong>all</strong> tools necessary to generate<br />
a stable and versatile producer cell line In order to circumvent the problems<br />
triggered by toxic proteins inevitably arising during rAAV formation, one<br />
objective of the project is to establish stable producer cells where rAAV<br />
production can be induced by temperature shift at the final production<br />
scale. To begin with, we first performed some general feasability studies to
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Figure 1(abstract P12) (A) Transfection- and infection-based generation of rAAV in HeLa cells at different temperatures. Cells were transfected<br />
by calcium phosphate using three plasmids encoding the rAAV vector, rep and cap, followed by AdV5 infection and subsequent incubation of the cells<br />
at three different temperatures. Genomic rAAV titers were determined 96 h post infection as previously described [4]. (B) Investigation of rAAV production<br />
in a “transfection only approach” applying plasmids encoding rep, cap, vector as well as adenoviral E1 and remaining AdV helper functions. Different<br />
variants of rep and cap were compared regarding rAAV productivity. 1: Approach implying function<strong>all</strong>y separated rep and cap genes on different<br />
plasmids, which are devoid of rep78 expression and lack an artificial Rep Binding Site (RBS) in the pUC19 plasmid backbones [6] (standard plasmids used<br />
in <strong>all</strong> preceding experiments). 2: Same rep and cap plasmids but modified to avoid the expression of non-functional and truncated viral gene products by<br />
deletion of various promoter and potential transcription start sites. Genome titre was analyzed 120 h post infection as previously described [4].<br />
investigate whether the generation of stable and inducible producer cell<br />
lines using proprietary constructs is a viable approach. For this purpose,<br />
experiments for rAAV manufacturing based on a transient packaging<br />
approach were conducted. Infection of rep, cap and rAAV vector<br />
plasmid transfected cells with wildtype Adenovirus was compared with<br />
co-tranfection of the cells with additional plasmids carrying the Adenoviral<br />
helper genes. The influence of different cultivation temperatures on<br />
Adenovirus replication kinetics and rAAV productivity in the transient<br />
packaging approaches were analyzed. Furthermore, we investigated<br />
differential gene expression in response to temperature downshifts.<br />
Results: In the first experiments, a transfection-/infection-based approach<br />
was chosen to produce rAAV. For this, HeLa cells were co-transfected with<br />
three plasmids encoding the AAV vector on one side and the rep and cap<br />
genes delivered on two separate constructs on the other side (trans-split<br />
packaging system, [6]). Subsequently, cells were infected with a helper virus.<br />
Cultivation of cells at 32 °C post infection resulted in significantly increased<br />
rAAV titres compared to 37 °C (Figure 1A). This could arise from an arrest of<br />
cells in G 2 /M phase, causing enhanced growth but decreased proliferation.<br />
Hence, cells exhibit enlarged size and elevated protein production, possibly<br />
supported by avoided degradation of rDNA as previously described for CHO<br />
cells [14]. Repressed adenoviral replication kinetics may trigger prolonged<br />
cellular viability and, thereby, further increase rAAV titres. In fact these<br />
results also suggest that high copy numbers of helper genes are not<br />
essential for efficient rAAV packaging being an important prerequisite for<br />
the generation of efficient producer cells by stable integration of only few<br />
copies of the Adenoviral helper genes. Importantly, rAAV production was<br />
also possible replacing the adenovirus infection step by co-transfection of<br />
rep-, cap- and rAAV vector transfected HeLa cells with two more plasmids<br />
coding for <strong>all</strong> known adenoviral helper genes. Considering that in such an<br />
approach cells have to be co-transfected by five different plasmids at the<br />
same time in order to produce rAAV, the yiels obtained in this “transfection<br />
only approach” were quite promising. Over<strong>all</strong> rAAV yields generated with<br />
the rep/cap trans-split packaging system [6] could be further increased by<br />
modifications of the rep and cap coding sequences in terms of avoidance of<br />
production of non-functional byproducts (Figure 1B).<br />
Differential gene expression analysis of HeLa cells cultivated at different<br />
temperatures gave rise to the identification of three genes up-regulated<br />
up to 7-fold and 16 miRNAs likely regulated more than 2-fold at lowered<br />
temperature (Table 1). Underlying genetic switches are subject to further<br />
investigations. Appropriate temperature-inducible switches will be used to<br />
control expression of the adenoviral helper gene E1a, thekeyinducerof<br />
thewholecascaderequiredforrAAV production. Applied in stable<br />
producer cells, such a system would <strong>all</strong>ow for timely-regulated induction<br />
of rAAV production. Making use of a temperature shift as primary switch<br />
for rAAV production, we would combine the inevitable induction event<br />
with conditions presumably enhancing rAAV production.<br />
Conclusions: Taken together, these first data provide the basis for a<br />
successful generation of temperature inducible stable producer cells<br />
Table 1(abstract P12) Analysis of differential gene expression in HeLa triggered by different cultivation temperatures<br />
Name Differential expression at Mode of regulation Microarray analysis RT qPCR<br />
Gene A 30°C Up 3.2-fold 6.9-fold<br />
Gene B 30°C Up 2.2-fold 2.6-fold<br />
Gene C 30°C Up 3.3-fold 2.3-fold<br />
miRNA A 32°C Up 3.1-fold -<br />
miRNA B 32°C Down 3.3-fold -<br />
miRNA C 32°C Up 3.0-fold -<br />
Cells were seeded at two different densities and cultivated at 37°C for two days. Subsequently, cells were incubated for another 6 hours at 30, 32, and 37°C,<br />
respectively, before mRNA was isolated from the cells. Microarray analysis (GeneChip® Human Exon 1.0 ST Array, Affymetrics) was performed to identify mRNAs<br />
differenti<strong>all</strong>y expressed more than 2-fold. Validation was done by RT qPCR analysis (EvaGreen® Mastermix, Biorad) and included controls of regulated and nonregulated<br />
mRNAs [12,11,1]. Differenti<strong>all</strong>y expressed miRNAs (>2-fold) were also identified by microarray analysis (GeneChip® miRNA 2.0 Array, Affymetrics). As<br />
validation is not yet completed, only an excerpt of the most promising miRNA candidates is shown.
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carrying <strong>all</strong> genetic elements required for rAAV production. A versatile<br />
and high-titre rAAV production platform based on such cells will be<br />
applicable for industrial-scale manufacturing and thus has the potential to<br />
open AAV-based gene therapy to a high number of patients.<br />
References<br />
1. Ars E, Serra E, de la Luna S, Estivill X, Lázaro C: Cold shock induces the<br />
insertion of a cryptic exon in the neurofibromatosis type 1 (NF1) mRNA.<br />
Nucl Acids Res 2000, 28(6):1307-1312.<br />
2. Asokan A, Schaffer D, Samulski JR: The AAV Vector Toolkit: Poised at the<br />
Clinical Crossroads. Mol Ther 2012, 20(4):699-708.<br />
3. Aucoin MG, Perrier M, Kamen AA: Critical assessment of current adenoassociated<br />
viral vector production and quantification methods.<br />
Biotechnol Adv 2008, 26(1):73-88.<br />
4. Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I,<br />
Nitschko H, Busch U, Sing A, Ehrhardt A, Baiker A: Universal real-time PCR<br />
for the detection and quantification of adeno-associated virus serotype<br />
2-derived inverted terminal repeats. Hum Gene Ther Methods 2012,<br />
23(1):18-28.<br />
5. Ayuso E, Mingozzi F, Bosch F: Production, purification and<br />
characterization of adeno-associated vectors. Curr Gene Ther 2012,<br />
10(6):423-436.<br />
6. Bertran J, Moebius U, Hörer M, Rehberger B: Host cells for packaging a<br />
recombinant adeno-associated virus (RAAV), method for the production<br />
and use thereof. World Intellectual Property Organization 2002, WO 02/<br />
20748 A2.<br />
7. Hauswirth WW, Aleman TS, Kaushal S, Cideciyan AV, Schwartz SB, Wang L,<br />
Conlon TJ, Boye SL, Flotte TR, Byrne BJ, Jacobson SG: Treatment of leber<br />
congenital amaurosis due to RPE65 mutations by ocular subretinal<br />
injection of adeno-associated virus gene vector: short-term results of a<br />
phase I trial. Hum Gene Ther 2008, 19:979-990.<br />
8. Manno CS, Chew AJ, Hutchison S, Larson PJ, Herzog RW, Arruda VR, Tai SJ,<br />
Ragni MV, Thompson A, Ozelo M, Couto LB, Leonard DG, Johnson FA,<br />
McClelland A, Sc<strong>all</strong>an C, Skarsgard E, Flake AW, Kay MA, High KA, Glader B:<br />
AAV-mediated factor IX gene transfer to skeletal muscle in patients with<br />
severe hemophilia B. Blood 2003, 101(8):2963-2972.<br />
9. Matsushita T, Okada T, Inaba T, Mizukami H, Ozawa K, Colosi P: The<br />
adenovirus E1A and E1B19K genes provide a helper function for<br />
transfection-based adeno-associated virus vector production. J Gen Virol<br />
2004, 85(8):2209-2214.<br />
10. Mingozzi F, High KA: Therapeutic in vivo gene transfer for genetic<br />
disease using AAV: progress and ch<strong>all</strong>enges. Nat Rev Genet 2011,<br />
12(5):341-355.<br />
11. Nishiyama H, Higashitsuji H, Yokoi H, Itoh K, Danno S, Matsuda T, Fujita J:<br />
Cloning and characterization of human CIRP (cold-inducible RNAbinding<br />
protein) cDNA and chromosomal assignment of the gene. Gene 1997,<br />
204:115-120.<br />
12. Sonna LA, Fujita J, Gaffin SL, Lilly CM: Molecular biology of<br />
thermoregulation invited review: Effects of heat and cold stress on<br />
mammalian gene expression. J Appl Physiol 2002, 92:1725-1742.<br />
13. Sonntag F, Schmidt K, Kleinschmidt JA: A viral assembly factor promotes<br />
AAV2 capsid formation in the nucleolus. Proc Natl Acad Sci USA 2010,<br />
107(22):10220-10225.<br />
14. Tait AS, Brown CJ, Galbraith DJ, Hines MJ, Hoare M, Birch JR, James DC:<br />
Transient production of recombinant proteins by chinese hamster ovary<br />
cells using polyethyleneimine/DNA complexes in combination with<br />
microtubule disrupting anti-mitotic agents. Biotechnol Bioeng 2004,<br />
88(6):707-721.<br />
15. UniQure BV:[http://www.uniqure.com/news/167/189/uniQure-s-Glybera-First-<br />
Gene-Therapy-Approved-by-European-Commission.html].<br />
16. Urabe M, Ding C, Kotin RM: Insect cells as a factory to produce adenoassociated<br />
virus type 2 vectors. Hum Gen Ther 2002, 13:1935-1943.<br />
P13<br />
Benchmarking of commerci<strong>all</strong>y available CHO cell culture media for<br />
antibody production<br />
David Reinhart 1* , Christian Kaisermayer 2 , Lukas Damjanovic 1 , Renate Kunert 1<br />
1 Dept. of Biotechnology, University of Natural Resources and Life Sciences,<br />
Muthgasse 11, 1190 Vienna, Austria;<br />
2 GE Healthcare Life Sciences AB,<br />
Björkgatan 30, 75184 Uppsala, Sweden<br />
E-mail: david.reinhart@boku.ac.at<br />
BMC Proceedings 2013, 7(Suppl 6):P13<br />
Introduction: Chinese hamster ovary (CHO) cells have become the<br />
preferred expression system for the production of complex recombinant<br />
proteins. Several suppliers offer CHO specific cell cultivation media and<br />
sometimes also media systems, which combine feeds and basal medium.<br />
We compared eight commerci<strong>all</strong>y available CHO cell culture media and feed<br />
supplements from three different vendors to evaluate their influence on cell<br />
growth and antibody production of a CHO cell line. In conclusion, ActiCHO<br />
Media System, with a matching base media and feeds, resulted in the<br />
highest cell growth and the highest productivity. Further nutrient additions<br />
did not have a profound effect on the process performance.<br />
Materials and methods: Cultivation media:<br />
ActiCHO P (GE Healthcare)<br />
CD CHO (Life Technologies)<br />
CD OptiCHO (Life Technologies)<br />
CD FortiCHO (Life Technologies)<br />
Ex-Cell CD CHO (Sigma Aldrich)<br />
ProCHO 5 (Lonza)<br />
BalanCD CHO Growth A (Irvine Scientific)<br />
Cellvento CHO-100 (EMD Millipore)<br />
• Anti-Clumping Agent (Life Technologies)<br />
• CHO DG44 cells expressing an IgG antibody<br />
• Cultivation conditions: 37°C, 7% CO 2 , 140 rpm<br />
• Batch and fed-batch cultivations were run in Erlenmeyer shake flasks<br />
(Corning, NY). The cultures were grown in a CO 2 incubator shaker<br />
(Kühner, Switzerland)<br />
• Batch cultures were run as single experiments, the method<br />
variability was determined by a triplicate reference experiment in<br />
ActiCHO P.<br />
• During fed-batch processes the cultures were fed with the<br />
corresponding feeds ActiCHO Feed A and Feed B (GE Healthcare),<br />
BalanCD CHO Feed 1 (Irvine Scientific) or EfficientFeed Aand/or<br />
FunctionMAX (both Life Technologies) according to the manufacturers<br />
inctructions [1]. The respective feeding regimens are shown in Table 1.<br />
• Fed-batch cultures were run in triplicates. The residual glucose<br />
concentration was maintained above 3 g/L by addition of glucose<br />
concentrate<br />
• Analytics: cell concentration, viability, selected metabolites, product<br />
concentration, amino acid concentrations<br />
Results and discussion: In batch cultures the highest cell concentrations<br />
were obtained in ActiCHO P and BalanCD as shown in Figure 1. In ActiCHO P<br />
the cells initi<strong>all</strong>y grew with a slightly higher specific growth rate (data not<br />
shown) and therefore the maximum cell concentration was reached 3 days<br />
earlier than in BalanCD. In ProCHO 5, Cellvento CHO-100 and CD OptiCHO,<br />
cell concentrations of 4 × 10 6 to 5 × 10 6 cells/mL were reached. Although<br />
initi<strong>all</strong>y the growth was similar in <strong>all</strong> three media, the culture in ProCHO<br />
5 was terminated on day 7 due to a viability below 60%. In the other two<br />
media the batch lasted for four days longer. In Ex-Cell CD CHO cells grew to<br />
2.6 × 10 6 cells/mL which was about 30% of the cell concentration reached in<br />
ActiCHO P. Fin<strong>all</strong>y in CD CHO and CD FortiCHO cells formed sm<strong>all</strong><br />
aggregates and rather low concentrations of 2.5 × 10 6 and 6.0 × 10 5 cells/<br />
mL were obtained, respectively. Cell adaptation in CD FortiCHO during seven<br />
passages and addition of Anti-Clumping Agent (1:250) did not resolve the<br />
aggregation problem or improve cell growth (data not shown). The antibody<br />
production in the different cultures followed the same ranking as the cell<br />
growth (Figure 1). The highest titers were achieved in ActiCHO P and<br />
BalanCDCHO.InCDOptiCHO,Ex-Cell CD CHO and Cellvento CHO-100<br />
product concentrations of about 500 mg/L were reached. The lowest titers<br />
were generated in ProCHO 5 and CD CHO with 380 mg/L and 330 mg/L,<br />
respectively. Fed-batch cultivations were then run in selected basal media<br />
with the respective feeds according to table 1. Again there was a strong<br />
correlation between cell concentration and antibody production. The highest<br />
cell and product concentrations were obtained in ActiCHO P (Table 1).<br />
Compared with the previous batch cultures, the cell concentrations were<br />
more than doubled and due to the extended process duration the titer<br />
was increased more than 6 fold, as shown in table 1. Feeding cultures in<br />
ActiCHO P with Feed A and B alone or addition<strong>all</strong>y with FunctionMAX,<br />
altered the process only margin<strong>all</strong>y. Supplementing the fed-batch only<br />
with ActiCHO Feeds A&B resulted in slightly higher cell concentrations and<br />
the process duration was reduced by 2 days (data not shown). A fed-batch<br />
culture in BalanCD medium and Feed 1 reached only 80% of the cell<br />
concentration achieved during the previous batch culture, however,
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Table 1(abstract P13) Feeding regimens in fed-batch cultures<br />
Basal medium ActiCHO Feed A ActiCHO Feed B EfficientFeed A FunctionMAX Feed 1 Peak cell conc.<br />
[10 6 c/ml]<br />
Harvest Titer<br />
[g/L]<br />
ActiCHO P daily; 3% daily; 0.3% - - 23.9 5.48<br />
ActiCHO P daily; 3% daily; 0.3% - 3, 5, 7; 3.3% 21.3 5.82<br />
CD OptiCHO - - 3, 5, 7, 9; 10% - 5.8 0.72<br />
CD OptiCHO - - 3, 5, 7; 10% - 5.2 0.80<br />
CD OptiCHO - - 3, 5, 7; 10% 3, 5, 7; 3.3% 6.3 1.74<br />
CD OptiCHO daily; 3% daily; 0.3% - - 9.0 1.46<br />
BalanCD CHO - - - - 1, 3, 5; 10% 7.1 1.30<br />
The time [d] for feed addition and the feed volume in % of the culture volume are indicated. Feed start for the culture in BalanCD CHO was day 1, <strong>all</strong> other<br />
cultures were fed from day 3 on. Values for peak cell concentration and harvest titer are mean values of triplicate experiments.<br />
feeding extended the process by five days and increased the antibody<br />
concentration by 60% compared with the previous batch culture to a final<br />
titer of 1.3 g/L (Table 1). Fed-batch cultures in CD OptiCHO achieved about<br />
40% of the cell concentrations in ActiCHO P. Similar cell concentrations<br />
were reached when feeding cultures in CD OptiCHO with ActiCHO feeds<br />
A and B or EfficientFeed A, independent if the feed was added during 7 or<br />
9 days or if additional feeding with FunctionMAX was performed (Table 1).<br />
However, the feeding had an impact on the product concentration.<br />
The lowest one was obtained when feeding cultures in CD OptiCHO with<br />
EfficientFeed A only. Further supplementation with FunctionMAX or<br />
feeding with ActiCHO Feed A&B substanti<strong>all</strong>y increased the product<br />
concentration (Table 1).<br />
Conclusions: • Batch cultivation in the different media resulted in peak<br />
cell concentrations from 2.5 × 10 6 to 9.0 × 10 6 cells/mL and a<br />
corresponding antibody titer from 220 to 860 mg/L. ActiCHO P and<br />
BalanCD CHO performed best in these cultures.<br />
Figure 1(abstract P13) Cell concentrations (upper panel) and product concentrations (lower panel) obtained in batch experiments with<br />
different commerci<strong>all</strong>y available CHO cell culture media. Titers in CD FortiCHO were not determined due to low cell concentrations. Error bars are<br />
one standard deviation.
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• Fed-batch cultivations substanti<strong>all</strong>y improved cell and product<br />
concentration. Feeding cultures in CD OptiCHO with EfficientFeed A<br />
and FunctionMAX or with Feed A and Feed B resulted in similar<br />
antibody concentrations and roughly doubled the antibody<br />
production compared to feeding with EfficientFeed A only.<br />
• The highest titer was achieved in ActiCHO P in combination with Feed<br />
A and Feed B. In this medium a 6.3-fold improvement, compared with<br />
the previous batch cultivation, was observed. Further addition of<br />
FunctionMAX to these cultures did not significantly improve the<br />
antibody production.<br />
Reference<br />
1. Barrett S, Boniface R, Dhulipala P, Slade P, Tennico Y, Stramaglia M, Lio P,<br />
Gorfien S: Attaining Next-Level Titers in CHO Fed-Batch Cultures.<br />
BioProcess International 2012, 10:56-62.<br />
P14<br />
Advanced off-gas measurement using proton transfer reaction mass<br />
spectrometry to predict cell culture parameters<br />
Timo Schmidberger 1,2* , Robert Huber 2<br />
1 Department of biotechnology, University of Natural Resources and Life<br />
Sciences, 1180 Vienna, Austria;<br />
2 Sandoz GmbH BU Biopharmaceuticals, 6336<br />
Langkampfen, Austria<br />
E-mail: timo.schmidberger@sandoz.com<br />
BMC Proceedings 2013, 7(Suppl 6):P14<br />
Background: Mass spectrometry is a well-known technology to detect O 2<br />
and CO 2 in the off-gas of cell culture fermentations. In contrast to classical<br />
mass spectrometers, the proton transfer reaction mass spectrometer (PTR<br />
MS) enables the noninvasive analysis of a broad spectrum of volatile<br />
organic compounds (VOCs) in real time. The thereby applied soft<br />
ionization technology generates spectra of less fragmentation and<br />
facilitates their interpretation. This gave us the possibility to identify<br />
process relevant compounds in the bioreactor off-gas stream in addition to<br />
O 2 and CO 2 . In our study the PTR-MS technology was applied for the first<br />
time to monitor volatile organic compounds (VOC) and to predict cell<br />
culture parameters in an industrial mammalian cell culture process.<br />
Materials and methods: The aptitude of PTR MS for advanced bioprocess<br />
monitoring was assessed by Chinese hamster ovary (CHO) cell culture<br />
processes producing a recombinant protein conducted in a modified 7L<br />
glass bioreactor (Applikon, Shiedam, Netherlands). The PTR MS-hs (Ionicon,<br />
Innsbruck, Austria) was equipped with a QMS422 quadrupole for mass<br />
separation and with a secondary electron multiplier detector to measure<br />
masses ranging from 18 to 200 m/z. The equipment set-up is illustrated in<br />
Figure 1. On a daily basis the glutamine concentration was determined<br />
with the BioProfile 100 plus (Nova Biomedical, Waltham, MA) and the<br />
viable cell density (VCD) was measured with the Vi-Cell XR cell counter<br />
(Beckham Coulter, Fullerton, CA). Samples for the product quantification<br />
were pulled daily and analyzed once at the end of a fermentation using<br />
affinity liquid chromatography. The PTR-MS data was first filtered with an<br />
adaptive online repeated median filter [1] and then correlated to the cell<br />
culture parameters with partial least square regression (PLS-R) using the<br />
software SIMCA P12+ (Umetrics, Umea, Sweden).<br />
Results: The applicability of the PTR-MS technique was studied using eight<br />
different fermentations conducted during process optimization to determine<br />
key cell culture parameters such as viable cell density, product titer and<br />
glutamine by partial least square regression models. Probably the most<br />
important parameter in industrial cell culture processes is the viable cell<br />
density. The R² of the PLS-R model for the VCD was 0.86 and hence, lower<br />
compared to other methods found in literature (such as 2D fluorescence<br />
[2]). Especi<strong>all</strong>y low values, which were observed only in the first few days of<br />
the fermentation, showed a high prediction error. At the beginning of the<br />
fermentation the VOC composition in the off-gas is characterized by VOCs<br />
from the media preparation (probably impurities of the raw materials used)<br />
and only a few VOC can be assigned to the cells. The media was prepared<br />
up to one week before the fermentations started and, depending on the<br />
storage time, the initial VOC content varied. Within the first days the media<br />
assigned VOCs were washed out and the cells started to produce VOCs.<br />
Accordingly the effect of the initial condition was weaker and prediction got<br />
better. In a second PLS-R model the product concentration was estimated<br />
based on the PTR-MS data. The model was better compared to the<br />
estimation of the VCD what is reflected in a R² of 0.94. The effect of the early<br />
Figure 1(abstract P14) Experimental set-up to monitor VOCs in<br />
mammalian cell culture.<br />
process phase on the prediction quality is not very distinct since almost no<br />
product was produced in the first days. The good model for the titer is a hint<br />
that producing the product is correlated with metabolic pathways involving<br />
VOCs. However distinct metabolic pathways could not be revealed within<br />
this study, since only a few VOC could be assigned to specific compounds<br />
yet. The third parameter assessed in this study was the glutamine<br />
concentration. The PLS-R model for glutamine concentration showed the<br />
lowest R² and Q² of this evaluation. Glutamine was added on demand and<br />
probably feeding corrupted the correlation. To overcome this problem, the<br />
glutamine related physiological parameter specific glutamine uptake (qGln)<br />
was used. The descriptive as well as the predictive power was higher when<br />
the specific consumption instead of the glutamine concentration was used<br />
(0.91 and 0.82). An explanation for this result is that the consumption of<br />
glutamine might be correlated to other metabolic pathways which can<br />
produce VOCs. In combination with an accurate online VCD measurement,<br />
the qGln can be used to estimate the over<strong>all</strong> glutamine demand of the<br />
culture in real-time. A summary of <strong>all</strong> PLS-R models is given in Table 1.<br />
Conclusions: In our study we showed that the VOC profile obtained with<br />
the PTR-MS can be used to predict important cell culture parameters, but<br />
compared to other on-line techniques such as near infrared spectroscopy<br />
the PLS-R models are currently less robust (expressed by a lower R²).<br />
Moreover the most important VOCs in the PLS-R model could be used to<br />
get deeper insights into the cellular metabolism. At the moment however,<br />
this is limited by the lack of identified VOCs and the sm<strong>all</strong> literature basis<br />
reporting of pathways including volatile metabolites. Fin<strong>all</strong>y, further<br />
experiments will be necessary to assess the most influential factors on the<br />
VOC production and to fully exploit the potential of the PTR-MS.<br />
Table 1(abstract P14) Summary PLS-R models<br />
Compound R² Q²<br />
VCD 0.86 0.76<br />
Product titer 0.94 0.88<br />
Glutamine 0.83 0.62<br />
Specific glutamine uptake 0.91 0.82
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Acknowledgements: We want to thank Rene Gutmann from Ionicon for<br />
the inst<strong>all</strong>ation and support for the PTR-MS and Karl Bayer for the fruitful<br />
discussions.<br />
References<br />
1. Schettlinger K, Fried R, Gather U: Real-time signal processing by adaptive<br />
repeated median filters. Int J Adapt Control 2009, 24:346-362.<br />
2. Teixeira AP, Portugal CA, Carinhas N, Dias JM, Crespo JP, Alves PM,<br />
Carrondo MJ, Oliveira R: In situ 2D fluorometry and chemometric<br />
monitoring of mammalian cell cultures. Biotechnol Bioeng 2009,<br />
102:1098-1106.<br />
P15<br />
New peptide-based and animal-free coatings for animal cell culture in<br />
bioreactors<br />
Youlia Serikova 1* , Aurélie Joly 1 , Géraldine Nollevaux 1 , Martin Bousmanne 2 ,<br />
Wafa Moussa 2 , Jonathan Goffinet 3 , Jean-Christophe Drugmand 3 ,<br />
Laurent Jeannin 2 , Yves-Jacques Schneider 1<br />
1 Laboratory of Cellular, Nutritional and Toxicological Biochemistry, Institute of<br />
Life Sciences, UCLouvain, 1348 Louvain-la-Neuve, Belgium;<br />
2 Peptisyntha sa,<br />
1120 Brussels, Belgium;<br />
3 ATMI, 1120 Brussels, Belgium<br />
E-mail: youlia.serikova@uclouvain.be<br />
BMC Proceedings 2013, 7(Suppl 6):P15<br />
Background: Anchorage dependent cells require an appropriate<br />
extracellular matrix for their survival, migration, proliferation, phenotyping<br />
and/or differentiation [1-3]. These cells interact with extracellular matrix<br />
proteins, primarily through integrins, which induces focal adhesion<br />
contacts assembly and activation of sign<strong>all</strong>ing pathways thatregulate<br />
diverse cellular processes [4].<br />
Culture supports usu<strong>all</strong>y include biochemical components <strong>all</strong>owing such<br />
cells to adhere and to reconstitute an extracellular environment close to<br />
that found in vivo. Currently, this artificial environment is achieved by<br />
extracellular matrix constituents deposition, adsorption or grafting; among<br />
them collagens, fibronectin, laminin, artificial lamina propria... [5]. However,<br />
such animal proteins used in cell culture may induce pro-inflammatory<br />
stress, be unstable against proteolysis or loose activity after adsorption<br />
[6,7]. Synthetic microenvironments should be more suitable for clinical<br />
purposes: (i) improved control of physicochemical and mechanical<br />
properties, (ii) limited risks of immunogenicity, (iii) increased biosafety<br />
(animal free) and (iv) facilitated scale-up [1].<br />
In this framework, research has recently focused on synthetic peptides or<br />
peptidomimetics that can mimic the extracellular matrix. Such molecules<br />
can be immobilized as recognition motifs on the surface of culture<br />
supports with a greater stability and easier surfaces characterization [5].<br />
Self-assembling peptide hydrogels could mimic the chemical and<br />
mechanical aspects of the natural extracellular matrix [8,9] by undergoing<br />
large deformations, as in mammalian tissues. They have an inherent<br />
biocompatibility and should be able to direct cell behaviour [10]. They also<br />
can be functionalized with various biologic<strong>all</strong>y active ligands constituting<br />
good candidates to a new range of smart biomaterials, able to ensure<br />
adhesion of different cell types [11-13].<br />
The range of biomimetic peptides that direct cell adhesion and are<br />
recognized by integrins is large. Recognition sequences derived from<br />
different extracellular matrix proteins include RGD [1], which are specific to<br />
different cell lines [1,5,6].<br />
In this context, this work aims at designing animal-free, chemic<strong>all</strong>y defined<br />
and industri<strong>all</strong>y scalable coatings for animal cell culture, as an alternative<br />
to collagen, fibronectin or Matrigel® for laboratory and industrial large<br />
scale applications. These are based on self-assembling short peptides<br />
bearing adhesion bioactive sequences like RGD-derived or other adhesion<br />
sequences developed to coat polystyrene or polyethylene terephthalate<br />
surfaces. Adhesion sequences should be recognized by cells, which should<br />
favour their anchorage and spreading.<br />
Experimental: Bioactive self-assembling peptide sequences were<br />
synthesized in liquid phase, purified, analytic<strong>all</strong>y characterised and<br />
manufactured by Peptisyntha (Brussels, Be) in GMP conditions, as sterile<br />
coating solutions. They were used to coat polystyrene flasks (Corning Inc.,<br />
NY) in comparison with animal-derived coatings i.e. collagen and fibronectin.<br />
Human Adipose Derived Stem Cells (hADSC) were purchased from Lonza<br />
(Verviers, Be); Caco-2, MRC-5 and CHO cells, obtained from ATCC.<br />
Cells were seeded at 8 000 cells/cm 2 and cultured until 7 days. After 60 h<br />
or 7 days of culture, cells were harvested and counted on Bürker cell in<br />
Trypan blue or fixed. Nuclei were then stained with DAPI and actin<br />
filaments with Rhodamin-Ph<strong>all</strong>oidin. Fluorescence microscopy was used<br />
to observe cell morphology and NIS software <strong>all</strong>owed cell-spreading<br />
determination.<br />
Results and discussion: The absence of cytotoxicity was assayed with<br />
necrosis (LDH) and cell metabolic activity (MTT) tests on different cell<br />
lines (Caco-2, MRC-5, CHO, hADSC). No cytotoxicity was detected.<br />
Two variants of bioactive self-assembling peptides, both containing RGDderived<br />
sequences, were compared with animal-derived coatings (collagen<br />
and fibronectin) in serum-poor of free medium. Cytocompatibility and<br />
dose dependent response studies revealed that peptides promote cell<br />
adhesion and growth.<br />
As for hADSC culture, these cells were first incubated in a serum-free<br />
medium during 6 to 24 h and the proportion of adherent cells and their<br />
spreading was evaluated. hADSC cells needed more than 6 h to fully<br />
adhere to the culture surface and the adhesion effectiveness appeared<br />
better for collagen and the first variant of peptide than for the other<br />
substrate coatings. Initial spreading was more marked on fibronectin, but<br />
then increased from 6 to 24 hours on <strong>all</strong> coatings.<br />
A second experiment consisted in a first cell incubation in DMEM<br />
supplemented with 1% Fetal Bovine Serum (FBS) and, after 24 h, the<br />
medium was replaced by DMEM supplemented with 10% FBS. After 7 days,<br />
the best cell growth was observed for substrates coated with collagen and<br />
peptide 1, fibronectin and peptide 2 being slightly less efficient. In par<strong>all</strong>el,<br />
cell spreading decreased or remained constant upon cell proliferation<br />
(Figure 1).<br />
As for Caco-2 cells culture, these cells were incubated in a serum-free,<br />
hormono-defined medium (BDM) during 6 to 24 h and the proportion of<br />
adherent cells and their spreading were evaluated. These cells required a<br />
shorter duration than hADSC to adhere on the surface and the adhesion<br />
effectiveness appeared a little bit better for collagen and fibronectin.<br />
Initial spreading was more marked on collagen and its importance varies<br />
between 6 and 24 h on different coatings.<br />
The second experiment consisted in a first cell incubation in a serum-free<br />
medium and, after 24 h, the nutritive medium was replaced by a medium<br />
supplemented with 1% FBS. After 60 h, there was almost no difference<br />
between the different coatings. Nevertheless, after 7 days, cells cultured<br />
on peptides reached the same effectiveness as on fibronectin, but slightly<br />
lower than collagen. As for hADSC, cell spreading decreased upon cells<br />
proliferation.<br />
Conclusion: Designed self-assembling bioactive peptides are not cytotoxic<br />
and are cytocompatible. Cell adhesion and growth on peptide coatings<br />
appear as effective as on animal-derived coatings and the peptide<br />
coatings <strong>all</strong>ow easy cell harvesting after culture.<br />
Glob<strong>all</strong>y, the results indicate that self-assembling bioactive peptides<br />
constitute chemic<strong>all</strong>y defined, entirely synthetic and effective promoters of<br />
cell adhesion, spreading and proliferation.<br />
Acknowledgements: This work is supported by Innoviris (Brussels<br />
Region) in the scope of a Doctiris PhD grant.<br />
References<br />
1. Petrie TA, Garcia AJ: Extracellular Matrix-derived Ligand for Selective<br />
Integrin Binding to Control Cell Function. Biol Interact Mater Surf 2009,<br />
1:133-156.<br />
2. Hynes RO: The Extracellular Matrix: Not Just Pretty Fibrils. Sci 2009,<br />
326:1216-1219.<br />
3. Rahmany MB, Van Dyke M: Biomimetic approaches to modulate cellular<br />
adhesion in biomaterials: A review. Acta Biomater 2013, 9:5431-5437.<br />
4. Badami AS, Kreke MR, Thompson MS, Riffle JS, Goldstein AS: Effect of fiber<br />
diameter on spreading, proliferation, and differentiation of osteoblastic<br />
cells on electrospun poly(lactic acid) substrates. Biomater 2006,<br />
27:596-606.<br />
5. Shin H, Jo S, Mikos AG: Biomimetic materials for tissue engineering.<br />
Biomater 2003, 24:4353-4364.<br />
6. Hersel U, Dahmen C, Kessler H: RGD modified polymers: biomaterials for<br />
stimulated cell adhesion and beyond. Biomater 2003, 24:4385-4415.<br />
7. Lin CC, Metters AT: Hydrogels in controlled release formulations: Network<br />
design and mathematical modeling. Adv Drug Deliv Rev 2006, 58:1379-1408.<br />
8. Wu EC, Zhang S, Hauser CAE: Self-Assembling Peptides as Cell-Interactive<br />
Scaffolds. Adv Funct Mater 2012, 22:456-468.<br />
9. Hamilton SK, Lu H, Temenoff JS: Micropatterned Hydrogels for Stem Cell<br />
Culture. Stud Mechanobiol Tissue Eng Biomater 2010, 2:119-152.
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Page 32 of 151<br />
Figure 1(abstract P15) Fluorescence micrographies of hADSC cultivated for 7 days on polystyrene substrates. After incubation and cell fixation,<br />
nuclei were stained with DAPI and actin filaments with rhodamin-ph<strong>all</strong>oidin. Pictures were taken at the centre of each flask. Upper left: collagen coating;<br />
upper right: fibronectin. Lower left: peptide 1; lower right: peptide 2.<br />
10. Jayawarna V, Ali M, Jowitt TA, Miller AF, Saiani A, Gough JE, Ulijn RV:<br />
Nanostructured Hydrogels for Three-Dimensional Cell Culture Through<br />
Self-Assembly of Fluorenylmethoxycarbonyl-Dipeptides. Adv Mater 2006,<br />
8:611-614.<br />
11. Bhat NV, Upadhyay DJ: Plasma-induced surface modification and<br />
adhesion enhancement of polypropylene surface. J Appl Polym Sci 2002,<br />
86:925-936.<br />
12. Varghese S, Elisseeff JH: Hydrogels for Musculoskeletal Tissue Engineering.<br />
Adv Polym Sci 2006, 203:95-144.<br />
13. Tessmar JK, Göpferich AM: Customized PEG-Derived Copolymers for<br />
Tissue-Engineering Applications. Macromol Biosci 2007, 7:23-39.<br />
P16<br />
An integrated synchronization approach for studying cell-cycle<br />
dependent processes of mammalian cells under physiological<br />
conditions<br />
Oscar B Platas 1 , Uwe Jandt 1 , Volker Sandig 2 , Ralf Pörtner 1 , An-Ping Zeng 1*<br />
1 Institute of Bioprocess and Biosystems Engineering, Hamburg University of<br />
Technology, Hamburg, D-21073, Germany;<br />
2 ProBioGen AG, Berlin, D-13086,<br />
Germany<br />
E-mail: aze@tuhh.de<br />
BMC Proceedings 2013, 7(Suppl 6):P16<br />
Introduction: The study of central metabolism and the interactions of its<br />
dynamics with growth, product formation and cell division is a key issue<br />
for decoding the complex metabolic network of eukaryotic cells. For this<br />
purpose, not only the quantitative determination of key cellular molecules<br />
is necessary, but also the variation of their expression rates in time, e.g.<br />
during cell cycle. The enrichment of cells within a specific cell cycle phase,<br />
referred to as cell synchronization, and their further cultivation <strong>all</strong>ow for<br />
the generation of a cell population with characteristics required for cell<br />
cycle related dynamic studies. Unfortunately, most of the synchronization<br />
methods used are not suitable for study under unperturbed physiological<br />
conditions.<br />
Physical selective methods appear to be a better choice. Among them, the<br />
method of countercurrent centrifugal elutriation <strong>all</strong>ows for an efficient<br />
separation of different cell subpopulations from an asynchronous cell<br />
population according to the cell size. Within an elutriated cell subpopulation<br />
high similarity in the size and DNA content of the cells can be achieved.<br />
Given the reproducibility of this method, high cell numbers can be obtained<br />
for inoculation of controlled bench-top bioreactors with synchronous cells.<br />
By integration of this method for synchronous cell generation and a culture<br />
method for further unperturbed growth, sampling of synchronous cells can<br />
be performed over many synchronous population doublings.<br />
Materials and methods: Using the combined approach mentioned<br />
above, centrifugal elutriation was employed for synchronization in
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different cell cycle phases of the industrial human cell line AGE1.HN®<br />
(ProBioGen AG, Berlin, Germany) and a CHO-K1 cell line (CeBiTec, Bielefeld,<br />
Germany). Cells were cultivated in bench-top bioreactors with culture<br />
volumes ranging between 200 mL and 1 L. A dialysis bioreactor<br />
(Bioengineering AG, Switzerland) with a total volume of 3.8 L was used for<br />
the cultivation of one cell line in order to <strong>all</strong>ow for a higher number of<br />
synchronous cell divisions. In this bioreactor cells are separated from the<br />
conditioning chamber, where pH and DO control takes place. In this way<br />
cells can’t be damaged neither by increase in stirring rate nor by bubble<br />
sparging. Furthermore, continuous nutrient exchange takes place through<br />
the dialysis membrane. Cell density values of 4.2 × 10 7 cells mL -1 have<br />
been reached in this system with AGE1.HN® cells without noticeable<br />
change in the cell specific growth rate.<br />
Results: Our first results had already demonstrated the successful<br />
separation of a heterogeneous AGE1.HN® cell population into synchronous<br />
subpopulations [1]. Independently of the targeted cell cycle phase, the<br />
countercurrent centrifugal elutriation <strong>all</strong>owed for a reproducible and<br />
scalable cell synchronization of AGE1.HN and CHO-K1 cells with high<br />
synchrony degrees, up to 95% in G 1 , 53% in S and 75% in the G 2 /M phases.<br />
After assessing the reproducibility of elutriation results, the process was<br />
scaled up successfully for inoculation of a dialysis bioreactor, where<br />
synchronous unperturbed growth was observed at least for 4 cell<br />
divisions (Figure 1). A very clear damped oscillation of the cell cycle<br />
phases could be observed during synchronous growth (Figure 1b and 1c).<br />
Moreover, a sawtooth-like oscillation of the cell diameters confirmed the<br />
successful synchronous growth of the cells. Bioreactor culture showed no<br />
noticeable perturbation in the doubling time of the population.<br />
Conclusions: With these results, one of the most important requirements<br />
for population-based research of mammalian cells was fulfilled. The<br />
dynamic behaviour of the synchronous growing cells was systematic<strong>all</strong>y<br />
studied not only based on cell growth, but also on the distribution of the<br />
cell size and the DNA content of the cells. Furthermore, dialysis culture<br />
<strong>all</strong>owed for a higher number of synchronous cell divisions without<br />
noticeable perturbations. With this contribution, we present an integrated<br />
approach for cell synchronization and further unperturbed cultivation<br />
which is useful for studying cell-cycle dependent processes under<br />
physiological conditions.<br />
Acknowledgements: This work is a part of SysLogics (FKZ 0315275A):<br />
Systems biology of cell culture for biologics, a project founded by the<br />
German Ministry for Education and Research (BMBF).<br />
Reference<br />
1. Platas Barradas O, Jandt U, Hass R, Kasper C, Sandig V, Pörtner R, Zeng AP:<br />
Physical methods for synchronization of a human production cell line.<br />
22nd European Society for Animal Cell Technology (ESACT) Meeting on<br />
Cell Based Technologies, Vienna, Austria.5(Supplement 8), Online:<br />
http://www.biomedcentral.com/1753-6561/5/S8/P49.<br />
Figure 1(abstract P16) Synchronous growth of AGE1.HN cells in a dialysis bioreactor. The cultured cells were elutriated with high synchrony in the<br />
G 2 /M phase. (a): viable cell density and viability, (b): percentage values of the cell cycle phase distribution, (c): distribution of the S phase, exhibiting a<br />
damped oscillation.
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P17<br />
Evaluation of process parameters in shake flasks for mammalian cell<br />
culture<br />
Oscar B Platas 1 , Volker Sandig 2 , Ralf Pörtner 1 , An-Ping Zeng 1*<br />
1 Institute of Bioprocess and Biosystems Engineering, Hamburg University of<br />
Technology, Hamburg, D-21073, Germany;<br />
2 ProBioGen AG, Berlin, D-13086,<br />
Germany<br />
E-mail: aze@tuhh.de<br />
BMC Proceedings 2013, 7(Suppl 6):P17<br />
Introduction: Shake flask cultivation is nowadays a routine technique<br />
during process development for mammalian cell lines. During shaken<br />
culture, changes in agitation velocity, shaking diameter or shake flask size<br />
affect the hydrodynamics in the shake flask. This might be reflected in the<br />
growth of the cultured cells.<br />
Process parameters such as power input, mixing time, fluid velocity etc.<br />
have been determined and described mathematic<strong>all</strong>y for shake flasks<br />
used for microbial cultivation, but onlytosomeextendformammalian<br />
cell culture. Especi<strong>all</strong>y the relationship between these parameters and<br />
growth characteristics of mammalian cells is still a relatively uncovered<br />
issue.<br />
In this work, process parameters like specific power input, mixing time,<br />
maximum fluid velocity and Reynolds number were determined for four<br />
different shake flasks (baffled and unbaffled) in a range of shaking velocities<br />
on a shaking machine. The specific growth rate (μ max ) of the human<br />
industrial cell line AGE1.HN® (ProBioGen AG, Berlin, Germany) was compared<br />
to the respective process parameters.<br />
Determination of process parameters: (1) Power input (P/V) was<br />
calculated according to experimental data, that have been published in<br />
correlations with the form of Np = f(Re), where Np is the power number<br />
and Re the Reynolds number of the culture. The first correlation is<br />
based on the work by Büchs et al. [1,2], who used a modified Np<br />
analog to bioreactors, and fited the experimental Np’ data to Re. The<br />
second correlation used is based on the work of Kato et al. [3]. Here,<br />
the calculation of the Reynolds number considers the diameter of the<br />
shaker (d o ) instead of the inner flask diameter (d i ).<br />
(2) Mixing time (Θ 95 ) was determined by means of the decolourization<br />
method (I/KI titrated with Na 2 S 2 O 3 ). Decolourization time course was<br />
video recorded and visu<strong>all</strong>y analyzed.<br />
(3) Maximum fluid velocity (u i ) was calculated at the maximum<br />
flask’s inner diameter.<br />
(4) Reynolds number (Re) was calculated as Re = rNd 2 /h, with d = d i ,<br />
and d=d o , for the methods published by Büchs et al., and Kato el al.<br />
respectively.<br />
A modified di (d i, mod ) was used for calculations of parameters in baffled<br />
flasks. This number considers the flask’s depth into the flask circumference.<br />
The average specific growth rate μ max wasemployedasindicatorfor<br />
growth performance.<br />
Relationship between cell growth and process transfer criteria:<br />
Figure 1 shows the dependency of the average specific growth rate μ max<br />
of AGE1.HN® cells on the process parameters of the cultures performed in<br />
shake flasks. A shaking velocity of 200-250 min -1 seems to be optimal for<br />
the cell growth rate. A maximal specific growth rate was observed in a<br />
close range of power input at 200-400 W m -3 according to the method of<br />
Büchs et al. and at 400-1000 W m -3 for the method of Kato et al. used for<br />
Re calculation. As has been shown for the culture of AGE1.HN® cells in<br />
bench-top bioreactors [4], a range of mixing time values between 8 and<br />
13 seconds can be identified here as common for <strong>all</strong> shake flasks too. The<br />
process operational windows identified in this work can lead to a<br />
significant reduction in the growth differences of mammalian cells in the<br />
context of standardization and reproducibility of shake flask cultures.<br />
Conclusions: Our results point to regions of the studied parameters, where<br />
common operation windows can be identified for μ max .Intheseprocess<br />
windows the cells show a similar μ max in different shake flask, making cell<br />
growth comparable. These process windows are common for the flasks,<br />
independently of their size and the number of baffles.<br />
The data obtained in this work can be used for process standardization and<br />
comparability of results obtained in shaken systems i.e. to guarantee<br />
consistency of results generated during laboratory studies with mammalian<br />
cells.<br />
Acknowledgements: This work is a part of SysLogics (FKZ 0315275A):<br />
Systems biology of cell culture for biologics, a project founded by the<br />
German Ministry for Education and Research (BMBF).<br />
References<br />
1. Büchs J, Maier U, Milbradt C, Zoels B: Power consumption in shaking flasks<br />
on rotary shaking machines: I. Power consumption measurement in<br />
unbaffled flasks at low liquid viscosity. Biotechnol Bioeng 2000, 68:589-593.<br />
2. Büchs J, Maier U, Milbradt C, Zoels B: Power consumption in shaking<br />
flasks on rotary shaking machines: II. Nondimensional description of<br />
specific power consumption and flow regimes in unbaffled flasks at<br />
elevated liquid viscosity. Biotechnol Bioeng 2000, 68:594-601.<br />
3. Kato Y, Hiraoka S, Tada Y, Shirai S, Koh ST, Yamaguchi T: Powerconsumption<br />
of horizont<strong>all</strong>y shaking vessel with circulating motion.<br />
Kagaku Kogaku Ronbunshu 1995, 21:365-371.<br />
4. Platas O, Jandt U, Phan LDM, Villanueva ME, Schaletzky M, Rath A, Freund S,<br />
Reichl U, Skerhutt E, Scholz S, Noll T, Sandig V, Pörtner R, Zeng AP:<br />
Evaluation of criteria for bioreactor comparison and operation<br />
standardization for mammalian cell culture. Eng Life Sci 2012,<br />
12:518-528.<br />
P18<br />
Online glucose-lactate monitoring and control in cell culture and<br />
microbial fermentation bioprocesses<br />
Henry Weichert * , Mario Becker<br />
Sartorius Stedim Biotech GmbH, August-Spindler-Strasse 11, 37079<br />
Goettingen, Germany<br />
E-mail: henry.weichert@sartorius-stedim.com<br />
BMC Proceedings 2013, 7(Suppl 6):P18<br />
Introduction: Conventional biopharmaceutical manufacturing is<br />
characterized by validated process steps and extensive lab testing<br />
procedures. The FDA PAT-Guidance recommends the use of potential for<br />
improving development, manufacturing, and quality assurance through<br />
innovation in product and process development, process analysis and<br />
process control.<br />
Measurement of glucose, as a major nutrient during cell cultivation and<br />
microbial fermentation, has a key role for controlling the status of the<br />
cultivation process. Together with the amount of lactate and additional<br />
process parameters, like pH and DO, it gives the possibility to calculate<br />
specific consumption rates of nutrients. The user gets information about the<br />
status of the culture and of the cells.<br />
BioPAT®Trace: Online Glc/Lac Analyser: BioPAT®Trace (Figure 1) is a<br />
dual-channel analyser for the simultaneously measurement of glucose and<br />
lactate which is based on an enzymatic detection of the two analytics.<br />
Special attention has been paid to the ease of use and hygienic issues<br />
related to cGMP environments. The system follows the plug & plays<br />
principle, can be fully integrated into <strong>all</strong> facility environment scenarios and<br />
is compliant with <strong>all</strong> relevant regulatory guidelines.<br />
Wide measuring range: The linear measuring range of the BioPAT<br />
®Trace extends from 0.01 to 40 g/l glucose and from 0.05 to 5 g/l lactate.<br />
The deviation from the average measurement value is less than 3% for a<br />
measurement of 5 g/l glucose and 2.5 g/l lactate.<br />
Fast measurement frequency: The measurement frequency is up to 60<br />
analyses per hour depending on the conditions. The service life of the<br />
sensor system ensures 30 days or 5000 analyses depending on the<br />
application. The ambient temperature of the BioPAT ®Trace can lie<br />
between 5 and 35°C due to internal temperature correction. The ambient<br />
humidity should not exceed 90%.<br />
Flexible system integration: The BioPAT ®Trace has a number of outputs<br />
making integration into data recording systems very flexible. Along with a<br />
standard analog output for signal ranges from 0 to 20 mA, 0 to 10 V or 4<br />
to 20 mA, the BioPAT ®Trace also has a USB interface, an Ethernet<br />
connection as well as a serial output for data recording.<br />
Connection to different fermenter scales by filtration or dialysis<br />
probes: The on-line analysing system BioPAT®Trace covers the different<br />
demands of long-term cell culture cultivations and fast microbial processes<br />
in different scales such as sm<strong>all</strong> volume cultivations and FDA-validated large<br />
scale productions. The sterile sampling systems based on filtration, dialysis<br />
or ContiTRACE disposable probes provide the perfect solution for reliable<br />
on-line sampling in bioreactors and bio disposables applied in industrial and<br />
laboratory facilities.
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Page 35 of 151<br />
Figure 1(abstract P17) Relationship between maximum specific growth rate μ max and the process parameters in shake flask culture.<br />
A) Shaking velocity, B) Power input calculated with the method by Büchs et al., C) Power input calculated by the method by Kato el al., D) Mixing time,<br />
E) Maximum fluid velocity, F) Reynolds number with d = d o .<br />
The simplest method is to directly measure a filtered sample of medium.<br />
However, because reactor medium is used, the range of applications is<br />
limited to processes for which there’s a sufficient reactor volume or which<br />
<strong>all</strong>ow continuous-feed. Dialysis sampling is an option when processes are<br />
involved for which reactor volume does not <strong>all</strong>ow enough sample material.<br />
This method only removes low molecular substances from the reactor<br />
medium, without reducing the volume of fluid.<br />
Automated control loop for glucose feed: Integrated in an automation<br />
platform enabled with a 2 point glucose controller, e.g. as part of an S88<br />
recipe module of the BioPAT®MFCS SCADA system, it is possible to realize<br />
a fully automated control loop for any kind of cultivation process.<br />
Conclusions: • Real Online system<br />
Fast & automated measurement<br />
SU tube sets and sensors<br />
• Direct culture control (24/7)<br />
Process knowhow<br />
Replace offline methods<br />
Real-time process monitoring<br />
Automated sampling<br />
• Setup of control loops and event based actions<br />
defined by using the S88 module<br />
• Different connections to automation systems possible<br />
• Automated feed control<br />
• Real-time Glucose and Lactate values<br />
P19<br />
Study of the improved Sf9 transient gene expression process<br />
Xiao Shen, David L Hacker, Lucia Baldi, Florian M Wurm *<br />
Laboratory of Cellular Biotechnology, Faculty of Life Sciences, Ecole<br />
Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland<br />
E-mail: florian.wurm@epfl.ch<br />
BMC Proceedings 2013, 7(Suppl 6):P19
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Figure 1(abstract P18) BioPAT®Trace equipped with the single use<br />
Tube set.<br />
Introduction: Insect cells have been widely used for the production of<br />
recombinant proteins using recombinant baculovirus for gene delivery [1].<br />
To simplify protein production in insect cells, we have previously described<br />
a method, based on transient gene expression (TGE) with cultures of<br />
suspension-adapted Sf9 cells using polyethylenimine (PEI) for DNA delivery<br />
[2]. Expression of GFP has been realized at high efficiency and a tumor<br />
necrosis factor receptor-Fc fusion protein (TNFR-Fc) was produced at a<br />
level of 40 mg/L. However, the efficiency of the insect cells TGE system has<br />
not been studied and further optimization may improve protein titers.<br />
Here, we studied the efficiency of PEI for plasmid delivery in Sf9 cells.<br />
Methods: Cell culture: Sf-9 cells were maintained in suspension in<br />
TubeSpin® bioreactor 600 at 28°C [3].<br />
Sf-9 cells Transfection: Sf9 cells were transfected as described before [2]<br />
using 25 kDa polyethylenimine PEI (Polysciences, Warrington, PA) and an<br />
expression vector for GFP or TNFR-Fc. GFP-specific fluorescence was<br />
measured 48 h post-transfection using the GUAVA EasyCyteTM flow<br />
cytometer (Millipore, Billerica MA, USA). TNFR-Fc was measured by<br />
sandwich ELISA [4].<br />
Estimation of plasmid copy number: Total DNA was isolated using<br />
DNeasy Blood & Tissue Kit (Qiagen AG, Hombrechtikon, Switzerland)<br />
according to the manufacturer’s protocol. PCR was executed using the<br />
Absolute qPCR SYBR Green ROX reaction mix (Axon Lab AG, Baden-Dättwil,<br />
Switzerland) with total cellular DNA as template. The PCR was performed<br />
using LightCycler® 480 real-time PCR system (Roche Applied Science, Basel,<br />
Switzerland). The plasmid copy number was estimated from the standard<br />
curve according to the threshold cycle (Ct) of each sample [4].<br />
Cell cycle analysis: Cells at different times post-transfection were<br />
centrifuged and washed with PBS before fixation in 70% ethanol. Fixed cells<br />
were washed with PBS and then stained with Guava Cell Cycle Reagent and<br />
analyzed by the GUAVA EasyCyteTM flow cytometer. Cells treated with<br />
nocodazole(50ng/mL,16h)andmimosine(1mM,24h)wereusedas<br />
references for determining the positions of the G1 and G2/M phases [5].<br />
Results: Plasmid delivery efficiency in Sf9 cells: To measure the time<br />
course of plasmid DNA delivery, cells were transfected with a GFP<br />
expression vector. At different times post-transfection, a complete medium<br />
exchange was performed. The percentage of GFP-positive cells was<br />
determined for <strong>all</strong> cultures including a control for which a medium<br />
exchange was not performed. All cultures exhibited similar levels of GFPpositive<br />
cells meaning that DNA uptake into cells occurred within 10 min of<br />
DNA addition (Figure 1A).<br />
To measure the amount of DNA uptake, Sf9 cells were transfected in two<br />
different ways with a TNFR-Fc expression vector and the amount of<br />
intracellular plasmid was measured by quantitative PCR. On the day of<br />
transfection more than 80% of the plasmid DNA was present within cells<br />
with the control transfection while 40% of the DNA was present within<br />
cells following a high-density transfection (Figure 1B). It has been reported<br />
that improved plasmid delivery can result in an increase in specific and<br />
volumetric productivity for HEK 293 cells transfected at high-density [6].<br />
However, in our high-density protocol, plasmid delivery was diminished in<br />
comparison to the control (Figure 1B).<br />
Plasmid delivery was not improved, but cell growth was inhibited in<br />
an optimized TGE process: Improvement in TGE yields from Chinese<br />
hamster ovary cells was achieved by reducing the cell growth rate [5,7].<br />
When the cell growth curve of the optimal TGE process with Sf9 cells was<br />
compared with that of the control protocol, we observed a significant<br />
decrease of viable cell number, within 24 h post-transfection (Figure 1C).<br />
This suggested a deregulation in the cell cycle in the initial phase of<br />
transfection. The cell cycle distribution was analyzed and an increase of the<br />
percentage of cells in the G2/M phase was observed for the high-density<br />
protocol early after transfection (Figure 1D). However, the growth inhibition<br />
was attenuated by 24 h post-transfection (Figure 1D). Nevertheless, the<br />
temporary cell growth inhibition contributed to yield improvement in our<br />
optimal protocol.<br />
Conclusion: A previously described method for the transient transfection<br />
of Sf9 cells was improved. The increase in recombinant protein yield was<br />
not due to an increased plasmid delivery after transfection. However,<br />
high-density transfection resulted in a significant percentage of cells<br />
being blocked in the G2/M phase of the cell cycle for the first 24 h posttransfection.<br />
References<br />
1. Kost TA, Condreay JP, Jarvis DL: Baculovirus as versatile vectors for<br />
protein expression in insect and mammalian cells. Nat Biotechnol 2005,<br />
23:567-575.<br />
2. Shen X, Michel PO, Xie Q, Baldi L, Wurm FM: Transient transfection of<br />
insect Sf-9 cells in TubeSpin® bioreactor 50 tubes. BMC Proc 2011, Suppl<br />
8: P37.<br />
3. Xie Q, Michel PO, Baldi L, Hacker DL, Zhang X, Wurm FM: TubeSpin<br />
bioreactor 50 for the high-density cultivation of Sf-9 insect cells in<br />
suspension. Biotechnol Lett 2011, 33:897-902.<br />
4. Matasci M, Baldi L, Hacker DL, Wurm FM: The PiggyBac transposon<br />
enhances the frequency of CHO stable cell line generation and yields<br />
recombinant lines with superior productivity and stability. Biotechnol<br />
Bioeng 2011, 108:2141-2150.<br />
5. Wulhfard S, Tissot S, Bouchet S, Cevey J, De Jesus M, Hacker DL, Wurm FM:<br />
Mild hypothermia improves transient gene expression yields several fold<br />
in Chinese hamster ovary cells. Biotechnol prog 2008, 24:458-465.<br />
6. Backliwal G, Hildinger M, Hasija V, Wurm FM: High-density transfection<br />
with HEK-293 cells <strong>all</strong>ows doubling of transient titers and removes need<br />
for a priori DNA complex formation with PEI. Biotechnol Bioeng 2008,<br />
99:721-727.<br />
7. Gorman CM, Howard BH, Reeves R: Expression of recombinant plasmids<br />
in mammalian cells is enhanced by sodium butyrate. Nucleic acids res<br />
1983, 11:7631-7648.<br />
P20<br />
Development of a Drosophila S2 insect-cell based placental malaria<br />
vaccine production process<br />
Wian A de Jongh 1 , Mafalda dos SM Resende 2 , Carsten Leisted 1 ,<br />
Anette Strøbæk 1 , Besim Berisha 2 , Morten A Nielsen 2 , Ali Salanti 2 ,<br />
Kathryn Hjerrild 3 , Simon Draper 3 , Charlotte Dyring 1*<br />
1 ExpreS 2 ion Biotechnologies, Horsholm, Denmark, 2970;<br />
2 Centre for Medical<br />
Parasitology, Copenhagen University, Copenhagen, Denmark, 1356;<br />
3 The<br />
Jenner Institute, University of Oxford, Oxford, UK, OX3 7DQ<br />
BMC Proceedings 2013, 7(Suppl 6):P20<br />
Background: Malaria during pregnancy is the cause of 1500 neonatal<br />
deaths a day. Moreover, 40% of <strong>all</strong> low weight births are caused by<br />
pregnancy associated malaria. Researchers at Copenhagen University have<br />
identified the VAR2CSA protein as a potential protective recombinant<br />
placental malaria vaccine. ExpreS 2 ion Biotechnologies is responsible<br />
establishment of cell lines expressing VAR2CSA variants and for developing<br />
the protein production process based on VAR2CSA.<br />
The ExpreS 2 System is a one-for-<strong>all</strong> protein expression system based on<br />
Drosophila S2 cells that is excellent in <strong>all</strong> phases of Drug Discovery, R&D and<br />
manufacturing due to high-level transient transfections, easy establishment<br />
of stable polyclonal pools that provides continuous high protein expression<br />
levels without selection pressure, and simple cloning procedure. It is a novel,<br />
non-viral, insect-cell based expression technology applied to the<br />
development of a critic<strong>all</strong>y needed vaccine. The VAR2CSA protein, which the<br />
vaccine is based on, is hard to express and comparison studies between
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Page 37 of 151<br />
Figure 1(abstract P19) Study of the Sf9 TGE process. (A) Sf9 cells were transfected with EGFP-coding plasmid DNA and PEI at a starting cell density of<br />
4×10 6 cells per ml. Media of the transfected culture were exchanged at 10, 30, 60, 90, 120, 180 minutes post-transfection. EGFP-positive cells were<br />
measured on day 2. (B) Average intracellular plasmid copy number on day of transfection and day 3 post-transfection of cultures transfected using control<br />
protocol and high-density TGE protocol were analyzed by quantitative PCR. (C) Cell growth of Sf9 cells transfected using the two different protocols were<br />
compared. Cell cycle distribution during the first 24 hours post-transfection of those two TGE culture were analyzed (D). C: control transfection at<br />
4×10 6 cells/mL; H: high-density Sf9 transfection; h: hours.<br />
insect, bacteria and yeast have shown that an insect cell system is the only<br />
one leading to a clinic<strong>all</strong>y useful immune response. Process optimization is<br />
also critic<strong>all</strong>y important, as the cost of manufacture must be as low as<br />
possible to <strong>all</strong>ow the vaccine to be used in the countries where it is most<br />
needed.<br />
Aim: The choice and cost of a manufacturing platform is one of the most<br />
important strategic decisions in recombinant subunit vaccine development.<br />
Furthermore, the geographic distribution of malaria and the philanthropic<br />
funding sources involved requires production to be as cost-effective as<br />
possible. Single-use provides manufacturing flexibility and economic<br />
advantages, both highly desirable in this type of process. We therefore aim<br />
to develop cost-effective Drosophila S2basedPlacentalandBlood-stage<br />
malaria vaccine production processes combining the ExpreS 2 constitutive<br />
insect cell expression system with single-use bioreactor technology.<br />
Materials and methods: Thirty-four truncation variants of the VAR2CSA<br />
placental malaria vaccine antigen and full-length PfRh5 were cloned into<br />
pExpreS 2 vectors and transfected into Drosophila S2 insect cells. Stable cell<br />
lines were established in three weeks in T-flask culture, which were then<br />
inoculated at 8E6 cells/ml in shake flasks, or batch or fed-batch production<br />
in DasGip Bioreactors and harvested after 3 and 7 days respectively. The<br />
cultures were harvested by centrifugation and filtration, where after the<br />
proteins were purified using Ni ++ affinity columns and gel filtration.<br />
Bioreactor optimisation were performed in 1L DasGip mini-bioreactors, 2L<br />
Braun glass bioreactor, and the single-use CellReady3L bioreactor.<br />
Alternating Tangential Flow (ATF) technology from Refine was also<br />
employed for perfusion production tests. The bioreactor conditions were<br />
25°C, pH6.5, Dissolved Oxygen 20%, 110 rpm stirrer speed using a Marine<br />
impeller. The perfusion rate was set to 0.5 to 3 Reactor Volumes (RV) per
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Page 38 of 151<br />
Table 1(abstract P21) Key compounds supplemented at<br />
0.01% (w/v) to CD media<br />
Key compound Specific IgG production (%)*<br />
Ferulic acid 154<br />
Syringic acid 194<br />
Galactarate 153<br />
Adenine 185<br />
Trigonelline 141<br />
SE50MAF-UF 204<br />
CD media 100<br />
All values are set relative to CD media (100%).<br />
Figure 1(abstract P20) Expression yields obtained for Rh5 in batch,<br />
fed-batch and ATF-perfusion modes. Using perfusion could<br />
significantly increase yields.<br />
day, but was increased significantly faster for the CellReady 3L perfusion<br />
run compared to the Braun runs, with 3 RV per day reached by day 6 vs.<br />
day 9 for the Braun runs.<br />
Results: Thirty-four protein variants of VAR2CSA were screened for<br />
expression level. Further process optimization was performed on the lead<br />
candidate in glass bioreactors, and >30% yield increase was achieved<br />
using a fed-batch approach (results not shown). The expression of Rh5 was<br />
compared in batch, fed-batch and perfusion using both CellReady3L and<br />
glass bioreactors. There was no significant difference between growth in<br />
the DasGip bioreactor and the disposable CellReady bioreactor.<br />
Comparable yields were obtained in both systems whether running in batch,<br />
fed-batch, or perfusion mode (e.g. Perfusion day 6: 190 vs. 210 mg/L, results<br />
not shown). Furthermore, 350E6 cells/ml were achieved in concentrated<br />
perfusion mode using the ATF and CellReady3L. Concentrated perfusion<br />
lead to final Rh5 yields of 210 mg/L and 500 mg/L after 6 or 9 days<br />
production runs (see Figure 1).<br />
Conclusions: The ExpreS 2 platform has demonstrated its robustness of<br />
expression ability, by expression of two complex malaria antigens; and in<br />
breadth of hardware adaptability, as it was shown to perform comparably<br />
in the single use CellReady3L and glass bioreactors. Furthermore,<br />
extremely high cell counts and yields of Rh5 were achieved in Fed-batch<br />
and perfusion modes. The results demonstrate how the ExpreS 2 expression<br />
system in conjunction with single-use technology can be used to produce<br />
cost-effective malaria vaccines.<br />
P21<br />
Understanding the complexity of hydrolysates<br />
Abhishek J Gupta 1,2 , Kathleen Harrison 2 , Dominick Maes 3*<br />
1 Laboratory of Food Chemistry, Wageningen University, Wageningen, The<br />
Netherlands;<br />
2 FrieslandCampina Domo, Delhi, NY 13753, USA;<br />
3 FrieslandCampina Domo, Wageningen, The Netherlands<br />
E-mail: dominick.maes@frieslandcampina.com<br />
BMC Proceedings 2013, 7(Suppl 6):P21<br />
Background: Hydrolysates are complex media supplements composed of<br />
many as well as different types of compounds. Within Frieslandcampina<br />
Domo’s Quality by Design project, detailed information of these compounds<br />
(annotation and quantification) has been generated. This was achieved for<br />
soy protein hydrolysates (Proyield Soy SE50MAF-UF) using metabolomics<br />
biochemical profiling. Biochemical profiling, together with peptide<br />
profiling and analysis of the inorganic compounds, resulted in complete<br />
characterization of this hydrolysate product. Addition<strong>all</strong>y, these lots of<br />
Proyield Soy SE50MAF-UF were tested for cell culture performance.<br />
Results and Discussion: The composition data was natural log transformed<br />
and functionality data was corrected for experiment-to-experiment<br />
variation. Consequently, the dataset was analyzed using statistical tools like<br />
two-mode cluster analysis, bootstrapped stepwise regression and 2D<br />
correlation analysis. These statistical tools were composed in-house using<br />
Matlab® R 2009b version 7.9.0.529.<br />
This resulted in identification of a series of key compounds in the<br />
hydrolysates that correlated with cell growth or IgG production in a CHO cell<br />
line. To validate these findings, pure preparations of these key compounds<br />
were supplemented to the chemic<strong>all</strong>y defined medium. Addition of these<br />
individual key compounds to chemic<strong>all</strong>y defined medium, in some cases,<br />
slightly improved cell growth or IgG production, but the effect was still<br />
much sm<strong>all</strong>er than the enhancing effect of the complete hydrolysate. The<br />
specific IgG production of key compounds supplemented to CD media, CD<br />
media alone, and soy protein hydrolysate supplemented to CD media is<br />
shown in Table 1.<br />
This suggests that the effect of a hydrolysate cannot by mimicked by<br />
adding certain key compounds. Alternatively, this suggests that these key<br />
compounds are biomarkers, which are interconnected with several other<br />
compounds, and that presence of <strong>all</strong> of these compounds is relevant/<br />
important for the enhancement in the functionality.<br />
The 2D correlation analysis reveals this complex network of compounds,<br />
in which these compounds are positively or negatively correlated with<br />
each other and with cell growth or IgG production (Figure 1).<br />
In hydrolysates, these compounds interact with several other compounds in<br />
a complex biochemical network. This network of compounds is a unique<br />
and native feature of hydrolysates and non-existent in chemic<strong>all</strong>y defined<br />
media.<br />
Working in close collaboration with our customers, we gain understanding<br />
about the relation between the complex composition of hydrolysates and<br />
their effect on cell growth and titer in the application.<br />
P22<br />
Developing a production process for influenza VLPs: a comparison<br />
between HEK 293SF and Sf9 production platforms<br />
Christine M Thompson 1,2 , Emma Petiot 1 , Marc G Aucoin 3 , Olivier Henry 2 ,<br />
Amine A Kamen 1,2*<br />
1 Human Health Therapeutics, Vaccine Program, NRC, Montréal, Québec, H4P<br />
2R2, Canada;<br />
2 Department of Chemical Engineering, École Polytechnique de<br />
Montréal, Montréal, Québec, H3C 3A7, Canada;<br />
3 Department of Chemical<br />
Engineering, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada<br />
E-mail: amine.kamen@cnrc-nrc.gc.ca<br />
BMC Proceedings 2013, 7(Suppl 6):P22<br />
Background: Influenza virus-like particle (VLP) vaccines are one of the most<br />
promising approaches to respond to the constant threat of the emergence<br />
of pandemic strains, as they possess the potentialforhigherproduction<br />
capabilities compared to traditional vaccines made in egg-based technology.<br />
VLPs are particles produced in cell culture utilizing recombinant protein<br />
technology composed of viral antigens that are able to elicit an immune<br />
response but lack viral genetic material. Thus far, influenza VLPs have been<br />
produced in mammalian, insect and plant based platforms [1], with<br />
production in insect cells being the most explored. Baculovirus with<br />
mammalian promoters (Bacmam) have been shown to efficiently transduce<br />
mammalian cells and further express genes but are unable to replicate,<br />
efficiently repressing baculovirus (BV) production that leads to contamination<br />
downstream [2]. Influenza VLP production was performed in HEK 293SF cells<br />
using the Bacmam gene delivery system. The proposed system was assessed
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Page 39 of 151<br />
Figure 1(abstract P21) 2D correlation map of compounds present in ProYield SE50MAF-UF that significantly influences IgG production by CHO<br />
cells. These key compounds are interconnected to other compounds of the hydrolysate, forming a complex biochemical network.<br />
for its ability to produce influenza VLPs composed of Hemagglutinin (HA),<br />
Neuraminidase (NA) and Matrix Protein (M1) and compared to VLPs<br />
produced in Sf9 cells through the lens of bioprocessing.<br />
Materials and methods: VLPs from both systems were characterized using<br />
currently available influenza quantification techniques such as Single Radial<br />
Immunodiffusion (SRID) assay, Hemagglutination (HA) assay, Negative<br />
Staining Electron Microscopy (NSEM) and western blot.<br />
Results: It was found that VLPs from the HEK 293SF system were present<br />
in the culture supernatant in a heterogeneous mixture in terms of particle<br />
shape and size. Particles were spherical and also pleomorphic in shape and<br />
ranged from sizes of 100-400 nm. Sucrose cushion concentrated samples<br />
contained broken particles and a lot of debris. Addition<strong>all</strong>y, it was found<br />
that VLPs were associated with the cell pellet after harvest in relatively the<br />
same amount as released into the supernatant in the form of unreleased<br />
VLPs from NSEM and HA assay analysis. This is possibly due to the sticky<br />
nature of the HA protein or from cell clumping during production that<br />
worked to trap the VLPs, preventing release into the supernatant. Sf9 cells<br />
produced more uniformly shaped VLPs that were spherical in shape,<br />
around 100 nm in size and were found to be mainly in the supernatant,<br />
not associated with the cell pellet. Sucrose cushion concentrated VLPs<br />
contained noticeably less debris than VLPs produced from HEK 293SF cells.<br />
It was found that VLP production in Sf9 cells produced 1.5 logs more VLPs<br />
than in HEK 293SF cells and had 30× higher HA activity. However, Sf9<br />
VLP samples contained 20× more baculovirus than VLPs, which can<br />
contribute to HA activity in both the HA and SRID assays which has to be<br />
acknowledged during process development stages. This is the first time to<br />
our knowledge that specific production values for influenza VLPs in terms<br />
of total particles/ml have been reported.<br />
Conclusions: From this study, the insect-cell baculovirus system produced a<br />
more homogeneous population of VLPs compared to its counterpart in HEK<br />
293SF cells. However, this study also highlights the major problem of<br />
baculovirus contamination in the Sf9 system, which requires removal for<br />
final vaccine formulations and to help ease the optimization of process<br />
production conditions.<br />
Acknowledgements: The authors would like to thank Dr. Ted M Ross of<br />
the University of Pittsburgh for kindly donating the Bacmam construct and<br />
NSERC for providing the Discovery Grant that supported this study. In<br />
addition, we’d like to thank Johnny Montes for his help with viral stock<br />
productions and the rest of the ACT group and graduate students at NRC in<br />
Montréal for their daily support.<br />
References<br />
1. Kang SM, Song JM, Quan FS, Compans RW: Influenza vaccines based on<br />
virus-like particles. Virus research 2009, 143:140-146.<br />
2. Tang XC, Lu HR, Ross TM: Baculovirus-produced influenza virus-like<br />
particles in mammalian cells protect mice from lethal influenza<br />
ch<strong>all</strong>enge. Viral immunology 2011, 24:311-319.<br />
P23<br />
Dynamic cyclin profiles as a tool to segregate the cell cycle<br />
David Garcia Munzer 1 , Margaritis Kostoglou 2 , Michalis C Georgiadis 3 ,<br />
Efstratios N Pistikopoulos 1 , Athanasios Mantalaris 1*<br />
1 Biological Systems Engineering Laboratory, Centre for Process Systems<br />
Engineering, Department of Chemical Engineering, Imperial College London,<br />
London, SW7 2AZ, UK;<br />
2 Department of Chemistry, Aristotle University of<br />
Thessaloniki, Thessaloniki, 54124 Greece;<br />
3 Department of Chemical<br />
Engineering, Aristotle University of Thessaloniki, Thessaloniki, 54124 Greece<br />
E-mail: amantalaris@imperial.ac.uk<br />
BMC Proceedings 2013, 7(Suppl 6):P23<br />
Background and novelty: Mammalian cells growth, productivity and cell<br />
death are highly regulated and coordinated processes. The cell cycle is at<br />
the centre of cellular control and should play a key role in determining<br />
optimization strategies towards improving productivity [1]. Specific<strong>all</strong>y, cell<br />
productivity is cell cycle, cell-line and promoter dependant [2]. The cyclins<br />
are key regulators that activate their partner cyclin-dependent kinases<br />
(CDKs) and target specific proteins driving the cell cycle. To our knowledge,<br />
there is no information on cyclin phase-dependent expression profiles of
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industrial relevant mammalian cell lines. We use the cyclin profiles as a tool<br />
to identify and quantify the landmarks of the cell cycle and implement a<br />
modelling approach to describe the bioprocess. Hereby, we introduce two<br />
possible experimental approaches to obtain such dynamic cyclin profiles.<br />
Experimental approach: Cyclin expression (cyclin E - G 1 class and cyclin B<br />
-G 2 class) was studied in GS-NS0 batch cultures by flow cytometry. Two set<br />
of experiments were performed: a) culture of cells under perturbed (cell<br />
arrest) and unperturbed growth (control run) and b) culture of cells for<br />
DNA labelling to perform a proliferation assay as well as a non-exposed<br />
cells (control run). The static profiles were obtained by direct cyclin<br />
staining and the dynamic profiles were reconstructed by either a) tracking<br />
a parti<strong>all</strong>y synchronized population or b) combining the timings from<br />
proliferation assays with the static profiles.<br />
Result discussion: Both cyclins showed a clear cell cycle phase-specific<br />
pattern (cyclin E was 10% higher at G 1 and cyclin B was 40% higher at G 2 ).<br />
These results were consistent among <strong>all</strong> the different culture conditions<br />
and were inferred from the static cyclin profiles. After the arrest release the<br />
dynamic cyclin profiles can be directly reconstructed by plotting the<br />
relevant cyclin content from the parti<strong>all</strong>y synchronized moving population<br />
traversing the cycle. An advantage of this approach is a clear view of the<br />
cyclin accumulation and transition threshold levels. However, this<br />
approach requires testing using different arrest agents, exposure levels<br />
and timings, which could have an effect on the cell behaviour.<br />
A second approach included an indirect dynamic cyclin profile<br />
reconstruction by combining the acquired proliferation times for different<br />
cell cycle phases (e.g. G 1 /G 0 ,G 2 /M) with the static cyclin profiles. If the static<br />
cyclin profiles are considered as the most representative cyclin values (and<br />
near to the transition threshold level), it is possible to reconstruct the<br />
dynamic profile by linking the threshold values with the cycling times (from<br />
the proliferation assay). The advantage of such approach is the ability to<br />
formulate different dynamic cyclin profiles such as constant functions, piecewise<br />
linear functions or more elaborated profiles. However, implementation<br />
of such an approach requires the tuning of the proliferation assay and the<br />
frequency of sampling since it will affect the quality of the assay.<br />
The two approaches showed comparable results both for the static cyclin<br />
profiles (also when compared to the control runs) and the dynamic cyclin<br />
profiles.<br />
Conclusions: The different approaches for deriving the dynamic<br />
cyclin profiles provide a versatile experimental toolbox for cell cycle<br />
characterization. Cyclins can be used as cell cycle distributed variables and be<br />
experiment<strong>all</strong>y validated (quantitatively), avoiding the use of weakly<br />
supported variables (e.g. age or volume). The observed patterns and timings<br />
provide a blueprint of the cell line’s cell cycle, which can be used for cell cycle<br />
modelling. The development of these models will aid the systematic study of<br />
the cell culture system, the improvement of productivity and product quality.<br />
Acknowledgements: The authors are thankful for the financial support from<br />
the MULTIMOD Training Network, European Commission, FP7/2007-2013,<br />
under the grant agreement No 238013 and to Lonza for generously<br />
supplying the GS-NS0 cell line.<br />
References<br />
1. Dutton RL, Scharer JM, Moo-Young M: Descriptive parameter evaluation<br />
in mammalian cell culture. Cytotechnol 1998, 26:139-152.<br />
2. Alrubeai M, Emery AN: Mechanisms and Kinetics of Monoclonal-Antibody<br />
Synthesis and Secretion in Synchronous and Asynchronous Hybridoma<br />
Cell-Cultures. J Biotechnol 1990, 16:67-86.<br />
P24<br />
Development and implementation of a global Roche cell culture<br />
platform for production of monoclonal antibodies<br />
Thomas Tröbs 1* , Sven Markert 1 , Ulrike Caudill 1 , Oliver Popp 2 , Martin Gawlitzek 3<br />
, Masaru Shiratori 3 , Chris Caffalette 3 , Robert Shawley 3 , Steve Meier 3 ,<br />
Abby Pynn 3 , Wendy Hsu 3 , Andy Lin 3<br />
1 Pharmaceutical Biotech Production & Development PTDE, Roche, 82377<br />
Penzberg, Germany;<br />
2 Pharma Research and Early Development pRED, Roche,<br />
82377 Penzberg, Germany;<br />
3 Early and Late Stage Cell Culture PTDU,<br />
Genentech, South San Francisco, CA 94061, USA<br />
E-mail: thomas.troebs@roche.com<br />
BMC Proceedings 2013, 7(Suppl 6):P24<br />
Introduction: Roche and Genentech both developed their first platform<br />
cell culture process using chemic<strong>all</strong>y-defined media independently.<br />
This resulted in significantly different processes with regards to operations<br />
and media formulations. The decision was made to evaluate both and<br />
decide for one existing platform. Drivers and benefits of a single upstream<br />
cell culture platform were the maximization of flexibility with regard to<br />
process development, clinical and commercial manufacturing by execution<br />
of any process at any network facility with standard transfer effort and by<br />
minimization of component lists and raw material inventories across sites.<br />
Furthermore capturing benefits of improvements made by <strong>all</strong> sites funneled<br />
into a common knowledge base benefits the whole organization. And<br />
process characterization and validation data could be leveraged across the<br />
entire organization what means less resource expenditure for PC/PV.<br />
The existing independent platforms were evaluated if there is a clear benefit<br />
in going forward with a given platform or certain aspects of a platform. The<br />
comparison consisted in a technical (cell culture performance, product<br />
quality, manufacturability) and a business case evaluation (product titer,<br />
timelines to launch, costs, IP. In result both platforms are capable of<br />
achieving sufficient titers for platform process (2-4 g/L) with acceptable<br />
product quality. There existed no major business driver to select one process<br />
over the other.<br />
Development: For development of new basal and feed media knowledge<br />
from two legacy efforts was leveraged and so potential synergies and<br />
performance benefits could be achieved (Figure 1). Based on platform<br />
Figure 1(abstract P24) Schematic diagram of major elements of the two legacy platforms and the optimization of medium and feed respective<br />
the leveraging of knowledge from two existing legacy platforms.
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evaluation results, decision was made to harmonize existent CHO host cell<br />
line, seed train medium and feeding strategy (chosen from the two<br />
existing platforms).<br />
Results: The cell culture media and feed optimization strategy started with<br />
a paper exercise to compare existing in-house chemic<strong>all</strong>y defined media<br />
formulations and identify components/component groups for further<br />
evaluation. Subsequently identified conditions were screened in highthroughput<br />
cell culture systems (HTS-CC) to identify beneficial components<br />
and remove components that are not required. Optimized best cases were<br />
confirmed in 2L bioreactors with 6 model cell lines and the final process<br />
was up-scaled to pilot scale.<br />
Promising results from HTS-CC media screening were confirmed in a<br />
2L-bioreactor experiment. The new platform medium and feed were<br />
finalized after a series of 2L optimization experiments. The process was<br />
successful up-scaled to 250L single-use bioreactor (SUB) and 400L stainless<br />
steel bioreactor with two model cell lines. Growth and titer were<br />
comparable to 2L satellites.<br />
In the course of the platform implementation four new GMP raw materials<br />
(dry powders and stock solution) were developed and tested. Raw material<br />
shelf life stability retesting and extension were initiated. Global specifications<br />
were established for equipment and site independent platform application<br />
and the applicability for global production units is given.<br />
High temperature/short time treatment (HTST) compatibility was tested.<br />
The sterile hold for liquid media was initiated.<br />
Summary: New chemic<strong>all</strong>y defined platform media (basal and feed) were<br />
developed by leveraging data and knowledge from the two Genentech<br />
and Roche legacy platform processes, and through a series of experiments<br />
including high-throughput systems for cell culture, shake flasks,<br />
2L bioreactors and pilot-scale bioreactors. An average increase in final titer<br />
of 30% was achieved compared to the two legacy platforms.<br />
The final process resulted in product quality attributes (glycans, charge<br />
variants, size) that were comparable to historical data. No new variants<br />
were detected. The final and fully harmonized platform process is specified<br />
and implemented.<br />
Acknowledgements: Thomas Tröbs on behalf of Technical Team for<br />
Global Cell Culture Platform development and Christine Jung, Josef<br />
Gabelsberger, Uli Kohnert, Josef Burg, Ralf Schumacher, Robert Kiss, John<br />
Joly, Brian Kelley, Alexander Jockwer, Nicola Beaucamp, Christian Walser,<br />
Carolin Lucia, Peter Harms, Pilot Plant Operations, Analytical Operations.<br />
P25<br />
Powerful expression in Chinese Hamster Ovary cells using bacterial<br />
artificial chromosomes: Parameters influencing productivity<br />
Wolfgang Sommeregger 1 , Andreas Gili 2 , Thomas Sterovsky 2 , Emilio Casanova 3 ,<br />
Renate Kunert 1*<br />
1 Vienna Institute of BioTechnology (VIBT), Department of Biotechnology,<br />
University of Natural Resources and Life Sciences, Vienna, 1190, Austria;<br />
2 Polymun Scientific Immunbiologische Forschung GmbH, Klosterneuburg, 3400,<br />
Austria; 3 Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, 1090,<br />
Austria<br />
E-mail: renate.kunert@boku.ac.at<br />
BMC Proceedings 2013, 7(Suppl 6):P25<br />
Background: CHO (Chinese Hamster Ovary) cells are the cell line of choice<br />
for therapeutic protein production. Although the achieved volumetric titers<br />
have increased significantly over the past two decades, the establishment of<br />
well-producing CHO cell lines is still difficult and not always successful [1].<br />
Factors influencing productivity are the chosen host cell line, the genetic<br />
vectors, applied media, the cultivation strategy as well as the product itself.<br />
Several CHO host strains are available for recombinant protein production,<br />
however, they are often quite diverse in terms of growth rate, maximal<br />
achieved cell concentrations and specific productivities. Specific productivity<br />
is also related to the locus of integration of the transgenes due to positional<br />
effects caused by the chromatin environment. Previously it was described<br />
that Bacterial Artificial Chromosomes (BACs) carrying the Rosa26 locus are<br />
advantageous for the recombinant protein production in CHO cells,<br />
enhancing the specific productivity compared to plasmid derived<br />
recombinant CHO cells [2-4]. In this project we aim to identify factors<br />
influencing volumetric productivity using different CHO hosts, Rosa 26 BACs<br />
as genetic constructs and suitable cell culture media. First, different<br />
commonly used CHO host cell lines were analyzed in various cell culture<br />
media to identify which host strain performs best. Secondly, we generated a<br />
recombinant cell line, producing the highly glycosylated HIV envelope<br />
protein gp140 as an example for a difficult to express model protein. Gp140<br />
expression was compared to an already existing gp140 cell line generated<br />
by a plasmid vector as expression system.<br />
Methods: Cell culture: CHO-DUKX-B11 (ATCC-CRL-9096) and CHO-DG44<br />
(life technologies) were serum-free cultivated in spinner flasks. CHO-K1<br />
(ATCC-CCL-61) and CHO-S (life technologies) were serum-free cultivated<br />
in in shaker flasks.<br />
BAC Recombineering: E.coli carryingtheRosa26BAC(~220kbp)were<br />
transformed with a plasmid coding for a recombinase. Consecutively, a<br />
plasmid carrying the gp140 (CN54) gene flanked by homologous regions<br />
to the BAC was used for the transformation of the recombinase positive<br />
E.coli cells. BAC positive colonies were selected and the BAC DNA was<br />
purified (NucleoBond Xtra BAC, Macherey Nagel).<br />
Transfection and selection: CHO-S host cells were transfected with linearized,<br />
lipid complexed (Lipofectin) CN54 Rosa26 BAC DNA. Recombinant clone<br />
selection was performed in 96-well plates using 0.5 mg/mL G418. BAC<br />
transfected CHO cells are able to express the transgene as well as a<br />
Neomycin resistance gene within the Rosa26 locus.<br />
Results: Host cell line comparison: CHO-DUKX-B11, CHO-DG44, CHO-K1<br />
and CHO-S were analyzed in batch culture in CD-CHO (life technologies),<br />
ActiCHO (GE-PAA), DMEM/Ham’s F12 (Biochrom) + supplements (Polymun<br />
Scientific), and CD-DG44 (life technologies) media in spinner and shaker<br />
flasks. CHO-DUKX-B11 and CHO-DG44 grew best in spinner flasks with<br />
CD-DG44 media, whereas CHO-K1 and CHO-S grew best in shaker flasks<br />
with ActiCHO media. The dhfr negative cell lines were growing to much<br />
lower viable cell densities than K1 and S. CHO-S reached the highest viable<br />
cell density (1.17 × 10 7 cells/mL) followed by CHO-K1 (8.39 × 10 6 cells/mL)<br />
(Table 1).<br />
Gp140 (CN54) recombinant cell lines: CHO-S was chosen for testtransfections<br />
and recombinant gp140 (CN54) producers were established<br />
using a Rosa 26 BAC construct carrying the gp140 (CN54) gene. The best<br />
clone was analyzed in a batch experiment and yielded 77 μg/mL which is<br />
~10 times the titer achieved with a recombinant plasmid derived CHO-<br />
DUKX-B11 (Figure 1). This 10-fold increase was related to the higher<br />
specific productivity (~18-fold) and the higher accumulated cell density<br />
(3.5-fold) in shorter batch duration.<br />
Conclusion: CHO-S and CHO-K1 have the potential to grow to high cell<br />
densities. The used dhfr deficient hosts (DUKX-B11 and DG44) are at least<br />
without a co-transfection of the dhfr gene not growing to high cell concentrations.<br />
Rosa 26 BAC derived clones need no amplification as they provide<br />
their own open chromatin region. Thus, higher specific productivity can be<br />
achieved by elevated transcript levels compared to conventional plasmid<br />
clones. The combination of cells growing to high cell densities and the<br />
transcriptional efficiency of the Rosa26 BAC system leads to accumulation of<br />
significantly increased volumetric titers for a difficult to express glyco-protein.<br />
Acknowledgements: This study was partly financed by Polymun Scientific<br />
Immunbiologische Forschung GmbH, Klosterneuburg, 3400, Austria; BioToP<br />
PhD Programme, University of Natural Resources and Life Sciences, Vienna,<br />
1190, Austria and the FWF Austrian Science Fund.<br />
References<br />
1. Kim JY, Kim YG, Lee GM: CHO cells in biotechnology for production of<br />
recombinant proteins current state and further potential. Appl Microbiol<br />
Biotechnol 2012, 93:917-930.<br />
2. Mader A, Prewein B, Zboray K, Casanova E, Kunert R: Exploration of BAC<br />
versus plasmid expression vectors in recombinant CHO cells. Appl<br />
Microbiol Biotechnol 2013, 97:4049-4054.<br />
3. Blaas L, Musteanu M, Grabner B, Eferl R, Bauer A, Casanova E: The use of<br />
bacterial artificial chromosomes for recombinant protein production in<br />
mammalian cell lines. Methods Mol Biol 2012, 824:581-593.<br />
4. Blaas L, Musteanu M, Eferl R, Bauer A, Casanova E: Bacterial artificial<br />
chromosomes improve recombinant protein production in mammalian<br />
cells. BMC Biotechnol 2009, 9:3.<br />
Table 1 Maximum achieved viable cell densities in batch<br />
experiments<br />
CHO cell line DUKX-B11 DG44 CHO-S CHO-K1<br />
Max. VCD (cells/mL) 2.00E+06 2.28E+06 1.17E+07 8.39E+06
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Figure 1(abstract P25) Titer and specific productivity comparison of a BAC derived recombinant CHO-S cell line producing gp140 (CN54) and<br />
an already existing recombinant plasmid derived CHO-DUKX-B11 cell line.<br />
P26<br />
INVect - a novel polycationic reagent for transient transfection of<br />
mammalian cells<br />
Sebastian Püngel 1 , Miklos Veiczi 2 , Tim Welsink 1 , Daniel Faust 1 , Vanessa Vater 1 ,<br />
Derek Levison 2 , Uwe Möller 2 , Wolfgang Weglöhner 1*<br />
1 InVivo BioTech Services GmbH, 16761 Hennigsdorf, Germany;<br />
2 emp Biotech<br />
GmbH, 13125 Berlin, Germany<br />
E-mail: wegloehner@invivo.de<br />
BMC Proceedings 2013, 7(Suppl 6):P26<br />
Background: For rapid recombinant protein production in sm<strong>all</strong> to medium<br />
size volumes, transient transfection of mammalian cells is still the method of<br />
choice in biotechnology [1]. However, due to the high costs of commerci<strong>all</strong>y<br />
available lipofectamines or polycationic transfection reagents such as<br />
polyethylenimine (PEI), the most widely used transfection reagents available<br />
present a substantial economic bottleneck. While these reagents produce<br />
seemingly high transient transfection rates [2], there is still a strong desire for<br />
transfection reagents providing both secure and easy handling and higher<br />
recombinant protein production. As part of our commitment to excellence,<br />
InVivo BioTech Services initiated a joint venture with emp Biotech and<br />
developed a novel polycationic reagent, named INVect, for transient<br />
transfection and recombinant protein production in mammalian cells.<br />
Materials and methods: Mammalian cells were cultured in CD-ACF media<br />
using shake flasks and standard culture conditions. Cells were transfected<br />
with 10 μg per mL of a GOI harboring plasmid at a cell density of 5 × 10 6<br />
cells per mL in FreeStyle Medium (Life Technologies) with INVect to DNA<br />
ratio of 6:1 (w/w) and PEI to DNA ration of 2:1 (w/w). Cultures were<br />
supplemented with same volume Protein Expression Medium (Life<br />
Technologies) 2 hours post transfection. GFP and SEAP expression took<br />
place in 8 mL culture volume in 50 mL bioreactor tubes. Expression of other<br />
reporter proteins were performed in 150 mL culture volume in 500 mL<br />
shake flasks. Transfection efficiency was determined 24 hours post<br />
transfection by counting green fluorescent positive cells using a FACSCalibur<br />
(Becton, Dickinson and Company). SEAP expression was determined in cell<br />
culture supernatant on day 6 post transfection by a photometric pNPP turnover<br />
assay. Quantification of IgG was performed by protein G affinity<br />
chromatography on day 6 post transfection. Thrombomodulin concentration<br />
was calculated from cell culture supernatant on day 6 post transfection by<br />
IMUBIND® Thrombomodulin ELISA Kit (american diagnostica). His-tagged<br />
recombinant protein was purified on day 6 post transfection by TALON®<br />
immobilized metal affinity chromatography system.<br />
Results: Cytotoxicity was tested over a broad range of concentrations.<br />
Results demonstrate several novel synthetic polymers exhibiting transfection<br />
efficiencies even higher than common PEIs after optimized ratios of DNA-topolymer<br />
were applied. Transfection efficiency of INVect was compared to<br />
PEI, currently the standard transfection reagent for transient gene<br />
expression. INVect was found to gener<strong>all</strong>y give better transfection<br />
efficiencies of greater 80% in a GFP assay (Figure 1A). Batch-to-batch<br />
reproducibility was shown on five independent INVect batches. Transfection<br />
results were highly consistent and in the range of 80-90% (Figure 1B).<br />
INVect successfully delivers genes to HEK293-F, CHO-S and CAP-T cells as<br />
shown in a SEAP expression system (Figure 1C). Post-transfection cell<br />
productivity was determined under TGE manufacturing conditions.<br />
Thrombomodulin (60 kDa) (Figure 1D), an IgG (144 kDa) (Figure 1E) and a<br />
HIS-tagged Protein of Interest (~40 kDa) (Figure 1F) were transiently<br />
expressed using INVect as transfection reagent and conventional 25 kDa PEI<br />
as control. Cells were transfected with a gene of interest harboring plasmid,<br />
with product concentration being measured on day 6 post transfection. The<br />
use of INVect provided a minimum 2-fold increase in protein production<br />
over PEI (25 kDa) based transfection.<br />
Conclusions: INVect is a novel polycationic transfection reagent which<br />
demonstrates low cell toxicity for transient transfection of mammalian cells<br />
and delivers extremely high transfection efficiencies of up to 90%, 24 h post<br />
transfection. The use of INVect for transfection under TGE conditions leads<br />
to exception<strong>all</strong>y high levels of protein expression and outperforms 25 kDa<br />
linear PEI by 2-fold. INVect can be used effectively with <strong>all</strong> common cell lines<br />
and is especi<strong>all</strong>y suited for HEK293-F and CAP-T cells.<br />
References<br />
1. Geisse S: Reflections on more than 10 years of TGE approaches. Protein<br />
Expr Purif 2009, 64:99-107.<br />
2. Fischer S, Charara N, Gerber A, Wölfel J, Schiedner G, Voedisch B,<br />
Geisse S: Transient recombinant protein expression in a human<br />
amniocyte cell line: the CAP-T® cell system. Biotechnol Bioeng 2012,<br />
109:2250-2261.<br />
P27<br />
Development of a chemic<strong>all</strong>y defined cultivation and transfection<br />
medium for HEK cell lines<br />
Sebastian Püngel 1 , T Tim Welsink 1 , Penélope Villegas Soto 1 ,<br />
Wolfgang Weglöhner 1 , Tim F Beckmann 2 , Ina Eickmeier 2 , Stefan Northoff 2 ,<br />
Christoph Heinrich 2*<br />
1 InVivo BioTech Services GmbH, 16761 Hennigsdorf, Germany;<br />
2 TeutoCell AG,<br />
33615 Bielefeld, Germany<br />
E-mail: Christoph.Heinrich@teutocell.de<br />
BMC Proceedings 2013, 7(Suppl 6):P27
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Figure 1(abstract P26) Transfection efficiency and 6 day post-transfection cell productivity of INVect. (A) Transfection efficiency of INVect<br />
compared to PEI. (B) transfection efficiency of 5 independent batches. Transfection efficiency was determined 24 hours post transfection by counting<br />
green fluorescent positive CAP-T cells using a FACSCalibur (Becton, Dickinson and Company). (C) CHO-S, HEK293-F and CAP-T cells were transfected with<br />
a SEAP harboring plasmid. Relative SEAP expression was determined in cell culture supernatant by a photometric pNPP turn-over assay. (D) CAP-T cells<br />
were transfected with a Thrombomodulin harboring plasmid. Thrombomodulin concentration was calculated from cell culture supernatant by IMUBIND®<br />
Thrombomodulin ELISA Kit (american diagnostica). (E) CAP-T cells were transfected with an IgG harboring plasmid. Antibody concentration was<br />
determined by protein G affinity chromatography. (F) CAP-T cells were transfected with a His-tagged protein harboring plasmid. Protein of interest was<br />
purified by TALON® immobilized metal affinity chromatography system.
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Background: In the process of generating a production cell, introduction<br />
of the gene of interest into the host cell can be performed by various<br />
physical, chemical or biological methods. Because of the greater scalability<br />
compared to physical methods and no safety concerns or restrictions that<br />
are associated with the use of viral systems, a transfection using chemical<br />
methods is of great interest. However, up to now up-scaling is limited by<br />
the ch<strong>all</strong>enge to transfect cells in conditioned media with the widely used<br />
reagent polyethylenimine (PEI). Considering the upscaling to gram yields, a<br />
culture medium that <strong>all</strong>ows both, transfection and production is required.<br />
In this work, the current status in the development of such media<br />
supporting cell growth, transfection and protein production in HEK cells is<br />
presented. By this, processes will no longer be limited by media exchange<br />
prior transient transfection.<br />
Materials and methods: Transfection was performed according to<br />
standard protocols described in the literature. Briefly, 5 × 10 6 cells/mL<br />
were transfected with 2 pg DNA/cell and 25 kDa PEI in 4 mL transfection<br />
volume. Transfection efficiency was determined 24 hours post transfection<br />
by counting green fluorescent positive cells using a FACSCalibur (BD<br />
Biosciences). All cultivations were carried out using shake flasks with<br />
standard conditions well known in the art. Automated viable cell counting<br />
was performed by a Cedex (Innovatis). Furthermore, the quantities of<br />
components like glucose, lactate, amino acids, salts and vitamins in the<br />
supernatant were measured. Based on this information, single ingredients<br />
or groups of components from the basal formulation were screened for<br />
their influence on transfection efficiency. To evaluate the effect of cellular<br />
proteins in conditioned medium, they were separated by chromatography<br />
and analyzed via MALDI-TOF/TOF mass spectrometry (MS) (ultrafleXtreme,<br />
Bruker). SEC was performed using the high resolution gel filtration medium<br />
Superdex 200 16/60 with the ÄKTAprime system (GE Healthcare).<br />
Results: Batch growth for an exemplary HEK host cell line in the latest basic<br />
growth medium formulation reached a maximum viable cell density of<br />
nearly 1 × 10 7 cells/mL. Direct adaption of three different adherent serumdepending<br />
host cell lines was also successfully implemented in this medium.<br />
The screening of basal medium components exhibited no significant<br />
influence on transient transfection efficiency of HEK cells (over<strong>all</strong> efficiency<br />
of 80% +/- 15%), as shown in Figure 1(A). In contrast, depending on the<br />
level of conditioning, the presence of proteins in the supernatant of these<br />
media reduced transfection efficiency up to 100% (Figure 1B).<br />
Separation and analysis of conditioned medium revealed that especi<strong>all</strong>y<br />
high molecular weight components have a negative impact on the<br />
transfection efficiency. Identification by MALDI-TOF/TOF-MS showed not<br />
only proteins of the basal lamina but also histones to be present in the<br />
analyzed high molecular weight fractions 1 and 2 (Table 1).<br />
Conclusions: The latest medium formulation supports cell growth and easy<br />
adaption to suspension of the three major HEK host cell lines and several<br />
producer cell lines originated from those. High transfection efficiencies of up<br />
to 80% 24 hours post transfection where reached in a basic medium<br />
formulation. In this context, the major ch<strong>all</strong>enge for combining a<br />
transfection- and growth medium in one formulation is to retain single cell<br />
growth, while avoiding commonly used anti-aggregation components,<br />
which are known to impair transfection efficiency. Beyond that, in this study<br />
basal medium components exhibited no influence on transient transfection,<br />
whereas high molecular weight fractions of conditioned media reduced<br />
transfection efficiency. Noticeably, these fractions contained histones which<br />
might be one factor with negative impact.<br />
Acknowledgements: This work was partly supported by ZIM (Zentrales<br />
Innovationsprogramm Mittelstand) and the German Federal Ministry of<br />
Economics and Technology.<br />
P28<br />
Automated substance testing for lab-on-chip devices<br />
Lutz Kloke 1* , Katharina Schimek 1 , Sven Brincker 1 , Alexandra Lorenz 1 ,<br />
Annika Jänicke 1 , Christopher Drewell 1 , Silke Hoffmann 1 , Mathias Busek 2 ,<br />
Frank Sonntag 2 , Norbert Danz 2 , Christoph Polk 2 , Florian Schmieder 2 ,<br />
Alexey Borchanikov 4 , Viacheslav Artyushenko 4 , Frank Baudisch 3 , Mario Bürger 3 ,<br />
Reyk Horland 1 , Roland Lauster 1 , Uwe Marx 1<br />
1 Technische Universität Berlin/Germany;<br />
2 Fraunhofer IWS, Dresden/Germany;<br />
3 GeSiM mbh, Großerkmannsdorf/Germany;<br />
4 ART Photonics GmbH, Berlin/<br />
Germany<br />
E-mail: lutz.kloke@tu-berlin.de<br />
BMC Proceedings 2013, 7(Suppl 6):P28<br />
Background: A smartphone-sized multi-organ-chip has been developed<br />
by TissUse. This platform consists of a microcirculation system which<br />
contains several fully endothelial-cell-coated micro- channels in which<br />
organ equivalents are embedded. Briefly, Human 3D organ equivalents<br />
such as liver and skin could be maintained functional over 28 days and<br />
treated with chemical entities in this microcirculation system.<br />
In order to automate the Multi-Organ-Chip (MOC) handling we developed<br />
with partners a robotic platform. The prototype is capable to maintain 10<br />
MOCs. Operations can be programmed individu<strong>all</strong>y by its user. For example<br />
OECD guidelines for acute toxicity testing could be performed. The robotic<br />
platform features also functions such as automatic media supply, sampling<br />
and storage, temperature control, fluorescence and microscopic monitoring,<br />
PIV, O2-measurement, etc. To display the functionality we performed a<br />
toxicity test with RPTEC cells treated with DMSO in different concentrations.<br />
Proof of concept study: RPTEC cells were used as cellular model system.<br />
The cells were cultivated in two Generation-4-MOCs as well as in 96-wellplates<br />
working as reference system. The systems were stained with<br />
CellTracker Red and cultivated at 37°C and 5% CO 2 saturation. After some<br />
hours of resting MOCs and MWPs were treated with 10% respectively 20%<br />
DMSO. Afterwards the fluorescence activity was measured in 20 minute<br />
intervals in order to detect potential cell death. The cells can be detected<br />
by the monitoring unit of the robot. A 20 μmol/L CellTracker Red staining<br />
provides a sufficient signal which can be monitored over time. The<br />
treatment with 10% DMSO shows a fluorescence signal decline of more<br />
than 50% and the following recovery of them.<br />
Summary: This project shows the successful development of a robotic<br />
platform to handle multi-organ-chips. Maintenance as well as user specific<br />
protocols, for example toxicity testing, can be accomplished with a<br />
minimum amount of labor time. The MOCs in combination with the robotic<br />
platform offer the plug-and-play solution to generate substance interaction<br />
data on a Lab-on-Chip system.<br />
Figure 1(abstract P27) A: Screening of media components and different concentrations thereof with regard to transfection efficiency.<br />
B: Transfection efficiency in conditioned media as well as in fractions from SEC.
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Table 1(abstract P27) Proteins identified with at least 2 peptides and a false discovery rate of 0% in up to<br />
5 biological replicates by MALDI-TOF/TOF-MS in the high molecular weight fractions 1 and 2<br />
High molecular weight fraction 1 High molecular weight fraction 2<br />
Group Protein name # Peptides Group Protein name # Peptides<br />
Histones Histone H2A 3 Histones Histone H2A 4<br />
Histone H2B 2 Histone H2B 3<br />
Histone H4 2 Histone H4 4<br />
Histone H3 3<br />
Cytoskeleton Tubulin alpha 2<br />
Tubulin beta 2 Cytoskeleton Tubulin alpha 2<br />
Actin 3 Tubulin beta 3<br />
Actin 6<br />
Other Galectin-3-binding protein 6<br />
Heat shock 70 kDa protein 1A/1B 5 Extracellular (matrix) Fibrillin-2 2<br />
Fibronectin 5<br />
Clusterin 3<br />
Cochlin 2<br />
Other Galectin-3-binding protein 10<br />
Heat shock 70 kDa protein 1A/1B 13<br />
Golgi membrane protein 1 6<br />
Alpha-enolase 2<br />
P29<br />
NIR-spectroscopy for bioprocess monitoring & control<br />
Marko Sandor 1 , Ferdinand Rüdinger 1 , Dörte Solle 1 , Roland Bienert 2 ,<br />
Christian Grimm 2 , Sven Groß 2* , Thomas Scheper 1<br />
1 Institut für Technische Chemie, Leibniz Universität Hannover, C<strong>all</strong>instraße 5,<br />
D-30167 Hannover, Germany;<br />
2 Sartorius Stedim Biotech GmbH, August-<br />
Spindler-Straße 11, D-37079 Göttingen, Germany<br />
E-mail: sven.gross@sartorius-stedim.com<br />
BMC Proceedings 2013, 7(Suppl 6):P29<br />
Introduction: The Quality by Design (QbD) approach shows significant<br />
benefit in classical pharmaceutical industry and is now on the cusp to a<br />
stronger influence on biopharmaceutical applications. Monitoring the<br />
critical process parameters (CPP) applying process analytical technologies<br />
(PAT) during biotechnological cell cultivations is of high importance in<br />
order to maintain a high efficiency and quality of a bioprocess. For<br />
parameters like glucose concentration, total cell count (TCC) or viability a<br />
robust online prediction is in many applications not yet possible. This gap<br />
can be closed with the help of NIR spectroscopy (NIRS), which provides<br />
quantitative prediction of single analytes in real-time.<br />
For accurate process control based on NIR spectroscopy, special care has to<br />
be taken while building the calibration model [1,2]. In cell cultivation almost<br />
<strong>all</strong> analytes are confounded and show large correlation coefficients.<br />
Therefore, partial least square (PLS) models are not able to discriminate<br />
between the signals of the different analytes. Especi<strong>all</strong>y, analytes like<br />
glucose or glutamine which are strongly confounded with cell growth need<br />
to be evaluated carefully as cell growth is the analyte causing the largest<br />
changes in NIR spectra throughout a cultivation run. Spiking experiments<br />
are the most efficient way in order to break correlations between critical<br />
analytes like glucose and other nutrients or TCC. This strategy should be<br />
followed in order to build robust calibration models without correlations<br />
[3,4]. Another very critical issue occurring in cell cultivation are batch-tobatch<br />
variations. As it is recommended in good modeling practice [5], for<br />
robust models it is crucial to use several complete batches for validation<br />
which are not part of the calibration set rather than cross validation [6].<br />
Materials and methods: CHO-K01 cells (Cell Culture Technology,<br />
University of Bielefeld), were cultivated in a BIOSTAT® C plus bioreactor<br />
(Sartorius Stedim Biotech) with a 7.5 L working volume. In total, eight<br />
cultivation runs were performed, each lasting six days on average. Sampling<br />
was performed every three to six hours, and reference analytics of the<br />
critical process parameters, such as TCC, viability (TC10 automated cell<br />
counter, Bio-Rad), glucose, lactate, glutamine, etc. (YSI 2700, YSI Inc.) were<br />
determined in the laboratory.<br />
Results: Table 1 gives an overview of the models and the accuracy of<br />
predictions for several analytes investigated. An excellent model could be<br />
obtained for total cell count (TCC). Viability can be predicted and glucose<br />
can be predicted as well. Correlations from glucose with other analytes have<br />
been reduced by spiking of glucose in one cultivation. Predictions for low<br />
concentration analytes like glutamine seem to be also predictable at the first<br />
glance, but are strongly related to correlations with other parameters, such<br />
as TCC. Models based on correlations are not recommended for process<br />
control since they show a lack of sensitivity to the analyte of interest and<br />
robustness. Whether a model is based on correlations can be easily<br />
demonstrated by spiking experiments. Glutamine, for example, was spiked<br />
in one cultivation at the end of the batch-phase up to 1 g/L. The glutamine<br />
model was not able to predict the spiking, which proves the strong<br />
correlation to other analytes. Glutamine cannot be measured directly in this<br />
Table 1(abstract P29) NIR results for calibration models and validation by external data sets<br />
Analyte Range No. Cal. No. Val. Batches (Samples) Reg. maths Factors SEC SEP<br />
TCC (·10 6 cell/mL) 0-16 5 (185) 3 (118) None 2 1.07 0.48<br />
Viability (%) 10-100 5 (193) 3 (110) None 4 4.2 4.2<br />
Glucose (g/L) 0-9 5 (198) 3 (105) None 4 1.2 0.48<br />
Glutamine (g/L) 0-1.1 5 (189) 3 (114) SNV 2 0.16 correlation<br />
(TCC: total cell count; No.Cal.: Number of batches (samples) of the calibration set. No.Val.: Number of batches (samples) of the validation set; SNV: standard<br />
normal variate; SEC: standard error of calibration; SEP: standard error of prediction)
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concentration range using NIRS. However, qualitative models on over<strong>all</strong><br />
nutrient consumption or metabolite accumulation yield promising results<br />
(data not shown).<br />
Additional benefit is generated via MSPC of NIR data. Batch trajectories have<br />
been generated from major variances of the NIR spectra. The Score values<br />
have been used and plotted over time using SIMCA 13. Figure 1 (top) shows<br />
the BEM build for the first principal component of the NIR spectra. Three<br />
batches contribute to this model, which showed optimal cell growth.<br />
All batches show almost an identical profile which indicates a high<br />
batch-to-batch reproducibility, both in terms of process operation and spectra<br />
acquisition. The mean trajectory (green dashed line) is c<strong>all</strong>ed golden batch<br />
and represent the profile of optimal performance. Moreover, process limits<br />
(red dashed lines) can be defined, which are calculated by three times the<br />
standard deviation of the batches involved in the model. Other batches can<br />
be compared to the model. As long as the trajectory of a new batch stays<br />
within the limits, it can be assigned as statistic<strong>all</strong>y identical to the golden<br />
batch. A relevant process deviation will be notified if the trajectory is outside<br />
of the limits. Significant process deviations are shown in Figure 1 (middle).<br />
The trajectory of batch 3 (blue line) surpasses the process limits after 30 h.<br />
The reason for this was a bacterial contamination during the process. In batch<br />
2 (black line) a different aeration strategy was applied which resulted in a<br />
lower cell growth rate. In Figure 1 (bottom) a BEM based on the third<br />
principal component is shown. The model (dashed lines) is again generated<br />
from high performance batches as seen in the model above.<br />
Summary: The Ingold port adaption of a free beam NIR spectrometer is<br />
tailored for optimal bioprocess monitoring and control. The device shows<br />
an excellent signal to noise ratio dedicated to a large free aperture and<br />
thereforealargesamplevolume.Thiscanbeseenparticularlyinthe<br />
batch trajectories which show a high reproducibility. The robust and<br />
compact design withstands rough process environments as well as SIP/<br />
CIP cycles.<br />
Robust free beam NIR process analyzers are indispensable tools within<br />
the PAT/QbD framework for real-time process monitoring and control.<br />
They enable multiparametric, non-invasive measurements of analyte<br />
concentrations and process trajectories. Free beam NIR spectrometers are<br />
an ideal tool to define golden batches and process borders in the sense<br />
of QbD. Moreover, sophisticated data analysis both quantitative and<br />
MSPC yields directly to a far better process understanding. Information<br />
can be provided online in easy to interpret graphs which <strong>all</strong>ow the<br />
operator to make fast and knowledge-based decisions. This fin<strong>all</strong>y leads<br />
to higher stability in process operation, better performance and less<br />
failed batches.<br />
References<br />
1. Cervera A, Petersen N: Application of near- infrared spectroscopy for<br />
monitoring and control of cell culture and fermentation. Biotechnology<br />
Progress 2009, 25:1561-1581.<br />
2. Rodrigues L, Vieira L, Cardoso J P, Menezes JC: The use of NIR as a multiparametric<br />
in situ monitoring technique in filamentous fermentation<br />
systems. Talanta 2008, 75:1356-1361.<br />
3. Arnold SA, Crowley J, Woods N, Harvey LM, McNeil B: In-situ near infrared<br />
spectroscopy to monitor key analytes in mammalian cell cultivation.<br />
Biotechnology and bioengineering 2003, 84:13-19.<br />
4. Vaidyanathan S, Macaloney G, Harvey LM, McNeil B: Assessment of the<br />
Structure and Predictive Ability of Models Developed for Monitoring Key<br />
Analytes in a Submerged Fungal Bioprocess Using Near-Infrared<br />
Spectroscopy. Applied Spectroscopy 2001, 55:444-453.<br />
5. Henriques JG, Buziol S, Stocker E, Voogd A, Menezes JC: Monitoring<br />
Mammalian Cell Cultivations for Monoclonal Antibody Production Using<br />
Near-Infrared Spectroscopy. Optical Sensor Systems in Biotechnology Place:<br />
Springer, Berlin, Heidelberg: Rao G 2010, 2010:29-72.<br />
6. Hakemeyer C, Strauss U, Werz S, Jose GE, Folque F, Menezes JC: At-line NIR<br />
spectroscopy as effective PAT monitoring technique in Mab cultivations<br />
during process development and manufacturing. Talanta 2012, 90:12-21.<br />
P30<br />
Case study: biosimilar anti TNFalpha (Adalimumab) analysis of Fc<br />
effector functions<br />
Carsten Lindemann * , Silke Mayer, Miriam Engel, Petra Schroeder<br />
EUFETS GmbH, 55743 Idar-Oberstein, Germany<br />
E-mail: Carsten.Lindemann@eufets.com<br />
BMC Proceedings 2013, 7(Suppl 6):P30<br />
Background: For the development of biosimilar monoclonal antibodies<br />
or related substances containing the IgG Fc part it is mandatory to fully<br />
compare immunological properties between originator and biosimilar in a<br />
“comparability exercise” [1]. The important Fc associated functions<br />
to mediate antibody dependent cellular cytotoxicity (ADCC) and<br />
complement dependent cytotoxicity (CDC) need to be characterized<br />
using both the active substance of the biosimilar and the comparator<br />
[2,3]. For testing anti TNFalpha antibodies target cells with stable<br />
expression of membrane TNFalpha (mTNFalpha) is required. Further<br />
prerequisites are test systems facilitating analysis with high precision and<br />
accuracy.<br />
Materials and methods: We generated a human transgenic NK-cell line<br />
(YTE756.V#26, effector cell line) with stable expression of Fc gammareceptor<br />
IIIA (CD16, high affinity variant, valine at position 159) and stable<br />
functional characteristics to replace primary effector cells in ADCC assays.<br />
Target cells for ADCC and CDC assays were genetic<strong>all</strong>y modified for<br />
stable expression of mTNFalpha without the capability to release soluble<br />
TNFalpha. Both target and effector cells were generated using retroviral<br />
vectors to facilitate high and stable transgene expression. Vector particles<br />
were generated by transient transfection of 293T cells with plasmids<br />
encoding gag, pol/env and an expression plasmid containing the<br />
packaging region and the sequences of promotor and the transgenes, i.e.<br />
selection marker and gene of interest. Multiple gene expression was<br />
achieved either by using a bicistronic design enabling transcription from<br />
two promotor sequences, or by using an internal ribosomal entry site.<br />
Transduction of cells in log phase was followed by a selection of<br />
transduced cells and clonal selection by limiting dilution. Cell clones were<br />
expanded for primary and secondary cell banks and further characterised<br />
with regard to transgene expression and functional characteristics.<br />
The more complex ADCC assays were developed employing design of<br />
experiments (DoE). To show assay suitability goodness of fit, ratio of<br />
upper to lower asymptote, slope and par<strong>all</strong>elism was determined for each<br />
dose-response curve compared to a standard. Hypo- and hyperpotent<br />
samples (50%, 100%, 150% and 200% potency) of Adalimumab and<br />
Infliximab were analysed in both ADCC and CDC assays to determine<br />
accuracy and linearity of each method.<br />
For ADCC assays HT1080 mTNFalpha+ cells were seeded into 96-well plates<br />
18 - 20 h before start of the assay. Anti TNFalpha dilution series were<br />
performed in separate plates and transferred into the assay plate together<br />
with YTE756.V#26 effector cells at an E:T ratio of 10:1 using the effectors<br />
cell medium as assay medium. After an incubation time of 17 ± 1 h<br />
effector cells were washed from the adherent target cells. Quantification of<br />
residual target cells was performed by staining with XTT and photometric<br />
measurement. Each assay consists of standard (the biosimilar) and sample<br />
(originator) concentrations ranging from 1000 to 4.69 ng/ml in duplicates.<br />
Comparison of dose-response curves in a 4 PL model and determination of<br />
potency was performed using PLA software (Stegmann Systems).<br />
For CDC assays CHO mTNFalpha+ cells were seeded into 96 well plates<br />
20 - 25 h before start of the assay. Antibody dilution series were transferred<br />
into the assay plate using cell culture medium containing 20% native<br />
human serum pool. After an incubation time of 2 ± 0.5 h medium nonadherent<br />
cells were removed by washing the MTP. Quantification of residual<br />
cells was performed as described for ADCC assays. Each assay consists of<br />
standard and sample (originator or accuracy item) concentrations ranging<br />
from 5000 to 130 ng/ml in duplicates. Comparison of dose-response curves<br />
in a 4 PL model and determination of the relative potency was performed<br />
using PLA software.<br />
Originator batches and the biosimilar were analysed by monosaccharide<br />
and sialic acid analysis, N-glycan profiling by MALDI-MS (permethylated<br />
glycans) and by HILIC-HPLC. N-Glycosylation site determination was done<br />
by MALDI and/or LC-ESI-MS and MS/MS (1 digestion).<br />
Results: Both ADCC and CDC assays show good accuracy (relative accuracy<br />
< 15%) and linearity (r squared < 0.97). Precision of CDC assays (CV < 8%)<br />
was better than that of the more complex ADCC assays (< 15%). Due to the<br />
distinctly lower actitivity of Adalimumab compared to that of Infliximab we<br />
evaluated the most influential factor for gaining a high asymptote ratio by<br />
DoE.Theincubationtimewasshowntobemostimportantcomparedto<br />
other factors as effector to target cell ratio and fetal bovine serum content.<br />
We analysed different batches of originators and a biosimilar candidate<br />
molecule for functional variability in ADCC and CDC assays (Table 1). In CDC<br />
assays (n = 3) the three originator batches of Adalimumab showed<br />
comparable potency in between batches and compared to the biosimilar.
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Figure 1(abstract P29) Batch evolution models (BEM) based on NIR spectra. (Top): Batch trajectories from three batches based on the first principal<br />
component of NIR spectra. The golden batch trajectory is shown in green (mean value of <strong>all</strong> contributing batches) and the process limits are shown in<br />
red (three times the standard deviation of the three contributing batches). (Middle): Compared to the BEM other batches show deviations which can be<br />
assigned to contaminations (blue line) or low cell growth rate (black line). (Bottom): Batch trajectories from three batches based on the third principal<br />
component of NIR spectra. Compared to the BEM other batches show deviations like contaminations (blue and violet line) or early glucose limitation<br />
which led to an early drop of viability (black, yellow and violet line).
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Page 48 of 151<br />
Table 1(abstract P30) Relative potency (compared to<br />
biosimilar) of originators in ADCC and CDC assays<br />
Assay Originator Relative potency CV<br />
ADCC 2 140% 9.9%<br />
3 141% 11.1%<br />
4 135% 16.8%<br />
CDC 2 92% 15.1%<br />
3 89% 10.2%<br />
4 89% 16.7%<br />
A higher variability of the originators was found in ADCC assays (n = 6)<br />
besides the potency was higher than that of the Adalimumab biosimilar.<br />
Major differences between originators with regard to glycosylation were not<br />
found. The biosimilar showed a high galactose content and consequently a<br />
higher percentage of galactosylated glycan structures than the originators.<br />
Conclusions: In summary we show the suitability of an ADCC potency<br />
assay for investigation of functional comparability of Adalimumab and<br />
biosimilar candidate substances. Differences between biosimilar and<br />
originators in glycosylation might contribute to differences found in the<br />
ADCC potency assay but not with the CDC potency assay.<br />
References<br />
1. Guideline in similar biological medicinal products containing<br />
monoclonal antibodies. , EMA/HCMP/BMWP/403543/2010.<br />
2. Guideline on development, production, characterisation and<br />
specifications for mnoclonal antibodies and related products. , EMEA/<br />
CHMP/BWP/157653/2007.<br />
3. ICHQ6B Test procedures and acceptance criteria for biotechnological/<br />
biological products. , CMP/ICH/365/96.<br />
P31<br />
Cellular tools for biosimilar mAb analysis<br />
Carsten Lindemann * , Silke Mayer, Miriam Engel, Petra Schroeder<br />
EUFETS GmbH, 55743 Idar-Oberstein, Germany<br />
E-mail: Carsten.Lindemann@eufets.com<br />
BMC Proceedings 2013, 7(Suppl 6):P31<br />
Background: For the development of biosimilar monoclonal antibodies<br />
(mAb) or related substances containing the IgG Fc part it is mandatory to<br />
fully compare immunological properties between originator and biosimilar<br />
in a “comparability exercise” [1]. The most complex Fc associated function<br />
to mediate antibody dependent cellular cytotoxicity (ADCC) needs to be<br />
characterized using the active substance of the biosimilar and the<br />
comparator. From a regulatory point of view potency assays should reflect<br />
the proposed mode of action but in vitro ADCC assays are considered<br />
difficult to validate due to the variability of the primary effector cells [2,3].<br />
The requirement to test for ADCC with high precision and accuracy is<br />
ch<strong>all</strong>enging. Design of cell lines to replace primary cells for effector or<br />
target cells is a solution to provide tools for standardized and extensive<br />
biosimilar testing.<br />
Materials and methods: Retroviral vectors were used to generate cell<br />
lines with stable genetic modification. Vector particles were generated by<br />
transient transfection of 293T cells with plasmids encoding gag, pol/env<br />
and an expression plasmid containing the packaging region and the<br />
sequences of promotor and the transgenes, i.e. selection marker and gene<br />
of interest. Multiple gene expression was achieved either by using a<br />
bicistronic design enabling transcription from two promotor sequences, or<br />
by using an internal ribosomal entry site. Transduction of cells in log phase<br />
was followed by a selection of transduced cells and clonal selection by<br />
limiting dilution. Cell clones were expanded for primary and secondary cell<br />
banks and further characterised with regard to transgene expression and<br />
functional characteristics. We developed a human transgenic NK-cell line<br />
(YTE756.V#26, effector cell line) with stable expression of Fc gammareceptor<br />
IIIA (CD16, high affinity variant, valine at position 159) and stable<br />
functional characteristics. Target cell lines were generated similarly using<br />
different expression plasmid constructs.<br />
ADCC assays were developed by using design of experiments (DoE) to<br />
determine experimental factors of importance for assay suitability. To show<br />
assay suitability goodness of fit, the amplitude of sigmoid curve, slope and<br />
par<strong>all</strong>elism was determined for each sample compared to a standard. Hypoand<br />
hyperpotent samples (50%, 100%, 150% and 200% potency) of<br />
Rituximab, Trastuzumab, Adalimumab and Infliximab were analysed to<br />
determine accuracy and linearity of each method. Optimisation of each<br />
assay requires determining the relative importance of factors including E:T<br />
ratio, incubation time, target cell density and pre-assay schedules for target<br />
and effector cells. Analysis of critical factor interaction was performed using<br />
Minitab software. A list of established ADCC assays is shown in Table 1.<br />
CD16 expression was analyzed and quantified by flow cytometry. Cells<br />
were stained using anti-CD16 PE-conjugated antibodies. PE-fluorescence<br />
was correlated to number of PE-molecules per cell using BD Quantibrite<br />
beads. Primary NK-cells were isolated using Dynal beads (purity > 95%)<br />
from 3 healthy donors and used immediately after isolation.<br />
Results: In order to prove genetic stability of the transgenic NK cell line<br />
CD16 expression was analysed by flow cytometry for up to 22 passages.<br />
More than 95% of cells were CD16 positive, viability of cells was >90%.<br />
CD16 expression level was stable (19.000 - 28.000 CD16 molecules/cell).<br />
Functional stability of the effector cell line was shown for more than 30<br />
passages. This was shown by a stable EC50 value obtained for a reference<br />
antibody in the Trastuzumab ADCC assay.<br />
The effector cell line was compared with primary NK-cells (purity > 95%)<br />
from 3 donors in a Trastuzumab ADCC assay. The data show high donor<br />
variability, mostly incomplete dose-response curves and a killing activity<br />
with a low dynamic range (baseline to top ratio: 3). For primary NK-cells<br />
the amplitude of the dose-response curve is dependent on both donor<br />
variability and the type of target cell. Using the effector cell line this is<br />
dependent on the target cell only. Assay variability was strongly reduced<br />
and sample throughput could strongly be increased by using the effector<br />
cell line in comparison to primary NK-cells. Optimization of each assay by<br />
DoE required determining the relative importance of various factors<br />
including effector to target cell ratio, incubation time, target cell density<br />
and pre-assay culture schedules for target and effector cells. Accuracy of<br />
these ADCC assays could be shown in between a range of 50% to 200%<br />
potency. Linearity was shown by a high coefficient of determination<br />
(>0.97) and other statistical methods. Inter-assay precision of <strong>all</strong> ADCC<br />
assays was
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Page 49 of 151<br />
Figure 1(abstract P31) Analysis of accuracy and linearity of the Infliximab ADCC assay. Data shown are sample dose response curves (left)<br />
determined by 4PL analysis and mean rel. potency +/- SD (dot, n = 3) compared to the standard (right).<br />
For precision analysis the relative potency of a sample was repeatedly<br />
analyzed on 4 days with 3 assays per day.<br />
Conclusions: Altogether these data show the feasibility of providing<br />
suitable tools for validation and routine testing of various mAbs in ADCC<br />
potency assays scalable to the analytical needs of biosimilar testing.<br />
References<br />
1. Guideline in similar biological medicinal products containing<br />
monoclonal antibodies. EMA/HCMP/BMWP/403543/2010.<br />
2. Guideline on development, production, characterisation and<br />
specifications for mnoclonal antibodies and related products.<br />
EMEA/CHMP/BWP/157653/2007.<br />
3. ICHQ6B Test procedures and acceptance criteria for biotechnological/<br />
biological products. CMP/ICH/365/96.<br />
P32<br />
The successful transfer of a modern CHO fed-batch process to different<br />
single-use bioreactors<br />
Sebastian Ruhl * , Ute Husemann, Elke Jurkiewicz, Thomas Dreher,<br />
Gerhard Greller<br />
Sartorius Stedim Biotech GmbH, D-37079 Göttingen, Germany<br />
E-mail: sebastian.ruhl@sartorius-stedim.com<br />
BMC Proceedings 2013, 7(Suppl 6):P32<br />
Introduction: Nowadays, single-use bioreactors are widely accepted in<br />
pharmaceutical industry. This is based on shorter batch to batch times,<br />
reduced cleaning effort and a significantly lower risk of cross contaminations<br />
[1,2]. One large field of the application of single-use bioreactors is the seed<br />
train cultivation of mammalian cells [1]. The focus is further extended to<br />
perform state of the art fed-batch production processes in such bioreactors.<br />
In this study an industrial proven CHO fed-batch process is established in<br />
different single-use and reusable bioreactors.<br />
Materials and methods: Cell line, medium and process strategy: For<br />
the fed-batch process the cell line CHO DG44 (Cellca, Germany) secreting<br />
human IgG1 was used. SMD5 medium (Cellca, Germany) was prepared for<br />
the seed train and PM5 medium (Cellca, Germany) as a basal medium for<br />
the fed-batch culture. The feeding procedure comprised the addition of<br />
three different feeds (feed medium A, feed medium B and concentrated<br />
glucose solution). After a 3 day batch phase, the 14 day fed-batch phase<br />
started. The automated discontinuous bolus feed of feed media A and B was<br />
supplemented by the glucose feed solution to keep the glucose<br />
concentration above 3 g/L.<br />
Bioreactors: The process was initi<strong>all</strong>y developed in a 5 L stirred glass<br />
bioreactor therefore the BIOSTAT® B with a UniVessel® 5 L was considered as<br />
a reference. Single-use bioreactors involved in this study were the stirred<br />
tank reactor BIOSTAT® STR 50 L with a CultiBag STR 50 L and the rocking<br />
motion bioreactor BIOSTAT® RM 50 optical with CultiBag RM 50 L.<br />
Process transfer: The used bioreactors were characterized in terms of<br />
process engineering [3]. Due to different agitation and gassing principles<br />
present in the BIOSTAT® STR and RM the k L a and mixing times were<br />
chosen as a scale-up criteria. The process conditions were specified to<br />
meet a k L a-value of > 7 h -1 [4] and a mixing time of < 60 s [5].<br />
Sampling procedure: A daily sampling procedure was performed before<br />
the bolus feed. Metabolites like glucose and lactate were analyzed by the<br />
Radiometer ABL800 basic (Radiometer, Germany). Viable cell density (VCD)<br />
and viability were determined by the Cedex HiRes (Roche Diagnostics,<br />
Germany).<br />
Results: The process transfer is considered successful, if comparable<br />
cellular proliferation activities and product titers are obtained.<br />
The initial viable cell density in <strong>all</strong> systems was 0.3 - 0.4 × 10 6 cells/mL. At the<br />
start of the fed-batch phase a viable cell density of 4 - 5 × 10 6 cells/mL could<br />
be achieved. As seen in Figure 1A the viable cell density peak of 27 - 28 ×<br />
10 6 cells/mL was reached in <strong>all</strong> systems after 8 - 9 days. At the point of<br />
harvest after 17 days viable cell densities between 12 - 17 × 10 6 cells/mL<br />
and viabilities of 57 - 82% were reached. The cell broth was harvested for<br />
further downstream operations.<br />
A well-controlled pH value is essential for a reproducible cell proliferation. As<br />
seen in Figure 1B exemplarily shown for the BIOSTAT® STR 50 L sm<strong>all</strong> peaks<br />
occurred due to the daily addition of feed medium B (pH 11). The offline<br />
measured pCO 2 trend shows a constant decrease during the batch phase<br />
followed by an increase during the fed-batch phase with a maximum value<br />
of 135 mmHg.
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Figure 1(abstract P32) Process trends.<br />
Shown in Figure 1D glucose concentration could be kept above 3 g/L in<br />
the fed-batch phase. Lactate had a peak accumulation of 0.9 g/L at the<br />
end of the batch phase and remained at low value afterwards.<br />
The product yield in <strong>all</strong> cultivations was comparable to the reference<br />
systems and exceeded 8 g/L IgG (Figure 1C).<br />
Conclusion: The high cell density CHO fed-batch process with industry<br />
relevant titers was successfully transfer from a reference bioreactor to a<br />
variety of single-use bioreactor systems.<br />
The k L a and mixing time were suitable as a scale-up criteria for systems<br />
with different agitation principles.<br />
Acknowledgements: My thanks go to the complete Upstream<br />
Technology-team at Sartorius Stedim Biotech Göttingen.<br />
References<br />
1. Brecht R: Disposable Bioreactors: Maturation into Pharmaceutical<br />
Glycoprotein Manufacturing. Adv Biochem Engin/Biotechnol 2009, 115:1-31.<br />
2. Eibl D, Peuker T, Eibl R: Single-use equipment in biopharmaceutical<br />
manufacture: a brief introduction. Wiley, Hoboken: Eibl R., Eibl D 2010,<br />
Single-use technology in biopharmaceutical manufacture.<br />
3. Löffelholz C, Husemann U, Greller G, Meusel W, Kauling J, Ay P, Kraume M,<br />
Eibl R, Eibl D: Bioengineering Parameters for Single-Use Bioreactors:<br />
Table 1(abstract P32) Bioreactor Setup and Process Parameters<br />
BIOSTAT® RM 50 L STR 50 L B 5 L<br />
Gassing principle Overlay Ring Sparger<br />
Sensors Single-use optical patches Reusable probes<br />
Working volume [L] 25 50 5<br />
Initial volume [L] 13 26 2.6<br />
pH set point 7.15<br />
pH control<br />
CO 2 gassing<br />
pO 2 set point<br />
60% sat.<br />
pO 2 control<br />
Multi stage cascade comprising N 2 , Air, O 2 - gassing<br />
Agitation [rpm] 30 @ 10° rocking angle 150 400
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Page 51 of 151<br />
Overview and Evaluation of Suitable Methods. Chem Ing Tech 2013,<br />
85:40-56.<br />
4. Ruhl S, Dreher T, Husemann U, Greller G: Design space definition for a<br />
stirred single-use bioreactor family from 50 to 2000 L scale. Poster ESACT<br />
Lílle 2013.<br />
5. Lara AR, Galindo E, Ramírez OT, Palomares LA: Living with Heterogeneities<br />
in Bioreactors. Mol Biotechnol 2006, 34:355-381.<br />
P33<br />
Differences in the production of hyperglycosylated IFN alpha in CHO<br />
and HEK 293 cells<br />
Agustina Gugliotta, Marcos Oggero Eberhardt, Marina Etcheverrigaray,<br />
Ricardo Kratje, Natalia Ceaglio *<br />
Cell Culture Laboratory, School of Biochemistry and Biological Sciences,<br />
Universidad Nacional del Litoral. Ciudad Universitaria - C.C. 242 - (S3000ZAA)<br />
Santa Fe, Provincia de Santa Fe, Argentina<br />
E-mail: nceaglio@fbcb.unl.edu.ar<br />
BMC Proceedings 2013, 7(Suppl 6):P33<br />
Background: IFN alpha is an important cytokine of the immune system. It<br />
has the ability to interfere with virus replication exerting antiviral activity.<br />
Moreover, it displays antiproliferative activity and can profoundly modulate<br />
the immune response. IFN4N (or hyperglycosylated IFN alpha) is an IFNalpha2b<br />
mutein developed in our laboratory using glycoengineering<br />
strategies. This molecule contains 4 potential N-glycosylation sites together<br />
with the natural O-glycosylation site in Thr106 [1]. The resulting N- and<br />
O-glycosylated protein shows higher apparent molecular mass and longer<br />
plasmatic half-life compared to the non-glycosylated IFN-alpha produced in<br />
bacterial systems and used for clinical applications. As a consequence, the<br />
correct glycosylation of our modified cytokine is very important for its<br />
in vivo activity. For this reason, it is of great relevance the evaluation of<br />
different mammalian host cells for its production. While hamster-derived<br />
CHO cells are widely used for large scale production of recombinant<br />
therapeutic glycoproteins, human HEK cells are a promising system because<br />
they are easy to grow and transfect [2]. In this work, we performed a<br />
comparison between both production systems in terms of cell growth,<br />
culture parameters and specific productivity of hyperglycosylated IFN alpha.<br />
Results: Lentiviral vectors containing the sequence of IFN4N were<br />
assembled and employed for the transduction of CHO-K1 and HEK 293T<br />
cells. The recombinant cell lines were subjected to a process of selective<br />
pressure using increasing concentrations of puromycin. The CHO-IFN4N<br />
and HEK-IFN4N producing cell lines resistant to the highest concentration<br />
of puromycin showed the highest productivity of IFN4N. In particular, the<br />
CHO-IFN4N cell line was resistant to 350 μg/ml of puromycin and it<br />
showed a specific productivity of 817 ± 134 ng.10 6 cell -1 .day -1 ,which<br />
represents an 8-fold increment compared to the parental line. The<br />
HEK-IFN4N cell line was resistant to 200 ug/ml of puromycin and<br />
showed a 15-fold increment in the specific productivity compared to the<br />
parental line, reaching a value of 1,490 ± 332 ng.10 6 cell -1 .day -1 .Inboth<br />
cases, complete culture death was achieved at higher puromycin<br />
concentrations. The specific productivity of IFN4N of HEK 293T cell line<br />
duplicated the value obtained for the CHO-K1 cell line, and it was<br />
achieved at a lower concentration of puromycin, making the selection<br />
process shorter (Figure 1).<br />
Both cell lines were cloned using the limiting dilution method, and after<br />
15 days of culture more than 100 clones were screened. To achieve the<br />
characterization and study both cell lines as recombinant protein expression<br />
hosts, the 6 best producer clones were isolated and amplified. The adherent<br />
clones were grown for 7 days in order to construct their growth curves. Cell<br />
density and viability were determined every 24 h by trypan-blue exclusion<br />
method and the culture supernatant was collected to determine IFN4N and<br />
metabolites concentration. The IFN4N production was assessed employing a<br />
sandwich ELISA assay developed in our laboratory. Glucose consumption<br />
and lactate production were evaluated using specific Reflectoquant® test<br />
strips (Merck Millipore) in a RQflex® Reflectometer (Merck Millipore). Levels<br />
of amonium in the culture supernatant were determined by the Berthelot<br />
reaction.<br />
As shown in Table 1, the average specific growth rates of CHO and HEK<br />
clones were similar. However, CHO clones reached higher maximum cell<br />
densities (between 7.10 5 -1.5.10 6 cell.ml -1 ) than HEK clones (between 6.10 5 -<br />
9.10 5 cell.ml -1 ), probably because of space limitation and higher glucose<br />
consumption, since average q gluc of HEK clones was higher (see Table 1). No<br />
differences were observed between lactate and ammonium production of<br />
both groups of clones. In contrast, specific production rate of IFN4N was<br />
higher for the clones derived from the human cell line. Moreover, higher<br />
average IFN4N cumulative production for HEK clones was achieved after<br />
7 days of culture (3,494 versus 5,961 ng.ml -1 ).<br />
Conclusion: CHO and HEK cells were genetic<strong>all</strong>y modified to produce<br />
IFN4N by using lentiviruses as a tool for the IFN4N gene transfer. Since both<br />
cell lines expressed high levels of IFN4N, 6 clones were amplified for an<br />
intensive characterization. Culture and production properties of both groups<br />
of clones were very different. On the one hand, CHO clones were easy to<br />
maintain in culture for a long period of time, reaching higher cell densities<br />
than HEK clones. On the other hand, the best specific productivity of IFN4N<br />
was achieved employing HEK cells. The behavior of CHO and HEK cells at<br />
large scale production should be analyzed in order to select the proper<br />
system for the cytokine’s production.<br />
Wide differences have been observed between the glycosylation profile<br />
of the same recombinant therapeutic protein produced in CHO and HEK<br />
systems [2]. Considering that glycosylation affects protein bioactivity,<br />
stability, pharmacokinetics and immunogenicity, it would be very<br />
important to evaluate the characteristics of the IFN4N produced in both<br />
hosts to determine their efficacy as therapeutic agents.<br />
Figure 1(abstract P33) Comparison between the specific productivity of the CHO-IFN4N (a) and HEK-IFN4N (b) producing cell lines as a function of<br />
puromycin concentration.
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Table 1(abstract P33) Determination of the specific cell growth rate, specific production rate of lactate, ammonium<br />
and IFN4N, and specific consumption rate of glucose of CHO-K1 (a) and HEK 293T (b) clones<br />
a)<br />
Clones μ(h -1 ) q IFN<br />
(ng.10 -6 cell.h -1 )<br />
q gluc<br />
(μg.10 -6 cell.h -1 )<br />
q lac<br />
(μg.10 -6 cell.h -1 )<br />
q amon<br />
(nmol.10 -6 cell.h -1 )<br />
P4D3 0,0182 ± 0,002 79 ± 8 36 ± 5 40 ± 5 0,027 ± 0,007<br />
P1E9 0,0196 ± 0,002 35 ± 4 24 ± 4 45 ± 5 0,027 ± 0,003<br />
P2A9 0,0249 ± 0,001 41 ± 4 30 ± 2 31 ± 1 0,014 ± 0,004<br />
P1B6 0,0240 ± 0,002 19 ± 3 26 ± 5 49 ± 5 0,013 ± 0,005<br />
P1B7 0,0191 ± 0,002 41 ± 3 32 ± 4 35 ± 2 0,017 ± 0,006<br />
P1B8 0,0277 ± 0,002 42 ± 3 21 ± 3 34 ± 2 0,015 ± 0,004<br />
b)<br />
Clones μ(h -1 ) q IFN<br />
(ng.10 -6 cell.h -1 )<br />
q gluc<br />
(μg.10 -6 cell.h -1 )<br />
q lac<br />
(μg.10 -6 cell.h -1 )<br />
q amon<br />
(nmol.10 -6 cell.h -1 )<br />
P2A5 0,020 ± 0,001 129 ± 10 56 ± 6 37 ± 4 0,014 ± 0,003<br />
P2C7 0,015 ± 0,002 122 ± 13 62 ± 11 38 ± 3 0,009 ± 0,003<br />
P2G11 0,017 ± 0,002 82 ± 6 47 ± 9 31 ± 3 0,008 ± 0,002<br />
P3B7 0,016 ± 0,002 99 ± 8 55 ± 8 31 ± 3 0,008 ± 0,003<br />
P3H8 0,027 ± 0,001 82 ± 11 46 ± 6 34 ± 3 0,008 ± 0,001<br />
P4B4 0,017 ± 0,002 63 ± 5 61 ± 15 32 ± 3 0,009 ± 0,001<br />
References<br />
1. Ceaglio N, Etcheverrigaray M, Conradt HS, Grammel N, Kratje R, Oggero M:<br />
Highly glycosylated human alpha interferon: An insight into a new<br />
therapeutic candidate. J Biotechnol 2010, 146:74-83.<br />
2. Croset A, Delafosse L, Gaudry JP, Arod C, Gleza L, Losbergera C, Beguea C,<br />
Krstanovicb A, Robertb F, Vilboisa F, Chevaleta L, Antonssona B: Differences<br />
in the glycosylation of recombinant proteins expressed in HEK and CHO<br />
cells. J Biotechnol 2012, 161:336-348.<br />
P34<br />
Developing an upstream process for a monoclonal antibody including<br />
medium optimization<br />
Sevim Duvar * , Volker Hecht, Juliane Finger, Matthias Gullans, Holger Ziehr<br />
Pharmaceutical Biotechnology, Fraunhofer Institute for Toxicology and<br />
Experimental Medicine (ITEM), Braunschweig, Germany<br />
E-mail: sevim.duvar@item.fraunhofer.de<br />
BMC Proceedings 2013, 7(Suppl 6):P34<br />
Background: Monoclonal antibodies have been established as important<br />
therapeutics in cancer and autoimmune diseases. Hence, there is a<br />
growing interest in the production of monoclonal antibodies in<br />
pharmaceutical industry. In order to reduce timelines and costs of<br />
production the process and medium development is of central importance.<br />
Perfusion processes are well known to achieve higher productivities<br />
compared with batch or fed batch. Major advantages of perfusion culture<br />
are that you can keep optimal culture medium conditions for the cells and<br />
realize higher performance. However, obtaining high performance requires<br />
the combination of process optimization as well as a well-balanced<br />
concentrated culture medium. Selecting the best system also depends on<br />
the shear sensitivity of the cell line, the robustness of the process and the<br />
scale used.<br />
In upstream processing batch, fed batch and perfusion mode were applied.<br />
Design of Experiments (DoE) was used to develop a feed protocol for fed<br />
batch cultivations. In shake flask experiments the influence of temperature,<br />
osmolality, and pH to improve antibody yield was examined.<br />
In a further study we compared different cell retention systems with regard<br />
to achieve high viable cell densities in a short time like required for a seed<br />
train application. The best results were achieved with the ATF system with<br />
cell densities up to 1.3 × 10 8 cells/mland4foldimprovedproduct<br />
concentration compared to batch culture.<br />
Materials and methods: A CHO cell line producing the antibody G8.8<br />
against Epithelial Cell Adhesion Molecule (Ep-CAM) was employed for the<br />
experiments performed in this study. The fermenters were Sartorius BBI<br />
Twin-System (2- and 5 L culture volume). We compared five different<br />
retention systems: SpinFilter (Sartorius BBI Systems), Cell Settler<br />
(Biotechnology Solutions), Centritech Lab III (Pneumatic Scale), Biosep<br />
(Applikon) and ATF (Alternate Tangential Flow; Refine Technology). The cell<br />
count was performed with CEDEX cell counter (Roche Diagnostics). The<br />
monoclonal antibody was quantified with HPLC-method using Protein A-<br />
column. Design of Experiments (DoE) was used to develop a feed protocol<br />
for Perfusion cultivations. In shake flask experiments we examined the<br />
influence of temperature, osmolality, and pH to improve antibody yield.<br />
Results: Fed batch development in shake flasks with DoE: For the<br />
development of fed batch in shake flasks we used D-optimal Design with<br />
18 runs. The examined factors were: Feed volume, time of feed start, time of<br />
temperature shift (33°C) and time of Osmolality shift (450 mOsmol/kg). The<br />
response was maximum antibody titer. The results show that the optimal<br />
feed volume is 15 ml/d. The time point for feeding start has almost no<br />
influence. The temperature shift and osmolality shift have negative influence<br />
(data not shown).<br />
Comparison of cultivations with different retention systems: We<br />
compared five different cell retention systems under same cultivation<br />
conditions. The best results could be achieved with the ATF system with cell<br />
densities up to 1.3 × 10 8 cells/ml. The next best retention systems were<br />
the Centrifuge and the Cell Settler with cell densities reached up to 3 ×<br />
10 7 cells/ml. Using BioSep and Spinfilter, cell densities up to 2 × 10 7 cells/ml<br />
were obtained (data not shown). The Spin filter and BioSep showed break<br />
through of cells at cell densities > 2 × 10 7 cells/ml. In contrast, the Cell<br />
Settler had the advantage of simplicity and robustness and no moving parts.<br />
The advantage of the centrifuge was the high flexibility concerning the<br />
reactor-volume to be perfused. The Spinfilter and BioSep showed the lowest<br />
performance.<br />
Comparison of cultivations with ATF: In a study we compared ATF<br />
cultivations with 0.2 μm membrane and with 50 kDa membrane. In<br />
cultivations with the 0.2 μm membrane a maximum cell density with 6.4 ×<br />
10 7 cells/ml could be achieved compared to a maximum cell density of 1.3 ×<br />
10 8 cells/ml with the 50 kDa membrane as shown in Figure 1. The increased<br />
cell densities resulted in a higher productivity compared to the other cell<br />
retention systems. Furthermore, the ATF with 50 kDa retended not only the<br />
cells but also the antibody within the reactor. Therefore, a higher volumetric<br />
productivity could be achieved with the 50 kDa membrane. The maximum<br />
titer in the reactor with the 50 kDa membrane was 4 fold higher compared<br />
with the 0.2 μm membrane.<br />
Viable cell densities (VCD) and product concentrations of the monoclonal<br />
antibody (MAB) are shown.
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Figure 1(abstract P34) Comparison of cultivations with ATF 0.2 μm and 50 kDa membrane.<br />
Conclusions: We have demonstrated that perfusion processes have a<br />
higher productivity compared to batch or fed batch processes. In our<br />
study the best retention system for perfusion culture was the ATF system<br />
compared with SpinFilter, Cell Settler, Centritech Lab III and Biosep. With<br />
the ATF system we realized cell densities up to 1.3 × 10 8 cells/ml and<br />
4foldimprovedproductconcentration compared to batch culture. Also,<br />
the ATF with a 50 kDa membrane retended not only the cells but also the<br />
antibody within the reactor. Therefore, a higher volumetric productivity<br />
couldbeachievedwiththe50kDamembrane. In perfusion culture the<br />
cells show constant specific productivity over the whole perfusion phase<br />
which shows that the cells are well fed.<br />
P35<br />
Development and evaluation of a new, speci<strong>all</strong>y tailored CHO media<br />
platform<br />
Tim F Beckmann * , Christoph Heinrich, Heino Büntemeyer, Stefan Northoff<br />
TeutoCell AG, Bielefeld, 33613, Germany<br />
E-mail: Tim.Beckmann@teutocell.de<br />
BMC Proceedings 2013, 7(Suppl 6):P35<br />
Background: Today’s biopharmaceutical industry is under increasing<br />
pressure considering cost efficient development. Short timeframes rule<br />
the progress starting from the generation of producer cell lines to the<br />
establishment of a final production process. Hence, the timescale for<br />
optimization of cell culture media is sm<strong>all</strong>, but on the other hand it<br />
contains high potential for global process improvement. In this scope, our<br />
speci<strong>all</strong>y tailored media development platform, which <strong>all</strong>ows a fast and<br />
reliable introduction of high-performance basis media and feeds,<br />
establishes new perspectives for an efficient process development.<br />
Materials and methods: For the design and development of TeutoCell’s<br />
new media platform various cell lines and expression systems were<br />
comprehensively analyzed and incorporated. The results gained from<br />
cultivations and extensive analysis of culture supernatant and e.g. product<br />
glycosylation were integrated in a cyclic development strategy, utilizing<br />
theoretical and empirical formulation optimizations. Special applications<br />
like single clone selection were integrated into our platform as well.<br />
The cell lines used for the development of our media platform include CHO-<br />
DG44, CHO-GS and CHO-K1 clones. Cultivations were carried out in shaking<br />
flasks as well as closed-loop controlled 0.5 - 2.0 L bioreactor systems in batch<br />
und fed-batch mode using standard conditions. An industri<strong>all</strong>y relevant,<br />
protein-free and chemic<strong>all</strong>y defined medium was used as a reference.<br />
Media development for single clone selection by limited dilution was<br />
performed with different CHO suspension cells in microtiter plates (from<br />
96- to 6-wells) up to shaking flaks. Analysis of single clone colonies was<br />
done with a Cellscreen System.
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Samples of a model antibody produced in commerci<strong>all</strong>y available CHO<br />
reference medium and TeutoCell’s platform medium using two different<br />
producer clones were desalted, denaturated and treated with PNGaseF.<br />
Glycanswereconcentratedviasolid-phaseextractionandanalyzedby<br />
MALDI-TOF mass spectrometry. Signal-to-noise ratios of specific masses<br />
were used for calculations of relative amounts.<br />
Results: The performance of the platform medium was evaluated using a<br />
set of eleven different CHO cell lines in comparison to an industri<strong>all</strong>y<br />
relevant, protein-free and chemic<strong>all</strong>y defined medium. For <strong>all</strong> tested cell<br />
lines, the maximum viable cell density (vcd) as well as the integrated viable<br />
cell density (ivcd) and the product titer were higher compared to the<br />
reference. In numbers, the improvement in vcd ranged between a factor of<br />
1.7 and 2.5, in ivcd between a factor of 1.2 and 4.2 and in product titer<br />
between a factor of 1.4 and 2.7 in batch cultures. By this improvement viable<br />
cell densities of up to 17.53·10 6 cells/mL and product titer of 1015 mg/L<br />
were reached. An overview of these results is illustrated in Figure 1.<br />
Furthermore, the potential influence of the utilized medium on product<br />
glycosylation was examined. For this, antibody harvest from two different<br />
clones cultivated in reference and platform medium was analyzed. The<br />
results of relative quantification of glycan structures by mass spectrometry<br />
showed highly comparable profiles for the reference and platform<br />
medium. An overview of the glycoanalysis is given in Table 1.<br />
As an additional application, the platform medium was successfully<br />
utilized as a basis for a chemic<strong>all</strong>y defined cloning medium in limited<br />
dilution experiments. For different cell lines single cell growth was<br />
achieved and cells were effectively expanded from 96-well plate format<br />
up to shaking flask cultures.<br />
Conclusions: Within this work a chemic<strong>all</strong>y defined and animal-component<br />
free media platform was successfully implemented, which supports high<br />
performance growth and productivity without supplementation of proteins<br />
or growth hormones. In addition, its streamlined formulation of less than<br />
50 components increases the design space for the efficient development of<br />
custom formulations. The suitability as a platform medium was verified by<br />
the successful cultivation of a wide range of cell lines including CHO-DG44,<br />
CHO-GS and CHO-K1 clones and the feature of easy adaption from serum<br />
containing and commerci<strong>all</strong>y available formulations. For <strong>all</strong> tested cell lines<br />
stable high performance cultivations with high product yields were<br />
achieved, with consistent glycosylation profiles. As a further field of<br />
application, the platform medium provides the basis for single cell growth<br />
following limited dilution.<br />
Acknowledgements: Parts of this work were financi<strong>all</strong>y supported by the<br />
German Federal Ministry of Education and Research - BMBF (#031A106).<br />
Responsibility for the content lies with the author.<br />
P36<br />
Streamlined process development using the Micro24 Bioreactor system<br />
Steve RC Warr * , John PJ Betts, Shahina Ahmad, Katy V Newell, Gary B Finka<br />
Upstream Process Research, GlaxoSmithKline, Stevenage, SG1 2NY, UK<br />
E-mail: steve.r.warr@gsk.com<br />
BMC Proceedings 2013, 7(Suppl 6):P36<br />
Introduction: The P<strong>all</strong> Micro24 Bioreactor system is one of several<br />
microbioreactor systems that have been commercialised in recent years<br />
in response to the demand to reduce costs and shorten process<br />
development time lines.<br />
We have previously demonstrated that the Micro24 Bioreactor system can<br />
be integrated successfully into the later stages of cell line screening<br />
programmes and that the results correlate well with those from more<br />
conventional methods [1]. Further process development for these<br />
selected cell lines tradition<strong>all</strong>y utilises bench top bioreactors to define<br />
appropriate process conditions giving the desired process outcomes<br />
Figure 1(abstract P35) Comparison of growth performance (A) and product titer (B) using the reference and platform medium. To illustrate the<br />
improvement in relation to the reference medium, the mean of 5 producer- and the corresponding parental cell line were normalized to the results<br />
obtained in the reference medium (C and D). The error bars show the deviation between the different cell lines. The development progress of the<br />
platform medium is represented by one major interstage.
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Table 1(abstract P35) Comparison of the glycosylation pattern of a model antibody produced in two different cell<br />
lines using the reference medium and the platform medium<br />
Structure Relative Amount of Glycan Structure [%]<br />
High Producer 1 High Producer 2<br />
Reference<br />
Shaker<br />
Platform<br />
Shaker<br />
Platform<br />
Bioreactor<br />
Reference<br />
Shaker<br />
Platform<br />
Shaker<br />
Platform<br />
Bioreactor<br />
Man3 - - - 4 ± 2 1 ± 2 -<br />
Man5 7 ± 2 3 ± 2 5 ± 2 12 ± 3 9 ± 2 4 ± 1<br />
G0F-GlcNAc 2 ± 2 2 ± 1 4 ± 2 10 ± 4 5 ± 0 2 ± 2<br />
G0F 46 ± 2 50 ± 5 47 ± 5 47 ± 5 47 ± 2 47 ± 5<br />
G1 6±3 6±2 8±2 3±1 9±1 5±1<br />
G1F 30 ± 3 32 ± 5 29 ± 7 19 ± 2 23 ± 3 35 ± 2<br />
G2F 9±2 7±2 7±2 5±1 6±1 7±2<br />
The relative amounts of detected structures are given in % with the standard deviation of four independent analyses.<br />
although this approach can be time consuming and resource intensive.<br />
However the Micro24 Bioreactor system <strong>all</strong>ows up to 24 different process<br />
conditions to be run concurrently thereby facilitating efficient process<br />
development.<br />
This work describes the use of the Micro24 Bioreactor system to identify<br />
improved process conditions for different cell lines and their subsequent<br />
validation in bioreactors.<br />
Micro-24 bioreactor system (P<strong>all</strong>): This system comprises 24 bioreactors<br />
(7 ml working volume) each capable of independent temperature,<br />
dissolved oxygen and pH control. The main limitation of the system is the<br />
lack of automation meaning that any feed additions or sample removal<br />
must be made manu<strong>all</strong>y and similarly, for mammalian cultures, upwards<br />
pH control is achieved by the manual addition of NaHCO 3 .<br />
Engineering characterisation studies carried out at UCL (data not shown)<br />
have shown how conditions within the individual Micro24 chambers<br />
compare with those in bioreactors and recent results also indicate that<br />
the selection of the Micro24 plate type is critical in ensuring good<br />
correlation with performance in traditional bioreactors.<br />
Within the Micro24 Bioreactor system cell cultures are carried out in<br />
presterilised polycarbonate mammalian cell culture cassettes which are<br />
inoculated manu<strong>all</strong>y in a laminar flow cabinet before sealing with Type A<br />
single use closures and incubation under experimental conditions.<br />
Methods: Chemic<strong>all</strong>y defined medium and feeds were used throughout<br />
this work. Unless otherwise stated standard experimental conditions were<br />
used. (35°C, pH 6.95, 30% Dissolved Oxygen (DO)). Viable cell numbers<br />
and viability were determined using a ViCell Cell Viability Analyser<br />
(Beckman Coulter) and antibody titres were determined using an Immage<br />
Immunochemistry System (Beckman Coulter).<br />
Process optimisation: Typical process relevant factors that can be tested<br />
in the Micro24 include feed regime, pH, DO and temperature. The effects<br />
of these types of factors are best tested using a Design of Experiments<br />
(DoE) approach to assess the effects not only of different factors but also<br />
of the interactions between them. Such data can then be used to build<br />
predictive models of process performance to specify the appropriate<br />
operating conditions in larger scale bioreactors. We have already<br />
developed and are using a similar approach for microbial dAb processes.<br />
The data below shows examples of how we have used this system to<br />
identify improvements to platform processes for specific cell lines.<br />
Case Study 1 - process conditions: In this experiment the effects of<br />
changes to the platform process pH and DO set points on the performance<br />
of a mAb producing cell line were assessed in the Micro24 using a DoE<br />
approach with different operating conditions in each well.<br />
This data demonstrated that although the dissolved oxygen level had<br />
little effect on viable cell numbers, titres and specific productivity,<br />
operating at a higher pH than the standard platform set point resulted in<br />
an increase in titre and in specific productivity. There was no significant<br />
interaction between the factors.<br />
Bioreactor validation (1) - 2 litre scale: The high pH process identified<br />
from the Micro24 was run in 2 litre bioreactors and compared to the<br />
standard platform process.<br />
At the high pH set point cell numbers during the later stages of the process<br />
were slightly reduced compared to the control and as in the Micro24 higher<br />
titres were produced under higher pH conditions. However, as in the<br />
Micro24 the greatest effect of increased pH was on specific productivity<br />
which in the bioreactors was increased by approximately 35% compared to<br />
the control.<br />
Bioreactor validation (2) - 50 litre scale: Similar results were achieved at<br />
the50litrescaleforadifferentcelllinerunninginthesameplatform<br />
process but producing a different molecule (Figure 1). There was little effect<br />
on the cell numbers but the higher pH condition resulted in increased titre,<br />
culture duration, volumetric productivity and specific productivity.<br />
Case study 2 - feeding regime: The Micro24 can be used to investigate<br />
the effect of different feeding regimes on culture performance. We have<br />
already demonstrated that the effect of feed addition on culture<br />
performance in the Micro24 is similar to that in shake flasks [1]; the data<br />
below (Table 1) shows that for a chemic<strong>all</strong>y defined process multiple feed<br />
additions have a similar effect in 2 litre bioreactors to the Micro24. In both<br />
systems the addition of the feed results in significant increases in cell<br />
numbers and titre. Culture duration is increased and the over<strong>all</strong> specific<br />
activity is increased by 63% in the Micro24 and 79% in the bioreactors.<br />
Discussion: Our previous work has demonstrated how the Micro24 system<br />
can be used for mammalian cell line selection [1] and the data presented<br />
here extends the application of the Micro24 into mammalian process<br />
development. The par<strong>all</strong>el nature of the Micro24 enables process relevant<br />
factors to be tested in DoE experiments and these data show that<br />
improved process conditions such as increased pH and feed additions<br />
identified in the Micro24 can be used to achieve process improvements in<br />
bioreactors.<br />
The validation of the Micro24 results in bioreactors suggests that the<br />
integration of this technology into mammalian process development could<br />
reduce significantly the numbers of bioreactors required to achieve process<br />
improvements which could result in reduced resource requirements and<br />
improved timelines.<br />
Reference<br />
1. Warr S, Patel J, Ho R, Newell K: Use of Micro Bioreactor systems to<br />
streamline cell line evaluation and upstream process development for<br />
monoclonal antibody production. BMC Proceedings 2011, 5(Suppl 8):P14.<br />
P37<br />
Temperature dependency of immunoglobulin production in novel<br />
human partner cell line<br />
Galina Kaseko * , Marjorie Liu, Edwin Hoe, Qiong Li, Mercedes B<strong>all</strong>esteros,<br />
Tohsak Mahaworasilpa<br />
The Stephen Sanig Research Institute, Sydney, NSW, 2015 Australia<br />
E-mail: g.kaseko@ssri.org.au<br />
BMC Proceedings 2013, 7(Suppl 6):P37<br />
Introduction: A number of immunoglobulin (Ig) secreting human hybrid<br />
cell lines were created using one-on-one somatic cell hybridization of a<br />
rare human tumor infiltrating B lymphocyte and a cell of a novel human<br />
cell line (WTM), developed in house and described earlier [1]. These hybrid<br />
cell lines secret various amounts of tumor-derived immunoglobulins (Igs)<br />
of different specificities. Current investigative efforts are directed towards
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Page 56 of 151<br />
Figure 1(abstract P36) Effect of pH on cell line performance in 50 litre bioreactors.<br />
determining the optimal culture conditions to ensure consistent cell<br />
growth and long-term stabilities of Ig productions by the hybrids. Based<br />
on previous literature reports [2,3], we investigated an effect of short- and<br />
long-term mild hypothermic conditions on Ig production, cell growth and<br />
cell size.<br />
Results: Three different hybrid cell lines each representing the highest,<br />
medium and lowest ranges of Ig productions, were subject to culture<br />
temperature drops from 37°C to 36°C, 35°C or 34°C for up to 168 hours with<br />
24-hour data point intervals. In case of prolonged mild hypothermia, the cell<br />
line with Ig production most susceptible to temperature drops was<br />
maintained at various temperatures below 37°C (e.g. 36°C, 35°C and 34°C)<br />
for at least 5 passages with each passage lasting 120 hours and the data<br />
taken at a 24-hour interval. At each data point for each of the hybrid cell<br />
lines at a given temperature interval, the sample was collected to determine<br />
cell concentration, cell size and Ig production.<br />
Whilst there was no observable effect of any of the short-term temperature<br />
drops on the cell growth or the cell size in any of the three hybrid cell lines,<br />
the level of Ig concentration consistently increased in <strong>all</strong> of them, with gains<br />
ranging from 67% and 320% and with Ig productivity peaking between 48<br />
and 72 hours after the exposure to lower temperatures (Figure 1).<br />
In contrast to short-temperature drop conditions, a prolonged exposure<br />
to mild hypothermic conditions (longer than 1 passage) led to a<br />
progressive decrease in cell size over 5 passages. This decrease in the cell<br />
size was accompanied by gradual 10-30% gains of Ig production with<br />
each passage after the initial 100 to 150% increase in Ig concentration<br />
immediately upon transfer to lower temperature (Table 1). When cultured<br />
at 36°C, it seems to generate the highest increase in Ig production. This<br />
temperature effect was not noticeable at log phase of cell growth.<br />
Conclusions: In conclusion, whilst lowering temperature in the culture<br />
resulted in over<strong>all</strong> increase in Ig concentration, our results suggest that<br />
Table 1(abstract P36) Comparison of the effect of feed on cell line key performance parameters in Micro24 and 2 litre<br />
bioreactors<br />
Effect of Feed on Key Performance Parameters in Micro 24 and 2 L Bioreactors<br />
Normalised VCC Normalised Culture Duration Normalised Peak Titre Normalised SPR<br />
Micro 24 Unfed 100 100 100 100<br />
Fed 147 113 206 163<br />
2 L Bioreactors Unfed 100 100 100 100<br />
Fed 171 133 379 179
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Figure 1(abstract P37) Effects of short-term temperature drops<br />
from 37°C to 36°C and 37°C to 35°C on Ig production by hybrid<br />
cell line 2.<br />
Table 1(abstract P37) Effects of prolonged mild<br />
hypothermia on Ig production by hybrid cell line 2 at<br />
day 5 of each passage over 5 passages<br />
Passage<br />
P0<br />
(ng/ml)<br />
P1<br />
(ng/ml)<br />
P2<br />
(ng/ml)<br />
P3<br />
(ng/ml)<br />
P4<br />
(ng/ml)<br />
P5<br />
(ng/ml)<br />
37°C 342 322 388 356 301 348<br />
36°C 0 712 758 929 1559 928<br />
35°C 0 605 452 514 586 716<br />
34°C 0 751 667 535 490 465<br />
there might be different mechanisms responsible for the increase in Ig<br />
productivity in response to short temperature drop and prolonged<br />
hypothermia.<br />
Acknowledgements: The project was financi<strong>all</strong>y supported in part by<br />
Anthrocell Pty Limited, an Australian biotechnology company located in<br />
Sydney, Australia.<br />
References<br />
1. Kaseko G, Liu M, Li Q, Mahaworasilpa T: Novel partner cell line for<br />
immortalisation of rare antigen-specific B cells in mAb development.<br />
BMC Proceedings 2011, 5(Suppl 8):P130.<br />
2. Chong SL, Mou DG, Ali AM, Lim SH, Tey BT: Cell growth, cell-cycle<br />
progress, and antibody production in hybridoma cells cultivated under<br />
mild hypothermic conditions. Hybridoma 2008, 27:107-111.<br />
3. Lloyd DR, Holmes P, Jackson LP, Emery N, Al-Rubeai M: Relationship<br />
between cell size, cell cycle and specific protein productivity.<br />
Cytotechnology 2000, 34:59-70.<br />
P38<br />
Strategies for clone detection, selection and isolation in Per.C6 cells -<br />
case for Rebmab100<br />
Fernanda P Yeda 1,2 , Mariana L dos Santos 1,2 , Lilian R Tsuruta 1,2 ,<br />
Bruno B Horta 1,2 , André L Inocencio 1 , Oswaldo K Okamoto 2,3 , Maria C Tuma 2 ,<br />
Ana M Moro 1*<br />
1 Lab. Biofármacos em Células Animais, Instituto Butantan, SP, 05503-900, Brazil;<br />
2 Recepta-biopharma, SP, 04533-014, Brazil; 3 Depto. Genética e Biologia Evolutiva,<br />
Instituto de Biociências, Universidade de São Paulo, SP, 05508-900, Brazil<br />
E-mail: ana.moro@butantan.gov.br<br />
BMC Proceedings 2013, 7(Suppl 6):P38<br />
Background: A successful monoclonal antibody (mAb) cell line development<br />
requires efficient clone detection and screening. Cloning by limiting dilution<br />
(LDC) is the traditional method to isolate mAbs expressing clones [1].<br />
Although effective, LDC is time-consuming, with limited workflow and<br />
therefore a critical step of cell line development. To compare to LDC in terms<br />
of timelines and productivities for Rebmab100 mAb cell line development we<br />
have implemented ClonePix FL (CP-FL), an automated system for high<br />
throughput clone detection. The robotic colony picker has the advantages of<br />
reducing the process time and increasing the probability to isolate highproducing<br />
clones. Moreover, we have combined these two approaches with<br />
high throughput screening assays for early detection of high productive<br />
clones.<br />
Rebmab100 mAb targets Lewis-Y, a blood group-related antigen expressed<br />
in over 70% of epithelial cancers, including breast, colon, ovary and lung<br />
carcinomas. The murine monoclonal 3S193 was generated in BALB/c mice<br />
by immunization with Le y -expressing cells from the MCF-7 breast carcinoma<br />
cell line [2]. The humanized version of anti- Le y 3S193 mAb was obtained by<br />
CDR-grafting method [3]. The hu3S193 (Rebmab 100) mAb has potent<br />
immune effector function (ADCC and CDC), is rapidly internalized into Le y<br />
expressing cancer cells, and has been shown to cause significant regressions<br />
in xenograft models in preclinical studies, alone or in conjunction with<br />
isotope and toxins [3,4]. Safety and desirable pharmacokinetic profiles of<br />
Rebmab100 were demonstrated in a Phase I clinical trial in patients with<br />
epithelial carcinomas [5] and promising results have been obtained in a<br />
Phase II clinical trial conducted in Brazil [6]. Very importantly, Rebmab100<br />
was granted orphan-drug status by the FDA for ovary cancer. Aiming the<br />
next step of Rebmab100 mAb development we generated a new<br />
Rebmab100 cell line that shows stability and high productivity <strong>all</strong>owing its<br />
scale-up to later clinical trials.<br />
Materials and methods: Suspension Per.C6® cells (Crucell, Netherlands)<br />
were transfected with a vector containing the genes coding for heavy and<br />
light chains of Rebmab100 mAb. After selection by G418 the cells from the<br />
stable pool were cloned by limiting dilution or plated in semi-solid<br />
medium (Molecular Devices, USA) for ClonePix FL screening.<br />
Cellular growth was assessed in plates, 96, 24 or 6-well plates, either by<br />
CloneSelect Imager (Molecular Devices) or Guava EasyCyte cytometer<br />
(Merck-Millipore). Antibody titers were measured by Biacore T100 (GE<br />
Healthcare, Sweden). The selected clones were transferred to T-flasks and<br />
subsequently to shaker flasks (SF). Clones were analyzed in 50 mL and 200<br />
mL SF fed-batch processes. The stability study was performed for at least<br />
50 generations in continuous culture and also starting batch runs with<br />
cells taken at different generations.<br />
Results: Generation of Rebmab100 stable pool: The transfection of<br />
Per.C6® cells with a vector containing the genes coding for heavy and light<br />
chains of Rebmab100 generated a stable pool through G418 selection.<br />
Cloning using two different approaches: The stable pool was cloned by<br />
LDC in liquid medium at 0.5 cell/well in 50 96-well plates, resulting in 261<br />
colonies transferred to 24-well plates in 3-4 weeks after screening with the<br />
CloneSelect Imager. Concomitantly the same pool was seeded at different<br />
concentrations (300 to 2000 cells/mL) in semi-solid medium. The plates<br />
were screened by light and fluorescence images about ten days after<br />
seeding. A total of 845 colonies were picked, from which 225 were<br />
transferred to 24-well plates. At the transference step to 24-well plates, 261<br />
out of 4800 wells seeded in LDC were transferred while 225 colonies out of<br />
845 colonies picked by CP-FL, representing 5.4% and 26.6% efficiency,<br />
respectively. Both approaches followed sequential steps as transfer of the<br />
clones to 6-well plates, T-flasks and SF, selecting them at each step for cell<br />
growth and productivity related to cell number.<br />
Fed-batch experiments and stability study: Thirty-one clones adapted<br />
to suspension cultures were assessed for productivity in fed-batch<br />
processes, being 15 originated from LDC and 16 from CP-FL. From the fedbatch<br />
in 50 mL SF 12 clones presented titers ranging from 1.3 to 3.0 g/L<br />
(Figure 1A). Out of 31 clones, 10 were selected for long-term stability study<br />
to determine growth and productivity along the time required for mAb<br />
production during a manufacturing process. The stability study performed<br />
with 6 LDC and 4 CP-FL originated clones ruled out 3 of them, two from<br />
LDC and one from CP-FL. Seven clones showed genetic and cellular<br />
stability (data not shown), 4 from LDC and 3 from CP-FL and were further<br />
analyzed in fed-batch in 200 mL SF. In this study we compared titers<br />
obtained after 2 weeks run for <strong>all</strong> clones, with results ranging from 0.9 to<br />
1.8 g/L to the maximum productivity attained by each clone, obtained at<br />
different lengths of culture (Figure 1B). Taken together the data for cell<br />
growth, productivity, kinetic and functional assays of the purified<br />
antibodies (data not shown), mainly the immune-effector activity<br />
characteristic<strong>all</strong>y displayed by Rebmab100, we identified 4 lead clones, the<br />
first and second originated by CP-FL screening. Final ranking will be<br />
evaluated after bioreactor runs.<br />
Conclusions: The CP-FL automated picking has the advantage of being<br />
less labor-intensive and time-consuming, while <strong>all</strong>owing the chance of
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Figure 1(abstract P38) Antibody titer measured by Biacore in SF fed-batch process (g/L). (A) 31 selected Rebmab100 clones measured on the last<br />
day of a 50 mL SF fed-batch culture. (B) Maximum (grey bars) and 2 weeks (black bars) mAb productivity obtained in a 200 mL SF fed-batch culture for<br />
the 7 stable Rebmab100 clones. The number above the grey bars indicates the day when maximum mAb productivity occurred.<br />
picking clones that would not grow isolated in LDC. Both CP-FL and LDC<br />
procedures proved efficient for generating high productive and stable<br />
cell clones. Over<strong>all</strong> productivity for individual clones depends on specific<br />
productivity, cell density and viability along time, <strong>all</strong>owing accumulation<br />
of the antibody. CP-FL clones reached maximum productivity at an earlier<br />
stage (2 weeks) of the 200 mL SF fed-batch experiment, which represents<br />
an advantage during the manufacturing process.<br />
The 4 lead clones will be submitted to bioreactor runs to evaluate the<br />
most suitable clone for the Rebmab100 mAb to be used in clinical trials<br />
and eventu<strong>all</strong>y to go under production.<br />
Acknowledgements: We acknowledge the excellent technical support of<br />
Denis N Aranha and José M Oliveira. WearegratefultoDr.MariaTA<br />
Rodrigues for logistics support. This work was supported by FAPESP, FINEP,<br />
CNPq, Fundação Butantan, and Recepta-biopharma.<br />
References<br />
1. Browne SM, Al-Rubeai M: Selection methods for high-producing<br />
mammalian cell lines. Trends Biotechnol 2007, 25:425-432.<br />
2. Kitamura K, Stockert E, Garin-Chesa P, Welt S, Lloyd KO, Armour KL,<br />
W<strong>all</strong>ace TP, Harris WJ, Carr FJ, Old LJ: Specificity analysis of blood group<br />
Lewis-y (Le(y)) antibodies generated against synthetic and natural Le(y)<br />
determinants. Proc Natl Acad Sci USA 1994, 91:12957-12961.<br />
3. Scott AM, Geleick D, Rubira M, Clarke K, Nice EC, Smyth FE, Richards EC,<br />
Carr FJ, Harris WJ, Armour KL, Rood J. Kypridis A, Kronina V, Murphy R,<br />
Lee FT, Liu Z, Kitamura K, Ritter G, Laughton K, Hoffman E, Burgess AW,<br />
Old LJ: Construction, production, and characterization of humanized<br />
anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of<br />
solid tumors. Cancer Res 2000, 60:3254-3261.<br />
4. Kelly MP, Lee FT, Smyth FE, Brechbiel MW, Scott AM: Enhanced efficacy of<br />
90Y-radiolabeled anti-Lewis Y humanized monoclonal antibody hu3S193<br />
and paclitaxel combined-modality radioimmunotherapy in a breast<br />
cancer model. J Nucl Med 2006, 47:716-725.<br />
5. Scott AM, Tebbutt N, Lee FT, Cavicchiolo T, Liu Z, Gill S, Poon AM, Hopkins W,<br />
Smyth FE, Murone G, MacGregor D, Papenfuss AT, Chappell B, Saunder TH,<br />
Brechbiel MW, Davis ID, Murphy R, Chong G, Hoffman EW, Old LJ: A phase I<br />
biodistribution and pharmacokinetic trial of humanized monoclonal<br />
antibody Hu3s193 in patients with advanced epithelial cancers that<br />
express the Lewis-Y antigen. Clin Cancer Res 2007, 13:3286-3292.<br />
6. Smaletz O, Diz MPD, Carmo CC, Sabbaga J, Cunha GF, Azevedo SJ,<br />
Maluf FC, Barrios CH, Costa RL, Fontana AG, Alves VA, Moro AM, Scott EW,<br />
Hoffman EW, Old LJ: Anti-LeY monoclonal antibody (mAb) hu3S193<br />
(Rebmab100) in patients with advanced platinum resistant/refractory<br />
(PRR) ovarian cancer (OC), primary peritoneal cancer (PPC), or f<strong>all</strong>opian<br />
tube cancer (FTC). ASCO Annual Meeting, 2011, Chicago. J Clin Oncol 2011,<br />
29:5078.<br />
P39<br />
Impact of single-use technology on continuous bioprocessing<br />
William G Whitford * , Brandon L Pence<br />
Thermo Fisher Scientific, 925 West 1800 South, Logan, Utah 84321, USA<br />
E-mail: bill.whitford@thermofisher.com<br />
BMC Proceedings 2013, 7(Suppl 6):P39<br />
Background: Single-use (SU) technologies supply a number of values to<br />
any mode of bioprocessing, but can provide some specific and enabling<br />
features in continuous bioprocessing (CB) implementations [1-3]. Most<br />
every operation in a CB process train is now supported by a commerci<strong>all</strong>y<br />
available single-use, or at least hybrid, solution (Figure 1). First of <strong>all</strong>,<br />
many of the SU equipment and solutions being developed for batch<br />
bioproduction have the same or related application in CB systems.<br />
Examples here include simple equipment such as tubings and connectors,<br />
to more complex applications such as the cryopreservation of large<br />
working stock aliquots in flexible bioprocess containers (BPCs). The list of<br />
CB-supporting SU technologies being developed is large and growing.<br />
Results: A SU advantage in process development is its supports of an<br />
open architecture approach and a number of hybrid designs. Such designs<br />
include combining reusable and single-use systems, or between divergent<br />
suppliers of particular equipment. Especi<strong>all</strong>y in bioproduction, the many<br />
flexibilities of SU support a manufacturing platform of exceptional<br />
efficiency, adaptability, and operational ease. Advances designs in SU<br />
transfer tubing, manifold design and container porting also supports<br />
creativity in process design. This is of particular value in designing a<br />
process with such demands as entirely new flow paths or lot designations,<br />
such for CB.<br />
SU systems upstream provide a reduced footprint and eliminate of the need<br />
for cleaning and sterilization service. This complements perfusion culture’s<br />
inherently sm<strong>all</strong>er size and independence from cleaning for extended<br />
periods of time.
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Page 59 of 151<br />
Figure 1(abstract P39) Hybrid intensified perfusion-based continuous bioproduction in a Thermo Scientific HyPerforma S.U.B. TK 250L supported<br />
by yhe Refine Technology ATF System.<br />
Several newer approaches to formulating process fluids support the concept<br />
of CB. Single-use mixing systems are typic<strong>all</strong>y constructed of a rigid<br />
containment system with a motor and controls driving radiation-sterilized<br />
single-use bags equipped with disposable impeller assemblies. From a<br />
variety of manufacturers there are a number of distinct approaches to<br />
motor/disposable impeller assembly linkages, tubing lines and connections.<br />
Also appearing are a number of exciting SU sampling, sensing, and<br />
monitoring solutions. Single-use powder containers permit seamless transfer<br />
between powder and liquid formulation steps, and the ridged mixing<br />
containers are available in jacketed stainless steel for heating and cooling<br />
requirements. Surprisingly, the “topping-up” of large-scale single-use fluid<br />
containers with newly prepared buffer to provide a virtu<strong>all</strong>y unlimited and<br />
constant supply of each buffer/media type can be validated for GMP<br />
manufacturing procedures.<br />
Process flexibility is a key feature in both SU and CB. CB contributes to<br />
over<strong>all</strong> process flexibility in that equipment tends to be easy to clean,<br />
inspect and maintain − and gener<strong>all</strong>y promotes simple and rapid product<br />
changeover. SU systems can provide similar flexibility and ease product<br />
changeover because they tend to be more modular and transportable<br />
than much of the older batch equipment. In fact the size, configuration<br />
and reduced service requirements of SU systems actu<strong>all</strong>y encourage<br />
diversity of physical location within a suite or plant, as well as re-location<br />
to other manufacturing sites.<br />
Due to its inherent demand for immediate process data and control<br />
capabilities, CB supports initiatives in continuous quality verification (CQV),<br />
continuous process verification (CPV), and real-time release (RTR). Although<br />
CB will not be feasible for <strong>all</strong> products and processes, many implementations<br />
well-support a “platform” approach, in which a single process<br />
supports more than one product. CB most always shortens the process<br />
stream, reduces downtime, and greatly reduces handling of intermediates.<br />
These features complement the operational efficiencies of SU systems,<br />
contributing to a greatly reduced cumulative processing time for the API.<br />
Furthermore, they greatly simplify production trains and inherently facilitate<br />
application of closed processing approaches to individual operations and<br />
even processes. Especi<strong>all</strong>y in bioproduction, the modularity and integral<br />
gamma irradiation sterility of SU combined with the sustained operation of<br />
CB promise the appearance of platforms of unpar<strong>all</strong>eled operational<br />
simplicity and convenience.<br />
The heart of a CB approach is the bioreactor. Perfusion bioreactors have<br />
been successfully employed in bioproduction, even biopharmaceutical<br />
production, for decades. And, rather remarkably, disposable bioreactors<br />
have been available for nearly 20 years. At the research scale there have<br />
even been single-use hollow fiber perfusion bioreactors available from a<br />
variety of vendors for over 40 years. However, only recently have<br />
commerci<strong>all</strong>y available SU and hybrid production-scale perfusion-capable<br />
equipment become available.<br />
The production-scale CB enabling SU bioreactor technologies now<br />
becoming commercial available include single-use and hybrid perfusioncapable<br />
reactors (Figure 1); a growing variety of SU and hybrid monitoring<br />
probes and sensors; SU pumps and fluid delivery automation of various<br />
design; and automated SU online sampling, interface, valving and feeding<br />
technologies. Their coordinated implementation in actual production<br />
settings with appropriate control is now beginning.<br />
Justified or not, concerns in the implementation of CB include performance<br />
reliability (incidence of failure), validation complexity, process control and<br />
economic justification. But for many processes, such previous limitations –<br />
or their perception – are being <strong>all</strong>eviated by advances in CB processing<br />
technology and OpEx driven advances bioprocess understanding, reactor<br />
monitoring and feedback control. However, while some CB attributes<br />
inherently provide immediate advantages (such as reduces reactor
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residency time) others do present ch<strong>all</strong>enges (such as cell-line stability<br />
concerns).<br />
Due to the limited contribution of API manufacturing to sm<strong>all</strong>-molecule<br />
pharmaceutical cost, the limited bottom-line financial savings of CB has<br />
been a concern. However, biopharma is a different animal in general, and<br />
as such trends as globalization and biosimilars alter the picture even<br />
further, the financial benefits of CB are becoming even stronger.<br />
The fact that many SU systems are constructed of standards compliant and<br />
animal product-free materials supports CB applications in a wide variety of<br />
product types and classification. In fact, SU systems are available to most<br />
any process format (eg, microcarriers and suspension), platform (eg, cell<br />
line, vectors, culture media), mode (eg, dialysis or enhanced perfusion) or<br />
scale (eg, through rapid, inexpensive scale-out). “Futureproofing”, or<br />
supporting the sustainability of a new CB process in the face of product<br />
lifecycle or emerging technology imperative, is supported by many SU<br />
features. Examples here include SUs low initial facility, service and<br />
equipment cost and especi<strong>all</strong>y SU’s undedicated manufacturing suits and<br />
ease of process train reconfiguration.<br />
Conclusion: As advanced single-use solutions are applied to single-use<br />
perfusion mode-capable reactors, the design of integrated closed,<br />
disposable and continuous upstream bioproduction systems are fin<strong>all</strong>y<br />
being realized.<br />
References<br />
1. Whitford WG: Supporting continuous processing with advanced singleuse<br />
technologies. BioProcess International 2013, 11:46-52.<br />
2. Whitford WG: Continued progress in continuous processing for<br />
bioproduction. Life Science Leader 2012, June:62-64.<br />
3. Whitford WG: Single-use systems support continuous processing in<br />
bioproduction. PharmaBioWorld 2012, 10:22-27.<br />
P40<br />
Comparison of BHK-21 cell growth on microcarriers vs in suspension at<br />
2L scale both in conventional bioreactor and single-use bioreactor<br />
(Univessel® SU)<br />
Lídia Garcia * , Elisenda Viaplana, Alicia Urniza<br />
Zoetis Manufacturung & Research Spain, S.L Pfizer Olot S.L.U., Ctra.<br />
Camprodon s/n, La Riba, 17813 V<strong>all</strong> de Bianya (Girona), Spain<br />
E-mail: Lidia.garcia@zoetis.com<br />
BMC Proceedings 2013, 7(Suppl 6):P40<br />
Background: BHK-21 cells are the most commonly used cells for vaccine<br />
production. Not <strong>all</strong> cell lines can be adapted to suspension growth. In<br />
general, anchorage-dependent cells (must be attached to a substrate to<br />
grow) will grow in suspension only with the use of microcarrier beads.<br />
However, some cell lines such as the BHK-21 can be adapted to grow in<br />
suspension.<br />
In recent years, the use of disposables in the pharmaceutical industry has<br />
increased extensively. The aim of this study is to evaluate the influence of<br />
a single use bioreactor on the final cell production of BHK-21 cells when<br />
they are growing with microcarriers or in suspension which can do an<br />
impact on the final product quality.<br />
Cultivations on conventional 2L-bioreactors were compared with results<br />
obtained from 2L single use bioreactor (UniVessel® SU).<br />
Materials and methods: Cell line: Two BHK-21 cell lines were used, BHK-<br />
21 clone C3 as an anchorage-dependent cell line and SBHK cells adapted<br />
to grow in suspension.<br />
Both cell lines were cultivated in MEM Glasgow medium supplemented<br />
with fetal bovine.<br />
BHK-21 cells were grown in microcarriers Cytodex-3.<br />
Cultivation system: The growth using two different bioreactors was<br />
analyzed: Conventional reusable bioreactor (Autoclaving glass vessel of 2L)<br />
and the UniVessel® SU as a single use bioreactor<br />
To control both bioreactors the BIOSTAT® B plus unit was used.<br />
Parameters as pH, temperature, stirring speed, aeration rate and viable<br />
cell number were analyzed.<br />
Cell growth was conducted at the optimal conditions determined previously<br />
on spinner flasks. Cells were seeded into the bioreactor at the following<br />
concentration:<br />
BHK-21: 5 × 10 5 cells/ml with a viability of ≥ 98%<br />
SBHK: 3 × 10 5 cells/ml with a viability of ≥ 97%<br />
Cell count: BHK-21 were counted using the crystal violet dye nucleus<br />
staining method.<br />
SBHK cells were counted using the NucleoCounter (ChemoMetec A/S).<br />
Results: Optimization, characterization of BHK cells culture processes and<br />
evaluation of microcarriers vs non-microcarrier processes at 2L scale were<br />
done.<br />
Processperformancewascomparedinconventionalglassvesselsto<br />
single use bioreactors.<br />
In Table 1 values of viability and final cell density are shown in single-use<br />
and conventional bioreactors (3 batches per bioreactor). The results<br />
obtained demonstrated that at 3 days of culture no significant differences<br />
were found using both bioreactors.<br />
BHK-21 attached and grew efficiently on microcarriers. Fully confluency<br />
and a maximum viable cell density (between 1.2 to 2.9 × 10 6 cells/ml)<br />
was obtained after 3 days of culture (Table 1, Figure 1). In <strong>all</strong> the cases,<br />
the viability was higher than 96.5%.<br />
SBHK cells reached higher yields comparing with the BHK-21. The<br />
maximum viable cell density (> 90% of viability) was obtained at 3 days<br />
of culture reaching a cell concentration between 1.95 to 3.5 × 10 6 cells/ml<br />
(Table 1, Figure 1).<br />
The variability on final cell density obtained between the different<br />
batches was similar in both types of bioreactors (Table 1).<br />
Conclusions: ✓ Comparable results between conventional glass vessels<br />
and single use bioreactors: cell density and viability.<br />
✓ Given the good results obtained with SBHK cells, elimination of<br />
microcarriers can decrease the cost of a large-scale operation.<br />
✓ The feasibility of transferring the BHK cells growth from a<br />
conventional bioreactor to single-use bioreactor has been<br />
demonstrated.<br />
✓ Benefits of single-use technology integration:<br />
• SU Bioreactors can replace conventional bioreactors without<br />
loss of process efficiency<br />
• The scale-up for both suspension and attached cell lines in SU<br />
bioreactors is guarantee. The flexibility and easy of use of this<br />
SU bioreactors enable rapid scale-up without any loss in<br />
product quality<br />
• SU Bioreactors increase easy of handling and offer advantages<br />
in the areas of cleaning, sterilization, validation, set-up, and<br />
turn-around time between runs.<br />
• SU Bioreactors are the best solution when containment is<br />
required (BL-3 and BL-4 laboratories).<br />
P41<br />
Size-dependent antioxidative activity of platinum nanoparticles<br />
Hidekazu Nakanishi 1* , Takeki Hamasaki 2 , Tomoya Kinjo 1 , Kiichiro Teruya 1,2 ,<br />
Shigeru Kabayama 3 , Sanetaka Shirahata 1,2<br />
1 Division of Life Engineering, Graduate School of Systems Life Sciences,<br />
Kyushu University, Fukuoka 812-0053, Japan;<br />
2 Department of Bioscience and<br />
Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-0053,<br />
Japan;<br />
3 Nihon Trim Co. Ltd, Osaka 531-0076, Japan<br />
BMC Proceedings 2013, 7(Suppl 6):P41<br />
Background: So far, most of studies on nanometer-sized metal particles<br />
have focused on biological safety and potential hazards. However, antioxidative<br />
activity of noble metal nanoparticles (NPs) attracts much<br />
attention, recently. Platinum nanoparticles (Pt NPs) are one of the most<br />
important noble metals in nanotechnology because Pt NPs have negative<br />
surface potential from negative charges and are stably suspended from<br />
an electric repulsion between the same charged particles [1]. We<br />
previously reported that Pt NPs of 2-3 nm sizes scavenged reactive<br />
oxygen species (ROS) such as superoxide anion radical, hydrogen<br />
peroxide and hydroxyl radical in vitro [2]. Here, we report the cytotoxicity<br />
and size-dependent antioxidative activity of Pt NPs on rat skeletal muscle<br />
cell line, L6.<br />
Materials and methods: Pt NPs were synthesized by a modified citrate<br />
reduction method of Hydrogen hexachloroplatinate (IV). Particle size and<br />
concentrations of Pt NPs were determined by a transmittance electron<br />
microscope (TEM) and ICP-MS, respectively. To find the toxic effect of Pt<br />
NPs rat myoblast L6 cells were pre-cultured for 24 hours in culture medium<br />
with a 10 -3 to 10 mg/l of Pt NPs and cell viability was determined by WST-1
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Table 1(abstract P40) Cell growth and viability at 3 days culture in a 2L conventional bioreactor and in a single use<br />
bioreactor<br />
Univessel SU (2L)<br />
Conventional Bioreactor (2L)<br />
BHK-21 SBHK BHK-21 SBHK<br />
Batch<br />
Viable Cells<br />
(cells/ml)<br />
Viability (%)<br />
Viable Cells<br />
(cells/ml)<br />
Viability (%)<br />
Viable Cells<br />
(cells/ml)<br />
Viability (%) Viable Cells<br />
(cells/ml)<br />
1 2.90 × 106 97 2.70 × 106 99.1 2.46 × 106 99 1.96 × 106 92.1<br />
2 1.20 × 106 96.5 1.95 × 106 90.5 1.80 × 106 98.9 2.36 × 106 98<br />
3 2.10 × 106 98 3.0 × 106 97 1.90 × 106 98.1 3.50 × 106 99.4<br />
Mean valors 2.08 × 10 6 97.5 2.55 × 10 6 97 2.05 × 10 6 99 2.61 × 10 6 99.4<br />
Viability (%)<br />
assay. To investigate the anti-oxidative effect of Pt NPs on L6 cells, the<br />
relative amount of intracellular H 2 O 2 was measured with a Bes-H 2 O 2 -AC<br />
florescent probe, which is designed to detect intracellular H 2 O 2 specific<strong>all</strong>y<br />
[3]. The intracellular ROS levels when treated with 1 mg/l of Pt NPs for<br />
2 hours were measured using IN Cell Analyzer 1000.<br />
Results and conclusions: The particle sizes we synthesized were<br />
determined to 1-2 nm, 2-3 nm and 4 nm respectively (data not shown).<br />
Cytotoxicity of Pt NPs of these sizes was not observed at a concentration<br />
below 10 mg/l (data not shown). Intracellular ROS levels are thought to<br />
result from a primary response to internalized NPs leading to decreased cell<br />
viability [4]. Thus, the suppression of excess ROS is of prime importance for<br />
cell survival. The intracellular ROS levels were decreased significantly by the<br />
whole sizes of Pt NPs treatment and 2-3 nm of Pt NPs scavenged the ROS<br />
most efficiently (Figure 1). The relative fluorescence level treated with 2-3<br />
nm of Pt NPs decreased significantly to about 60% (*** P < 0.001) compared<br />
with that of non-treated cells. Sm<strong>all</strong>er NPs should be more taken up by the<br />
cells efficiently and might more scavenge ROS effectively [5]. However, the<br />
Pt NPs of 1-2 nm less scavenged the intracellular ROS than that of 2-3 nm.<br />
The one reason might be that 1-2 nm of Pt NPs is rather too sm<strong>all</strong> to<br />
activate intracellular anti-oxidant defense pathways than 2-3 nm of Pt NPs<br />
because of their less cytotoxicity. However, we have no data to show.<br />
Therefore, we have to make more effort to investigate the relationship<br />
between the sizes of Pt NPs and ROS scavenging activity.<br />
Our results suggest Pt NPs of 2-3 nm sizes have no cytotoxity below 10 mg/l<br />
and are useful materials to scavenge ROS. In this regard Pt NPs are<br />
expected as redox regulation factors for suppression of various ROSrelated<br />
diseases.<br />
References<br />
1. Aiuchi T, Nakajo S, Nakaya K: Reducing activity of colloidal platinum<br />
nanoparticles for hydrogen peroxide, 2,2-diphenyl-1-picrylhydrazyl<br />
radical and 2,6-dichlorophenol indophenol. Biol Pharm Bull 2004,<br />
27:736-738.<br />
2. Hamasaki T, Kashiwagi T, Imada T, Nakamichi N, Aramaki S, Toh K,<br />
Morisawa S, Shimakoshi H, Hisaeda Y, Shirahata S: Kinetic analysis of<br />
superoxide anion radical-scavenging and hydroxyl radical-scavenging<br />
activities of platinum nanoparticles. Langmuir 2008, 24:7354-7364.<br />
3. Maeda H, Fukuyasu Y, Yoshida S, Fukuda K, Saeki K, Matsuno H, Yamauchi Y,<br />
Yoshida K, Hirata K, Miyamoto K: Fluorescent probes for hydrogen<br />
peroxide based on a non-oxidative mechanism. Angew Chem Int Ed Engl<br />
2004, 43:2389-2391.<br />
4. Long TC, Tajuba J, Sama P, Saleh N, Swartz C, Parker J, Hester S, Lowry GV,<br />
Veronesi B: Nanosize titanium dioxide stimulates reactive oxygen species<br />
in brain microglia and damages neurons in vitro. Environ Health Perspect<br />
2007, 115:1631-1637.<br />
5. Hirn S, Semmler-Behnke M, Schleh C, Wenk A, Lipka J, Schäffler M,<br />
Takenaka S, Möller W, Schmid G, Simon U, Kreyling WG: Particle sizedependent<br />
and surface charge-dependent biodistribution of gold<br />
nanoparticles after intravenous administration. Eur J Pharm Biopharm<br />
2011, 77:407-416.<br />
P42<br />
Sampling and quenching of CHO suspension cells for the analysis of<br />
intracellular metabolites<br />
Judith Wahrheit * , Elmar Heinzle<br />
Biochemical Engineering Institute, Saarland University, D-66123 Saarbrücken,<br />
Germany<br />
E-mail: j.wahrheit@mx.uni-saarland.de<br />
BMC Proceedings 2013, 7(Suppl 6):P42<br />
Background: Metabolic studies are of fundamental importance in<br />
metabolic engineering approaches to understand cell physiology and to<br />
pinpoint metabolic targets for process optimization. Knowledge on<br />
intracellular metabolites, in particular in combination with powerful dynamic<br />
metabolic flux analysis methods will substanti<strong>all</strong>y expand our basic<br />
understanding on metabolism, e.g. about metabolic compartmentation [1].<br />
Few protocols for quantitative analysis of intracellular metabolites in<br />
mammalian suspension cells have been proposed in the literature. However,<br />
due to limited validation of sampling and quenching procedures provided<br />
in previous publications, we thoroughly investigated the associated critical<br />
issues, such as (a) cellular integrity, (b) quenching efficiency, (c) cell<br />
separation at different centrifugation conditions and its influence on cell<br />
fitness, and (d) different washing procedures to prevent carryover of<br />
extracellular metabolites. Many metabolites of interest are also contained in<br />
the medium in large amounts, e.g. amino acids, making their intracellular<br />
quantification critical.<br />
Materials and methods: Cell cultivation: Two CHO cell lines were used,<br />
T-CHO ATIII cells (GBF, Braunschweig, Germany) cultivated in serum-free<br />
CHO-S-SFM II medium (GIBCO, Invitrogen, Karlsruhe, Germany) and CHO<br />
Figure 1(abstract P40) Comparison of cell growth and viability at 3 days culture in a 2L conventional bioreactor and in a single use bioreactor.
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Figure 1(abstract P41) The scavenging effect of several sized Pt NPs on intracellular hydrogen peroxide in L6 cells. Asterisks donate significant<br />
difference from the untreated control cells. (***P < 0.001).<br />
K1 cells (University of Bielefeld, Germany) cultivated in amino acid rich<br />
TC-42 medium (TeutoCell, Bielefeld, Germany) in baffled shake flasks in a<br />
shaking incubator. Cell counting and determination of cell diameters<br />
were performed using an automated cell counter (Invitrogen, Darmstadt,<br />
Germany). Cell viability was verified using the trypan blue exclusion<br />
method.Cellrecoverywasdefinedas(totalviablecellnumberafter<br />
quenching)×100/(total viable cell number in initial sample).<br />
Determination of the energy charge: ATP, ADP, and AMP were analyzed<br />
in a luminometer (Promega, Mannheim, Germany) using the CellTiter-Glo,<br />
the ADP-Glo Kinase, and the AMP-Glo assays (Promega, Mannheim,<br />
Germany), respectively. For the ADP- and AMP-Glo assays, cells were lysed<br />
using the CelLytic M reagent (Sigma-Aldrich, Germany) before adding the<br />
assay reagents. The energy charge value was calculated as ([ATP] + ½ ×<br />
[ADP])/([ATP] + [ADP] + [AMP]).<br />
Evaluation of different washing procedures and carryover of media<br />
components: Carryover of extracellular metabolites from the culture<br />
medium was investigated without washing and after applying different<br />
washing procedures. Cell pellets were either resuspended in 50 ml<br />
quenching solution or rinsed once or twice with 50 ml quenching solution<br />
without re-suspension. After another centrifugation step and re-suspension<br />
in a sm<strong>all</strong> volume PBS, cell numbers were determined and extracellular<br />
metabolite amounts analyzed via HPLC as described previously [2] and<br />
related to the initial sample.<br />
Final protocol: (1) Precooling of 45 ml and 50 ml 0.9% saline quenching<br />
solution in an ice-water bath to 0°C for at least 1 hour. (2) Adding of 5 ml<br />
cell suspension to 45 ml 0.9% quenching solution and immediate mixing<br />
by inverting the tube. (3) Centrifugation at 2000 × g in a precooled<br />
centrifuge at 0°C for 1 min. (4) Careful decanting of the supernatant<br />
followed immediately by suction of residual liquid using a vacuum pump<br />
without touching the cell pellet. (5) Washing once by careful pouring of<br />
50 ml precooled QS 50 ml on top of the cell pellet without resuspending<br />
the cells followed by repetition of steps (3) and (4). (6a) Immediate<br />
freezing by placing the tube in liquid nitrogen or (6b) determination of<br />
cell recovery.<br />
Results: Ice-cold 0.9% saline is a suitable quenching solution maintaining<br />
cellular integrity as reported previously [3]. However, longer incubation<br />
times at 0°C reduce cellular viability and should be avoided. The time from<br />
taking the sample (final protocol, step 2) to freezing the cell pellet in liquid<br />
nitrogen (final protocol, step 6a) is critical and should be kept to a minimum.<br />
A rapid temperature shift and in addition a significant dilution of extracellular<br />
metabolites was achieved using a nine-fold excess of quenching<br />
solution. Efficient inactivation of metabolism was proven by a high and<br />
representative energy charge value of 0.82 (± 0.01, n =3).<br />
Separation of cells via centrifugation was incomplete due to required short<br />
centrifugal times. Thus, it is necessary to determine the cell recovery after<br />
quenching. However, from the average cell size estimation we conclude that<br />
centrifugation at short times provides a representative sample, although<br />
sampling was incomplete. Centrifugation time and speed, total volume and<br />
even the initial cell density in the cell suspension have an impact on the cell<br />
recovery after quenching. Centrifugation at 1000 × g and 2000 × g did not<br />
affect cell integrity. Higher centrifugal accelerations (3000 × g, 4000 × g)<br />
reduce cell viability. Above 2000 × g no further improvement in the cell<br />
recovery was obtained. Thus, centrifugation should be limited to 2000 × g to<br />
prevent unnecessary stress to the cells. Due to highly reproducible<br />
centrifugation, the cell recovery can be determined from a biological replicate<br />
(final protocol, step 6b).<br />
Washing steps further reduce cell recovery. Rinsing the cell pellet affects cell<br />
recovery only little and much less than resuspending the cell pellet. Cell<br />
integrity was not impaired by different washing procedures. Reducing the<br />
carryover of metabolites contained in the medium is a prerequisite for their<br />
intracellular analysis. Using a nine-fold excess of quenching solution,<br />
contamination with medium components was very low (less than 0.3% of<br />
the initial metabolite amount was found for glucose, lactate, pyruvate, citrate,<br />
and <strong>all</strong> proteinogenic amino acids). Rinsing the cell pellet without resuspending<br />
the cells further reduces the carryover of medium components<br />
efficiently. However, washing cannot completely prevent medium carryover.<br />
Washing by resuspending does not remove more metabolites than rinsing<br />
and should be avoided due to substanti<strong>all</strong>y reduced cell recovery.
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Conclusions: Ice-cold 0.9% saline was shown to be a suitable quenching<br />
solution maintaining cellular integrity. A rapid temperature shift was<br />
achieved using a nine-fold excess of quenching solution resulting in<br />
efficient inactivation of metabolism. The applied conditions result in a very<br />
low level of medium contamination. Rinsing the cell pellet without<br />
re-suspending the cells reduced medium carryover effectively. Separation<br />
of cells via centrifugation was incomplete due to required short centrifugal<br />
times. Thus, it is necessary to determine the cell recovery after quenching.<br />
References<br />
1. Wahrheit J, Nicolae A, Heinzle E: Eukaryotic metabolism: measuring<br />
compartment fluxes. Biotechnol J 2011, 6:1071-1085.<br />
2. Strigun A, Wahrheit J, Beckers S, Heinzle E, Noor F: Metabolic profiling<br />
using HPLC <strong>all</strong>ows classification of drugs according to their mechanisms<br />
of action in HL-1 cardiomyocytes. Toxicol Appl Pharmacol 2011,<br />
252:183-191.<br />
3. Dietmair S, Timmins NE, Gray PP, Nielsen LK, Krömer JO: Towards<br />
quantitative metabolomics of mammalian cells: Development of a<br />
metabolite extraction protocol. Anal Biochem 2010, 404:155-164.<br />
P43<br />
13 C labeling dynamics of intra- and extracellular metabolites in CHO<br />
suspension cells<br />
Judith Wahrheit * , Averina Nicolae, Elmar Heinzle<br />
Biochemical Engineering Institute, Saarland University, D-66123 Saarbrücken,<br />
Germany<br />
E-mail: j.wahrheit@mx.uni-saarland.de<br />
BMC Proceedings 2013, 7(Suppl 6):P43<br />
Background: Isotope labeling techniques have become a most valuable<br />
tool in metabolomics and fluxomics [1]. In particular the dynamics of label<br />
incorporation provide rich information about metabolism. A thorough<br />
understanding of CHO metabolism is crucial for metabolic engineering and<br />
process optimization.<br />
Materials and methods: Experimental set-up: CHO-K1 cells were<br />
cultivated in protein free TC-42 medium (TeutoCell, Bielefeld, Germany) in<br />
250 ml baffled shake flasks. For the non-stationary experiment the cultures<br />
were inoculated at a start cell density of 2 × 10 6 cells/ml in a start volume<br />
of 120 ml. Four par<strong>all</strong>el cultivations were performed, two with 100%<br />
[U- 13 C 6 ]glucose and two with 100% [U- 13 C 5 ]glutamine, respectively.<br />
Extracellular samples were taken from <strong>all</strong> four cultivations every 6 h for cell<br />
counting and determination of extracellular metabolite concentrations and<br />
extracellular labeling dynamics. Intracellular samples were taken alternately<br />
from the two replicates. After 2 min, 10 min, 20 min, 30 min, 60 min, 2 h,<br />
4 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, and 48 h, a sample of 5 ml cell<br />
suspension was quenched in 45 ml ice-cold 0.9% sodium chloride solution,<br />
centrifuged for 1 min at 2000 × g, washed once by rinsing the cell pellet<br />
with 50 ml ice-cold 0.9% sodium chloride solution, and frozen in liquid<br />
nitrogen. Intracellular metabolites were extracted in methanol and water<br />
by repeated freeze-thaw cycles, as described previously [2]. Extracts were<br />
dried in a centrifugal evaporator.<br />
Analytics: Cell counting and viability determination was carried out using<br />
an automated cell counter (Invitrogen, Darmstadt, Germany). Quantification<br />
of extracellular glucose, organic acids and amino acids via HPLC was carried<br />
out as described recently [3]. For determination of extracellular labeling<br />
dynamics, lyophilized supernatants were resolved in dimethylformamid<br />
(0.1% pyridine) and derivatized with MBDSTFA (Macherey-Nagel, Düren,<br />
Deutschland). Dried cell extracts were resolved in pyridine (20 mg/ml<br />
methoxylamine) and derivatized with MSTFA (Macherey-Nagel, Düren,<br />
Deutschland). Samples were analyzed by GC-MS. Unique fragments<br />
containing the whole carbon backbone were chosen for excreted<br />
extracellular metabolites and selected intracellular metabolites of the central<br />
metabolism.<br />
Results: We observed a monotonic cultivation profile during short-term<br />
cultivation for 48 h. Metabolic steady state was confirmed by exponential<br />
growth and constant metabolite yields. The two tracers, glucose and<br />
glutamine, were the major carbon sources. Lactate, alanine, glycine, and<br />
glutamate were excreted, <strong>all</strong> other metabolites were consumed. Although<br />
serine, aspartate, and glutamine were only consumed, we found significant<br />
extracellular labeling of these metabolites indicating simultaneous<br />
consumption and excretion.<br />
Label incorporation into intracellular pyruvate and lactate was very fast on<br />
[U- 13 C 6 ]glucose (mainly m3). Isotopic steady state in extracellular lactate<br />
was reached after 12 h. Labeling in pyruvate and lactate was also found<br />
using [U- 13 C 5 ]glutamine as tracer (mainly m1) indicating a significant reflux<br />
from TCA cycle via anaplerotic reactions. Label incorporation into alanine<br />
was slower than for pyruvate and lactate and had a different labeling<br />
pattern. A significantly higher m2 fraction on labeled glucose indicates<br />
synthesis after pyruvate has entered the mitochondria. Significant labeling<br />
of serine and glycine was found on labeled glucose but not on labeled<br />
glutamine indicating the absence of gluconeogenesis.<br />
Label incorporation into TCA cycle metabolites was fast on both tracers<br />
approaching steady state in citrate within 6 h of cultivation. Nearly<br />
identical labeling patterns were found for fumarate, malate and aspartate<br />
indicating a tight connection between these metabolite pools. After 24 h a<br />
metabolic shift takes place. Glutamine was synthesized in significant<br />
amounts. Labeling in TCA cycle metabolites decreased and labeling in<br />
pyruvate, lactate, and alanine further increased.<br />
Conclusions: We present the very first study of 13 Clabelingdynamicsin<br />
CHO suspension cells. We were able to capture labeling dynamics in<br />
excreted extracellular metabolites as well as in intracellular organic acids<br />
and amino acids providing a representative overview of the central<br />
metabolism in CHO cells. Furthermore, we could draw some first<br />
qualitative conclusions. These transient labeling data is currently used in a<br />
non-stationary 13 C metabolic flux analysis in order to obtain an in-depth<br />
understanding of CHO central metabolism, e.g. about reversibilities and<br />
the connection between glycolysis and TCA cycle.<br />
References<br />
1. Klein S, Heinzle E: Isotope labeling experiments in metabolomics and<br />
fluxomics. Wiley Interdiscip Rev Syst Biol Med 2012, 4:261-272.<br />
2. Sellick CA, Hansen R, Stephens GM, Goodacre R, Dickson AJ: Metabolite<br />
extraction from suspension-cultured mammalian cells for global<br />
metabolite profiling. Nat Protocols 2011, 6:1241-1249.<br />
3. Strigun A, Wahrheit J, Beckers S, Heinzle E, Noor F: Metabolic profiling<br />
using HPLC <strong>all</strong>ows classification of drugs according to their mechanisms<br />
of action in HL-1 cardiomyocytes. Toxicol Appl Pharmacol 2011,<br />
252:183-191.<br />
P44<br />
Investigation of glutamine metabolism in CHO cells by dynamic<br />
metabolic flux analysis<br />
Judith Wahrheit * , Averina Nicolae, Elmar Heinzle<br />
Biochemical Engineering Institute, Saarland University, D-66123 Saarbrücken,<br />
Germany<br />
E-mail: j.wahrheit@mx.uni-saarland.de<br />
BMC Proceedings 2013, 7(Suppl 6):P44<br />
Background: Glutamine metabolism represents one of the major targets in<br />
metabolic engineering and process optimization due to its importance as<br />
cellular energy, carbon and nitrogen source. Metabolic flux analysis<br />
represents a powerful method to investigate the physiology and<br />
metabolism of cells [1]. Classical metabolic flux analysis methods require<br />
steady state conditions. However, industri<strong>all</strong>y relevant cultivation conditions,<br />
i.e. batch and fed-batch cultivations, are characterized by changing<br />
environmental conditions and metabolic shifts [2]. We used dynamic<br />
metabolic flux analysis to study the impact of glutamine availability or<br />
limitation on the physiology of CHO K1 cells capturing metabolic dynamics<br />
during batch- and fed-batch cultivations.<br />
Materials and methods: Cell cultivation: CHO-K1 cells were cultivated in<br />
protein free TC-42 medium (TeutoCell, Bielefeld, Germany) in 50 ml filtertube<br />
bioreactors (TPP, Trasadingen, Switzerland) at a start cell density of 2 ×<br />
10 5 cells/ml and a start volume of 20 ml. Cell counting and viability<br />
determination was carried out using an automated cell counter (Invitrogen,<br />
Darmstadt, Germany). Quantification of glucose, organic acids and amino<br />
acids via HPLC was carried out as described recently [3]. Ammonia was<br />
quantified using an ammonia assay kit (Sigma-Aldrich, Steinheim, Germany)<br />
in 96-well plates. Six different batch cultivations with 0 mM, 1 mM, 2 mM,<br />
4 mM, 6 mM or 8 mM glutamine start concentration and two different fedbatch<br />
cultivations starting at 1 mM glutamine and feeding 1 mM every 24 h<br />
or starting at 2 mM and feeding 2 mM every 48 h were performed.<br />
Metabolic flux analysis: Splines were fitted to the cell density and the<br />
extracellular metabolite profile using the SLM curve fitting tool in Matlab
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2012b (The Mathworks, Natick, MA, USA). Using a stoichiometric model of<br />
the CHO metabolism the intracellular fluxes were calculated by flux<br />
balancing.<br />
Results: Glutamine has an initial growth stimulating effect. With increasing<br />
glutamine concentration, the specific growth rate was initi<strong>all</strong>y higher but<br />
dropped earlier. However, increased accumulation of waste products at high<br />
glutamine levels, e.g. ammonia, inhibited growth later on and decreased<br />
culture longevity. The highest viable cell densities were reached in the<br />
1 mM glutamine batch and 8 × 1 mM glutamine fed-batch cultivations.<br />
Substantial dose-dependent flux rearrangements were observed for<br />
different glutamine availabilities. Initi<strong>all</strong>y, no significant impact on the<br />
glycolytic fluxes and lactate excretion was found. In later phases, glycolytic<br />
and lactate excretion rates were higher in the glutamine free cultivation.<br />
Waste product excretion of ammonia, alanine and glutamate increased<br />
with increasing glutamine concentration. The highest glutamate excretion<br />
was, however, found in the glutamine free cultivation. Uptake of pyruvate<br />
and serine as well as their importance as substrates increased with<br />
decreasing glutamine concentration and were highest under glutamine<br />
free conditions. This was accompanied by increasing excretion rates<br />
for glycine. At high glutamine concentrations, glutamate is converted to<br />
a-ketoglutarate feeding TCA cycle fluxes from a-ketoglutarate to<br />
oxaloacetate. However, due to an increased flux from oxaloacetate to<br />
phosphoenol pyruvate, fluxes from oxaloacetate to a-ketoglutarate were<br />
only significantly increased at 8 mM glutamine, but not at lower glutamine<br />
levels. The flux from oxaloacetate to phosphoenol pyruvate was reversed<br />
(phosphoenol pyruvate to oxaloacetate) at glutamine free conditions,<br />
resultinginanapleroticfeedingofcarbonintotheTCAcycle.The<br />
glutamate dehydrogenase flux was reversed (a-ketoglutarate to glutamate)<br />
at glutamine free conditions to produce glutamate for glutamine synthesis.<br />
Waste product excretion was reduced in the fed-batch cultivations<br />
compared to respective batch cultivations with 1, 2, or 8 mM glutamine.<br />
TCA cycle fluxes decreased over time in cultivations with high glutamine<br />
start concentrations and increased for cultivations with low initial<br />
glutamine levels and the glutamine free cultivation. With glutamine<br />
feeding, less variation of TCA cycle fluxes was observed.<br />
Conclusions: Dynamic metabolic flux analysis is a suitable method to<br />
describe the dynamics of growth and metabolism during batch and fedbatch<br />
cultivations with changing environmental conditions. For the batch<br />
cultivations, we observed dose-dependent effects of 1 to 8 mM glutamine<br />
start concentration. The fed-batch cultivations showed an intermediate<br />
response. The glutamine free cultivation had a very different physiology.<br />
Feeding of glutamine resulted in a reduced waste product excretion<br />
compared to respective batch cultivations and TCA cycle fluxes showed<br />
less variation during the cultivation process.<br />
References<br />
1. Niklas J, Heinzle E: Metabolic Flux Analysis in Systems Biology of<br />
Mammalian Cells. Adv Biochem Eng Biotechnol 2011, 127:109-132.<br />
2. Niklas J, Schräder E, Sandig V, Noll T, Heinzle E: Quantitative<br />
characterization of metabolism and metabolic shifts during growth of<br />
the new human cell line AGE1.HN using time resolved metabolic flux<br />
analysis. Bioprocess Biosyst Eng 2011, 34:533-545.<br />
3. Strigun A, Wahrheit J, Beckers S, Heinzle E, Noor F: Metabolic profiling using<br />
HPLC <strong>all</strong>ows classification of drugs according to their mechanisms of<br />
action in HL-1 cardiomyocytes. Toxicol Appl Pharmacol 2011, 252:183-191.<br />
P45<br />
The optimization of a rapid low-cost alternative of large-scale medium<br />
sterilization<br />
Dominique T Monteil 1 , Cédric A Bürki 2 , Lucia Baldi 1,2 , David L Hacker 1 ,<br />
Maria de Jesus 2 , Florian M Wurm 1,2*<br />
1 Laboratory of Cellular Biotechnology, Faculty of Life Sciences, Ecole<br />
Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland;<br />
2 ExcellGene SA, 1870 Monthey, Switzerland<br />
E-mail: florian.wurm@epfl.ch<br />
BMC Proceedings 2013, 7(Suppl 6):P45<br />
Background: One of the most important unit operations in upstream<br />
animal cell bioprocesses at scales over 100 L is the preparation and<br />
sterilization of the medium. This complex, sensitive, and expensive process<br />
requires a considerable investment in both material and time [1].<br />
Tradition<strong>all</strong>y, large-scale medium sterilization is performed with costly<br />
single-use dead-end filters. To optimize and reduce the cost of this unit<br />
operation, we investigated the sterilization of mammalian cell culture<br />
medium at volumes larger than 100 L.<br />
Materials and methods: In this study, an optimization of the cost and<br />
time for the sterilization of cell culture medium at volumes larger than 100 L<br />
was investigated. Pressure-volume diagrams were completed for both a<br />
positive displacement pump (Watson-Marlow 620, Cornw<strong>all</strong>, England) and a<br />
bearingless centrifugal pump (Levitronix PuraLev 600 MU, Zurich,<br />
Switzerland) to determined optimal pumping speeds and pressures. The<br />
study was completed using 0.25” ID tubing with a gate valve downstream of<br />
the pump. The pressure (SciLog SciPres, Madison, WI, USA) and flow<br />
rate (Equflow flowsensor, Ravenstein, Netherlands) were measured at<br />
diffeFinarent closures of the valve. Independently, a range of different size<br />
glass microfiber (GF) pre-filters were tested in combination with and without<br />
the dead-end filters by measuring the turbidity (TN100, Eutech Instruments,<br />
Singapore). A range of different 0.2 μm dead-end membrane filter materials<br />
including polyethersulfone (PES), polyvinylidene fluoride (PVDF), and mixed<br />
cellulose ester (ME) were tested using a positive displacement pump. In<br />
addition, tangential flow filtration (TFF) was examined with both PES and ME<br />
0.2 μm membranes in comparison to the dead-end filters. A mammalian cell<br />
culture medium was filter sterilized at a starting pressure of 500 mbar. The<br />
pressure and flow rate were recorded during the filtration until the<br />
transmembrane pressure increased to 1200 mbar. The filtration was then<br />
stopped at the pressure limit of the tubing connections. Specific filtered<br />
medium volume, filter liquid flux rate, and filtrate turbidity were determined<br />
for each membrane type.<br />
Results: The pressure-volume diagram displayed a higher flow rate for the<br />
bearingless centrifugal pump (6 to 7 Lpm) in comparison to the peristaltic<br />
pump (2.5 Lpm) at the desired pressure of 1000 mbar (data not shown). The<br />
turbidity for unfiltered, pre-filtered, and filtered medium was 2.5, 0.75, and<br />
0.2 NTU, respectively, demonstrating the possible benefits of using a prefilter<br />
(data not shown). The filter liquid flux rates ranged from 3 to<br />
25 L/min/m 2 for the range of different filters. The PES hollow fiber TFF<br />
filters (Spectrum Labs, Breda, Netherlands) displayed a flux rate of 10 L/min/m 2<br />
(Figure 1B). The specific filtered volume for the dead-end filters was up<br />
to 300 L per m 2 of filter surface, while the TFF filter was able to achieve over<br />
1000 L of sterilely filtered medium per m 2 of filter surface (Figure 1A).<br />
Conclusions: The optimization of pumps for the sterile filtration of<br />
mammalian cell culture was completed. Our results indicate that a<br />
bearingless centrifugal pump could provide twice the flow rate at the<br />
desired filtration pressure in comparison to a peristaltic pump. In addition,<br />
the bearingless centrifugal pump was able to provide a constant flow in<br />
comparison to the peristaltic pump. Pre-filters were found to clarify the<br />
medium and thus could further reduce the cost of the filtration. The PES<br />
hollow fiber TFF filter was able to filter over three times the sterile medium<br />
volume in comparison to the dead-end filters. The TFF filters displayed a<br />
similar range of filter liquid flux rates in comparison to the different filters<br />
types. This study showed that a hollow fiber TFF coupled with the use of a<br />
bearingless centrifugal pump provides a low-cost technology for the rapid<br />
large-scale 0.2 μm sterilization of mammalian cell culture medium.<br />
Acknowledgements: We gratefully acknowledge Stéphane Itart-<br />
Longueville from Spectrum labs and Juerg Burkart from Levitronic GmbH<br />
for their considerable support of equipment and material. This work has<br />
been supported by the KTI-Program of the Swiss Economic Ministry and by<br />
the Swiss National Science Foundation (SNSF).<br />
Reference<br />
1. Zhang X, Stettler M, De Sanctis D, Perrone M, Parolini N, Discacciati M, De<br />
Jesus M, Hacker D, Quarteroni A, Wurm F: Use of orbital shaken<br />
disposable bioreactors for Mammalian cell cultures from the milliliterscale<br />
to the 1,000-liter scale. Adv Biochem Eng Biotechnol 2010, 115:33-53.<br />
P46<br />
Improved fed-batch bioprocesses using chemic<strong>all</strong>y modified amino<br />
acids in concentrated feeds<br />
Ronja Mueller 1 , Isabell Joy-Hillesheim 1 , Karima El Bagdadi 1 , Maria Wehsling 1 ,<br />
Christian Jasper 2 , Joerg von Hagen 1 , Aline Zimmer 1*<br />
1 Merck Millipore, Pharm-Chemical Solutions - Research & Development<br />
Upstream, Darmstadt, Germany;<br />
2 Merck KGaA, Performance Materials -<br />
Advanced Technologies Synthesis, Darmstadt, Germany<br />
E-mail: aline.zimmer@merckgroup.com<br />
BMC Proceedings 2013, 7(Suppl 6):P46
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Figure 1(abstract P45) The calculated specific filtered volume displayed over the changing transmembrane pressure for a range of different<br />
filter types (A). The calculated filter liquid flux rate for different filter types (B). The filter pore sizes were as followed: A - PVDF 0.45/0.22 μm, B - PES<br />
0.2 μm, C - PES/PVDF 0.2/0.1 μm, D - GF/PVDF 0.5/0.2 μm, E - PES 0.8/0.2 μm, and F - PES 0.2 μm.<br />
Background: Fed-batch culture bioprocesses are essential for the<br />
production of therapeutic proteins [1]. In these cultures, concentrated<br />
feeds are added during cultivation to prevent nutrient depletion and to<br />
extend the growth phase, thus increasing product concentration [2]. One<br />
limitation arises from the low solubility of some amino acids at high<br />
concentrations, in particular tyrosine [3]. This amino acid is commonly<br />
solubilized in separate feeds at basic pH [4] inducing pH spikes and<br />
precipitation when added in the bioreactor. This work describes the<br />
evaluation of several chemic<strong>all</strong>y modified tyrosines as alternative to<br />
simplify fed-batch bioprocesses by using single feeding strategies at<br />
neutral pH.<br />
Materials and methods: For solubility experiments, increasing concentrations<br />
of modified tyrosines were solubilized in Merck Millipore proprietary<br />
feed at pH 7,0 until reaching the maximum solubility. Stability was assessed<br />
during 6 months in Merck Millipore proprietary medium and amino acids<br />
(including modified tyrosine) were quantified by ultra performance liquid<br />
chromatography using ACQ·Tag Ultra reagent (Waters).<br />
For batch cultures, modified tyrosines were solubilized at a concentration of<br />
4,5 mM in Merck Millipore proprietary medium depleted in unmodified<br />
tyrosine. The control medium contained 1 mM tyrosine di-sodium salt. CHO-<br />
S cells were seeded at 1.10 5 cells/ml in 50 ml spin tubes and incubated at<br />
37°C, 5% CO 2 , 80% humidity and a rotation speed of 320 rpm. Growth and<br />
viability were monitored during 11 days using Beckman Coulter ViCell®.<br />
For fed-batch cultures, CHO-S cells expressing a human monoclonal<br />
antibody were seeded at 2.10 5 cells/ml in medium containing tyrosine<br />
di-sodium salt. Feeds were added every other day starting at day 3. In the<br />
control, tyrosine di-sodium salt was added in a separate feed at pH 11<br />
whereas modified tyrosines were solubilized in the main feed at pH 7,0.<br />
Glucose was maintained at 4 g/L using a separate feed. Growth and viability<br />
were monitored during 14 days using Beckman Coulter ViCell®.<br />
For antibody analysis, IgG concentrations were determined by a<br />
turbidometric method using Roche Cedex bio HT®. Intact mass analysis,<br />
peptide mapping and glycan analyses were performed on samples from<br />
day 14 using mass spectrometry and 2-aminobenzamide labeling followed<br />
by ultra performance liquid chromatography.<br />
Results: Solubility and stability experiments: Chemic<strong>all</strong>y modified<br />
tyrosines demonstrated an increased solubility in concentrated feed at<br />
neutral pH in comparison with tyrosine or tyrosine di-sodium salt (Table 1).<br />
The highest solubility was achieved for the modified tyrosine 4 with a value<br />
of 75 g/L. The stability was assessed by quantification of the modified amino<br />
acid through ultra performance liquid chromatography. Moreover, no<br />
precipitation was detected over a 6 months period indicating that the<br />
chemical modification was stable in the tested conditions.<br />
Batch and fed-batch cultures: The performance in batch culture was<br />
determined using tyrosine depleted media and supplementation with the<br />
different derivates. The growth of CHO-S cells with medium supplemented<br />
with modified tyrosine 2 reached only 50% of the growth of the control<br />
indicating that this molecule may not be able to be taken up by the cells<br />
or to promote growth through alternative mechanisms. This derivate was<br />
not evaluated further. Both modified tyrosines 3 and 4 induced a growth<br />
comparable to the control culture until day 6 and were then able to<br />
extend the growth during 2 additional days indicating that both derivates<br />
can be used successfully in batch cultures.<br />
In fed-batch mode, modified tyrosines 3 and 4 were solubilized in a<br />
single concentrated feed at pH 7,0 and added to the culture every other<br />
day starting at day 3. The growth of recombinant CHO-S cells obtained<br />
with the derivates was similar to the control (where tyrosine di-sodium<br />
salt was added through a separate feed at pH 11) reaching a maximum<br />
viable cell density of 14.10 6 at day 7 (Figure 1A). The titer obtained after<br />
14 days was equivalent in the two feeds and the new single feed process<br />
with final titers around 1,5 g/l (Figure 1B) indicating no negative effect of<br />
the chemical modification on productivity.<br />
Impact of modified tyrosines on the monoclonal antibody quality<br />
attributes: Intact mass, peptide mapping and glycosylation analyses were<br />
performed on the monoclonal antibody to study the impact of modified<br />
tyrosines on the final molecule. No significant difference could be established<br />
in either the intact mass of the antibody or the detailed analysis of the<br />
tryptic peptides by mass spectrometry. Glycosylation analysis indicated the<br />
same over<strong>all</strong> glycosylation pattern with 8,2% GlcNac3Man3Fuc, 72,3% G0F,<br />
7,4% Man5 and 8,5% G1F glycans. Altogether these data indicated that the<br />
Table 1(abstract P46) Maximum solubility and stability of tyrosine derivates in Merck Millipore proprietary feed or<br />
medium at pH 7,0<br />
Molecule Tyrosine Tyrosine di-sodium salt Modified Tyrosine 2 Modified Tyrosine 3 Modified Tyrosine 4<br />
Solubility in concentrated Not soluble < 1 g/l 10 g/l 70 g/l 75 g/l<br />
feed at pH 7,0<br />
Stability in medium at pH 7,0 - > 6 months > 6 months > 6 months > 6 months
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Figure 1(abstract P46) Performance of the modified tyrosines in fed-batch culture. A: Viable cell density and viability during the fed-batch process.<br />
B. IgG production during the fed-batch culture.<br />
use of chemic<strong>all</strong>y modified tyrosines in concentrated feeds did not induce<br />
any detectable modification of the monoclonal antibody.<br />
Conclusions: The chemical modification of tyrosine can improve the<br />
solubility of the amino acid by up to 70 fold.<br />
Modified tyrosines are stable in chemic<strong>all</strong>y defined media and feeds and can<br />
be used in batch and fed-batch mode. The use of these modified amino<br />
acids in fed-batch bioprocesses has no detectable impact on the<br />
monoclonal antibody or the recombinant protein produced. Altogether, this<br />
study demonstrates that modified amino acids can be used successfully in<br />
highly concentrated neutral feeds to improve and simplify next generation<br />
fed-batch processes.<br />
References<br />
1. Butler M, Meneses-Acosta A: Recent advances in technology supporting<br />
biopharmaceutical production from mammalian cells. Appl Microbiol<br />
Biotechnol 2012, 96:885-894.<br />
2. Wlaschin KF, Hu WS: Fedbatch culture and dynamic nutrient feeding. Adv<br />
Biochem Eng Biotechnol 2006, 101:43-74.<br />
3. Hitchcock DI: The Solubility of Tyrosine in Acid and in Alkali. J Gen Physiol<br />
1924, 6(6):747-757.<br />
4. Yu M, Hu Z, Pacis E, Vijayasankaran N, Shen A, Li F: Understanding the<br />
intracellular effect of enhanced nutrient feeding toward high titer<br />
antibody production process. Biotechnol Bioeng 2011, 108:1078-1088.<br />
P47<br />
Approaches for automized expansion and differentiation of human<br />
MSC in specialized bioreactors<br />
Anne Neumann 1,2* , Antonina Lavrentieva 2 , Dominik Egger 2 , Tim Hatlapatka 1 ,<br />
Cornelia Kasper 1<br />
1 Department for Biotechnology, University of Natural Resources and Life<br />
Sciences, 1190 Vienna, Austria;<br />
2 Institute for Technical Chemistry, Leibniz<br />
University of Hannover, 30167 Hanover, Germany<br />
E-mail: anne.neumann@boku.ac.at<br />
BMC Proceedings 2013, 7(Suppl 6):P47<br />
Background and experimental approach: A main ch<strong>all</strong>enge in cell<br />
therapies and other tissue regeneration approaches is to produce a<br />
therapeutic<strong>all</strong>y significant cell number. For expansion of mesenchymal stem<br />
cells (MSC) the cultivation on 2D plastic surfaces is still the conventional<br />
procedure, even though the culture conditions differ significantly from the<br />
3D environment in vivo. Addition<strong>all</strong>y, static amplification of MSC is a labourintensive<br />
procedure. We therefore used a specialized rotating bed bioreactor<br />
in order to maximize ex vivo expansion of MSC. MSC were isolated from<br />
umbilical cord (UC) by explant method approach under xeno-free<br />
conditions. UC-MSC were thereafter expanded under dynamic conditions in<br />
a novel rotating bed bioreactor. The bioreactor system was designed to<br />
enable integration of sensors for online monitoring of various parameters (e.<br />
g. pH, pO 2 ,pCO 2 ) and hence, <strong>all</strong>ow ensured cultivation under well<br />
controlled and reproducible conditions. Beside cell expansion, directed<br />
differentiation of MSC was also achieved in bioreactors. MSC lack the ability<br />
to grow in 3D direction and build functional tissue in vitro. Thus, it is<br />
necessary to seed and culture MSC on 3D matrices to obtain functional<br />
implants. For guided differentiation towards the osteogenic lineage, MSC<br />
were cultivated on ceramic porous matrices under dynamic conditions.<br />
Custom-made miniaturized perfusion bioreactors for par<strong>all</strong>el testing were<br />
designed and optimized for that purpose.<br />
Methods: MSC isolation was achieved as described previously [1]. Briefly,<br />
umbilical cord tissue is cut into pieces (approx. 0.5 cm 2 ) and cultivated for<br />
10 days in aMEM containing 15% human serum in cell culture flasks. Cells<br />
grow out of the tissue pieces and adhere to the cell culture plastic.<br />
Subsequently, cells are harvested and subcultivated in aMEM containing<br />
10% human serum.<br />
UC-MSC were expanded in a rotating bed bioreactor (Figure 1A). The<br />
bioreactor chamber is a cylindrical bioreactor shell, comprising an inlet<br />
(bed) fixed to a magnet whereas the bioreactor chamber is hold by that<br />
magnet to an engine. The inlay is rotating, while the shell is fixed. The inlay<br />
comprises cell culture plastic slides with an <strong>all</strong> over surface of 2000 cm 2 ,<br />
requiring approximately 130 ml cell culture medium to be completely<br />
covered. The bioreactor is equipped with a feed circulation for fresh medium<br />
and removal of waste. An additional circulation to pH and pO 2 sensor<br />
electrodes enables online monitoring. Sampling is performed through a<br />
septum in the bioreactor shell. Gas mixture of air and CO 2 is supplied by an<br />
overlay stream. The whole bioreactor set up is situated in a GMP conform<br />
breeder, enabling sterile handling as well as an environmental temperature<br />
of 38°C. The system is connected to a control unit, which comprises gas<br />
regulation, pumps and software for parameter set up and monitoring.<br />
UC-MSC were seeded (1,500 cells/cm 2 ) in the bioreactor for 24 h hours<br />
and expanded for 5 days under dynamic conditions. Medium feed was<br />
adjusted depending on glucose consumption. After 5 days of cultivations<br />
UC-MSC were harvested by flushing the bioreactor with accutase for 20 min.<br />
MSC were counted, examined regarding their senescence (b-galactosidase),<br />
proliferation capacity (glucose/lactate) and differentiation potential<br />
(Oil Red O, Alizarin Red, Von Kossa, Alcian Blue), as well as surface markers.<br />
Perfusion bioreactors consist of a stainless steel tube in which the material is<br />
inserted and a piston, which closes the reactors (Figure 1B). As the piston<br />
can be adjusted in height a bioreactor can host materials with a diameter of<br />
10 mm and a high of max. 10 mm. MSC-seeded ceramic materials (10 mm ×<br />
3 mm) were inserted into the bioreactor. The bioreactors are connected to
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Figure 1(abstract P47) A) Rotation bed bioreactor for expansion of MSC and B) Perfusion bioreactors.<br />
a medium reservoir, equipped with a sterile filter for gas exchange. The<br />
volume of the bioreactor containing the ceramic material is 1,5 ml, the<br />
over<strong>all</strong> volume of medium used for the cultivation is 10 ml. Dynamic<br />
cultivation was achieved using flow rates of 0.3 and 1.5 ml/min. Viability was<br />
examined using MTT Assay. Cell distribution throughout the scaffold was<br />
investigated using DAPI staining. The status of differentiation was examined<br />
using different histological stainings (e.g. Von Kossa, Calcein, Alizarin Red).<br />
Results and discussion: UC-MSC isolated using explant method approach<br />
fulfils MSC criteria, such as adherence to plastic surfaces, specific surface<br />
marker pattern and differentiation potential towards at least the<br />
adipogenic, chondrogenic and osteogenic lineage [1].<br />
MSC expanded under dynamic conditions in a rotating bed bioreactor<br />
also fulfil these MSC criteria. Furthermore it could be shown, that MSC<br />
consume glucose and produce lactate during dynamic cultivation in the<br />
rotating bed bioreactor and consequently proliferate. After 5 days of<br />
cultivation MSC were investigated regarding their specific surface marker.<br />
They express CD44, CD73, CD90 and CD 105 and lack CD 31, CD34 and<br />
CD45.<br />
MSC on ceramic materials could be shown to differentiate towards the<br />
osteogenic lineage under static conditions. Also after dynamic cultivation<br />
with a medium perfusion of 0.3 ml/min and even 1.5 ml/min cells adhere<br />
on the macro porous ceramic material, were viable and equ<strong>all</strong>y distributed<br />
throughout the scaffold. Seeding efficiency was found to be approximately<br />
20%. Osteogenic differentiation could be achieved by cultivation in<br />
perfusion bioreactors.<br />
Conclusion: MSC could be successfully isolated from human umbilical<br />
cord tissue. MSC expansion in the rotation bed bioreactor provides a high<br />
number of cells, maintaining their stem cell properties such as specific<br />
surface markers, proliferation capacity and differentiation potential.<br />
Cultivation of MSC in perfusion bioreactors have been shown to support<br />
and improve osteogenic differentiation as mechanical plays an important<br />
role in directing MSC fate. Our results support the argument that the<br />
application of tailor-made bioreactors are an essential step toward the<br />
production of stem cell based therapeutics and tissue engineering<br />
products.<br />
Reference<br />
1. Moretti P, Hatlapatka T, Marten D, Lavrentieva A, Majore I, Hass R, Kasper C:<br />
Mesenchymal Stromal Cells Derived from Human Umbilical Cord Tissues:<br />
Primitive Cells with Potentisl for Clinical and Tissue Engineering<br />
Applications. Adv Biomedical Engin/Biotechol 2010, 123:29-45.<br />
P48<br />
Cell cycle and apoptosis in PER.C6® cultures<br />
Sarah M Mercier 1* , Bas Diepenbroek 1 , Dirk E Martens 2 , Rene H Wijffels 2 ,<br />
Mathieu Streefland 2<br />
1 Crucell, Leiden, The Netherlands;<br />
2 Bioprocess Engineering, Wageningen<br />
University, Wageningen, The Netherlands<br />
E-mail: smercier@its.jnj.com<br />
BMC Proceedings 2013, 7(Suppl 6):P48<br />
Background: PER.C6® is a human cell line designed for virus production,<br />
which was immortalized by transformation with adenoviral E1A and E1B<br />
genes. Expression of E1A is known to inhibit negative regulators of cell<br />
cycle and E1B protein function analogously to an apoptosis inhibitor. As<br />
changes in cell cycle and apoptosis are likely to affect cell’s ability for<br />
viral infection and propagation, the study of these parameters in PER.C6®<br />
cultures is essential to develop optimum virus production processes.<br />
Materials and methods: Cell cycle distribution and apoptosis were<br />
measured in batch and perfusion PER.C6® cultures using flow cytometry.<br />
Propidium iodide was used to measure cell cycle distribution. Three<br />
methods were used to measure apoptosis: staining of externalized<br />
phosphatidylserine (PS) using annexinV, staining of activated caspases<br />
using a fluorochrome-conjugated inhibitor of caspases, and staining of<br />
fragmented DNA using BrdU incorporation and specific fluorescent<br />
labeling. 7-ADD was used to stain dead cells with a permeable membrane.<br />
Results: Significant cell death occurred in 14-days batches, when the main<br />
carbon sources were depleted. Apoptosis was initi<strong>all</strong>y not detected by the<br />
annexinV staining. However, activated caspases were detected after 6 days,<br />
suggesting that apoptosis occurred in batch. In perfusion, where the<br />
required nutrients were constantly supplied, no significant cell death or<br />
induction of apoptosis occurred, showing that the cultures were maintained<br />
in healthy conditions. At the end of batches, the portion of cells in S phase<br />
increased drastic<strong>all</strong>y and the one in G0/G1 decreased. In perfusion, cell cycle<br />
distribution was stable until 10 days, when a similar trend as the end of<br />
batch was observed.<br />
This is the first research describing apoptosis and cell cycle distribution in<br />
PER.C6® batch and perfusion cultures. Our data are in accordance with the<br />
theoretical effect of immortalization by the E1A/B system, which inhibits<br />
apoptosis when nutrients are in excess and promotes the entry into the cell<br />
division cycle.
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P49<br />
Scale-up considerations for monoclonal antibody production process:<br />
an oxygen transfer flux approach<br />
Laura Gimenez * , Claire Simonet, Laetitia Malphettes<br />
BioTech Sciences, UCB Pharma SA, Braine l’Alleud, Belgium<br />
E-mail: Laura.Gimenez@ucb.com<br />
BMC Proceedings 2013, 7(Suppl 6):P49<br />
Background: When scaling up a monoclonal antibody (mAb) production<br />
process in stirred tank bioreactor, oxygen transfer is probably one of the<br />
most ch<strong>all</strong>enging parameters to consider. Approaches such as keeping<br />
constant specific power input or tip speed across the scales are widely<br />
described in the literature and are often based on the assumption that<br />
mammalian cells are sensitive to shear stress.<br />
However, with the high cell densities reachedinmodernprocesses,such<br />
scale-up strategies can lead to relatively high gas flow rate to compensate<br />
low agitation speed which could be detrimental to cells in its own right.<br />
As an alternative, we explored a scale-up strategy based on the over<strong>all</strong><br />
oxygen transfer flux (OTF) required by the cell culture process. OTF was<br />
defined as directly proportional to oxygen transfer coefficient (k L a) and<br />
oxygen enrichment in the gas mix. This way the over<strong>all</strong> gas flow can be<br />
kept at relatively low values, while satisfying the oxygen requirements of a<br />
high cell density culture.<br />
Materials and methods: Process scale-up between 3 different stirred tank<br />
bioreactors was studied: a 2 L glass bioreactor (Sartorius Stedim Biotech)<br />
equipped with one 3-segment blade impeller, a 10 L glass bioreactor<br />
(Sartorius Stedim Biotech) equipped with two 3-segment blade impellers<br />
and a 80 L stainless steel bioreactor (Zeta Biopharma) equipped with two<br />
elephant ear impellers.<br />
Oxygen transfer coefficients (k L a) were determined for the chemic<strong>all</strong>y<br />
defined production medium, using the dynamic technique of oxygen<br />
adsorption. The statistical analysis software JMP (SAS) was then used in<br />
order to express k L a’s according to the following equation: k L a = A * (P/V) a<br />
*Vs b , P/V being volumetric power input [W.m -3 ] and Vs being superficial air<br />
velocity [m.s -1 ], and to analyze our results.<br />
Oxygen transfer flux was defined as followed: OTF = k L a*(%O 2 in the<br />
gas mix/% O 2 in air).<br />
For cell culture experiments, bioreactors were inoculated with a CHO cell line<br />
producing a mAb. Cells were cultivated in chemic<strong>all</strong>y defined media for a<br />
14-day fed-batch process. The culture was controlled to maintain the desired<br />
process parameters (temperature, pH, dO 2 and glucose concentration). dO 2<br />
level was maintained using a cascade aeration. Viable cell density (VCD) and<br />
viability were monitored by Trypan blue dye exclusion using a Vicell XR<br />
(Beckman Coulter). Glucose and lactate concentrations were determined<br />
using a Nova Bioprofile 400 analyzer (Nova Biomedical). Offline dissolved CO 2<br />
and osmolality were measured with a Nova Bioprofile pHox (Nova<br />
Biomedical) and Osmo 2020 (Advanced Instrument) analyzers respectively.<br />
mAb concentrations were determined by Protein A HPLC.<br />
Results: k L a mapping of 2 L, 10 L and 80 L bioreactors: The 2 L and<br />
10 L bioreactors were characterized for a range of superficial gas velocity<br />
going from 5.0 × 10 -5 to 4.0 × 10 -4 m.s -1 and the 80 L for a range going from<br />
2.0 × 10 -4 to 1.2 × 10 -3 m.s -1 . Specific power input was ranged from 10 to<br />
90 W.m -3 for the 2 L bioreactor, 20 to 130 W.m -3 for the 10 L bioreactor and<br />
5 to 80 W.m -3 for the 80 L bioreactor. Models were generated with JMP and<br />
gave the following equations for k L a[s -1 ]:<br />
2 L bioreactor: k L a = 6.37 × 10 -2 * (P/V) 0.28 *Vs 0.59 (R 2 = 0.98, Prob>F:<br />
F: F:
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Figure 1(abstract P49) Cell culture process performance at 2 L, 10 L and 80 L scale. a) Impact of agitation speed on VCD and mAb titer at 2 L scale.<br />
b) Comparison of VCD, viability and mAb titer obtained in 2 L, 10 L and 80 L bioreactors. c) Comparison of osmolality, glucose and lactate profiles<br />
obtained in 2 L, 10 L and 80 L bioreactor. d) Online pH and dCO 2 levels obtained in 2 L, 10 L and 80 L bioreactors.<br />
G3000SWXL, TOSOH), in which the Tris buffer (pH = 7.4) containing 0.05%<br />
CHAPS was used as elution buffer.<br />
Results: All batch cultivations were carried out until viable cells become<br />
equal to zero. Cells grew well at more than 33 °C, however cells didn’t grow<br />
at 30 °C. Compared to 37 °C-cultivation, lower specific growth rates were<br />
observed in the lower temperature cultivations. The specific production rate<br />
of sCR1, q s CR1 , was obtained by the slope of relationship between sCR1<br />
concentration and time integrated cell concentration within a linear range.<br />
The q s CR1 at each temperature were the almost same except at 30 °C.<br />
The final sCR1 concentrations at 33 °C was rather higher than those at 37<br />
and 35 °C. The cell concentration in stationary phase, X S , at 33 °C was lower<br />
than those at 37 and 35 °C. Thus the ratio of the final sCR1 concentration to<br />
X S at 33 °C was the highest in case of more than 33 °C. The final sCR1<br />
concentration to X S at 30 °C is rather higher than that at 33 °C, however it<br />
makes no sense because of the extremely low specific growth rate at 30 °C.<br />
In order to increase the final sCR1 concentration, we proposed a two-stage<br />
culture that at first cultivation temperature was set to 37 °C and then a<br />
culture temperature became lower at late logarithm phase. Thus the final<br />
sCR1 concentration by using a two-stage culture, in which the temperature<br />
was 37 °C initi<strong>all</strong>y and changed to 33 °C after 120 h-cultivation, increased<br />
by 1.75 and 1.99, compared as a flat temperature culture at 33 °C and<br />
37 °C, respectively (Figure 1, Table 1).<br />
Conclusions: The conclusions are as follows:<br />
1. It was shown that the ratio of the final sCR1 concentration to the cell<br />
concentration in stationary phase was rather higher at lower temperature<br />
than that in 37 °C-cultivation.<br />
2. A two-stage cultivation with temperature change from 37 °C to lower<br />
temperature was proposed and it was shown that the final product<br />
concentration was considerably improved.<br />
References<br />
1. Yoon SK, Song Ji Y, Lee GM: Effect of low temperature on specific<br />
productivity, transcription level, and heterogeneity of erythropoietin in<br />
Chinese hamster ovary cells. Biotechnol Bioeng 2003, 82:289-298.<br />
2. Kato H, Inoue T, Ishii N, Murakami Y, Matsumura M, Seya T, Wang PC:<br />
A novel simple method to purify recombinant soluble human<br />
complement receptor type 1 (sCR1) from CHO cell culture. Biotechnol<br />
Bioprocess Eng 2002, 7:67-75.<br />
P51<br />
HEK293 cell culture media study: increasing cell density for different<br />
bioprocess applications<br />
Leticia Liste-C<strong>all</strong>eja * , Martí Lecina, Jordi Joan Cairó<br />
Chemical Engineering Department, Universitat Autònoma de Barcelona,<br />
Cerdanyola del V<strong>all</strong>ès, 08193, Spain<br />
E-mail: Leticia.Liste@uab.cat<br />
BMC Proceedings 2013, 7(Suppl 6):P51<br />
Background: The increasing demand for biopharmaceuticals produced in<br />
mammalian cells has lead industries to enhance bioprocess volumetric<br />
productivity through different strategies. Among them, media development<br />
is of major interest [1]. According to the increasing constraints regarding the<br />
use of animal derived components on industrial bioprocesses but also the<br />
drawbacks of its depletion from cell culture [2], the main goal of the present<br />
work was to provide different cell culture platforms which are suitable for a<br />
wide range of applications depending on the type and the final use of the<br />
product obtained.
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Figure 1(abstract P50) Time courses of cell-cultivation: (a) 37 °C, (b) two-stage cultivation (37 °C to 33 °C after 120 h).<br />
Table 1(abstract P50) Comparison of culture parameters at various temperatures<br />
30 °C 33 °C 35 °C 37 °C 37 °C®33 °C<br />
specific growth rate [h -1 ] >0.0002 0.0072 0.0107 0.0136 -<br />
q CR1 s [10 9 g cells -1 h -1 ] 0.0304 0.0416 0.0407 0.0446 -<br />
final sCR1 [mg/mL] (a) 3.04 8.68 8.11 7.67 15.2<br />
X S [10 6 cells/mL] (b) 0.223 0.788 1.09 1.15 1.20<br />
(a)/(b) 13.6 11.0 7.43 6.68 12.7<br />
Materials and methods: The cell line HEK293SF-3F6 employed in this<br />
studywaskindlyprovidedbyDr.A.Kamen,NRC-BRI.Thebasalmedia<br />
tested were CDM4HEK293, SFM4HEK293 and SFMTransFx-293 (Hyclone,<br />
Thermo Scientific) supplemented -when indicated- with FBS (Invitrogen)<br />
and/or Cell Boost 5 (80 g/L) (Hyclone, Thermo Scientific). Viable cell<br />
density and viability were determined by trypan blue exclusion method<br />
and manual counting using an haemocytometer. The adenovirus strain<br />
HAdV-5(ΔE1/E3) encoding pCMV-GFP was used for infection experiments.<br />
All infections were carried out at MOI≈1 TOI≈0.5 × 10 6 cell/mL in 6-wellplate.<br />
Harvesting was performed 48 hpi.<br />
Viral titration was performed by Flow cytometry on a FACS Canto<br />
(Becton and Dickinson, Bioscience) by adaption of a protocol previously<br />
described [3].<br />
Results: The first part of this work was focused on screening different<br />
serum-free cell culture media specific<strong>all</strong>y recommended for HEK293<br />
cell line. As shown in Figure 1A top panel, cultures performed in HyQ<br />
SFM4HEK293 and HyQ SFMTransFx-293 showed better cell growth than<br />
HyQ CDM4HEK293, reaching maximum cell densities of about 3.5 × 10 6<br />
cell/mL, 2 × 10 6 cell/mL and less than 1 × 10 6 cell/mL respectively. In order<br />
to evaluate whether the substitution of critical serum components have<br />
satisfactorily been performed in the media tested without affecting cell<br />
growth, the addition of fetal bovine serum (FBS) was assessed. FBS<br />
depletion was acceptable only in HyQ SFM4HEK293 as the other cell media<br />
reached higher cell densities when FBS was added (up to 7-fold increment<br />
of Xv max ). Regarding the screening of Animal derived component free<br />
supplements, three chemic<strong>all</strong>y defined supplements were tested but only<br />
one (Cell Boost 5, onwards CB5) significantly enhanced cell growth. This<br />
supplement enabled to reach higher cell densities in <strong>all</strong> media tested:<br />
2-fold up in HyQ SFM4HEK293 and CDM4HEK293 and 5-fold increment in<br />
HyQ SFMTransFx-293 (Figure 1A, bottom panel).<br />
The results obtained so far showed that supplementation of <strong>all</strong> cell media<br />
tested is recommended in order to achieve higher cell density cultures.<br />
Among <strong>all</strong> the conditions, HyQSFMTransFx-293 was the media which<br />
supported the highest Xv max with both supplements (FBS and CB5).<br />
Therefore, this medium was selected for tuning the final concentration<br />
of each supplement. Among the studied concentration range for FBS<br />
(2.5-10% v/v) and for CB5 (2.5-20%) it was determined that the best<br />
conditions were 5% for FBS and 10% for CB5 solution. At these<br />
concentrations, Xv max achieved were (7.14 ± 0.56*10 6 cell/mL) and (12.63 ±<br />
1.76*10 6 cell/mL) respectively (Figure 1B). Interestingly, CB5 enabled to<br />
extend μ max phase while FBS increased μ max value, as previously detected in<br />
the initial media screening (Table 1). The combination of supplements (5%<br />
FBS and 10%CB5) resulted in an Xv max as high as 16.77 ± 0.70 × 10 6 cell/mL<br />
in batch culture, with an increment in specific growth rate of 15% in<br />
comparison to those cultures in which FBS was deprived. Specific growth<br />
rate was maintained for 144 h of cell culture.<br />
From the range of applications in which HEK293 can be used, the work<br />
carried out in this work was directed to recombinant adenovirus production.<br />
Hence, the evaluation of the effect of supplementation in the cell media<br />
selected on adenovirus infection efficiency and final titer obtained was<br />
evaluated (Figure 1C). Efficiency of infection was around 63% as expected<br />
for an effective infection [4] in <strong>all</strong> conditions. In regards to adenovirus<br />
production, FBS increased it up to fivefold, whereas CB5 supplementation<br />
did not affect significantly, and the addition of both supplements almost<br />
doubled the viral production in comparison to basal medium. It is proposed<br />
that an increment of osmolarity due to the addition of both supplements<br />
might explain the slight reduction on productivity in comparison to the<br />
addition of FBS solely [5].<br />
Conclusions: Two culture platforms are proposed for two possible<br />
scenarios in basis of the Xv max reached: (1) HyQSFMTransFx-293 CB5
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Figure 1(abstract P51) (A) Comparison of cell growth profiles of HEK293 cell cultures in serum-free cell media (top panel) and in the same cell media<br />
FBS supplemented (middle panel) or CB5 supplemented (bottom panel). (B) HyQ SFMTransFx-293 cell cultures with the best concentrations encountered<br />
for FBS and CB5 and combination of supplements. (C) Evaluation of the effect of supplement addition on efficiency of infection and Viral Titer obtained.<br />
Table 1(abstract 51) Kinetic parameters for HEK293 cell cultures corresponding to the profiles depicted in Figure 1<br />
HyQ CDM4HEK293 HyQ SFM4HEK293 HyQ SFMTransFx-293<br />
No adition Xv max (×10 6 cell·mL -1 ) 0.85 ± 0.0 3.53 ± 0.21 2.1 ± 0.12<br />
μ max (×10 -2 h -1 ) 1.06 ± 0.01 2.46 ± 0.14 2.43 ± 0.03<br />
t μ (h) 96 74 74<br />
5% FBS Xv max (×10 6 cell·mL -1 ) 6 ± 0.0 4.67 ± 0.48 7.02 ± 0.06<br />
μ max (h -1 ) 2.61 ± 0.04 2.8 ± 0.05 2.67 ± 0.01<br />
t μ (h) 95 71 72<br />
5%CB5 Xv max (×10 6 cell·mL -1 ) 4.11 ± 0.33 7.29 ± 0.18 9.75 ± 0.25<br />
μ max (h -1 ) 2.1 ± 0.06 2.06 ± 0.03 2.17 ± 0.03<br />
t μ (h) 92 69 116<br />
supplemented -10% v/v- for animal derived component Free required<br />
bioprocesses (Xv max = 12.6 × 10 6 cell/mL) and (2) HyQSFMTransFx-293 FBS<br />
and CB5 supplemented -5% and 10% v/v respectively- for animal derived<br />
component containing bioprocesses (Xv max =16.7×10 6 cell/mL). In both<br />
cases, μ max and t μ values were preserved or even improved with respect to<br />
basal media and any of the supplements negatively affected the adenovirus<br />
production when compared to non-supplemented infections.<br />
Acknowledgements: We would like to thank Dr. Amine Kamen (BRI-NRC,<br />
Canada) for kindly providing the HEK 293 cell line.<br />
References<br />
1. Burgener A, Butler M: Medium Development. Cell Culture Technology For<br />
Pharmaceutical And Cell-Based Therapies Boca Ratón, FL: CRC Press: Ozturk S,<br />
Hu WS, 1 2006, 41-80.<br />
2. Keenan J, Pearson D, Clypes M: The role of recombinant proteins in the<br />
development of serum-free media. Cytotechnology 2006, 50:49-56.<br />
3. Gálvez J, Lecina M, Solà C, Cairó JJ, Gòdia F: Optimization of HEK-293S cell<br />
cultures for the production of adenoviral vectors in bioreactors using<br />
on-line OUR measurements. J Biotech 2012, 157:214-222.<br />
4. Condit RC: Principles of Virology. Fields Virology Lippencott: Williams and<br />
Wilkins: Knipe DM, Howley PM , 5 2007, 25-58.<br />
5. Dormond E, Perrier M, Kamen A: From the first to the third generation<br />
adenoviral vector: what parameters are governing the production yield?<br />
Biotechnology advances 2009, 27:133-144.<br />
P52<br />
Preliminary studies of cell culture strategies for bioprocess<br />
development based on HEK293 cells<br />
Leticia Liste-C<strong>all</strong>eja * , Jonatan López-Repullo, Martí Lecina, Jordi Joan Cairó<br />
Chemical Engineering Department, Universitat Autònoma de Barcelona,<br />
Cerdanyola del V<strong>all</strong>ès, 08193, Spain<br />
E-mail: Leticia.Liste@uab.cat<br />
BMC Proceedings 2013, 7(Suppl 6):P52<br />
Background: The use of human embryonic kidney cells (HEK293) for<br />
recombinant protein or virus production has gained relevance along the
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last years. They are speci<strong>all</strong>y recommended for transient gene expression<br />
and adenovirus or adeno-associated virus generation [1,2]. To achieve<br />
high volumetric productivities towards bioprocess optimization, the<br />
concentration of biocatalizer (i.e. animal cells) must be enhanced. The<br />
limits for cell growth are mainly related to the accumulation of metabolic<br />
by-products, or the depletion of nutrients [3]; therefore, cell cultures<br />
strategies must be developed. In this work, we have explored Punctual<br />
Feeding and Media Replacement cell culture strategies to over perform<br />
the limit on Xv max encountered on batch culture mode. Fin<strong>all</strong>y, we scaled<br />
up cell culture in order to control other parameters (i.e. pO 2 )thatcould<br />
be limiting cell growth.<br />
Materials and methods: The cell line used in this study was HEK293SF-3F6<br />
(kindly provided by Dr. A.Kamen, NRC-BRI). The basal medium for <strong>all</strong> cell<br />
cultures was SFMTransFx-293 (Hyclone, Thermo Scientific) supplemented<br />
with 5% (v/v) of FBS and 4 mM GlutaMAX (Gibco, Invitrogen). For Punctual<br />
Feeding and FedBatch Fementation Cell Boost 5 (Hyclone, Thermo Scientific)<br />
was used. Batch, media replacement and punctual feeding experiments<br />
were performed in 125-ml plastic shake flasks (Corning Inc.) shaken at<br />
110rpminanorbitalshakerat37°C,95%humidity,5%CO 2 incubator.<br />
FedBatch Fermentation was carried out in Bioreactor Braun-MCD (2 L) with<br />
mechanical agitation at 80 rpm, pH set point 7.1 and pO 2 set point 50%.<br />
Viable cell density and viability were determined by trypan blue exclusion<br />
method and manual counting using a haemocytometer. Glucose and lactate<br />
were analysed in an automatic analyser, YSI (Yellow Springs Instrument,<br />
2700 Select).<br />
Results: Characterization of HEK293 cell culture in batch operation was<br />
initi<strong>all</strong>y performed. It was encountered that cell growth was extended for<br />
168 h reaching approximately 7·10 6 cell/mL of cell density (Figure 1.1).<br />
Nevertheless, maximal cell growth rate (μ max ) was only maintained for<br />
96 h. As glucose and lactate were not at limiting concentrations [4],<br />
Figure 1(abstract P52) Comparison of HEK293 cell growth, viability, glucose and lactate profiles in different cell culture strategies.
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nutrient limitation different from glucose arose as the first hypothesis for<br />
this decrease on cell growth rate. Therefore, punctual additions of<br />
nutritional supplement for HEK293 were carried out. Xv max was<br />
significantly increased in comparison to basal media, reaching cell<br />
densities as high as 17·10 6 cell/mL (Figure 1.2). Nevertheless, we could not<br />
overcome this limit on Xv max regardless the number of punctual feedings<br />
performed. Moreover, nutrient addition did not elongate μ max period (t μ ).<br />
These results suggested that by-product accumulation different from<br />
lactate could be limiting cell growth. In order to validate the hypothesis,<br />
complete media replacement (up to three replacements) was studied<br />
(Figure 1.3). Although this strategy enabled to extend t μ up to 168 h of cell<br />
culture, the maximal cell density reached was similar to nutrient addition<br />
strategy (1MR: 12·10 6 cell/mL; 2MR: 16·10 6 cell/mL; 3MR: 18·10 6 cell/mL).<br />
This limit on Xv max encountered on shake flask might be related to a<br />
limitation on pO 2. Thus, the cell culture system was changed towards a<br />
bioreactor with controlled pO 2 (maintained between 20-60% of air<br />
saturation). In addition, a continuous feeding using a pre-fixed profile<br />
addition was implemented. As it can be noticed in Figure 1.4, FedBatch<br />
operation in bioreactor enabled to beat the limit encountered in shake<br />
flask system, reaching cell densities of 27·10 6 cell/mL.<br />
Conclusions: Punctual feeding and media replacement overcame the limit<br />
of 7·10 6 cell/mL encountered in batch mode operation indicating that<br />
nutrient depletion was one of the causes of that limit. Nevertheless, the<br />
elongation of t μ found out performing MR suggests that the accumulation<br />
of by-products might not be ruled out.<br />
The new limit on Xv max (≈17-18·10 6 cell/mL) encountered regardless the<br />
cell culture strategy, was outperformed by transferring O 2 more<br />
efficiently in bioreactor system, reaching cell densities as high as Xv max =<br />
27·10 6 cell/mL. The monitoring and control of cell culture parameters (i.e.<br />
pO 2 , pH) will enable to develop more accurate feeding strategies in<br />
order to achieve higher cell densities than those presented here (on<br />
going work).<br />
Acknowledgements: We would like to thank Dr. Amine Kamen (BRI-NRC,<br />
Canada) for kindly providing the HEK 293 cell line.<br />
References<br />
1. Nadeau I, Kamen A: Production of adenovirus vector for gene therapy.<br />
Biotechnology advances 2003, 20:475-489.<br />
2. Geisse S, Fux C: Recombinant protein production by transient gene<br />
transfer into Mammalian cells. Methods in Enzymology 2009,<br />
463:223-238.<br />
3. Butler M: Animal cell cultures:recent achievements and perspectives in<br />
the production of biopharmaceuticals. Appl Microbiol Biotechnol 2005,<br />
68:283-291.<br />
4. Petiot E, Jacob D, Lanthier S, Lohr V, Ansorge S, Kamen A: Metabolic and<br />
Kinetic analyses of influenza production in perfusion HEK293 cell<br />
culture. BMC Biotechnol 2011, 11:84-96.<br />
P53<br />
Adhesion and colonization of mesenchymal stem cells on polylactide or<br />
PLCL fibers dedicated for tissue engineering<br />
Frédérique Balandras 1 , Caroline Ferrari 1 , Eric Olmos 1 , Mukesh Gupta 2 ,<br />
Cécile Nouvel 2 , Jérôme Babin 2 , Jean-Luc Six 2 , Nguyen Tran 3 ,<br />
Isabelle Chevalot 1 , Emmanuel Guedon 1* , Annie Marc 1<br />
1 CNRS, Laboratoire Réactions et Génie des Procédés, UMR 7274, Université<br />
de Lorraine-ENSAIA, 2 avenue de la forêt de Haye, TSA 40602, F-54518<br />
Vandoeuvre-lès-Nancy Cedex, France; 2 CNRS, Laboratoire de Chimie Physique<br />
Macromoléculaire, FRE 3564, Université de Lorraine-ENSIC, 1 rue Grandville,<br />
54000 Nancy Cedex, France;<br />
3 École de Chirurgie, Faculté de Médecine,<br />
Université de Lorraine, F-54500 -Vandœuvre-lès-Nancy, France<br />
E-mail: emmanuel.guedon@univ-lorraine.fr<br />
BMC Proceedings 2013, 7(Suppl 6):P53<br />
Background: Tissue engineering covers a broad range of applications<br />
dedicated to the repair or the replacement of part or whole tissue such as<br />
blood vessels, bones, cartilages, ligaments, etc [1]. Practic<strong>all</strong>y, a bio<br />
substitute, made with cells cultivated on scaffold, is needed. Mesenchymal<br />
stem cells (MSC) are gener<strong>all</strong>y the most suitable cells for such application<br />
since they are self-renewable with a great potential for differentiation and<br />
immuno suppression [2]. However, materials used for bio functional<br />
scaffold synthesis have to meet several criteria, such as biocompatibility<br />
and biodegradability. Thus, the aim of the study was to screen several<br />
Table 1(abstract 53) Composition of co-polymers used in<br />
this study<br />
Commercial PLCL 70% L-LA 30% CL<br />
MKG58 70% D, L-LA 30% CL<br />
MKG64 - 100% CL<br />
MKG70 50%L-LA 50% CL<br />
MKG71 100%D, L-LA -<br />
MKG74 100%L-LA -<br />
LA: lactic acid; CL: ε-caprolactone<br />
biopolymers differing in their composition for their capability to promote<br />
adhesion and growth of MSC.<br />
Materials and methods: Porcine MSC were cultivated in a-MEM<br />
supplemented with 10% serum and FGF2. For cell adhesion experiments,<br />
6(co)polymers (Table 1) were synthesized and tested.<br />
Fibres of polymers were electrospun on 4 cm 2 cover glasses. Briefly, the<br />
polymer solutions are introduced into a syringe with various flow rates and<br />
an electrical field is applied, resulting in the formation of a polymer jet on<br />
cover glasses or on any surfaces. Then, cover glasses were put onto 6 wells<br />
plate before to be seeded with MSC. Then, cell adhesion and colonization<br />
of polymer fibres were monitored by microscopy and counted using<br />
Guava Viacount assay after trypsine treatment as already described [3].<br />
Results: With the aim of studying and identifying new materials dedicated<br />
to scaffold manufacturing for tissue ingineering, MSC were cultivated on<br />
various (co) polymers. These polymers, made with lactic acid (L and/or D)<br />
and/or caprolactone (blue bars; MKG 58, 64, 70, 71 and 74) in comparison<br />
with a commercial PLCL (red bars), were electrospun on cover glasses in<br />
order to functionalize them. Then MSC were cultivated on theses<br />
functionalized cover glasses at two initial cell seeding (10 000 and 60 000<br />
cells) during 200 hours (Figure 1).<br />
Whatever the polymer used and the initial cell seeding, cells were able to<br />
adhere and to colonize fibres. A cell multiplication factor ranging from 6.5 to<br />
22 was measured after 200 hours of culture depending on the polymer<br />
composition and the initial seeding. However, compared to the commercial<br />
PLCL, the total cell number was strongly increased with MKG 71 (21 and<br />
50%), MKG 74 (34 and 34%) and MKG 58 (15 and 40%) whereas a moderate<br />
growth was observed with MKG 64 (9 and 30%) at an initial seeding of<br />
10 000 and 60 000 cells respectively. MKG 70 did not improve the cell<br />
growth compared to the commercial polymer (> 5% for both seeding).<br />
Conclusion: In this study, porcine MSC were cultivated on various (co)<br />
polymers made with lactic acid (L and/or D) and/or caprolactone. Our results<br />
demonstrated that composition of these (co)polymers strongly influences<br />
MSC growth and colonization. Indeed, polymers such as MKG 58, 71 and 74<br />
appeared to promote MSC growth contrary to other polymers tested, i;e<br />
MKG 64 and MKG 70, compared to the commercial one. Therefore, MKG 58,<br />
71 and 74 could be favoured for further scaffold synthesis.<br />
References<br />
1. Caplan AI: Adult mesenchymal stem cells for tissue engineering versus<br />
regenerative medicine. J Cell Physio 2007, 213:341-347.<br />
2. Chamberlain G, Fox J, Ashton B, Middleton J: Concise review:<br />
Mesenchymal stem cells: Their phenotype, differentiation capacity,<br />
immunological features, and potential for homing. Stem Cells 2007,<br />
25:2739-2749.<br />
3. Ferrari C, Balandras F, Guedon E, Olmos E, Chevalot I, Marc A: Limiting cell<br />
aggregation during mesenchymal stem cell expansion on microcarriers.<br />
Biotechnol Prog 2012, 28:780-787.<br />
P54<br />
Cell cycle and apoptosis: a map for the GS-NS0 cell line at the genetic<br />
level and the link to environmental stress<br />
Chonlatep Usaku, David Garcia Munzer, Efstratios N Pistikopoulos,<br />
Athanasios Mantalaris *<br />
Biological Systems Engineering Laboratory, Centre for Process Systems<br />
Engineering, Department of Chemical Engineering, Imperial College London,<br />
London, SW7 2AZ, UK<br />
E-mail: a.mantalaris@imperial.ac.uk<br />
BMC Proceedings 2013, 7(Suppl 6):P54
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Figure 1(abstract P53) Quantitative evaluation of MSC growth on poly lactide/caprolactone polymers. Two initial MSC seeding, 10 000 and 60 000<br />
cells, were carried out. The red stars indicate a significant increase in final cell number compared to the control (commercial PLCL).<br />
Background: Large scale mammalian cell culture systems, especi<strong>all</strong>y fedbatch<br />
systems, are currently utilised to manufacture monoclonal<br />
antibodies (MAbs) in order to meet the continuously growing global<br />
demand [1]. Nutrient deprivation and toxic metabolite accumulation<br />
commonly encountered in such systems influence the cell cycle and<br />
trigger apoptosis, resulting in shorter culture times and a lower final MAb<br />
titre. Control of the cell cycle has been previously studied in order to<br />
achieve higher titre through apoptosis inhibition by bcl-2 overexpression<br />
and cell cycle arrest in G 1 /G 0 by p21 transfection. However, the above<br />
mentioned strategies have not always been successful; no improvement in<br />
titre was often observed though bcl-2 over-expression helped prolong the<br />
culture viability whereby the majority of cells were arrested at G 1 /G 0 to<br />
avoid apoptosis [2-4]. Thus, a systematic insight of the dynamic relation<br />
between metabolic stress, cell cycle and apoptosis is still required. To this<br />
end, we aim to establish a novel map of the dynamic interplay between<br />
cell cycle and apoptosis at the genetic level, and provide a link with the<br />
culture conditions at the metabolic level.<br />
Materials and methods: Batch culture of GS-NS0 producing a cB72.3<br />
MAb was performed. Cell density and viability was quantified using the<br />
dye exclusion method. Extracellular glucose, glutamate, lactate and<br />
ammonium were quantified using Bioprofile 400 (Nova Biomedical,<br />
Waltham, USA). The extracellular antibody was measured using ELISA. DNA<br />
staining and Annexin V/PI assay was used to quantify the fraction of cells<br />
in each cell cycle phase as well as the degree of apoptosis. The<br />
measurement of both apoptosis and cell cycle related gene expression was<br />
conducted using real-time PCR.<br />
Results and discussion: Our results showed a clear link between the<br />
environmental factors and gene expression. The batch cultures started<br />
with a high fraction of cells in the G 1 /G 0 phase, which rapidly left this state<br />
in order to join the proliferating population. Soon after, glutamate<br />
deprivation occurred at around 50 h of culture, whereby atf5 upregulation<br />
peaked (50% higher) suggesting that glutamate deprivation is among the<br />
first factors that introduce metabolic stress, in agreement with previous<br />
results [5]. The upregulation of atf5 triggered the upregulation of bcl-2<br />
(which followed at around 90 h). After the batch cultures reached their<br />
maximum cell density (which occurred roughly the same time as the<br />
glutamate exhaustion), the onset of an increasing early apoptotic cell<br />
population was observed - around 10%. Together with the high cell<br />
density, casp8 was upregulated (100% increase). Consequently, the<br />
expression of casp3 followed a similar trend with a lag of few hours as its<br />
protein, caspase-3, is one of downstream targets of caspase-8 and a final<br />
executor of the apoptosis pathways [6]. In addition, trp53bp2 showed a<br />
similar trend to casp3. These results suggest that apoptosis could initi<strong>all</strong>y<br />
occur via the death receptor pathway as marked by the casp8 upregulation,<br />
which might be induced by the glutamate exhaustion and/or the cell<br />
density peak. However, given that the trp53bp2 upregulation happened later<br />
than that of casp8 suggests that apoptosis in the later stages of culture<br />
might also occur through the mitochondrial pathway and it could also be<br />
triggered by other lethal signals (e.g. high level of lactate accumulation).<br />
As soon as the onset of apoptosis occurred, the upregulation of p21 was<br />
also observed (300% increase) and this happened simultaneously with the<br />
bcl-2 upregulation. Since it was reported that Bcl-2 protein helps facilitate<br />
cell cycle arrest at G 1 /G 0 phase and an increase in G 1 /G 0 cell fraction was<br />
observed later in the death phase of culture, this could suggest that<br />
the bcl-2 upregulation may underlie the p21 upregulation and the cell<br />
cycle arrest at G 1 /G 0 phase and this could be a mechanism to avoid<br />
apoptosis [7].<br />
Conclusions: These findings set a map of the cell cycle and apoptotic<br />
timing and magnitudes of the events from the genetic level and their links<br />
to the environmental conditions, which can be used to gain insight of the<br />
GS-NS0 cultures. By looking at the map, we can systematic<strong>all</strong>y analyse<br />
cellular responses to the environmental conditions which may have<br />
detrimental effect on the culture and utilise the result of the analysis to<br />
tackle the culture issues way before the final executors, but at the genetic<br />
level. Ultimately, the goal is to utilize mathematical models that will help<br />
to establish new strategies in order to achieve a longer cultivation period,<br />
high viability and increased MAb titre.<br />
Acknowledgements: We would like to thank Lonza Biologics (Slough,<br />
UK) for kindly providing the cell line and members of Biological<br />
Systems Engineering Laboratory (BSEL) for help with the analytical<br />
techniques.<br />
References<br />
1. Elvin JG, Couston RG, van der W<strong>all</strong>e CF: Therapeutic antibodies: Market<br />
considerations, disease targets and bioprocessing. International Journal of<br />
Pharmaceutics 2013, 440:83-98.<br />
2. Simpson NH, Singh RP, Emery AN, Al-Rubeai M: Bcl-2 over-expression<br />
reduces growth rate and prolongs G1 phase in continuous chemostat<br />
cultures of hybridoma cells. Biotechnology and Bioengineering 1999,<br />
64:174-186.<br />
3. Tey BT, Singh RP, Piredda L, Piacentini M, Al-Rubeai M: Bcl-2 mediated<br />
suppression of apoptosis in myeloma NS0 cultures. Journal of<br />
Biotechnology 2000, 79:147-159.
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4. Watanabe S, Shuttleworth J, Al-Rubeai M: Regulation of cell cycle and<br />
productivity in NS0 cells by the over-expression of p21CIP1.<br />
Biotechnology and Bioengineering 2002, 77:1-7.<br />
5. Browne SM, Al-Rubeai M: Analysis of an artifici<strong>all</strong>y selected GS-NS0<br />
variant with increased resistance to apoptosis. Biotechnology and<br />
Bioengineering 2011, 108:880-892.<br />
6. Hengartner MO: The biochemistry of apoptosis. Nature 2000, 407:770-776.<br />
7. Janumyan YM, Sansam CG, Chattopadhyay A, Cheng N, Soucie EL, Penn LZ,<br />
Andrews D, Knudson CM, Yang E: Bcl-xL/Bcl-2 coordinately regulates<br />
apoptosis, cell cycle arrest and cell cycle entry. EMBO J 2003,<br />
22:5459-5470.<br />
P55<br />
Design space definition for a stirred single-use bioreactor family from<br />
50 to 2000 L scale<br />
Thomas Dreher * , Ute Husemann, Sebastian Ruhl, Gerhard Greller<br />
Sartorius Stedim Biotech GmbH, Göttingen, Germany, D-37079<br />
E-mail: thomas.dreher@sartorius-stedim.com<br />
BMC Proceedings 2013, 7(Suppl 6):P55<br />
Background: Single-use bioreactors continue to gain large interest in the<br />
biopharmaceutical industry. They are excessively used for mammalian cell<br />
cultivations, e.g. production of monoclonal antibodies and vaccines [1]. This<br />
is motivated by several advantages of these bioreactors like reduced risk of<br />
cross contaminations or shortening lead times [2]. Single-use bioreactors<br />
differ in terms of shape, agitation principle and gassing strategy [3]. Hence, a<br />
direct process transfer or scale-up between different systems can be a<br />
ch<strong>all</strong>enge. Reusable bioreactors are still regarded as gold standard due to<br />
their well-known and defined geometrical properties. Based on this<br />
knowledge a stirred single-use bioreactor family from 50 to 2000 L scale was<br />
developed with similar geometrical ratios like commonly used reusable<br />
systems. To follow a Quality by Design approach the key process parameters<br />
for a modern mammalian cell cultivation were specified. Therefore, the k L a-<br />
value, mixing time and the power input per volume were evaluated by<br />
using process engineering methods for <strong>all</strong> scales.<br />
Stirred single-use bioreactor family: The used stirred single-use<br />
bioreactor family (BIOSTAT® STR, Sartorius Stedim Biotech, Germany) has<br />
design criteria similar to conventional reusable systems. The bioreactors<br />
have a cylindrical cultivation chamber, two impellers mounted on a rigid<br />
shaft and a submerged sparger. The H/D ratio of 2:1 and the impeller to bag<br />
ratio of 0.38 was kept constant for <strong>all</strong> scales [4]. There is the possibility to<br />
select between the impeller configuration 2 × 3-blade segment impeller<br />
(downward mixing) and 6-blade disk (bottom) + 3-blade segment (top)<br />
impeller. For the process engineering characterization 2 × 3-blade segment<br />
impellers were used. The aeration was performed by a combi sparger, which<br />
consists a ring sparger part (hole diameter 0.8 mm) and a micro sparger part<br />
(hole diameter 0.15 mm).<br />
Process engineering characterisation: Design space approach: The<br />
field of application of the stirred single-use bioreactor family is the<br />
cultivation of mammalian cells. To verify the single-use bioreactors a<br />
modern CHO process was considered with a peak cell density of 27 - 28 ×<br />
10 6 cells/mL. This process defines the key process parameters relevant for<br />
the design space definition [3,5], which are a moderate shear rates (tip<br />
speeds < 2.0 m/s), a sufficient oxygen transfer rate (k L a>7h -1 , supply pure<br />
oxygen assumed), a suitable homogeneity (mixing times < 60 s) and a<br />
power input per volume (P/V L ) between 10 and 250 W/m 3 (from lab to<br />
production scale).<br />
Power input per volume: Energy has to be transferred to a bioreactor to<br />
ensure cell suspension, homogenization and gas dispersion [6]. For the<br />
quantification the dimensionless Newton number (Ne) was determined by<br />
torque measurements [3]. From the results the power input per volume<br />
was calculated for tip speeds between 0.6 and 1.8 m/s. Ne for the selected<br />
configuration was 1.3. Figure 1a shows the P/V L characteristics, which<br />
increased for <strong>all</strong> scales with the tip speed. With increasing size of the<br />
CultiBag STR the power input per volume decreases at a defined tip speed.<br />
Mixing time: Appropriate mixing is important to avoid concentration or<br />
temperature gradients inside the cultivation chamber. The mixing time of<br />
the stirred single-use bioreactor was determined by the decolourization<br />
method [7]. The mixing times as a function of the tip speed are illustrated<br />
in Figure 1b. As the tip speed increases, expectedly the mixing times<br />
decrease. For <strong>all</strong> scales mixing times below 30 s are achieved.<br />
Oxygen transfer capabilities: The oxygen transfer efficiency of a<br />
bioreactor can be described by the k L a-value, which was determined by<br />
the gassing-out method (1xPBS, room temperature) [8]. The aeration was<br />
carried out through the holes with 0.8 mm (ring sparger part) (Figure 1c)<br />
and in another trial through the holes with 0.15 mm diameter (micro<br />
sparger part) (Figure 1d). The volumetric mass transfer coefficients were<br />
determined as a function of the tip speed for a constant gas flow rate of<br />
0.1 vvm. With increasing tip speed the k L a-value characteristics increased<br />
for <strong>all</strong> scales. For larger scales higher k L a-values were achieved presumably<br />
due to longer residence times of the gas bubbles. By using aeration<br />
through the holes with the sm<strong>all</strong>er diameter the k L a-value can be<br />
significantly increased.<br />
Conclusions: The main application of the presented single-use bioreactor<br />
family is the cultivation of mammalian and insect cells. These cells have<br />
special demands on the cultivation system for their optimal growth. To<br />
verify the suitability of the bioreactor family different process engineering<br />
parameters were determined. Based on the results the process<br />
engineering parameters are in the desired ranges of the defined design<br />
space regarding the power input per volume, mixing efficiency and the<br />
k L a-value. This indicates that the stirred single-use bioreactor family is<br />
suitable for cell culture applications. The design criteria of the CultiBag<br />
STR family directly relates to those from reusable systems. Therefore,<br />
existing ch<strong>all</strong>enges for a scale-up or process transfer are removed due to<br />
the improved design. Consequently, this technology represents an<br />
important step towards further maturity of single-use bioreactors and<br />
their acceptance.<br />
References<br />
1. Brecht R: Disposable Bioreactors: Maturation into Pharmaceutical<br />
Glycoprotein Manufacturing. Adv Biochem Engin/Biotechnol 2009, 115:1-31.<br />
2. Eibl D, Peuker T, Eibl R: Single-use equipment in biopharmaceutical<br />
manufacture: a brief introduction. Single-use technology in<br />
biopharmaceutical manufacture Wiley, Hoboken: Eibl R, Eibl D 2010, 3-11.<br />
3. Löffelholz C, Husemann U, Greller G, Meusel W, Kauling J, Ay P, Kraume M,<br />
Eibl R, Eibl D: Bioengineering Parameters for Single-Use Bioreactors:<br />
Overview and Evaluation of Suitable Methods. Chem Ing Tech 2013,<br />
85:40-56.<br />
4. Noack U, Verhoeye F, Kahlert W, Wilde D de, Greller G: Disposable stirred<br />
tank reactor BIOSTAT® CultiBag STR. Single-use technology in<br />
biopharmaceutical manufacture Wiley, Hoboken: Eibl R, Eibl D 2010, 225-240.<br />
5. Ruhl S, Dreher T, Husemann U, Jurkiewicz E, Greller G: The successful<br />
transfer of a modern CHO fed-batch process to different single-use<br />
bioreactors. Poster ESACT Lillé 2013.<br />
6. Storhas W: Aufgaben eines Bioreaktors. Bioreaktoren und periphere<br />
Einrichtungen Vieweg & Sohn Verlagsgesellschaft, Braunschweig/Wiesbaden<br />
1994, 15-86.<br />
7. Zlokarnik M: Bestimmung des Mischgrades und der Mischzeit.<br />
Rührtechnik, Theorie und Praxis, Springer-Verlag Berlin Heidelberg New York<br />
2002, 97-99.<br />
8. Wise W S: The measurement of the aeration of culture media. J Gen<br />
Microbiol 1951, 5:167-177.<br />
P56<br />
Full transcriptome analysis of Chinese Hamster Ovary cell lines<br />
producing a dynamic range of Coagulation Factor VIII<br />
Christian S Kaas 1,2* , Claus Kristensen 1 , Jens J Hansen 1 , Gert Bolt 1 ,<br />
Mikael R Andersen 2<br />
1 Department of Mammalian cell technology, Novo Nordisk A/S, Maaloev,<br />
2760, Denmark;<br />
2 Center for Microbial Biotechnology, Technical University of<br />
Denmark, Kgs Lyngby, 2800, Denmark<br />
E-mail: csrk@novonordisk.com<br />
BMC Proceedings 2013, 7(Suppl 6):P56<br />
Background and novelty: Coagulation Factor VIII (FVIII) is an essential<br />
cofactor in the blood coagulation cascade. Inability to produce functional<br />
FVIII results in haemophilia A which can be treated with recombinant<br />
FVIII [1]. Chinese Hamster Ovary (CHO) cells are the most used cell line for<br />
producing complex biopharmaceuticals due to its ability to perform<br />
complex post-translational modifications. When mammalian cells<br />
overexpress a protein like FVIII they will adapt by regulating various proteins<br />
and pathways to support synthesis/production of this protein. Yields of FVIII<br />
produced in CHO are low and for this reason a greater understanding of
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Figure 1(abstract P55) Process engineering parameters of the CultiBag STR family. (a) Characteristics of the power input per volume, (b) mixing<br />
time characteristics, (c) k L a-values for the aeration with the ring sparger part, (d) k L a-values for the aeration with the micro sparger part.<br />
what constitute a high producing cell line is desired. In this study a full<br />
transcriptome analysis was undertaken in order to analyze the differences<br />
between high and low producers of FVIII<br />
Experimental approach: The FVIII gene was introduced into CHO-DUKX-<br />
B11 cells and a stable pool was generated by selection with MTX.<br />
A number of subclones were analysed and 3 high producing clones,<br />
3 medium producers and 3 low (~0) producer clones were isolated. These<br />
9 clones were grown in shake flasks in batch culture. During the<br />
cultivation essential metabolites were monitored as well as cell number<br />
and viability. RNA was extracted after 48 hours of cultivation and<br />
sequenced using the Illumina HiSeq system. Reads were processed and<br />
aligned to the CHO-K1 genome [2] using Tophat2 and expression levels<br />
were deduced using htseq<br />
Results and discussion: Experiments showed that 48 hours into the<br />
cultivation cells were seen to grow in the exponential phase in media still<br />
containing sufficiently high amounts of glutamine and low amounts of<br />
lactate. Furthermore, a significant difference in FVIII levels was detected<br />
at this time in the media of cells from the different groups and for this<br />
reason this time point was chosen for extraction of RNA. 1677 genes<br />
were found to be differenti<strong>all</strong>y expressed in high vs non-producing<br />
clones. Among these, genes involved in oxidative stress were seen to be<br />
enriched (p = 1.74 × 10 -6 ). This finding is strengthened by the work by<br />
Malhotra et al [3] showing that CHO cell lines activate the oxidative stress<br />
response when producing FVIII, which might induce apoptosis. The non-<br />
FVIII-producing clones were seen to express predominantly truncated<br />
FVIII-DHFR mRNAs (Figure 1) explaining the phenotype for growth in<br />
media containing MTX selection but no functional FVIII expressed. Further<br />
analyses are ongoing.<br />
References<br />
1. Thim L, Vandahl B, Karlsson J, Klausen NK, Pedersen J, Krogh TN, Kjalke M,<br />
Petersen JM, Johnsen LB, Bolt G, Nørby PL, Steenstrup TD: Purification<br />
and characterization of a new recombinant factor VIII (N8).<br />
Haemophilia. The official journal of the World Federation of Hemophilia<br />
2010, 16:349-359.<br />
2. Xu X, Nagarajan H, Lewis NE, Pan S, Cai Z, Liu X, Chen W, Xie M, Wang W,<br />
Hammond S, Andersen MR, Neff N, Passarelli B, Koh W, Fan HC, Wang J,<br />
Gui Y, Lee KH, Betenbaugh MJ, Quake SR, Famili I, Palsson BO, Wang J: The<br />
genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line.<br />
Nature biotechnol 2011, 29:735-741.<br />
3. Malhotra JD, Miao H, Zhang K, Wolfson A, Pennathur S, Pipe SW,<br />
Kaufman RJ: Antioxidants reduce endoplasmic reticulum stress and<br />
improve protein secretion. PNAS 2008, 105:18525-18530.
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Figure 1(abstract P56) Depth of sequenced reads at every position of the FVIII gene. It is seen that the 3 non-producing clones transcribe<br />
5’-truncated RNA species. This would explain the phenotype of no FVIII protein production but growth under MTX selection as the IRES element<br />
containing DHFR is still transcribed.<br />
P57<br />
Production of monoclonal antibody, Anti-CD3 by hybridoma cells<br />
cultivated in Basket Spinner under free and immobilized conditions<br />
Elsayed A Elsayed 1,2* , Hoda Omar 3 , Hasnaa R Shahin 4 , Hamida Abou-Shleib 3 ,<br />
Maha El-Demellawy 4 , Mohammad Wadaan 1 , Hesham A El-Enshasy 4,5<br />
1 Bioproducts Research Chair, Zoology Department, Faculty of Science, King<br />
Saud University, 11451 Riyadh, Kingdom of Saudi Arabia;<br />
2 Natural & Microbial<br />
Products Department, National Research Centre, Dokki, Cairo, Egypt;<br />
3 Microbiology Department, Faculty of Pharmacy, Alexandria University, Egypt;<br />
4 City for Scientific Research and Technology Applications, New Burg Al Arab,<br />
Alexandria, Egypt;<br />
5 Institute of Bioproducts Development, Universiti<br />
Teknologi Malaysia, Skudai, Johor, Malaysia<br />
E-mail: eaelsayed@ksu.edu.sa<br />
BMC Proceedings 2013, 7(Suppl 6):P57<br />
Background: Monoclonal antibodies (Mabs) have been recently used for<br />
the treatment of virtu<strong>all</strong>y every debilitating disease. Packed-bed<br />
bioreactors have been used for the cultivation and production of a wide<br />
range of cell lines and biologics including MAbs. The principle behind a<br />
Packed-bed bioreactor is that the cells are being immobilized within a<br />
suitable stationary matrix which is represented by the bed. Packed-bed<br />
bioreactors also have the advantage of being capable of generating high<br />
cell densities with a low concentration of free cells in suspension; hence,<br />
simplifying downstream processing. Thepresentworkwasdesignedto<br />
compare between the cultivation of hybridoma cells as well as the<br />
production of OKT3 MAb in free and immobilized culture conditions.<br />
Materials and methods: Hybridoma cell line (OKT3), producing IgG2a<br />
monoclonal antibodies against CD3 antigen of human T lymphocyte cells<br />
were adapted to grow in serum free medium. The specificity of the produced<br />
MAbs was determined through the use of indirect immunofluorescence
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staining of T lymphocytes from peripheral blood followed by flowcytometeric<br />
analysis using cell quest software and FACSCalibur. The MAb was<br />
continuously produced by the cultivation of hybridoma cells in Basket<br />
Spinner. The cells were immobilized within the Fibra-Cel® disks. For<br />
comparison, two Basket Spinners were used in par<strong>all</strong>el, one of them was<br />
packed with 5 gm of Fibra-Cel® disks, and the other was used as a control for<br />
the cultivation of free cells. Samples were daily collected throughout the<br />
cultivation for the determination of cell viability using Trypan blue exclusion<br />
method. Glucose/lactate concentrations were determined using automatic<br />
glucose/lactate analyzer. The concentration of MAb was determined by direct<br />
ELISA assay.<br />
Results: Determination of MAb specificity: Secondary fluorescence<br />
antibodies bounded to the produced antibody which in turn is bound to<br />
CD3 positive lymphocytes (T-cells) showed a percentage of CD3 positive<br />
lymphocytes of 76.68%. This was proved using indirect immunofluorescence<br />
staining of healthy volunteer T lymphocytes from peripheral blood. Forward<br />
scatter (FSC) versus side scatter (SSC) can <strong>all</strong>ow for the differentiation of<br />
blood cells in a heterogeneous cell population.<br />
When the “gated” cells were analyzed for their emitted fluorescence upon<br />
stimulation by the laser beam, high fluorescence is produced from the<br />
cells that react with FITC- anti-mouse specific antibody which reflects CD3<br />
antibody content in the added culture supernatant. Histogram statistics<br />
showed that there were 2513 events inside the gated lymphocytes; the<br />
percentage of lymphocytes that were CD3 positive was 76.68%.<br />
Continuous production of MAb by the cultivation of hybridoma<br />
cells in Basket spinner: In this work two Basket Spinners were used in<br />
par<strong>all</strong>el, one of them was packed with 5 gm of Fibra-Cel disks (Figure 1), and<br />
the other was used as control without packing (free living cells). For the free<br />
Basket Spinner, the growth and viability of the hybridoma cells as well as their<br />
metabolic activities and mAb productivity were determined after 120 h. Viable<br />
cell concentration increased only during the first 72 h of cultivation reaching<br />
9.2 × 10 5 Cells mL -1 . On the other hand, mAb production reached its<br />
maximum concentration of 206.5 mg L -1 also at 72 h. For the immobilized<br />
Basket Spinner, the growth and viability of the hybridoma cells as well as their<br />
metabolic activities and mAb productivity were determined for 288 h. The<br />
Culture medium was perfused through the bed to supply cells with nutrients.<br />
This <strong>all</strong>owed the spinner to run as repeated batch, enabling long term<br />
cultivation of cells. The number of viable, and dead cells determined over the<br />
12 days of the cultivation corresponded to the cells detached from Fibra-CelR<br />
disks and does not reflect the actual cell number. On the other hand, the<br />
mAb titer increased in each batch reaching its maximum concentration of<br />
298.5 mg L -1 at batch number VI (after 216 h of cell inoculation).<br />
It was found that the rates of glucose consumption and lactate<br />
production increased for each batch where the medium was changed<br />
once after the first 72 h and then the batch time was further reduced to<br />
only 48 h in the subsequent batches, then once each 24 h over the<br />
remaining 12 days of the cultivation period. The maximum production of<br />
lactate was 2.74 g L -1 occurred at batch number VII (after 240 h).<br />
Upon comparing at 72 h of cultivation, it was found that the produced<br />
mAb in case of the immobilized Basket Spinner was higher than that<br />
produced in case of the free Basket Spinner, however, the rate of glucose<br />
consumption and lactate production at the same time interval for the<br />
former was lower than the later (2.2, 1.825 g L -1 for glucose and 1.27,<br />
2.075 g L -1 for lactate, respectively).<br />
Conclusion: The results obtained revealed that upon using flow cytometry<br />
and the fluorochrome-conjugated secondary antibody attached specific<strong>all</strong>y<br />
to MAb present in the supernatant from the cells adapted to serum free<br />
medium succeeded in sorting 76.8% of the gated cells (lymphocytes). This<br />
confirmed the binding of MAb of the adapted cells to CD3 positive<br />
lymphocytes. Which means that, stable hybridoma cells adapted to grow in<br />
serum free medium (SMIF-6) were successfully obtained. It was also<br />
observed upon using the backed spinner basket, the MAb titer increased in<br />
each successive batch to reach to 298.5 mg L -1 after 216 h. This might be<br />
due to the protection of the cells against shear stress and air/O 2 sparging by<br />
their immobilization on the microcarriers, promoting the use of serum- or<br />
protein-free medium. Moreover, the microcarrier is designed to ensure<br />
sufficient nutrient supply and also to remove toxic metabolites. On the other<br />
hand, the rate of glucose consumption and lactate production increased for<br />
each repeated batch. This explains why the decrease in the batch period.<br />
This indicated the good physiological state of the cells.<br />
P58<br />
Using Rice Bran Extract (RBE) as Supplement for Mescenchymal Stem<br />
Cells (MSCs) in Serum-free Culture<br />
Rinaka Yamauchi 1 , Ken Fukumoto 1 , Satoko Moriyama 1 , Masayuki Taniguchi 2 ,<br />
Shigeru Moriyama 3 , Takuo Tsuno 3 , Satoshi Terada 1*<br />
1 University of Fukui, Fukui, 910-8507, Japan;<br />
2 Niigata University, Niigata, 950-<br />
2102, Japan;<br />
3 Tsuno Food Industrial Co., Ltd, Katsuragi-cho, Wakayama, 649-<br />
7122, Japan<br />
E-mail: terada@u-fukui.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):P58<br />
Figure 1(abstract P57) Kinetics of cell growth, metabolism, and<br />
MAb production during cultivation of hybridoma cells in packed<br />
Basket Spinner.<br />
Introduction: Currently, therapies using multipotent mescenchymal stem<br />
cells (MSCs) are tested clinic<strong>all</strong>y for various disorders, including cardiac<br />
disease [1]. However, conventional culture media contain fetal bovine<br />
serum (FBS) and so the concern about amphixenosis remains. Therefore,<br />
developing animal derived factor-free media are desired [2].<br />
We previously reported that rice bran extract (RBE) significantly improved<br />
the proliferation of various cell lines and the cellular functions. In this<br />
study, we tested the effect of RBE on MSCs in serum-free culture.<br />
Materials and methods: Effect of RBE on osteogenic differentiation:<br />
MSCs obtained from the bone marrow of Wistar rats were cultured under<br />
conventional a-MEM with 15% FBS medium, supplemented with or without<br />
RBE for three days at passage 1 - 3. After treatment with RBE for three days,<br />
the media were replaced by RBE-free osteogenic medium composed of<br />
a-MEM containing 10% FBS, 10 mM b-glycerol phosphate (Merck, USA),<br />
0.05 mM L-ascorbic acid 2 phosphate (Sigma, USA), 10 nM dexamethasone<br />
(Sigma), 1% penicillin-streptomycin solution and the cells were cultured in<br />
the medium for 24 days. To evaluate the differentiation ability, the cells<br />
were stained with Alizarin Red S and analyzed by using Image J.
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Page 79 of 151<br />
Figure 1(abstract P58) Effect of RBE on osteogenic differentiation.<br />
Effect of RBE on cell proliferation: After MSCs were cultured in the<br />
presence of RBE for three days, viable cell number was measured by the<br />
trypan blue dyeing assay on a hemocytometer.<br />
Effect of RBE on gene expression after expansion: After treatment<br />
with RBE for three days, cells were lysed to be analyzed the maintaining<br />
MSC markers with real-time PCR. Total RNA from the cells was isolated by<br />
Acid Guanidinium Phenol Chloroform method and cDNA was synthesized<br />
with supersucriptTM (Invitologen, USA). These cDNAs were analyzed by<br />
LightCycler R480 (Roche, Germany) using primers: MSC markers, CD44,<br />
CD105 and CD166, and osteogenic genes, BMP2, ALPL, OCN. The results<br />
were normalized with respect to GAPDH or HPRT. Relative mRNA quantify<br />
was calculated using the comparative ΔΔCT.<br />
Results and discussion: AsshowninFigure1,thresholdarea(%)was<br />
significantly increased in MSCs expanded in the presence of RBE in<br />
comparison with in absence (*P < 0.03), suggesting that the cells expanded<br />
in RBE-containing medium differentiated into bone superior to the negative<br />
control cells.<br />
The viable cell densities in the culture with and without RBE were quite<br />
similar, suggesting that increase in osteogenisis with RBE is not due to the<br />
population of the cells. Expression levels of MSC markers such as CD44,<br />
CD105 and CD166, were not up- nor down-regulated in the presence of RBE<br />
during expansion, whereas that of osteogenic gene BMP2 was remarkably<br />
reduced. These results suggest that RBE does not induce osteogenesis<br />
during expansion and imply that RBE could keep MSCs undifferentiatiated.<br />
Treatment with RBE during expansion up-regulated the expression levels of<br />
osteogenic genes including ALPL and OCN in MSCs during osteogenic<br />
differentiation.<br />
Conclusion: Decreased osteogenic differentiation ability of MSCs after<br />
expansion could be maintained by addition of RBE into expansion<br />
medium. RBE is a candidate for the novel supplement for maintaining<br />
differentiation ability of MSCs in expansion culture.<br />
References<br />
1. Amado CLuciano, Saliaris PAnastasios, Schuleri HKarl, St John Marcus ,<br />
Xie Jin-Sheng, Cattaneo Stephen, Durand JDaniel, Fitton Torin, Kuang Jin<br />
Qiang, Stewart Garrick, Lehrke Stephanie, Baumgartner WWilliam,<br />
Martin JBradley, Heldman WAlan, Hare MJoshua: Cardiac repair with<br />
intramyocardial injection of <strong>all</strong>ogeneic mesenchymal stem cells after<br />
myocardial infarction. PNAS 2005, 102:11474-11479.<br />
2. Leopold G, Thomas RK, Sonia N, Manfred R: Emerging trends in plasmafree<br />
manufacturing of recombinant protein therapeutics expressed in<br />
mammalian cells. Biotechnol J 2009, 4:186-201.<br />
P59<br />
Viral vector production in the integrity® iCELLis® single-use fixed-bed<br />
bioreactor, from bench-scale to industrial scale<br />
Alexandre Lennaertz * , Shane Knowles, Jean-Christophe Drugmand, Jose Castillo<br />
ATMI LifeSciences, Brussels, 1120, Belgium<br />
E-mail: alennaertz@atmi.com<br />
BMC Proceedings 2013, 7(Suppl 6):P59<br />
Introduction: Wild-typeorrecombinantvirusesusedasvaccinesand<br />
human gene therapy vectors are an important development tool in<br />
modern medicine. Some have demonstrated high potential such as<br />
lentivirus, paramyxovirus and adeno-associated-virus (AAV). These vectors<br />
are produced in adherent and suspension cell cultures (e.g. HEK293T,<br />
A549, VERO, PER.C6, Sf9) using either transient transfection (e.g PEI, calcium<br />
phosphate precipitation) or infection (e.g. modified or recombinant viruses)<br />
strategies. Most of these processes are currently achieved in static mode<br />
on 2-D systems (Roller Bottles, Cell Factories, etc.) or on suspended microcarriers<br />
(porous or non-porous). However, these two systems are timeconsuming<br />
(large numbers of manipulation, preparation of equipment, etc.)<br />
and hardly scalable. In regards to process simplification and traceability,<br />
Integrity® iCELLis® bioreactors offer a new solution for scalability and<br />
monitoring of adherent cell cultures.<br />
The Integrity iCELLis Bioreactor: Integrity® iCELLis® bioreactors from<br />
ATMI LifeSciences were designed for adherent cell culture applications<br />
such as recombinant protein, viral vaccine and gene therapy vector<br />
production. Using PET carriers trapped into a fixed-bed, cells grow in a 3-D<br />
environment with temperature, pH and dissolved oxygen controls. The<br />
iCELLis technology can be used at sm<strong>all</strong>-scale (the iCELLis nano from 0.5 to<br />
4m 2 ) and manufacturing scale (iCELLis 500 from from 66 to 500 m 2 ) which<br />
eases process scale-up and its over<strong>all</strong> utilization.<br />
Materials and methods: All the experiments described here have been<br />
performed in the bench-scale and pilot scale iCELLis bioreactors<br />
containing iPack carriers made of 100% pure non-woven PET fibers.<br />
Crystal violet was used for cell nuclei counts from carriers.<br />
Recombinant viral vectors production: Some recombinant entities are<br />
produced in the iCELLis bioreactors using hybrid vectors. For example,<br />
A549-stable packaging cell line, maintained in Optipro medium + 1% FBS,<br />
can deliver recombinant AAV vectors frequently used in gene transfer<br />
applications (Inserm UMR 649, Institut de Recherche Thérapeutique).<br />
Alternatively, other rAAV vectors are obtained by transient transfection. In<br />
this case, HEK293-T cells are regularly found to be sensitive to the viral<br />
DNA and transfection reagent complex (gener<strong>all</strong>y polyethylenimine - PEI<br />
or phosphate calcium precipitate). The transfer of the transfection process<br />
from static or dynamic systems to the iCELLis bioreactors requires some<br />
adaptation in order to fully benefit of both technologies. Using a<br />
fluorescent protein marker, the transfected cells can be observed during<br />
the culture and the viral vectors can be quantified after the harvest.<br />
Transfection method using the PEI/DNA complexes is frequently found in<br />
cell suspension processes due to its high efficiency and adaptability to<br />
high-throughput systems. The circulation pattern of the medium through<br />
the fixed-bed of the iCELLis system <strong>all</strong>ows a good contact between cells<br />
and transfection complexes.<br />
The transfection by phosphate precipitation is a static method where the<br />
DNA precipitates settle on the cells. For this reason, it is difficult to apply<br />
this technic in dynamic conditions. To be able to implement it in the<br />
iCELLis bioreactor, the agitation speed has to be minimal to get a<br />
medium circulation through the fixed-bed. This maintains the precipitate<br />
in suspension while giving the longest contact time between these<br />
precipitates and the cells. The iCELLis system with its pH regulation and<br />
low-shear circulation is well adapted for this method sensitive to sm<strong>all</strong><br />
pH changes and reagent mix.<br />
Results: Recombinant adeno-associated virus vector production:<br />
Recombinant AAV vectors were produced in an A549 based stable<br />
packaging cell line containing the AAV2 rep and cap genes from various<br />
AAV serotypes. Using a dual adenovirus infection (wild-type Ad5 followed<br />
by hybrid Ad/AAV) in the iCELLis nano bioreactor under perfusion mode,<br />
recombinant particles were harvested up to 96 hours post-infection. The<br />
expression levels of the AAV2 rep and cap genes from various AAV<br />
serotypes were assessed by western-blot and qPCR. This 8-days process<br />
demonstrated higher vector particles production in the iCELLis bioreactor<br />
compared to CS-5 control (4.5 × 10 8 vs 3.1 × 10 8 vg/cm 2 ,72hafterthe<br />
first infection) (Inserm UMR649, Institut de Recherche Thérapeutique).<br />
Triple transient transfection using PEI was performed in the iCELLis nano<br />
system (0.53 m 2 , 40 mL fixed-bed) for the production of serotype 5 AAV<br />
in HEK 293T cells. Cells were seeded at 80,000 cells/cm 2 in the CS10 and<br />
the iCELLis bioreactor. Twenty-four hours post-inoculation, the DNA-PEI<br />
mix containing the GFP gene was added to fresh medium inside the<br />
bioreactor. Cells were still growing on the carriers after the transfection.<br />
The expression of GFP by cells demonstrated that the transfection had a<br />
high efficiency rate in both vessels (FACS analysis on sampled carriers for
BMC Proceedings 2013, Volume 7 Suppl 6<br />
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• Paramyxovirus production in Vero cells<br />
• Undisclosed lytic virus in Vero cells<br />
Transfer and scale-up of a HEK293 cell culture process for<br />
production of adenovirus: Sm<strong>all</strong> Scale Development<br />
An existing process using HEK293 cells for the production of adenovirus<br />
was first transferred from multi-tray systems to an iCELLis nano bioreactor<br />
(0.53 m 2 , 40 ml of fixed-bed) by keeping equivalent cell culture<br />
parameters:<br />
• Temperature, pH, DO (% saturation with air)<br />
• Multiplicity of infection (pfu/cell)<br />
• Time of infection<br />
• Cell seeding density (cells/cm 2 and cells/mL)<br />
• Culture duration<br />
Figure 1(abstract P59) Comparison of Green Fluorescent Units and<br />
Viral Genome/cm 2 and VG/GFYU ratio in the CS10 and iCELLis<br />
nano 0.53 m 2 .<br />
the iCELLis bioreactor). Green Fluorescent Units (GFU) and Viral Genome<br />
(VG) were measured for the CS10 control and the iCELLis nano bioreactor.<br />
Viral particles were harvested using a freeze/thaw method, suboptimal in<br />
the case of the iCELLis system. The GFU and VG titers/cm 2 in the iCELLis<br />
bioreactor were about 53% of the control (Figure 1) (Dept of Biochemical<br />
Eng. - UCL).<br />
Conclusions: We demonstrated that the iCELLis system could be very<br />
useful for production of viral vaccine and gene therapy vectors. The<br />
iCELLis platform facilitates handling and scale-up, high biomass<br />
amplification and sterile containment within a closed system. Moreover,<br />
in many cases, the specific culture environment enhances virus<br />
production yields.<br />
Specific<strong>all</strong>y, after some optimization of the culture parameters, it was<br />
demonstrated that rAAV vectors were produced by modified A549 cells in<br />
high viral level in the 0.53 m 2 iCELLis bioreactor. The maximum viral yield<br />
achieved in the bioreactor was 4.5 × 10 8 vg/cm 2 , which was higher than<br />
the yield per cm 2 obtained in a CellSTACK vessel (3.1 × 10 8 vg/cm 2 ).<br />
Fin<strong>all</strong>y, the preliminary results of transfection demonstrated that the<br />
method using PEI is applicable in the iCELLis bioreactors, with<br />
optimization of the viral recovery at harvest yet to be performed. This<br />
also demonstrated that the iCELLis can be considered as a solution for<br />
transient transfection processes at large scales.<br />
P60<br />
Linear scalability of virus production in the integrity® iCELLis®<br />
single-use fixed-bed bioreactors from bench to industrial scale<br />
Shane Knowles * , Jean-Christophe Drugmand, Nicolas Vertommen,<br />
Jose Castillo<br />
ATMI LifeSciences, Rue de Ransbeek 310, Brussels, 1120, Belgium<br />
E-mail: sknowles@atmi.com<br />
BMC Proceedings 2013, 7(Suppl 6):P60<br />
Introduction: In order to maximize cell growth within a compact space<br />
and retain cells for easy medium exchange, the iCELLis bioreactors from<br />
ATMI LifeSciences contain macro-carriers trapped in a fixed-bed, creating<br />
a 3-D matrix within which cells adhere and replicate. These bioreactors<br />
also enable precise temperature, pH and dissolved oxygen control which<br />
cannot be done in 2-D cultures.<br />
The iCELLis technology can be used at sm<strong>all</strong> and large scales with<br />
straightforward process scale-up, easy single-use operations and minimal<br />
space requirement.<br />
Here we present a summary of adherent cell process development in<br />
iCELLis bioreactors, including:<br />
• HEK 293 cell expansion for production of adenovirus<br />
• MVA virus production in CEF cells<br />
• Bovine Herpes Virus production in MDBK cells<br />
• Recombinant Adeno-Associated Virus in A549 cells<br />
• Adenovirus production in A549 cells<br />
• Influenza virus production in Vero cells<br />
Additional experiments were performed with lower cell densities at<br />
inoculation in order to reduce the number of pre-culture steps at large<br />
scale. The following parameters were also optimized for cell growth and<br />
virus productivity:<br />
• Compaction of carriers inside the fixed-bed (96 g/L or 144 g/L)<br />
• Linear velocity of medium through the fixed-bed (cm/s).<br />
• Fixed-bed height (2,4 or 10 cm)<br />
Industrial scale-up: The scale-up of iCELLis technology is similar to that<br />
of chromatography columns. The difference in fixed bed geometry from<br />
sm<strong>all</strong> to large scale is that the cross-sectional area increases, while the<br />
fixed-bed (FB) height remains constant. Therefore, cell seeding, nutrient<br />
and oxygen delivery throughout the fixed bed are comparable at sm<strong>all</strong><br />
and large scale.<br />
After determining optimal parameters at sm<strong>all</strong> scale, HEK293 cell culture<br />
batches were performed in duplicate with sm<strong>all</strong> and large scale<br />
bioreactors. Inoculation density, medium volume ratios, culture duration,<br />
pH, DO and temperature set points were kept identical. Consistent cell<br />
densities of 2.7 to 3.8 cells/cm2 were achieved in multiple experiments at<br />
both sm<strong>all</strong> and large scale. Analysis of glucose and lactate (Figure 1) at<br />
both scales in comparison to a 5-tray Cell Factory control indicated that<br />
cell metabolism was comparable between sm<strong>all</strong> and large scale iCELLis<br />
bioreactors and the standard 2D process.<br />
Additional Virus Production Process Development: Results of<br />
experiments performed for production of several viruses in various cell<br />
lines at various bioreactor scales are shown in Table 1. Bench scale<br />
bioreactors were used for each process to determine what conditions and<br />
feeding strategies sustained the highest growth rates and cell densities.<br />
Bench scale bioreactors were used for each process to determine what<br />
conditions and feeding strategies sustained the highest growth rates and<br />
cell densities.<br />
For chicken embryonic fibroblasts (CEF) and production of Modified<br />
Vaccinia Ankara (MVA), a prototype “Artefix” bioreactor (the predecessor<br />
of iCELLis) with a 0.07 m 2 fixed-bed surface area was tested.<br />
Intermediate “pilot” scale prototype iCELLis bioreactors with surface areas<br />
of 20 or 40 m 2 were used to test Vero and MDBK cell processes.<br />
The Vero cell process was scaled up to a 660 m 2 bioreactor. In this case,<br />
cells were inoculated at only 3200 cells/cm 2 using two 40-tray Cell<br />
Factories (2.5 m 2 each), equivalent to fifteen roller bottles (1700 cm 2<br />
each). With such a low seeding density the seed train required for<br />
inoculation is simplified extensively compared to standard 2D cell culture<br />
processes. The Vero cell density reached 2.3 × 10 5 cells/cm 2 for a total<br />
biomass of 1.5 × 10 12 cells in 11 days. A complete medium exchange was<br />
then performed, followed by virus infection. Continuous perfusion of<br />
medium was used during the production phase. While the virus type and<br />
productivity data is confidential, the results indicated that virus output<br />
was equivalent or better than expected based on the standard 2D<br />
process.<br />
Conclusions: This summary of experiments demonstrates that the fixedbed<br />
design of the iCELLis bioreactor enables high cell densities to be<br />
achieved and maintained in both sm<strong>all</strong> and large bioreactor volumes.<br />
Different processes have been easily scaled up by keeping cell culture<br />
conditions and process parameters identical to the standard 2-D cell<br />
culture process.<br />
The iCELLis bioreactor can be inoculated at a very low cell density,<br />
leading to a dramatic simplification of seed train operations and a<br />
significant reduction of development timelines.
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Figure 1(abstract P60) Comparability of Glucose (Top Panel) and Lactate (Bottom Panel) Profiles of HEK293 culture in iCELLis 133 m 2 (Blue),<br />
iCELLis nano 1.06 m 2 (Green) and 5-tray Cell Factory (Red).<br />
In conclusion, large biomass amplification and excellent virus productivities,<br />
combined with the advantages of a fully closed disposable system with low<br />
shear stress, make the iCELLis fixed-bed bioreactor a simple and<br />
straightforward solution for industrial production of viruses.<br />
P61<br />
Scale-up of hepatic progenitor cells from multitray stack to 2-D<br />
bioreactors<br />
Matthieu Egloff 1* , Florence Collignon 1 , Jean-François Michiels 1 ,<br />
Jonathan Goffinet 1 , Sarah Snykers 2 , Philippe Willemsen 2 , Christophe Gumy 2 ,<br />
Claude Dedry 2 , Jose Castillo 2 , Jean-Christophe Drugmand 1<br />
1 ATMI LifeSciences, Brussels, Belgium, 1120;<br />
2 Promethera Biosciences, Mont-<br />
Saint-Guibert, 1435, Belgium<br />
E-mail: megloff@atmi.com<br />
BMC Proceedings 2013, 7(Suppl 6):P61<br />
Introduction: Promethera Biosciences (Mont-St-Guibert, BE) is developing<br />
cell therapies to treat several liver genetic metabolic diseases, such as the<br />
Crigler-Najjar syndrome. Human heterologous adult liver progenitors cells<br />
(HHALPCs) were initi<strong>all</strong>y cultivated in 2D standard cultivation devices. The<br />
present study is investigating the feasibility to cultivate HHALPCs in<br />
Xpansion bioreactors, with the following objectives:<br />
➢ The process must be closed<br />
➢ The growth rate and population-doubling level (i.e. the number of<br />
times the cells in the population has doubled) must be at least<br />
equivalent to the current process in multilayer trays<br />
➢ The process must comply to the cGMP rules<br />
➢ The cells must succeed the quality control (QC) test specifications<br />
at the end of cultivation, i.e. cells must remain undifferentiated and<br />
show the presence of HHAPLCs markers, while exhibiting the<br />
capacity to differentiate toward functional hepatocytes.<br />
Integrity® Xpansion multiplate bioreactors have been specific<strong>all</strong>y<br />
designed to enable an easy transfer from existing multiple-tray-stack<br />
processes by offering the same cell growth environment on 2-D<br />
hydrophylized Polystyrene (PS) plates in a fully closed system. To make<br />
the bioreactors compact, the headspace between each plate has been<br />
reduced to a minimum (1.3 mm). Gas transfer is made through a semipermeable<br />
silicone tubing mounted in the central column. Addition<strong>all</strong>y,<br />
critical cell culture parameters such as pH and DO are controlled and the<br />
cell density is automatic<strong>all</strong>y monitored via a specific holographic<br />
microscope developed by Ovizio<br />
Materials and methods: Cell culture parameters: ✓ pH set-point: 7.5<br />
✓ DO regulated > 50%<br />
✓ No agitation during the first 8 hours after plating<br />
Table 1(abstract 60) Summary of results of virus production processes tested in various cell lines in iCELLis<br />
bioreactors (or predecessors)<br />
Cells Virus Bioreactor Surface<br />
Area (m 2 )<br />
Average Cell Density<br />
at TOI (cells/cm 2 )<br />
Specific Virus<br />
Productivity<br />
Total Virus<br />
CEF Modified Vaccina Ankara Artefix 0.07 3.9E+05 3.0E+06 pfu/cm 2 2.1E+09 pfu<br />
MDBK Bovine Herpes Virus iCELLis nano 4 1.2E+05 2.2E+07 pfu/cm 2 8.7E+11 pfu<br />
iCELLis pilot 20 1.4E+05 1.7E+07 pfu/cm 2 3.4E+12 pfu<br />
iCELLis 500 66 3.3E+05 3.3E+07 pfu/cm 2 2.2E+13 pfu<br />
A549 rAAV iCeLLis nano 0.53 6.0E+04 5.3E+08 vg/cm 2 2.8E+12 vg<br />
Adenovirus iCELLis nano 2.67 2.3E+05 1.1E+10 TCID50/cm 2 3.0E+14 TCID50<br />
Vero Influenza iCELLis nano 4 1.0E+05 3.8E+06 TCID50/cm 2 1.5E+11 TCID50<br />
iCELLis pilot 20 7.5E+04 2.5E+06 TCID50/cm 2 5.0E+11 TCID50<br />
Paramyxovirus iCELLis nano 2.67 2.7E+05 6.4E+05 TCID50/cm 2 1.7E+10 pfu<br />
Vero Undisclosed Lytic Virus iCELLis pilot 40 1.5E+05 Confidential Confidential<br />
iCELLis 500 133 1.5E+05<br />
iCELLis 1000 660 2.3E+05
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Table 1(abstract 61) Scale-up feasibility of stem cells growth in Xpansion bioreactor<br />
QUALITY CONTROL TEST<br />
Xpanion10<br />
(Five runs)<br />
Xpansion 50<br />
(Two runs)<br />
Xpansion 180<br />
(Three runs)<br />
CELL CULTURE SURFACE (CM 2 ) 6.120 30.600 110.160 /<br />
AVERAGE CELL QUANTITY AT HARVEST 1.8 × 10 8 9×10 8 3.3 × 10 9 /<br />
VIABILITY ≥90% ≥90% ≥90% ≥90%<br />
GROWTH PROFILE Normal Normal Normal Normal<br />
CONFLUENCY √ √ √ √<br />
HOMOGENEOUS CELL DISTRIBUTION &<br />
MORPHOLOGY<br />
√ √ √ √<br />
IDENTITY<br />
CYP3A4 Activity<br />
IDENTITY<br />
Phenotype<br />
PURITY<br />
POTENCY<br />
(Urea secretion)<br />
POTENCY<br />
(Bilirubin Conjugation)<br />
Conform<br />
>10 -8 pmol/cell/4 h<br />
Conform<br />
CD73, CD90>60%<br />
ALB+, vim+, ASMA+<br />
Conform<br />
CD31+CD133+<br />
CD45+CK19 < 15%<br />
ConforM<br />
4/5*<br />
Conform<br />
4/5*<br />
Conform<br />
>10 -8 pmol/cell/4 h<br />
Conform<br />
CD73, CD90>60%<br />
ALB+, vim+, ASMA+<br />
Conform<br />
CD31+CD133+<br />
CD45+CK19 < 15%<br />
ConforM<br />
1/2*<br />
Conform<br />
2/2<br />
Conform<br />
>10 -8 pmol/cell/4 h<br />
Conform<br />
CD73, CD90>60%<br />
ALB+, vim+, ASMA+<br />
Conform<br />
CD31+CD133+CD45+<br />
CK19 < 15%<br />
Conform<br />
3/3<br />
Conform<br />
(pending)<br />
In-Line Centrifugation<br />
Three runs<br />
Conform<br />
>10 -8 pmol/cell/4 h<br />
Conform<br />
CD73, CD90>60%<br />
ALB+, vim+, ASMA+<br />
Conform<br />
CD31+CD133+CD45+<br />
CK19 < 15%<br />
Conform<br />
3/3<br />
Conform<br />
(pending)<br />
Cell properties are checked throughout the scale-up process and results are expressed in terms of cell viability, confluence, morphology, growth and cell<br />
characterization (identity/purity/potency). * 1 QC failed in the Xpansion 10 & Xpansion 50 bioreactors but QC were similar to their respective CS control (not<br />
related to the bioreactor).<br />
Stem cells expansion and harvesting: ✓ Inoculation: 5,000 cells/cm 2<br />
✓ Harvest: 20,000-40,000 cells/cm 2<br />
✓ 10% serum-containing medium<br />
Results: Xpansion 10 was used to prove feasibility of stem cell growth in<br />
Xpansion multiplate bioreactor and to optimize cell culture parameters. The<br />
goal was to perform a simple process transfer from multitray stack (e.g.<br />
Corning CellStack (CS)) to the Xpansion by mimicking cell culture conditions.<br />
All Xpansion runs achieved similar results to their control in terms of cell<br />
density, homogenous distribution, viability and morphology. Additional<br />
quality control (QC) analysis revealed that cell characteristics were<br />
maintained (identity/purity/potency) (table 1)<br />
Scale-up from the Xpansion 10 to the Xpansion 180: Cultures were<br />
directly transferred from the Xpansion 10 bioreactor to the larger scales<br />
Xpansion 50 and Xpansion 180 bioreactors, where cells reached similar<br />
levels of growth and confluence (Table 1). Further analysis of the cultures<br />
at <strong>all</strong> scales showed compliancy with the QC specifications. In order to<br />
keep the process within a closed system, cells harvested from Xpansion<br />
180 were directly centrifuges. The in-line continuous centrifugation step<br />
achieved 80% yields while maintaining cells characteristics (Table 1).<br />
Xpansion bioreactor regulation: Figure 1 shows the pH and DO<br />
regulation profiles of cultures in Xpansion 10 and Xpansion 180. The<br />
trends of both bioreactors are highly similar, except that the duration of<br />
a regulation cycle is longer in the Xpansion 180 compared to the<br />
Figure 1(abstract P61) Regulation parameters in XP-10 (A) or XP-180 (B) in the course of time, pH (green), D.O. (blue) and T° (red) evolution.<br />
Set points (dashed lines) were fixed at 7.5 for pH and D.O. >50%. T° peaks are due to Xpansion disconnection for microscopic observation or samplings.
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Xpansion10.Thisisduetothelongerhomogenizationtime.Thegas<br />
diffusion system through the silicone tubing is efficient.<br />
Cell observation using the holographic microscope - iLine: The iLine<br />
holographic microscope and the Xpansion bioreactors are designed to<br />
<strong>all</strong>ow cell observation on the first ten plates of each bioreactor. The<br />
microscope software enables an automatic cell counting of the cell<br />
confluency. Cell confluence assessment through DDHM microscope is a<br />
key element for defining cell harvest time given that cell confluence<br />
levels are critical to guarantee cell characteristics.<br />
Conclusions: The Integrity Xansion multiplate bioreactors demonstrated<br />
their efficiency for the growth of progenitor of hepatocyte cells at large<br />
scale while keeping the cell therapeutic potency.<br />
The use of a robust process control system and the iLine microscope<br />
enabled to record the evolution of the culture:<br />
➢ Sampling port that can be used for dosing of nutrients, growth<br />
factors, etc.<br />
➢ On-line pH and D.O. tracking<br />
➢ Off-line microscopic observations<br />
The Xpansion 10 bioreactor proved to be a useful tool for determining<br />
optimal cell culture parameters. Actu<strong>all</strong>y, several runs could be performed<br />
using this scaled-down, while sparing time and money and extrapolating<br />
the cell behavior, the pH and DO trends in the Xpansion 50 and<br />
Xpansion 180. The new Xpansion bioreactor offers a valuable technology<br />
for large-scale production while meeting GMP compliancy. Moreover, the<br />
in-line centrifugation step guarantees a closed manufacturing process,<br />
from seeding to freezing.<br />
P62<br />
Characterization and quantitation of fluorescent Gag virus-like particles<br />
Sonia Gutiérrez-Granados, Laura Cervera, Francesc Gòdia,<br />
María Mercedes Segura *<br />
Departament d’Enginyeria Química, Universitat Autònoma de Barcelona,<br />
Bellaterra, Barcelona, 08193, Spain<br />
E-mail: mersegura@gmail.com<br />
BMC Proceedings 2013, 7(Suppl 6):P62<br />
Background: Upon expression, the Gag polyprotein of HIV-1<br />
spontaneously assembles giving rise to enveloped virus-like particles<br />
(VLPs). These particulate immunogens offer great promise as HIV-1<br />
vaccines. In order to develop robust VLP manufacturing processes, the<br />
availability of simple, fast and reliable quantitation tools is crucial.<br />
Tradition<strong>all</strong>y, commercial p24 ELISA kits are used to estimate Gag VLP<br />
concentrations. However, this quantitation technique is time-consuming,<br />
laborious, costly and prone to methodological variability. Reporter<br />
proteins are frequently used during process development to <strong>all</strong>ow a<br />
straightforward monitoring and quantitation of labeled products. This<br />
alternative was evaluated in the present work by using a Gag-GFP fusion<br />
construct.<br />
Materials and methods: Generation of fluorescent VLPs was carried out<br />
by transient transfection of HEK 293 suspension cells with a plasmid<br />
coding for Gag fused to GFP (NIH AIDS Reagent Program). VLP budding<br />
from producer cells was visualized by electron microscopy (JEM-1400, Jeol)<br />
and confocal fluorescence microscopy (Fluoview® FV1000, Olympus,<br />
Japan). A purified standard of Gag-GFP VLP material was obtained by<br />
ultracentrifugation through a sucrose cushion and fully characterized. SDS-<br />
PAGE,Westernblot,size-exclusionchromatography (SEC), nanoparticle<br />
tracking analysis (NTA, NanoSight®, UK) and transmission electron<br />
microscopy (TEM) were used for VLP characterization. The standard VLP<br />
material was used for the development and validation of a Gag-GFP VLP<br />
quantitation technique based on fluorescence. Viral particle titers<br />
estimated using this method were compared with those obtained by p24<br />
ELISA (Innotest®, Innogenetics, Belgium), densitometry, TEM and NTA.<br />
Results: Upon transfection, Gag-GFP was expressed in the cytoplasm of<br />
the producer cells and accumulated in the vicinity of the plasma<br />
membrane where the budding process takes place. Upon staining with<br />
Cell Mask, co-localization of green (Gag-GFP molecules) and red (lipid<br />
membrane) fluorescence was observed in yellow (Figure 1A). VLP<br />
budding was also visualized in TEM images of ultrathin sectionsofHEK<br />
293 producer cells (Figure 1B).<br />
A purified Gag-GFP VLP standard material was obtained by harvesting<br />
VLPs from cell culture supernatants of transfected HEK 293 cells by low<br />
speed centrifugation followed by VLP pelleting through a 30% sucrose<br />
cushion. The purity of the standard material was analyzed by SEC. The<br />
SEC chromatogram showed a single peak eluting in the column void<br />
volume (V 0 = 44 mL) as determined by UV and fluorescence analyses of<br />
collected fractions (Figure 1D). The A260/A280 ratio was 1.24 which is<br />
consistent with reported ratios for purified retroviral particles [1]. The<br />
standard VLP material was further characterized using different<br />
techniques. Particle morphology was analyzed by TEM. Roughly spherical<br />
viral particles surrounded by a lipid envelope and containing an electrodense<br />
core could be observed (Figure 1C). The mean VLP diameter<br />
according to TEM analysis was determined to be 141 ± 22 nm (n = 100),<br />
which is the expected size of Gag-GFP VLPs as they resemble immature<br />
HIV particles that are larger than wild-type HIV-1 virions [2]. NTA analyses<br />
of the standard material showed that the most frequent particle size<br />
value (statistical mode) was 149 ± 5 nm, which is consistent with our<br />
TEM results. SDS-PAGE analysis of the standard VLP material (Figure 1E)<br />
was performed. Approximately, 65% of the total protein loaded in the gel<br />
corresponded to Gag-GFP (Figure 1E, full arrow), the major HIV-1 VLP<br />
structural protein. The other minor bands should correspond to cellular<br />
proteins derived from host cells as retroviral particles are known to<br />
promiscuously incorporate a significant amount of host proteins [3,4]. A<br />
Gag-GFP band of the expected molecular weight (~81 kDa) was<br />
specific<strong>all</strong>y detected using an anti-p24 mAb by Western blot analysis<br />
(Figure 1E, full arrow). The presence of a Gag-GFP fragment (Figure 1E,<br />
empty arrow), representing only 5% of the total Gag-GFP loaded, was<br />
also observed in the gel.<br />
A fluorescence-based quantitation method for Gag-GFP VLPs was<br />
developed [5]. Validation of the quantitation assay was carried out<br />
according to International Conference Harmonization (ICH) guidelines [6].<br />
The validation parameters evaluated included specificity, linearity,<br />
quantitation range, limit of detection, precision, and accuracy [5]. All<br />
validation parameters met the criteria for analytical method validation.<br />
Some parameters were also studied in par<strong>all</strong>el for p24 ELISA for<br />
comparison purposes (Table 1). Both techniques specific<strong>all</strong>y detected<br />
Gag-GFP. Even though the p24 ELISA assay showed to be more sensitive<br />
for Gag-GFP detection, the fluorescence-based method was more precise<br />
and showed to be linear in a wider range. In addition, the developed<br />
quantitation method required less time and was considerably less<br />
expensive than the traditional p24 ELISA method used for Gag VLP<br />
quantitation. Fin<strong>all</strong>y, the standard VLP material was quantified using<br />
several methods. In order to compare the concentration of Gag-GFP in<br />
μg/mL as determined by the fluorescence-based method, ELISA and<br />
densitometry with the titers obtained by TEM and NTA analyses which<br />
are given in particles/mL, it was assumed that a Gag VLP contains 2500<br />
Gag molecules as previously reported [7]. All concentration values,<br />
regardless of the quantitation technique used, were in close agreement<br />
within an expected range. These results support the reliability of the<br />
fluorescence-based method developed [5].<br />
Conclusions: Due to the flexibility of the retrovirus particle assembly<br />
process, fluorescently tagged Gag VLPs can be easily generated by<br />
expressing Gag as a fusion construct with GFP. Although fluorescently<br />
labeled Gag has mainly been used to study retrovirus replication in<br />
living cells, this attractive feature is exploited in our laboratory to<br />
facilitate the monitoring and quantitation of Gag VLPs. A purified<br />
standard VLP material was obtained and fully characterized. VLPs in the<br />
standard material showed to be of the expected size, morphology and<br />
with a composition consistent with immature HIV-1 particles. A fast,<br />
reliable and cost-effective quantitation method based on fluorescence<br />
was developed and validated using the standard VLP material. The<br />
fluorescence-based quantification method should facilitate the<br />
development and optimization of bioprocessing strategies for Gagbased<br />
VLPs.<br />
Acknowledgements: We would like to thank Dr. Amine Kamen<br />
(National Research Council of Canada) for helpful discussions about this<br />
projectandforkindlyprovidingthecGMPcompliantHEK293SF-3F6<br />
cell line. The pGag-GFP plasmid was obtained through the NIH AIDS<br />
reagent program (Cat #11468). The contribution of Dr. Julià Blanco and<br />
Dr. Jorge Carrillo (IrsiCaixa, Spain) to this work is greatly appreciated.<br />
This project was financi<strong>all</strong>y supported by MINECO-SEIDI, reference<br />
BIO2012-31251.
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Figure 1(abstract P62) Characterization of the purified standard Gag-GFP VLP material. (A) Confocal fluorescence microscopy image of a HEK 293<br />
producer cell expressing green fluorescent Gag-GFP molecules. The lipid membrane is stained with Cell Mask (red) and the cell nucleus with Hoechst<br />
(blue). (B) TEM image of an ultrathin section showing VLP budding from HEK 293 producer cells. (C) Negatively stained Gag-GFP VLPs in the purified<br />
standard material. (D) Size exclusion chromatogram of the standard Gag-GFP VLP material. (E) SDS-PAGE and Western-blot analyses of the standard VLP<br />
material. Full and empty arrows represent Gag-GFP protein and Gag-GFP fragment, respectively. Abbreviations: MW, molecular weight standard.<br />
References<br />
1. McGrath M, Witte O, Pincus T, Weissman IL: Retrovirus purification:<br />
method that conserves envelope glycoprotein and maximizes infectivity.<br />
J Virol 1978, 25:923-927.<br />
2. V<strong>all</strong>ey-Omar Z, Meyers AE, Shephard EG, Williamson AL, Rybicki EP:<br />
Abrogation of contaminating RNA activity in HIV-1 Gag VLPs. Virol J<br />
2011, 8:462.<br />
3. Ott DE: Cellular proteins in HIV virions. Rev Med Virol 1997, 7:167-180.<br />
4. Segura MM, Garnier A, Di Falco MR, Whissell G, Meneses-Acosta A,<br />
Arcand N, Kamen A: Identification of host proteins associated with<br />
retroviral vector particles by proteomic analysis of highly purified vector<br />
preparations. J Virol 2008, 82(3):1107-1117.<br />
5. Gutierrez-Granados S, Cervera L, Godia F, Carrillo J, Segura MM:<br />
Development and validation of a quantitation assay for fluorescently<br />
tagged HIV-1 virus-like particles. J Virol Methods 2013, 193:85-95.<br />
6. ICH: Validation of Analytical Procedures:Text and Methodology Q2(R1).<br />
2005.<br />
7. Chen Y, Wu B, Musier-Forsyth K, Mansky LM, Mueller JD: Fluorescence<br />
fluctuation spectroscopy on viral-like particles reveals variable gag<br />
stoichiometry. Biophys J 2009, 96:1961-1969.<br />
Table 1(abstract 62) Comparison between the<br />
fluorescence-based quantitation method and the p24<br />
ELISA assay<br />
Fluorescence-based p24 ELISA assay<br />
method<br />
Specificity Gag-GFP fusion protein Gag-GFP fusion protein<br />
Linear range<br />
7 to 1000 RFU<br />
(10 to 3600 ng of p24/mL)<br />
10 to 300 pg of<br />
p24/mL<br />
Precision ~2% CV ~10% CV<br />
Limit of detection 10 ng/mL of p24 10 pg/mL of p24<br />
Time (96 samples) ~1.5 h ~4 h<br />
Price (96 samples) ~10 € ~400 €<br />
RFU: Relative fluorescence units<br />
CV: Coefficient of variation<br />
P63<br />
BI-HEX®-GlymaxX® cells enable efficient production of next generation<br />
biomolecules with enhanced ADCC activity<br />
Anja Puklowski, Till Wenger, Simone Schatz, Jennifer Koenitzer,<br />
Jochen Schaub, Barbara Enenkel, Anurag Khetan, Hitto Kaufmann,<br />
Anne B Tolstrup *<br />
Boehringer-Ingelheim, Biberach an der Riss, Germany, 88397<br />
E-mail: Anne.Tolstrup@boehringer-ingelheim.com<br />
BMC Proceedings 2013, 7(Suppl 6):P63<br />
Background: Despite the succes story of therapeutic monoclonal<br />
antibodies (mAbs), a medical need remains to improve their efficacy. One<br />
possibility to achieve this is to modulate important effector functions<br />
such as the antibody dependent cellular cytotoxicity (ADCC).<br />
The advantage of highly active biotherapeutic molecules is - apart from<br />
the enhanced efficacy - the reduction of side effects due to lower<br />
administered doses. Furthermore, these therapeutic antibodies may
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enable treatment of current non-responders, e.g. patients with low<br />
antigen bearing tumors. Enhancement of the effector functions of<br />
antibodies can be achieved either by directlymutatingtheantibody’s<br />
amino acid sequence or by modifying its glycosylation pattern, e.g. by<br />
using a novel host cell line able to attach a desired glycostructure to the<br />
product. The latter approach has the advantage of not impacting the<br />
antibody structure itself, thereby avoiding negative effects on the PK/PD<br />
of the molecule. During the last decade it has been shown that<br />
antibodies with a reduced level of glycan fucosylation are much more<br />
potent in mediating ADCC, a mode of action particularly relevant for<br />
cancer therapeutics. Therefore, defucosylated antibodies are of major<br />
interest for biotherapeutics developers. To produce such antibodies,<br />
Boehringer Ingelheim has inlicensed the GlymaxX® system from<br />
ProBioGen, Germany. This technology utilises the bacterial protein RMD<br />
(GDP-6-deoxy-D-lyxo-4-hexulose reductase) which, when stably integrated<br />
into host cell lines, inhibits fucose de-novo biosynthesis. The enzyme<br />
deflects the fucosylation pathway by turning an intermediate (GDP-4-<br />
Keto-6-Deoxymannose) into GDP-Rhamnose, a sugar that cannot be<br />
metabolised by CHO cells. As a consequence, recombinant antibodies<br />
generated by such host cells exhibit reduced glycan fucosylation and<br />
20-100 fold higher ADCC activity. Here, we show the establishment of a<br />
new host cell line, termed BI-HEX®-GlymaxX® which is capable of<br />
producing highly active therapeutic antibodies. We furthermore present<br />
data on the cell line properties concerning cell culture performance (e.g.<br />
titer, growth, transfection efficiency), process robustness and product<br />
quality reproducibility.<br />
Methods: The BI-HEX® host cell line was transfected with the bacterial<br />
RMD enzyme and stably expressing clones were selected. The presence<br />
of RMD was confirmed by Western blotting. The clones were analysed for<br />
stability of RMD expression over time in continous culture (>100 days),<br />
glycoprofile structure, CD16 binding and ADCC activity of mAbs produced<br />
by these clones before selection of the final new BI-HEX®-GlymaxX® host<br />
cell. Furthermore, we examined the growth and cultivation properties of<br />
the modified BI-HEX®GlymaxX® cells to ensure that the engineered host<br />
cell maintained the favourable manufacturability properties of BI-HEX®<br />
and we tested the reproducibility of key product quality attributes of the<br />
generated antibodies.<br />
Results: Up to date seven different antibodies were produced in our new<br />
BI-HEX®-GlymaxX®host cell line. All molecules showed a very significant<br />
reduction of fucosylation down to 1-3% compared to the control.<br />
Correlating with the low fucose levels, antibodies produced in BI-HEX®-<br />
GlymaxX® exhibited a 20-100× increased ADCC activity (Figure 1A). This<br />
enhancement also correlated well with an increase in CD16 binding. For<br />
the routine cell line and process development we investigated the<br />
robustness of the defucosylation and its resulting activity enhancement.<br />
The results indicated a high reproducibility between independent<br />
production runs. The ADCC level as well as the CD16 binding was robust<br />
for <strong>all</strong> analysed mAbs (Figure 1B). Investigating the cell culture behaviour<br />
of the BI-HEX®-GlymaxX®and its parental BI-HEX® cell line, we saw<br />
comparable results for their transfection efficiencies, doubling times, titer<br />
and production run performance. Depletion studies of RMD showed that<br />
this enzyme can be efficiently depleted during downstream purification<br />
of the mAb.<br />
Conclusions: Our new BI-HEX®-GlymaxX®cell line is capable of producing<br />
>90% defucosylated antibodies which exhibit a 20-100 fold higher ADCC<br />
activity compared to a normal CHO production cell line like BI-HEX®. This<br />
increase in ADCC activity correlated with a stronger CD16 binding in<br />
those molecules. Furthermore, the BI-HEX®-GlymaxX® cells show the same<br />
manufacturing properties (transfection efficiency, doubling times, titer,<br />
peak cell density) to its originator cell line. For the depletion of RMD<br />
we’ve established a sensitive depletion assay and measured a complete<br />
reduction of RMD after the first purification step (protein A capture).<br />
P64<br />
Effects of perfusion processes under limiting conditions on different<br />
Chinese Hamster Ovary cells<br />
Anica Lohmeier 1* , Tobias Thüte 1 , Stefan Northoff 2 , Jeff Hou 3 , Trent Munro 3 ,<br />
Thomas Noll 1,4<br />
1 Institute of Cell Culture Technology, Bielefeld University, Germany;<br />
2 TeutoCell AG, Bielefeld, Germany;<br />
3 The Australian Institute for<br />
Bioengineering and Nanotechnology (AIBN), University of Queensland,<br />
Brisbane, Australia;<br />
4 Center for Biotechnology (CeBiTec), Bielefeld University,<br />
Germany<br />
E-mail: anica.lohmeier@uni-bielefeld.de<br />
BMC Proceedings 2013, 7(Suppl 6):P64<br />
Background: The use of perfusion culture to generate biopharmaceuticals<br />
is an attractive alternative to fed-batch bioreactor operation. The process<br />
<strong>all</strong>ows for generation of high cell densities, stable culture conditions and a<br />
short residence time of active ingredients to facilitate the production of<br />
sensitive therapeutic proteins.<br />
However, ch<strong>all</strong>enges remain for efficient perfusion based production at<br />
industrial scale, primarily complexity of required equipment and<br />
strategies adopted for downstream processing. For perfusion systems to<br />
be industri<strong>all</strong>y viable there is a need to increase product yields from a<br />
perfusion-based platform.<br />
We have shown previously that one effective way to enhance the cell<br />
specific productivity is via glucose limitation [1,2]. The mechanisms leading<br />
to an increased productivity under these glucose limiting conditions are<br />
still under investigation. Preliminary studies using proteomic analysis have<br />
indicated changes in histone acetylation [2].<br />
In this work, we investigated the influence of glucose limited conditions on<br />
the production of two different recombinant proteins in perfusion processes.<br />
Materials and methods: CHO-MUC2 and CHO-XL99 cell lines were<br />
cultivated perfusion based in a 2 L pO 2 - and pH-controlled bioreactor<br />
using an internal spin filter (20 μm) for cell retention. In addition these<br />
cell lines were cultivated both under limiting and non-limiting glucose<br />
conditions in fed-batch mode in a four vessel par<strong>all</strong>el single-use system<br />
(Bayshake, Bayer Technology Services GmbH).<br />
Figure 1(abstract P63) A) Comparison of ADCC activity of Rituximab produced in either BI-HEX® or BI-HEX®-GlymaxX®. B) ADCC activity of 3<br />
different mAbs produced in BI-HEX®-GlymaxX®. Three independent production runs were performed for each mAb. The mAbs were individu<strong>all</strong>y purified<br />
by protein A capture before ADCC activity determination.
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Perfusion mode was started three days after inoculation; flow rate was<br />
adjusted between 0.3 d -1 and 0.6 d -1 . For fed-batch cultivation the<br />
limiting range for glucose concentration was chosen between 0.2 and<br />
0.5 g/L. Reference cultivation was performed between 1.5 and 3.0 g/L.<br />
Both cultures were fed with similar volumes.<br />
All cultivations were performed in chemic<strong>all</strong>y-defined, animal-component<br />
free CHO growth media (TeutoCell AG).<br />
Viable cell density and viability were determined using the automated cell<br />
counting system CEDEX (Roche Diagnostics), glucose and lactate<br />
concentrations were detected via YSI (YSI life sciences). Amounts of IgG1<br />
were quantified via Protein A HPLC, anti IL-8 mAb purified from a CHO<br />
DP-12 cell clone was used as a standard. Mucin-2 quantity was measured<br />
via photometric quantification of eGFP coupled to the Mucin 2.<br />
Results: Using perfusion mode with a 20 μm spin filter as cell retention device<br />
we have reached viable cell densities of 1.4·10 7 cells/mL in a 24 day perfusion<br />
run of CHO-MUC2 (Figure 1A). During perfusion the average viability remained<br />
higher than 85% was attained. After 6 days of cultivation glucose reached a<br />
limiting concentration below 1 mM (Figure 1B). Meanwhile a relative eGFP<br />
concentration of 5 mg/L was achieved (Figure 1C) and cell specific productivity<br />
increased by 90% during glucose limitation (data not shown).<br />
A further 34 day perfusion cultivation using a CHO-XL99 clone reached a<br />
viable cell density of 2.6·10 7 cells/mL with an average viability of 90%<br />
(Figure 1A). Glucose and Lactate concentrations of CHO-XL99 were below<br />
detectable limits on day 8 and 17 post-inoculation respectively (Figure 1B).<br />
Simultaneously, cells were able to reach an IgG1 titer of 326 mg/L, with<br />
significant increases in product titer observed after 24 days of culture<br />
(Figure 1C). Simultaneously, cell specific productivity showed a slight<br />
increase after 25 days (data not shown).<br />
Neither the CHO-MUC2, nor the CHO-XL99 cells showed any limitations<br />
concerning other substrates, e.g. amino acids (data not shown).<br />
In two par<strong>all</strong>el fed-batch cultivations of the CHO-XL99 clone the glucose<br />
limited culture showed similar growth characteristics as the unlimited<br />
reference culture. Viable cell densities of 1.9·10 7 cells/mL (reference) and<br />
2.9·10 7 cells/mL (-Glc), respectively, were observed (Figure 1D). The<br />
limited culture reached an IgG1 concentration of 610 mg/L, in contrast to<br />
292 mg/L produced by the reference culture (Figure 1D). Under glucose<br />
limitation the cells consumed lactate while under non-limiting conditions<br />
lactate accumulated (Figure 1E).<br />
Conclusions: During perfusion processes under glucose limitation three<br />
characteristic phases appear: At first glucose concentration is high and<br />
lactate is below detection limit. Afterwards glucose is metabolized into<br />
lactate with an increasing lactate formation rate. In the end both<br />
metabolites are consumed and an increase in product concentration and<br />
cell specific productivity occurs.<br />
Reduced lactate formation was observed during the perfusion run as<br />
CHO-MUC2 cells shift towards a more efficient glucose metabolism.<br />
Figure 1(abstract P64) A Viable cell counts and cell viabilities for the time course of CHO-MUC2 and CHOXL99 cells during perfusion process; B: glucose<br />
and lactate concentrations during CHO-XL99 and CHO-MUC2 perfusion cultivation; C: Concentration of IgG1 mAb and eGFP during CHO perfusion<br />
cultivations; D: Viable cell counts and mAb concentration for the time course of CHO-XL99 fed-batch cultivations; E: Glucose and lactate concentrations<br />
for the time course of CHO-XL99 under limiting (-Glc) and non-limiting conditions.
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Thereby cell specific productivity of CHO-MUC2 cells increased by 90%<br />
during glucose limitation.<br />
CHO-XL99 cells showed a similar metabolic shift during perfusion along<br />
with increased mAb production as well as in fed-batch cultivation.<br />
Resulting from this fed-batch cultivations <strong>all</strong>ow predictions concerning<br />
cell behavior under glucose limitation in perfusion.<br />
To analyse the impact of limiting conditions on transcriptome level of<br />
CHO cells, a microarray will be used. This proprietary CHO microarray<br />
contains 41.304 different probes to elucidate reasons for the increase in<br />
cell specific productivity.<br />
Acknowledgements: We gratefully acknowledge to the Australian<br />
Institute for Bioengineering and Nanotechnology, University of<br />
Queensland-Brisbane, Australia (AIBN) for providing the CHO-XL99 clone.<br />
We would also thank Bayer Technology Services for providing the<br />
Bayshake system.<br />
References<br />
1. Link T, Bäckström M, Graham R, Essers R, Zörner K, Gätgens J: Bioprocess<br />
development for the production of a recombinant MUC1 fusion protein<br />
expressed by CHO-K1 cells in protein-free medium. J Biotechnol 2004,<br />
110:51-62.<br />
2. Wingens M, Gätgens J, Hoffrogge R, Noll T: Proteomic characterization<br />
of a glucose-limited CHO-perfusion process-analysis of metabolic<br />
changes and increase in productivity. ESACT proceedings Springer: Noll T<br />
4:265-269.<br />
P65<br />
Development of 3D human intestinal equivalents for substance testing<br />
in microliter-scale on a multi-organ-chip<br />
Annika Jaenicke 1* , Dominique Tordy 3 , Florian Groeber 3 , Jan Hansmann 3 ,<br />
Sarah Nietzer 4 , Carolin Tripp 4 , Heike W<strong>all</strong>es 3,4 , Roland Lauster 1 , Uwe Marx 1,2<br />
1 TU Berlin, Institute for Biotechnology, Faculty of Process Science and<br />
Engineering, 13355 Berlin, Germany;<br />
2 TissUse GmbH, 15528 Spreenhagen,<br />
Germany;<br />
3 Fraunhofer Institute for Interfacial Engineering and Biotechnology<br />
IGB, 70569 Stuttgart, Germany;<br />
4 Chair of Tissue Engineering and<br />
Regenerative Medicine, Julius-Maximilians-Universität Würzburg, 97070<br />
Würzburg, Germany<br />
E-mail: a.jaenicke@tu-berlin.de<br />
BMC Proceedings 2013, 7(Suppl 6):P65<br />
Background: Robust and reliable dynamic bioreactors for long term<br />
maintenance of various tissues at milliliter-scale on the basis of a<br />
biological, vascularized matrix (BioVaSc®) have been developed at the<br />
Fraunhofer IGB in Stuttgart, Germany. As an intestinal in vitro equivalent,<br />
seeding of the matrix with CaCo-2 cells yielded in the self-assembly of a<br />
microenvironment with the typical histological appearance of villus-like<br />
structure and morphology [1]. We modified this matrix (BioVaSc®) - cell<br />
(CaCo-2) system to some extent with the aim to develop 3D intestinal<br />
equivalents for systemic preclinical testing of or<strong>all</strong>y applied drug<br />
candidates in microliter-scale on a human Multi-Organ-Chip (MOC), which<br />
consists of different organ equivalents important for ADMET (adsorption,<br />
distribution, metabolism, excretion, toxicity) testing.<br />
Materials and methods: For the generation of biological, vascularized<br />
matrices (rBioVaSc®), jejunal segments of the sm<strong>all</strong> intestine of Wistar rats<br />
including the corresponding capillary bed were explanted and decellularized<br />
by perfusion with 1% sodium deoxycholate. Characterization of the matrix<br />
wasdonebyhistologicalanalysisaswellas2-photonmicroscopy(2PM)<br />
and immunofluorescent stainings. After sterilization by g-irradiation, the<br />
rBioVaSc® could be used to built up a 3D intestinal equivalent. Punch<br />
biopsies of the matrix were fixed on the frame of a 96-well transwell insert<br />
and seeded with CaCo-2 cells (2*10^6 cells) on the former luminal side of<br />
the matrix following static cultivationfor48hoursandintegrationina<br />
perfused MOC device. Our MOC device consists of an integrated micropump,<br />
a microfluidic channel system and inserts for the cultivation of<br />
different organ equivalents (Figure 1e). For the generation of the intestinal<br />
equivalent, the generated matrix-cell construct was placed in the MOC<br />
device and perfused for up to one week with cell culture medium<br />
(supplemented MEM), following histological as well as immunofluorescence<br />
(IF) analysis of the growth behavior of the cells. As a control, matrix-cell<br />
constructs were cultivated static<strong>all</strong>y. Daily medium samples have been<br />
analyzed to monitor metabolic activity and the absorption properties of the<br />
intestinal equivalent. Immunohistostaining of cryo-preserved tissue slices<br />
have been analyzed to compare self-assembled organoid tissue structures<br />
with their corresponding in vivo counterparts.<br />
Results: Decellularization of jejunal segments of rats together with the<br />
corresponding capillary bed yielded in a biological, vascularized matrix<br />
which was free of non-human cells but with the preserved 3D structure<br />
of the former intestinal extracellular matrix (ECM) (Figure 1a-d). Those<br />
ECM components were used for the resettlement of human intestinal<br />
Figure 1(abstract P65) a-d) Characterization of the decellularization procedure. a) Explanted jejunal segment with the preserved capillary bed after<br />
decellularization. b) H/E staining of the decellularized matrix. c) Feulgen staining of the decellularized matrix. d) immunofluorescent stainings for collagen<br />
I on rBioVaSc. e) The multi-organ-chip (MOC) device consisting of an integrated micro-pump, a microfluidic .channel system and inserts for the cultivation<br />
of different organ equivalents. f+g) Characterization of the intestinal in vitro equivalent. f) H/E staining of the recellularized matrix after one week of<br />
dynamic culture in the MOC device. g) Second Harmonic Generation by 2 PM, nuceli were stained with Hoechst 33342.
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cells (CaCo-2) which resulted in the formation of characteristical villus-like<br />
structures on the matrix after one week of perfused cultivation (Figure 1f+g).<br />
Cells expressed typical intestinal epithelial markers, e.g. CK8/18, EpCAM and<br />
Na/K-ATPase. Process parameters, such as nutrient perfusion rate and<br />
culture time, have been optimized to qualify the system for repeated dose<br />
testing of or<strong>all</strong>y administered drug candidates.<br />
Conclusions: Asshownbyhistologicalaswellasimmunofluorescent<br />
stainings, we succeeded in the development of self-assembled 3D organ<br />
equivalents which have a characteristical intestinal architecture. Those<br />
organ equivalents can be used as an in vitro system for the evaluation of<br />
adsorption properties of or<strong>all</strong>y administered drugs in microliter-scale on a<br />
multi-organ-chip (MOC). Further improvements of the MOC device are<br />
necessary, e.g. the integration of a second circulation, representing the<br />
intestinal lumen. In addition, reseeding the matrix with primary intestinal<br />
cells as well as co-cultures of epithelial and endothelial cells are planned.<br />
Acknowledgements: The work has been funded by the German Federal<br />
Ministry for Education and Research, GO-Bio Grand No. 0315569.<br />
Reference<br />
1. Pusch J, Votteler M, Göhler S, Engl J, Hampel M, W<strong>all</strong>es H, Schenke-<br />
Layland K: The physiological performance of a three-dimensional model<br />
that mimics the microenvironment of the sm<strong>all</strong> intestine. Biomaterials<br />
2011, 32:7469-7478.<br />
P66<br />
A robust RMCE system based on a CHO-DG44 platform enables<br />
efficient evaluation of complex biological drug candidates<br />
Thomas Rose 1,2* , Annette Knabe 1 , Rita Berthold 1 , Kristin Höwing 1 ,<br />
Anne Furthmann 1 , Karsten Winkler 1 , Volker Sandig 1<br />
1 ProBioGen AG, 10439 Berlin, Germany;<br />
2 Freie Universität Berlin, 14195 Berlin,<br />
Germany<br />
E-mail: thomas.rose@probiogen.de<br />
BMC Proceedings 2013, 7(Suppl 6):P66<br />
Background: In early development stages of biologicals there is often<br />
more than one molecule against a specific target. A careful candidate<br />
evaluation is crucial to choose an optimal lead variant for further<br />
development. Complex biologicals are typic<strong>all</strong>y produced in CHO cells<br />
and host cells as well as the process are known to influence important<br />
molecule features such as glycan patterns or activity. To streamline the<br />
generation of stable producer cell lines we have established an Flp-based<br />
RMCE system in our CHO-DG44 platform. RMCE application <strong>all</strong>ows for<br />
multi-par<strong>all</strong>el production of candidate material in the host cell and<br />
process background used for the pharmaceutical cell lines. Therefore, the<br />
molecular features of this material are expected to match with material<br />
that will be derived from a future producer cell line.<br />
Generation of the RMCE host cell line: A replaceable gfp gene cassette<br />
was established at random chromosomal integration sites in CHO-DG44<br />
cells. This clone pool was subjected to a primary RMCE with a secreted<br />
and complex glycosylated alpha1-antitrypsine (A1AT) reporter. Resulting<br />
cells were screened for A1AT producers that have undergone a successful<br />
cassette exchange. This strategy <strong>all</strong>ows for selection of a RMCE host cell<br />
line that combines transgene expression from highly active genomic loci<br />
with superior processing and secretion capabilities.<br />
Strategy for routine RMCE application: The selected RMCE host cell<br />
line is susceptible for cassette exchange with any desired target gene<br />
and candidate protein. Successful cassette exchange is enforced by<br />
promoter trap and a well defined selection system (Figure 1A). For RMCE<br />
application the promoterless target gene encoding for the candidate<br />
protein is cloned into a target vector where it is linked to a selection<br />
marker via an IRES element. Upon successful cassette exchange, the<br />
target and marker gene will be activated by a promoter residing at the<br />
targeting locus. In addition, a second inactive marker gene (lacking an<br />
ATG) that resides also at the host genome, but downstream of the<br />
replaceable gene cassette will be activated. The target vector is<br />
introduced together with a vector encoding the flp recombinase into the<br />
RMCE host cell line. The use of heterospecific FRT sites prevents from<br />
simple re-excision of the gene cassette.<br />
A robust protocol provides for efficient RMCE: RMCE application<br />
results in cell populations showing comparable expression levels of the<br />
newly introduced genes as exemplified for individual RMCEs with a gfp<br />
reporter and different selection formats (Figure 1B). Also, a homogenous<br />
expression was observed within the individual RMCE derived populations<br />
after drug selection. Efficient RMCE application is supported by a fine<br />
tuned and robust protocol that can be applied in T-flasks or multiwell<br />
formats.<br />
Evaluation studies: RMCE application with monoclonal antibody and<br />
fusion proteins: RMCE was applied to a monoclonal antibody and single<br />
cell clones have been generated from the RMCE derived population. Those<br />
clones were analyzed together with the original population in fed batch<br />
culture using ProBioGen’s chemical defined platform medium and process<br />
(Figure 1C). The RMCE derived population yielded in harvest titers of 0.5 g/L<br />
matching the titers obtained for individual clones. Consequently, after drug<br />
selection the cells can be directly used for material production. Single cell<br />
cloning is not required!<br />
In a second study two variants of a soluble receptor-Fc fusion protein<br />
were analyzed for manufacturability. Over a number of individual RMCEs<br />
variant #1 was expressed at a ~2-fold higher rate. In a fed batch process<br />
the difference was maintained yielding in final titers of 1.2 g/L for variant<br />
#1 (Figure 1D). The 2-3-fold outperformance of variant #1 was confirmed<br />
in classic cell line development.<br />
RMCE facilitates streamlined generation of stable cell lines and POC<br />
material production: At minimal effort RMCE application <strong>all</strong>ows for<br />
streamlined generation of stable cell lines and production of POC<br />
material (Figure 1E). Applying a single RMCE within only 2 weeks a<br />
suspension culture is available for scale-up and production. Compared to<br />
transient protocols production runs can easily be repeated at any time<br />
and scale.<br />
Conclusions: A robust protocol provides for efficient and reproducible<br />
RMCE application for antibodies and single chain proteins.<br />
At minimal effort RMCE application enables fast and multi-par<strong>all</strong>el<br />
evaluation of complex biological drug candidates.<br />
RMCE application <strong>all</strong>ows for streamlined production of candidate material<br />
in the background of ProBioGen’s CHO-DG44 platform.<br />
P67<br />
Systems biology of unfolded protein response in recombinant CHO<br />
cells<br />
Kamal Prashad Segar, Vikas Chandrawanshi, Sarika Mehra *<br />
Department of Chemical Engineering, Indian Institute of Technology<br />
Bombay, Mumbai - 400076, India<br />
E-mail: sarika@che.iitb.ac.in<br />
BMC Proceedings 2013, 7(Suppl 6):P67<br />
Background: Productivity of recombinant therapeutics is a coordinated<br />
effort of multiple pathways in the cell [1]. The protein processing<br />
pathway in endoplasmic reticulum has been the target of many cell<br />
engineering studies but with mixed results [2]. We have observed the<br />
induction of UPR genes in recombinant CHO cells (data not shown). In<br />
this work, we attempt to increase their productivity further by inducing<br />
ER stress using a known UPR inducer.<br />
Materials and methods: Cell culture: Suspension CHO cells secreting<br />
anti rhesus IgG were grown in a media containing 50% PF-CHO<br />
(Hyclone) and 50% CDCHO (Invitrogen) supplemented with 4 mM L-<br />
Glutamine (Invitrogen), 0.10% Pluronic (Invitrogen), 600 μg/ml G418<br />
(Sigma) and 250 nM Methotrexate (Sigma) in a total culture volume of<br />
20 ml. All cultures were run in replicates in 125 ml Erlenmeyer flasks<br />
(Corning). Cells were treated with tunicamycin (Sigma) for 12 hours and<br />
were harvested for RNA isolation. Cell densities and viabilities were<br />
determined by a hemocytometer using the tryphan blue exclusion<br />
method.<br />
Quantitative real time PCR: Primers were designed based on consensus<br />
sequences from human, mouse and rat and checked against the CHO<br />
genome database wherever available. Total RNA was isolated using Tri<br />
reagent (Sigma) and converted to cDNA using the Reverse Transcription<br />
kit (Thermo). 100 ng of cDNA was used for qPCR to quantify the mRNA<br />
levels of different UPR genes with Actin as the house keeping genes<br />
following the ΔΔCT method.<br />
Antibody quantification: Antibody titres were quantified using the<br />
protocol as described earlier by Chusainow et.al.,[3] and their specific<br />
productivities (qP) were also calculated.<br />
Results: Induction of different ER stress genes was observed at peak<br />
productivities in these recombinant CHO cell lines (data not shown).
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Figure 1(abstract P66) A: RMCE Strategy for routine RMCE application in the selected CHO-DG44 RMCE host cell line. M = selection marker, haat =<br />
A1AT gene. B: GFP expression of cell populations derived from multiple RMCEs and selection formats. C: RMCE with a monoclonal antibody. Fed batch of<br />
the direct RCME derived population and three individual RMCE clones derived from original RMCE population. D: RMCE with two variants of a soluble<br />
receptor-Fc fusion protein. Exemplary fed batch process for the both RMCE derived Fc fusion protein variants. E: Timescale of routine RMCE Application.<br />
Therefore, we hypothesized that increasing the ER stress to higher levels<br />
may have an additive effect on IgG productivity in these cell lines.<br />
Tunicamycin a known ER stress inducer was used to induce ER stress in<br />
these cells. CHO cells were treated with tunicamycin (2.5 mM) for 12<br />
hours and harvested for RNA isolation. qPCR was performed to quantitate<br />
the expression levels of different ER stress genes. IgG HC and LC mRNA<br />
were also quantified and their fold changes were also calculated. IgG<br />
titers in the supernatant were quantified using ELISA.<br />
The IgG titers and cumulative productivities in the tunicamycin treated and<br />
control cells are presented in Figures 1a and 1b. 12 hours post-treatment<br />
with tunicamycin, the IgG titers increased to 460 μg/ml. Productivity in<br />
treated cells was found to be 25 pg/cell/day, corresponding to a 1.7 fold<br />
increase compared to control cells. Interestingly, both the IgG HC and LC<br />
mRNA were not induced in treated cells (Figure 1c, d). To elucidate the<br />
role of UPR pathway in the observed increase in productivity, expression of<br />
many chaperones and UPR genes was measured. In response to<br />
tunicamycin, chaperones including GRP78 and GRP94 were induced to a<br />
maximum of 17-fold (Figures 1e, f). Co-chaperone ERDJ4, involved in<br />
the translocation of nascent proteins inside ER and activation of ERAD<br />
pathway [4], was also induced in response to tunicamycin treatment<br />
indicating increase in ER load. Figure 1g and 1h show the mRNA profiles<br />
of ERDJ4 and EDEM in control and treated cells. No significant difference<br />
in expression of UGGT1 mRNA was observed, suggesting that there may<br />
be negligible mis-folded proteins (Figure 1i) which can be recycled for<br />
refolding while most of them are continuously degraded by the ERAD<br />
machinery. Highly active transcription factors of the UPR pathway viz.,<br />
GADD34, CHOP and XBP1s were also induced in response to tunicamycin<br />
treatment. Figures 1j-1l show the mRNA profiles of different UPR genes.<br />
GADD34 was induced to 38-folds on treatment while CHOP mRNA<br />
induced to about 30-folds. Spliced XBP1 mRNA was also induced to a<br />
maximum of 5.5-folds in treated cells leading to increased expression of<br />
GRP78 mRNA.<br />
Conclusion: Engineering cells towards high productivity by exploiting<br />
their cellular pathways has been gaining importance recently in<br />
biopharmaceutical industries. The unfolded protein response (UPR)<br />
pathway has also been targeted to develop a high producing clone.<br />
However, the results from previous engineering studies on this pathway<br />
are either cell line or product dependent.
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Figure 1(abstract P67) Effect of Tunicamycin on the IgG titres, productivities and mRNA levels of different UPR genes.<br />
In this study, with prior knowledge on the induction of different UPR genes<br />
at peak productivities, we attempted to increase productivity by increasing<br />
ER stress using a known UPR inducer, tunicamycin. Tunicamycin induced<br />
the expression of chaperones and key UPR transcription factors including<br />
GADD34 and XBP1s mRNA.<br />
Increase in the levels of GRP78 and GRP94 mRNA with no change in the<br />
levels of the UGGT1 mRNA suggests that the treated cells may possess a<br />
highly active folding pathway. Increase in the productivities with no<br />
change in the levels of IgG HC and LC mRNA support our hypothesis of<br />
an increased folding capacity in treated cells. Hence, we suggest that the<br />
UPR pathway can be modulated to increase the productivity.<br />
Acknowledgements: This work was parti<strong>all</strong>y supported by a grant from<br />
Department of Biotechnology, Government of India. We would like to<br />
thank Dr. Miranda Yap and Dr. Niki Wong, Bioprocessing Technology<br />
Institute, Singapore for providing the CHO cell lines.<br />
References<br />
1. Seth G, Charaniya S, Wlaschin KF, Hu WS: In pursuit of a super produceralternative<br />
paths to high producing recombinant mammalian cells. Curr<br />
Opin Biotechnol 2007, 18:557-564.<br />
2. Seth G, Hossler P, Yee JC, Hu WS: Engineering cells for cell culture<br />
bioprocessing–physiological fundamentals. Adv Biochem Eng Biotechnol<br />
2006, 101:119-164.<br />
3. Chusainow J, Yang YS, Yeo JHM, Toh PC, Asvadi P, Wong NSC, Yap MGS: A<br />
Study of Monoclonal Antibody-Producing CHO Cell Lines: What Makes a<br />
Stable High Producer? Biotechnology 2009, 102:1182-1196.<br />
4. Lai CW, Otero JH, Hendershot LM, Snapp E: ERdj4 protein is a soluble<br />
endoplasmic reticulum (ER) DnaJ family protein that interacts with ERassociated<br />
degradation machinery. The Journal of biological chemistry<br />
2012, 287:7969-7978.<br />
P68<br />
Chemical chaperone suppresses the antibody aggregation in CHO cell<br />
culture<br />
Masayoshi Onitsuka 1 , Miki Tatsuzawa 2 , Masahiro Noda 2 , Takeshi Omasa 1*<br />
1 Institute of Technology and Science, The University of Tokushima,<br />
Tokushima, 770-8506, Japan;<br />
2 Graduate School of Advanced Technology and<br />
Science, The University of Tokushima, Tokushima, 770-8506, Japan<br />
E-mail: omasa@bio.tokushima-u.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):P68<br />
Background: Aggregation of therapeutic antibodies could be generated<br />
at different steps of the manufacturing process, posing the problem for<br />
quality control of produced antibodies. It has been well known that<br />
secreted antibodies from recombinant mammalian cells into culture<br />
medium can aggregate due to the physicochemical stresses such as<br />
media pH and osmolality, cultivation temperature [1,2]. The antibody<br />
aggregation during the cell culture process is difficult to suppress<br />
because the cell culture conditions for antibody production are gener<strong>all</strong>y<br />
optimized for cell culture and growth and not for suppressing the<br />
aggregateformation.Hereweshowthe novel strategy to suppress the<br />
antibody aggregation; application of chemical chaperone to the cell<br />
culture process. It is well established that an addition of some cosolutes<br />
serves as chemical chaperone to suppress the protein aggregation.<br />
Trehalose, non-reducing sugar formed from two glucose units with a-1,1<br />
linkage, is known as an effective chemical chaperone. In this study, we<br />
investigated the anti-aggregation effect of trehalose in the culture process<br />
of recombinant Chinese hamster ovary cell (CHO) line producing Ex3-<br />
humanized IgG-like bispecific single-chained diabody with Fc (Ex3-scDb-Fc).
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Table 1(abstract 68) Kinetic parameters of cell culture in<br />
Erlenmeyer flasks<br />
Specific growth rate<br />
(μ; ×10 -2 1/h)<br />
Specific<br />
antibody production rate<br />
(r Ab ; pg/cell/day)<br />
Without trehalose 3.07 ± 0.18 a 0.39 ± 0.02 a<br />
150mM trehalose 1.51 ± 0.04 a 1.55 ± 0.03 a<br />
a Mean ± S.D. (n = 3).<br />
Ex3-scDb-Fc shows the remarkable anti-tumor activity based on anti-EGFR<br />
and anti-CD3 bispecificity [3]. However, our in-house results showed that<br />
Ex3-scDb-Fc shows aggregation tendency, demonstrating the necessity of<br />
developing a bioprocess for suppressing the aggregation of the bispecific<br />
diabody.<br />
Materials and methods: CHO Top-H cell line producing the Ex3-scDb-Fc [4]<br />
was cultivated in 500mL Erlenmeyer flask and 2L-glass bioreactor with serumfree<br />
medium containing 150mM trehalose. Viable cell densities and antibody<br />
concentrations were determined with Vi-Cell XR cell viability analyzer<br />
(Beckman Coulter) and by ELISA, respectively. Ex3-scDb-Fc was purified with<br />
Hi-Trap protein A column (GE Healthcare). 1M Arg-HCl (pH4.2) was used as<br />
eluting solution, which make it possible to prevent the aggregation of the<br />
antibody in the affinity purification process. Antibody aggregation was<br />
analyzed by sephacryl S-300 column (GE healthcare). Solution structure of<br />
Ex3-scDb-Fc was assessed by circular dichroism spectroscopy.<br />
Results and discussion: Cell culture performance in trehalose<br />
containing medium: We cultivated CHO Top-H cell line in 150mM<br />
trehalose containing medium. The media osmolalities with and without<br />
trehalose (150 mM) were 480 mOsm/kg and 319 mOsm/kg, respectively.<br />
Estimated kinetic parameters of cell culture are listed in Table 1. Cell<br />
culture in Erlenmeyer flasks demonstrated that cell growth was strongly<br />
affected by trehalose; the specific cell growth rate and the maximum cell<br />
density were decreased compared to those in the absence of trehalose.<br />
On the other hand, both the specific antibody production rate and<br />
volumetric production were largely enhanced by trehalose addition. The<br />
results in Erlenmeyer flask mentioned above were reproduced in 2L-glass<br />
bioreactor culture. Observed properties of the cell culture in the presence<br />
of trehaose, suppressed cell growth and enhanced antibody production,<br />
were similar to those reported for mammalian cell cultures under<br />
hyperosmotic condition [5], although the underlying mechanisms<br />
responsible for the enhanced antibody production are largely unknown.<br />
Anti-aggregation effects by trehalose during the cell culture process:<br />
The scDb-Fc was purified from the culture supernatant by protein A<br />
affinity chromatography, and the aggregation states were analyzed by<br />
size exclusion chromatography. We observed the 3 states of scDb-Fc,<br />
monomer, dimer, and large aggregates, which were included in the<br />
culture supernatant when harvested (Figure 1). The peak area of the large<br />
Figure 1(abstract P68) Size-exclusion chromatography showing the<br />
aggregation status of Ex3-scDb-Fc.<br />
aggregates in the presence of trehalose was one-third that in the<br />
absence of trehalose, indicating that trehalose suppressed the formation<br />
of large aggregates in the CHO cell culture. Circular dichroism (CD)<br />
spectroscopy showed that the large aggregates were misfolded state<br />
with non-native b-strand. Trehalose is expected to suppress the<br />
accumulation of misfolded state and the intermolecular interactions<br />
leading to the aggregate formation in cell culture.<br />
Conclusions: We demonstrated the potential application of chemical<br />
chaperon in the culture of antibody-producing mammalian cells. Trehalose<br />
can be incorporated in the culture media for CHO cells, and can suppress<br />
the antibody aggregation, especi<strong>all</strong>y high-order aggregates. In addition,<br />
trehalose may be involved in the enhancement of antibody production.<br />
Acknowledgements: This study was supported by the Advanced<br />
research for medical products Mining Programme of the National Institute<br />
of Biomedical Innovation (NIBIO). Trehalose was kindly supplied by<br />
HAYASHIBARA Biochemical Laboratories, Inc. (Okayama, Japan). This work was<br />
collaboration with Assoc. Prof. Ryutaro Asano and Prof. Izumi Kumagai<br />
(Tohoku University, Japan).<br />
References<br />
1. Cromwell ME, Hilario E, Jacobson F: Protein aggregation and<br />
bioprocessing. AAPS J 2006, 8:E572-579.<br />
2. Vázquez-Rey M, Lang DA: Aggregates in monoclonal antibody<br />
manufacturing processes. Biotechnol Bioeng 2011, 108:1494-1508.<br />
3. Asano R, Kawaguchi H, Watanabe Y, Nakanishi T, Umetsu M, Hayashi H,<br />
Katayose Y, Unno M, Kudo T, Kumagai I: Diabody-based recombinant<br />
formats of humanized IgG-like bispecific antibody with effective<br />
retargeting of lymphocytes to tumor cells. J Immunother 2008,<br />
31:752-761.<br />
4. Onitsuka M, Kim WD, Ozaki H, Kawaguchi A, Honda K, Kajiura H, Fujiyama K,<br />
Asano R, Kumagai I, Ohtake H, Omasa T: Enhancement of sialylation on<br />
humanized IgG-like bispecific antibody by overexpression of a2,6-<br />
sialyltransferase derived from Chinese hamster ovary cells. Appl Microbiol<br />
Biotechnol 2012, 94:69-80.<br />
5. Rodriguez J, Spearman M, Huzel N, Butler M: Enhanced production of<br />
monomeric interferon-beta by CHO cells through the control of culture<br />
conditions. Biotechnol Prog 2005, 21:22-30.<br />
P69<br />
Dynamical analysis of antibody aggregation in the CHO cell culture<br />
with Thermo Responsive Protein A (TRPA) column<br />
Masahiro Noda 1 , Masayoshi Onitsuka 2 , Miki Tatsuzawa 1 , Ichiro Koguma 3 ,<br />
Takeshi Omasa 2*<br />
1 Graduate School of Advanced Technology and Science, The University of<br />
Tokushima, Tokushima, 770-8506, Japan;<br />
2 Institute of Technology and<br />
Science, The University of Tokushima, Tokushima, 770-8506, Japan;<br />
3 New<br />
Products Development Department, Asahikasei Medical Co., LTD., Bioprocess<br />
Division, Fuji, 416-8501, Japan<br />
E-mail: omasa@bio.tokushima-u.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):P69<br />
Background: Aggregation of therapeutic antibody is gener<strong>all</strong>y occurred<br />
in its manufacturing process, and should be suppressed and removed<br />
because its potential risk for unexpected immune response [1,2]. Protein<br />
A affinity chromatography is the first purification step in the monoclonal<br />
antibody manufacturing. Although the affinity purification is a powerful<br />
technique, high affinity between protein A and antibody requires acidic<br />
condition (below pH 3.0) to elute the captured antibody molecules.<br />
Exposure to acidic condition can induce the denaturation and aggregation<br />
of antibody molecules, demonstrating the necessity of novel strategy to<br />
reduce the antibody aggregation in the affinity purification process. Here we<br />
introduced a novel affinity purification strategy, thermo responsive protein A<br />
(TRPA) resin. TRPA is an engineered protein A ligand which adopts folded<br />
structure under 10°C and unfolds at moderate temperature, above 25°C.<br />
TRPA resin can control capture and elution of antibody by changing column<br />
temperature, making it possible to elute antibody molecules without low pH<br />
condition. In this study, we applied the TRPA column to the purification of<br />
Ex3 humanized IgG-like single-chained bispecific diabody-Fc (Ex3-scDb-Fc)<br />
[3]. The bispecific diabody is the promising candidate for next-generation<br />
therapeutic antibody, whereas it shows aggregation tendency. Furthermore,<br />
we observed the time-dependent formation of antibody aggregation in the
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culture process of the recombinant Chinese hamster ovary (CHO) cell line<br />
with TRPA column.<br />
Materials and methods: CHO Top-H cell line producing the Ex3-scDb-Fc<br />
[4] was cultivated in a 1L-glass bioreactor with working volume of 750 mL<br />
serum-free medium. Viable cell densities and antibody concentrations in<br />
the medium was determined with Vi-Cell XR cell viability analyzer<br />
(Beckman Coulter) and by ELISA, respectively. The bispecific diabody was<br />
purified with conventional protein A (PA) column or thermo responsive<br />
protein A (TRPA) column, which were connected with AKTA prime plus (GE<br />
Healthcare). Elution of antibody was performed by acidic pH solution<br />
(pH2.7) for PA column and by raising column temperature to 45°C for TRPA<br />
column. Aggregate formation was analyzed with Superdex 200 10/30 GL<br />
column (GE Healthcare).<br />
Results and discussion: Performance of TRPA column in the affinity<br />
purification of bispecific diabody-Fc: We purified the Ex3-scDb-Fc from<br />
the culture supernatant of CHO Top-H cell line with PA and TRPA column.<br />
Compared to the conventional protein A column (PA), purification with<br />
TRPA column showed no precipitation of the aggregated scDb-Fc after the<br />
elution. Figure 1A is the size exclusion chromatography (SEC) profiles,<br />
showing that TRPA purification substanti<strong>all</strong>y reduced the formation of<br />
soluble large aggregates as compared to the PA purification including the<br />
exposure to acidic pH condition. Collectively, the above results demonstrate<br />
that TRPA column is highly effective in preventing the formation of<br />
precipitated and soluble aggregates in the affinity purification of the<br />
bispecific diabody-Fc.<br />
Dynamical aggregation analysis in the cell culture process: SEC profile<br />
of TRPA-purified Ex3-scDb-Fc would correctly reflect the status of antibody<br />
aggregation in CHO cell culture, because no further aggregation was<br />
induced in the affinity purification process with TRPA column as compared<br />
with that with conventional PA. Although secreted antibody is known to<br />
aggregate during cell culture process [1,2], the underlying mechanism is<br />
still poorly understood due to the lack of observation of the aggregation<br />
process. We applied the TRPA column to dynamical aggregation analysis of<br />
Ex3-scDb-Fc in CHO cell culture. Culture supernatants from exponential to<br />
stationary growth phase in a bioreactor operation were sampled, and the<br />
bispecific diabody was purified with TRPA column and analyzed by Size<br />
exclusion chromatography. The procedure makes it possible to observe the<br />
time-dependent formation of antibody aggregates in CHO cell culture. In<br />
Figure 1B, the peak areas of large aggregates were plotted as a function of<br />
cultivation time, showing that after 250 hours the amounts of aggregated<br />
Ex3-scDb-Fc were abruptly increased in time dependent manner. The<br />
results suggest a nucleation-dependent aggregation model for antibody<br />
aggregation, where the accumulation of aggregation nucleus is the rate<br />
limiting step and then the nucleus induces the formation of large<br />
aggregates in CHO cell culture. The bispecific diabody in this study has a<br />
tendency to aggregate during the CHO cell culture process, demonstrating<br />
the necessity of the novel cell culture strategy to suppress the aggregates<br />
formation.<br />
Conclusions: We propose the Thermo Responsive Protein A (TRPA)<br />
column as a novel strategy to reduce the antibody aggregation in an<br />
affinity purification process and to analysis the aggregation during the<br />
cell culture process.<br />
Acknowledgements: This study was supported by the Advanced<br />
research for medical products Mining Programme of the National Institute<br />
of Biomedical Innovation (NIBIO). This work was collaboration with Assoc.<br />
Prof. Ryutaro Asano and Prof. Izumi Kumagai (Tohoku University, Japan).<br />
References<br />
1. Cromwell ME, Hilario E, Jacobson F: Protein aggregation and<br />
bioprocessing. AAPS J 2006, 8:E572-579.<br />
2. Vázquez-Rey M, Lang DA: Aggregates in monoclonal antibody<br />
manufacturing processes. Biotechnol Bioeng 2011, 108:1494-1508.<br />
3. Asano R, Kawaguchi H, Watanabe Y, Nakanishi T, Umetsu M, Hayashi H,<br />
Katayose Y, Unno M, Kudo T, Kumagai I: Diabody-based recombinant<br />
formats of humanized IgG-like bispecific antibody with effective<br />
retargeting of lymphocytes to tumor cells. J Immunother 2008,<br />
31:752-761.<br />
4. Onitsuka M, Kim WD, Ozaki H, Kawaguchi A, Honda K, Kajiura H, Fujiyama K,<br />
Asano R, Kumagai I, Ohtake H, Omasa T: Enhancement of sialylation on<br />
humanized IgG-like bispecific antibody by overexpression of a2,6-<br />
sialyltransferase derived from Chinese hamster ovary cells. Appl Microbiol<br />
Biotechnol 2012, 94:69-80.<br />
P70<br />
Fucoidan extract enhances the anti-cancer activity of chemotherapeutic<br />
agents in breast cancer cells<br />
Sanetaka Shirahata 1,2* , Zhonguan Zhang 1 , Toshihiro Yoshida 1 , Hiroshi Eto 3 ,<br />
Kiichiro Teruya 1<br />
1 Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu<br />
University, Fukuoka 812-8581, Japan;<br />
2 Yosida Clinic, Osaka 532-0002, Japan;<br />
3 Daiichi Sangyo Co. Ltd., Osaka 530-0037, Japan<br />
E-mail: sirahata@grt.kyushu-u.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):P70<br />
Background: Fucoidan, a fucose-rich polysaccharide isolated from brown<br />
alga, is currently under investigation as a new anti-cancer compound<br />
[1-4]. In the present study, fucoidan extract (FE) from Cladosiphon navaecaledoniae<br />
Kylin was prepared by enzymatic digestion. We investigated<br />
whether a combination of FE with chemotherapeutic agents had the<br />
potential to improve the therapeutic efficacy of cancer treatment.<br />
Materials and methods: Estrogen receptor (ER)-positive MCF-7 and<br />
ER-negative MDA-MB-231 breast cancer cells were cultured in DME<br />
Figure 1(abstract P69) (A) Size-exclusion chromatography showing the elution profiles of Ex3-scDb-Fc purified with TRPA (red) and PA (blue).<br />
(B) Time-dependent formation of aggregates in CHO cell culture.
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medium supplemented with 10% fetal bovine serum in a humidified<br />
atmosphere of 5% CO 2 at 37 °C. The abalone glycosidase-digested<br />
fucoidan extract (FE) was obtained from Daiichi Sangyo Corporation (Osaka,<br />
Japan). The cells were treated with FE and chemotherapeutic agents like<br />
cisplatin, tamoxifen or paclitaxel. The cell growth was determined by MTT<br />
assay. Apoptosis was evaluated using annexin V binding assay and flow<br />
cytometry analysis. Signaling proteins were analyzed by western blot.<br />
Intracellular reactive oxygen species (ROS) were determined using DCFH-DA<br />
and determined using IN Cell Analyzer 1000. The reduced glutathione (GSH)<br />
concentration was measured by the GSH assay kit.<br />
Results: The co-treatments significantly induced cell growth inhibition,<br />
apoptosis, as well as cell cycle modifications in MDA-MB-231 and MCF-7<br />
cells. FE enhanced apoptosis in cancer cells that responded to treatment<br />
with cisplatin, tamoxifen, or paclitaxel after 48 h of treatment (Figure 1).<br />
FE enhanced the downregulation of the anti-apoptotic proteins Bcl-xL<br />
and Mcl-1 by these chemotherapeutic drugs. The combination treatments<br />
led to an obvious decrease in the phosphorylation of ERK and Akt in<br />
MDA-MB-231 cells, but increased the phosphorylation of ERK in MCF-7<br />
cells. In addition, we observed that combination treatments enhanced<br />
intracellular ROS levels and reduced glutathione (GSH) levels in breast<br />
cancer cells, suggesting that induction of oxidative stress was an<br />
important event in the cell death induced by the combination treatments.<br />
FE protected normal human fibroblast TIG-1 cells from apoptosis by<br />
cisplatin and tamoxifen, suggesting its favorable characteristic for<br />
application to cancer therapy.<br />
Conclusions: • Combination of FE and three chemotherapeutic agents<br />
exhibit highly synergistic inhibitory effects on the growth of breast<br />
cancer cells.<br />
• Combination treatments induced modifications in cell cycle<br />
distribution.<br />
Figure 1(abstract P70) Synergistic induction of apoptosis by cotreatmentAnalysis<br />
of apoptotic cells by annexin/PI double-staining<br />
using theIN Cell Analyzer 1000. MDA-MB-231 and MCF-7 cells were<br />
treatedfor different times with 200 μg/mL FE alone or 200 μg/mL FEin<br />
combination with 5 μM CDDP, 10 μM TAM or 2.5 nM TAXOL after 48 h<br />
of treatment. All results were obtained from three independent<br />
experiments. A significant difference from control is indicated by p <<br />
0.05 (#) or p < 0.01 (##); a significant difference from single treatments is<br />
indicated by p < 0.05 (*) or p < 0.01 (**).<br />
• Combination treatments modified the Bcl-2 expression, and ERK<br />
and Akt phosphorylation induced by FE, demonstrating different<br />
effects on apoptotic pathways in MDA-MB-231 cells and MCF-7 cells.<br />
• Generation of intracellular ROS and depletion of GSH are related to<br />
the cell death in combination treated -breast cancer cells.<br />
References<br />
1. Ye J, Li Y, Teruya K, Katakura Y, Ichikawa A, Eto H, Hosoi M, Hosoi M,<br />
Nishimoto S, Shirahata S: Enzyme-digested fucoidan extracts derived from<br />
seaweed Mozuku of Cladosiphon novae-caledoniae kylin inhibit invasion<br />
and angiogenesis of tumor cells. Cytotechnology 2005, 47:117-126.<br />
2. Zhang Z, Teruya K, Eto H, Shirahata S: Fucoidan extract induces apoptosis in<br />
MCF-7 Cells via a mechanism involving the ROS-dependent JNK activation<br />
and mitochondria-mediated pathways. PLoS ONE 2012, 6:e27441.<br />
3. Zhang Z, Teruya K, Eto H, Shirahata S: Induction of apoptosis by lowmolecular<br />
weight fucoidan through calcium- and caspase-dependent<br />
mitochondrial pathways in MDA-MB-231 breast cancer cells. Biosci<br />
Biotechnol Biochem 2012, 77:235-242.<br />
4. Zhang Z, Teruya K, Yoshida T, Eto H, Shirahata S: Fucoidan extract<br />
enhances the anti-cancer activity of chemotherapeutic agents in MDA-<br />
MB-231 and MCF-7 breast cancer cells. Marine Drugs 2013, 11:81-98.<br />
P71<br />
Assessment of troglitazone induced liver toxicity in a dynamic<strong>all</strong>y<br />
perfused two-organ Micro-Bioreactor system<br />
Eva-Maria Materne 1 , Caroline Frädrich 1 , Reyk Horland 1 , Silke Hoffmann 1 ,<br />
Sven Brincker 1 , Alexandra Lorenz 1 , Mathias Busek 2 , Frank Sonntag 2 ,<br />
Udo Klotzbach 2 , Roland Lauster 1 , Uwe Marx 1 , Ilka Wagner 1*<br />
1 TU Berlin, Institute for Biotechnology, Faculty of Process Science and<br />
Engineering, Gustav-Meyer-Allee 25, 13355 Berlin, Germany;<br />
2 Fraunhofer IWS<br />
Dresden, Winterbergstraße 28, 01277 Dresden, Germany<br />
E-mail: ilka.wagner@tu-berlin.de<br />
BMC Proceedings 2013, 7(Suppl 6):P71<br />
Background: The ever-growing amount of new substances released to the<br />
market and the limited predictability of current in vitro test systems has led<br />
to an ample need for new substance testing solutions. Many drugs like<br />
troglitazone, that had to be removed from the market due to drug<br />
induced liver injury, show their toxic potential only after chronic long term<br />
exposure. But for long-term multiple dosing experiments, a controlled<br />
microenvironment is pivotal, as even minor alterations in extracellular<br />
conditions may greatly influence the cell physiology. Within our research<br />
program, we focused on the generation of a micro-engineered bioreactor,<br />
which can be dynamic<strong>all</strong>y perfused by an on-chip pump and combines at<br />
least two culture spaces for multi-organ applications. This circulatory<br />
systems better mimics the in vivo conditions of primary cell cultures and<br />
assures steadier, more quantifiable extracellular signaling to the cells.<br />
Materials and methods: Liver microtissues (aggregates of HepaRG+human<br />
hepatic stellate cells) and skin biopsies were cultured in separate inserts of<br />
a 96-well Transwell® unit (Corning), which were hung inside the chip with<br />
the membrane fitting directly over the circuit. The tissues were cultivated<br />
either air/liquid interfaced (skin) or submerged in media (liver equivalent) for<br />
a culture period of 28 days. Exposing the tissues to troglitazone, the cultures<br />
were cultured for one day in normal medium and were, subsequently,<br />
exposed to 0 μM, 5 μM and50μM troglitazone, respectively for further<br />
6 days. Application of troglitazone was repeated at 12 h intervals<br />
simultaneously with the medium change. In a further experiment co-cultures<br />
of liver and skin equivalents were cultured in a fully vascularized chip.<br />
Therefore, HDMECs isolated from human foreskin were seeded into the<br />
microfluidic channel system using a syringe. After even cell infusion inside<br />
thecircuitthedevicewasincubatedin5%CO 2 at 37°C under static<br />
conditions for 3 h to <strong>all</strong>ow the cells to attach to the channel w<strong>all</strong>s.<br />
A frequency of 0.476 Hz was applied for continuous dynamic operation, after<br />
10 days of monoculture, skin and liver tissue were added for co-cultivation<br />
for another 15 days.<br />
Results: Co-cultures of human artificial liver microtissues and skin biopsies<br />
have successfully proven the long-term performance of the novel<br />
microfluidic multi-organ-chip device. The metabolic activity of the co-culture<br />
analysed in media supernatants reached a steady state at day 7 of<br />
co-culture and stayed constant for the rest of the culture period (Figure 1A).<br />
Furthermore, the co-cultures revealed a dose-dependent response to a<br />
6-day exposure to the toxic substance troglitazone. Liver microtissues
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Figure 1(abstract P71) Multi-tissue culture in the MOC device. (A) Liver and skin tissue performance over 28-day MOC co-culture. Metabolic activity of the<br />
co-culture analysed in media supernatants. (B) LDH values (C) Real-time qPCR of the cytochrome P450 3A4. Statistical analysis was performed by one-way<br />
analysis of variance (ANOVA), followed by post-hoc Dunnett’s pairwise multiple comparison test. * P < 0.05 versus control. Data are means ± SEM (n = 4).<br />
showed sensitivity at different molecular levels. LDH levels measured in the<br />
media supernatants increased significantly with increasing troglitazone<br />
concentration (Figure 1B). Furthermore, an induction of Cyp450 3A4 levels<br />
on RNA level were observed (Figure 1C). In addition, a robust procedure<br />
applying pulsatile shear stress has been established to cover <strong>all</strong> fluid contact<br />
surfaces of the system with a functional, tightly closed layer of HDMECs and<br />
co-cultivation of liver, skin and endothelial cells for 15 days was successful.<br />
Conclusion: A unique chip-based tissue culture platform has been<br />
developed enabling the testing of drugs or chemicals on a set of miniaturized<br />
human organs. This “human-on-a-chip” platform is designed to generate high<br />
quality in vitro data predictive of substance safety in humans. Tissue<br />
co-cultures can be exposed to pharmaceutical substances at regimens<br />
relevant to respective guidelines, currently used for subsystemic substance<br />
testing in animals.<br />
Acknowledgements: The work has been funded by the German Federal<br />
Ministry for Education and Research, GO-Bio Grand No. 0315569.<br />
P72<br />
Dynamic culture of human liver equivalents inside a micro-bioreactor<br />
for long-term substance testing<br />
Eva-Maria Materne 1* , Ilka Wagner 1 , Caroline Frädrich 1 , Ute Süßbier 1 ,<br />
Reyk Horland 1 , Silke Hoffmann 1 , Sven Brincker 1 , Alexandra Lorenz 1 ,<br />
Matthias Gruchow 2 , Frank Sonntag 2 , Udo Klotzbach 2 , Roland Lauster 1 ,<br />
Uwe Marx 1,3<br />
1 TU Berlin, Institute for Biotechnology, Faculty of Process Science and<br />
Engineering, Gustav-Meyer-Allee 25, 13355 Berlin, Germany;<br />
2 Fraunhofer IWS<br />
Dresden, Winterbergstraße 28, 01277 Dresden, Germany;<br />
3 TissUse GmbH,<br />
Markgrafenstraße 18, 15528 Spreenhagen, Germany<br />
E-mail: eva-maria.materne@tu-berlin.de<br />
BMC Proceedings 2013, 7(Suppl 6):P72<br />
Background: Current in vitro and animal tests for drug development are<br />
failing to emulate the organ complexity of the human body and, therefore, to<br />
accurately predict drug toxicity. In this study, we present a self-contained,<br />
bioreactor based human in vitro tissue culture test system aiming to support<br />
predictive substance testing at relevant throughput. We designed a<br />
microcirculation system interconnecting several tissue culture spaces within a<br />
PDMS-embedded microfluidic channel circuit. The bioreactor is reproducibly<br />
perfused by a peristaltic on-chip micro-pump, providing a near physiologic<br />
fluid flow and volume to liquid ratio.<br />
Materials and methods: Liver microtissue aggregates containing 4.8 × 10 4<br />
HepaRG cells and 0.2 × 10 4 human hepatic stellate cells (HHSteC) were<br />
formed in Perfecta3D® 384-Well Hanging Drop Plates (3D Biomatrix, USA).<br />
After two days of hanging drop culture, 20 aggregates were loaded into a<br />
single tissue culture compartment of the micro-bioreactor. Each circuit of<br />
the micro-bioreactor device contained 700 μl medium in total. During the<br />
first 7 days, a 40% media exchange rate was applied at 12 h intervals. From<br />
day 8 onwards, a 40% exchange rate was applied at 24 h intervals. Daily<br />
samples were collected for respective analyses. Experiments were stopped<br />
at day 14 and 28 and tissues were subjected to immunohistochemical<br />
stainings and qRT-PCR analyses. Experiments were conducted with four<br />
replicates. To expose the chip-cultures to troglitazone, liver microtissues<br />
were cultured for one day in normal medium and were, subsequently,
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Figure 1(abstract P72) 14-day tissue performance of the micro-bioreactor culture compared to static control Cell functionality shown by<br />
immunostaining of (A) phase I enzyme CYP450 3A4 (red) and CYP450 7A1 (green), (B) collagen I (red) and vimentin (green), (C) MRP2, an ABC<br />
transporter located at the apical membrane, (green) and (D) tight junction protein ZO-1 (red). Cell viability shown by TUNEL KI67 staining of<br />
(E) liver equivalents cultivated for 28 days in the micro-bioreactor and (F) liver equivalents cultivated for 28 days under static conditions. Nuclei are<br />
stained with hoechst 33342. Scale bar: 100 μm.<br />
treated with 0 μM, 5 μM and 50 μM substance, respectively. Application of<br />
troglitazone was repeated at 12 h or 24 h intervals simultaneously with the<br />
medium change.<br />
Results: Cultures of human artificial liver microtissues have successfully<br />
been cultivated over 28 days in the novel microfluidic bioreactor. Glucose<br />
consumption and lactate production indicated an aerobic metabolism which<br />
reached a steady state after 7 days. Immunohistochemical staining revealed<br />
the expression of phase I metabolic enzymes CYP450 3A4 and CYP450 7A1,<br />
extracellular matrix component collagen I, apical transporter MRP2 and tight<br />
junction protein ZO-1 (Figure 1A-D). Cell viability over 28 days was increased<br />
in the bioreactor culture compared to static control (Figure 1E, F).<br />
Furthermore, the cultures revealed a dose-dependent response to a 7-day<br />
exposure to the toxic substance troglitazone. Liver microtissues showed<br />
sensitivity at different molecular levels. Concentration of LDH released to the<br />
medium increased with troglitazone concentration and gene expression of<br />
selected marker genes varied. An induction of CYP450 3A4 by troglitazone<br />
treatment was also recorded on protein level by immunhistochemistry.<br />
Conclusion: A promising tool for long term culture of human liver<br />
equivalents has been developed. The simple MOC design presented,<br />
assisted the culture of human liver equivalents over a period of up to<br />
28 days. The cultures, operated at a total on-chip volume of 700 μl medium<br />
at recirculation rates of 40 μl/minassistedbyanon-chipmicropump,<br />
stabilize approximately within a week at a metabolic steady state. The<br />
prediction of toxicology profiles of compounds metabolised by the liver was<br />
demonstrated possible by exposing the cells to different concentrations of<br />
troglitazone. This platform is designed to generate high-quality in vitro data<br />
predictive of substance safety in humans. Tissue cultures can be exposed to<br />
pharmaceutical substances at regimens relevant to respective guidelines,<br />
currently used for subsystemic substance testing in animals.<br />
Acknowledgements: The work has been funded by the German Federal<br />
Ministry for Education and Research, GO-Bio Grand No. 0315569.<br />
P73<br />
Evaluation of the advanced micro-scale bioreactor (ambr) asa<br />
highthroughput tool for cell culture process development<br />
Frédéric Delouvroy * , Guillaume Le Reverend, Boris Fessler, Gregory Mathy,<br />
Mareike Harmsen, Nadine Kochanowski, Laetitia Malphettes<br />
Cell Culture Process Sciences, Biotech Sciences, UCB Pharma S.A., Chemin du<br />
Foriest, Braine l’Alleud, Belgium<br />
E-mail: frederic.delouvroy@ucb.com<br />
BMC Proceedings 2013, 7(Suppl 6):P73<br />
Introduction: Bio-pharmaceutical industries face an increasing demand to<br />
accelerate process development and reduce costs. This ch<strong>all</strong>enge requires<br />
high throughput tools to replace the traditional combination of shake<br />
flasks and sm<strong>all</strong>-scale stirred tank bioreactors. A conventional and widely<br />
used process development tool is the stirred tank reactor (STR) ranging<br />
from approximately 1L to 10L in working volume. Physical culture<br />
parameters such as pH, temperature and pO 2 can be easily controlled in<br />
such systems.<br />
However preparation and operation of these systems are time and resource<br />
consuming. The ambr system from TAP Biosystems has the capabilities for<br />
automated sampling, feed addition, and control for pH, dissolved oxygen,<br />
gassing, agitation, and temperature.<br />
Here, through the evaluation of parameters including cell growth,<br />
viability, metabolite concentration and production titer during a fed-batch<br />
process using CHO cells producing a recombinant mAb, we assessed the<br />
reproducibility of the ambr system for standard conditions compared to<br />
2L stirred tank bioreactors and the effects of parameter ranging between<br />
both culture systems, namely feed rate and pH ranging.<br />
Material and methods: A CHO cell line expressing a recombinant<br />
monoclonal antibody was used. Cells were carried out for 14 days in a fedbatch<br />
mode in a chemic<strong>all</strong>y defined medium and fed according to process<br />
description.<br />
Culture systems: ambr48 is an automated system with 48 disposable<br />
microbioreactor vessels. Results of ambr 48 workstation (TAP Biosystems)<br />
were compared to the results obtained with 2L stirred tank bioreactors<br />
with Biostat B-DCUII control systems (Sartorius Stedim).<br />
Commerci<strong>all</strong>y available production media and feeds were used as per<br />
manufacturer’s recommendations. pH (7.0 +/- 0.2 for standard conditions).<br />
All fed-batch cultures lasted 14 days.<br />
For the scale down model, parameters were divided in two groups. 1. The<br />
scale dependent factors: culture start volume, feed volumes that are<br />
linearly dependent and agitation speed and gazing that are theoretic<strong>all</strong>y<br />
or by experiences determined. 2. The scale independent factors: Media,<br />
temperature, seeding densities, pH, dissolved O 2 , culture duration.<br />
Product quality of the monoclonal antibody produced was analyzed as<br />
follows: Cell culture fluid samples were centrifuged and filtered to remove<br />
cell debris. The monoclonal antibody was purified by ÄKTA-express (GE<br />
Healthcare) Protein-A purification. The neutralized eluate was used for<br />
product quality analysis.<br />
Sample analysis: Viable Cell Concentration (VCC) and cell viability were<br />
measured using a ViCell XR cell counter (Beckman Coulter). Metabolite<br />
concentrations were measured by enzymatic assay using a UV-method
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Table 1(abstract P73) Design of the experiment<br />
pH set point Feed rate Number of replicates in ambr run Number of replicates in 2L bioreactor run<br />
7.0 -30% 2 0<br />
7.0 -20% 2 1<br />
7.0 -10% 2 1<br />
7.0 Control feed rate 6 1<br />
7.0 +10% 2 1<br />
7.0 +20% 2 1<br />
7 +30% 2 0<br />
6.9 Control feed rate 2 1<br />
7.1 Control feed rate 2 1<br />
(R-Biopharm) for the ambr vessels and by a BioProfile Analyzer 400<br />
(Nova Biomedical) for stirred tank bioreactors. For both systems, pH<br />
measurement was obtained with a BioProfile pHOx pH/Gas Analyzer (Nova<br />
Biomedical), Osmolality was obtained using a Omometer (Advanced<br />
Instruments). Production titers were measured throughout the culture<br />
using an Octet QK (ForteBio) and after 14 days with protein A HPLC<br />
(Agilent) after purification.<br />
Design of experiment: A 3x7-factorial design was implemented using JMP<br />
software (SAS). Parameter ranging included pH (6.9, 7.0, and 7.1) and feed<br />
rate addition (±30%, ±20% and ±10% compared to standard conditions)<br />
see Table 1.<br />
Results and discussion: The ambr run was performed in par<strong>all</strong>el to a 2L<br />
bioreactor run. Both experiments were inoculated with the same pool of<br />
cells, same batches of media and feeds were used in both systems.<br />
Different pH setpoints and feed rates were assessed to determine the<br />
impact on cell growth (see Table 1), viability and mAb titers. Each<br />
condition was tested in duplicates in the ambr minibioreactors and<br />
singlet in 2L bioreactors. The design of experiment is described in Table 1.<br />
The aim of this experiment was to test the reproducibility within ambr<br />
and the comparability between the minibioreactors and the 2L.<br />
Cell growth and cell viability were monitored daily throughout the cultures<br />
in2L(controlruns,n=4).Intheambr system, cell density and viability<br />
were measured every two days to avoid excessive sampling on control runs<br />
(n = 6). Cell viabilities were maintained at acceptable values (>80%)<br />
throughout the cultures in the established culture conditions.(Figure 1). Cell<br />
growth and viability performances observed in the ambr minibioreactors<br />
and 2L bioreactors were comparable (Figure 1). Final mAb titer obtained<br />
using ambr showed slightly (15%) lower concentration than the 2L<br />
bioreactors. Osmolality profiles showed the same trend in 15 mL and 2L<br />
bioreactors (between and 300 mOsm/kg at the beginning and 420 mOsm/kg<br />
at the end of the run). Online pH profiles were also comparable in both<br />
ambr minibioreactors and in 2L bioreactors.<br />
The impact of different feed rates were assessed and compared between<br />
2L bioreactors stirred tank bioreactors and ambr minibioreactors.<br />
Obtained results show similar profiles of viable cell density, cell viability,<br />
pre-harvest Mab titer at day 14 and osmolality profiles with different feed<br />
rates.<br />
High feed rates and low feed rates impact cell growth profiles and<br />
osmolality profiles. The different feed rates applied do not show any<br />
significant impact on the final mAb titer. Profiles observed in 2L<br />
bioreactors and ambr are comparable in both systems, except viability<br />
at the end of the ambr run due to a lack of glucose.<br />
The impact of different pH setpoints on cell growth, viability, final mAb<br />
titer and osmolality didn’t showed significant impact on those parameters<br />
in both systems. mAb titer at day 14 was comparable in 2L stirred<br />
bioreactors than in the ambr system.<br />
Conclusions: Our evaluation of the ambr system showed there is good<br />
reproducibility within the 6 ambr controls. There is good comparability<br />
Figure 1(abstract P73) Viable cell concentration (VCC) and viability average comparison between ambr and 2L bioreactors (control runs)
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in terms of cell growth, product titer, pH, pO2 and osmolality profiles as<br />
well as PQA obtained between ambr and bioreactors despite the fact<br />
ambr used a bolus feeding regimen and the stirred tank bioreactors<br />
used a continuous feeding strategy. The impact of feed rate on cell<br />
growth and osmolality upon feed rate ranging was observed in both<br />
culturing systems, but has no impact on PQA. pH set point ranging did<br />
not have an impact on the measured output parameters in either scale.<br />
ambr provides a predictive and resource-efficient tool to do process<br />
development especi<strong>all</strong>y media testing, feeding strategy screening and cell<br />
culture production conditions.<br />
P74<br />
Optimized fermentation conditions for improved antibody yield in<br />
hybridoma cells<br />
Martina Stützle 1,2* , Alina Moll 1 , René Handrick 1 , Katharina Schindowski 1<br />
1 Institute of Applied Biotechnology, University of Applied Sciences Biberach,<br />
Biberach, 88400, Germany; 2 Medical Faculty, Ulm University, Ulm, 89081,<br />
Germany<br />
E-mail: martina.stuetzle@hochschule-bc.de<br />
BMC Proceedings 2013, 7(Suppl 6):P74<br />
Background: Tradition<strong>all</strong>y antibody producing cells like hybridoma cells<br />
sank into oblivion since other suspension cell lines have captured the<br />
biopharmaceutical production market. However, they are still of particular<br />
interest in academic and industrial diagnostic research. Hence, fast and<br />
sufficient antibody production is needed as proof of concept, for toxicology<br />
and in vivo studies. Although, hybridoma cultivation in fetal bovine serum<br />
(FBS) containing animal derived ingredients, like contaminating IgG, is<br />
undesirable and leads to difficulties in purification. When reducing the<br />
serum to a minimum other key components of the FBS have to be replaced.<br />
Therefore, human insulin-like growth factor (IGF) [1] and the surfactant<br />
Pluronic F68 were supplemented to improve over<strong>all</strong> cell performance and to<br />
reduce shear stress during shaking respectively employing Design of<br />
Experiment (DoE)[2]. Compared to the original basal medium an<br />
improvement in cell growth, viability and antibody titer was achieved. These<br />
optimized inoculum conditions were used for subsequent bioreactor<br />
fermentations. Furthermore, these conditions were used in order to test<br />
feeding strategies. For this purpose a fed-batch process with a double bolus<br />
feed was simulated in shake flasks with two different glucose feeding<br />
strategies - with and without Hyclone Cell Boost 6 (CB6). Fin<strong>all</strong>y, the result<br />
from shake flasks could be verified and improved antibody yield was<br />
achieved in a controlled 2L fed-batch process.<br />
Material and methods: DoE approach: DoE (Modde, Umetrics) was<br />
used to optimize the cultivation medium by varying the three factors,<br />
FBS (1-10%), IGF (10 - 100 μg/L) and Pluronic (0.2 - 1 g/L). The central<br />
composite face-centered design was applied to test 24 different medium<br />
compositions. Cells were cultivated with a seeding density of 2 × 10 5<br />
cells/mL for five days in these media in 40 mL working volume in 125 mL<br />
shaker flask. Cell concentration and viability was quantified every day<br />
using an image-based cell counter (Cedex XS, Roche) and were defined<br />
as response factors for DoE analysis (table 1). Cultures grown with<br />
optimized conditions were used as inoculum for subsequent bioreactor<br />
fermentations.<br />
Feeding strategy: Cells were seeded with 3 × 10 5 cells/mL in 35 mL<br />
working volume in 125 mL shake flasks in optimized medium (DMEM, 4.5 g/L<br />
glucose, 2 mM stable glutamine, 6% FBS, 100 μg/L IGF and 0.2 g/L Pluronic).<br />
The 1 st triplicate was cultivated without feeding as batch control. The 2 nd<br />
triplicate was fed with 20 mM glutamine and 20 g/L glucose. The 3 rd<br />
triplicate was fed with 14 g/L glucose in CB6 (Hyclone, Thermo Scientific)<br />
instead of usual glucose feeding in medium. Substrates and metabolites, cell<br />
concentration and antibody titer were measured with a chemical analyzer<br />
(Konelab, Thermo Scientific), an image-based cell counter (Cedex XS, Roche)<br />
and Protein A HPLC (Agilent), respectively.<br />
Fed-batch with and without Cell Boost 6: Both feeding strategies with<br />
and without CB6 were performed again in a 2L bioreactor. The incolumn<br />
density was 3 × 10 5 cells/mL. The main parameters were kept constant at<br />
1 mM glutamine and at 2 g/L glucose.<br />
Table 1(abstract P74) <strong>Central</strong> composite face-centered result<br />
Exp No FBS [%] Pluronic [g/L] IGF [ug/L) Viability [%] Viable cell concentration [cells/mL]<br />
1 -1 (1) -1 (0.2) -1 (10) 75.8 736000<br />
2 1 (10) -1 (0.2) -1 (10) 79.1 1.468e+006<br />
3 -1 (1) 1 (1) -1 (10) 66.6 575000<br />
4 1 (10) 1 (1) -1 (10) 82 1.401e+006<br />
5 -1 (1) -1 (0.2) 1 (100) 71.6 696000<br />
6 1 (10) -1 (0.2) 1 (100) 77.9 1.545e+006<br />
7 -1 (1) 1 (1) 1 (100) 59.3 554000<br />
8 1 (10) 1 (1) 1 (100) 78.7 1.319e+006<br />
9 -1 (1) 0 (0.6) 0 (55) 69.1 632000<br />
10 1 (10) 0 (0.6) 0 (55) 79.8 1.455e+006<br />
11 0 (5.5) -1 (0.2) 0 (55) 82.5 1.461e+006<br />
12 0 (5.5) 1 (1) 0 (55) 78.9 1.442e+006<br />
13 0 (5.5) 0 (0.6) -1 (10) 79.1 1.326e+006<br />
14 0 (5.5) 0 (0.6) 1 (100) 81.6 1.336e+006<br />
15 0 (5.5) 0 (0.6) 0 (55) 81.8 1.27e+006<br />
16 0 (5.5) 0 (0.6) 0 (55) 80.2 1.194e+006<br />
17 0 (5.5) 0 (0.6) 0 (55) 81.3 1.188e+006<br />
18 0 0.6 55 28.5 13300<br />
19 5.5 0 55 78.4 1.255e+006<br />
20 5.5 0.6 0 81.25 1.2685e+006<br />
21 5.5 0.2 100 83.7 1.533e+006<br />
22 10 0 0 83.6 1.28e+006<br />
23 6 0 0 81.9 1.305e+006<br />
24 1 0 0 57.2 464000
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Results and discussion: DoE approach: A simple DoE approach with the<br />
three factors FBS, IGF and Pluronic led to improved hybridoma cultivation<br />
conditions. In Table 1 viability and viable cell concentration are depicted<br />
from exponential phase for <strong>all</strong> 24 media on day 3. Additional controls were<br />
run to improve the model like zero values for each factor and various FBS<br />
concentrations. FBS could be reduced from 10% to 6% by adding 100 μg/L<br />
human insulin-like growth factor and 0.2 g/L Pluronic. Compared to the<br />
original base medium an improvement in cell growth and viability was<br />
achieved.<br />
Three concentration levels for each variable including a maximum (1) a<br />
minimum (-1) and a center point (0) were used. Values shown in parenthesis<br />
are concentrations. Exp no 15-17 shows the central points for the medium,<br />
which were repeated three times. Exp no 18-20 shows the zero controls for<br />
each factor. Exp no 21-24 are additional controls for FBS at different<br />
concentrations. The concentrations in the yellow and red box are not in<br />
brackets. Viability and viable cell concentration were determined as<br />
response factors and used for fitting and evaluating the model.<br />
Based on the DoE results, the optimized medium was compared to the<br />
original culture conditions with FBS (10%, 6% and 1%) subsequently in<br />
125 mL shake flasks in triplicates. Reduction of FBS without supplementation<br />
results in decreased viability and cell concentration. The optimized medium,<br />
compared to 10% FBS supplementation, showed a significant impact in<br />
viable cell concentration and antibody titer by 1.2 fold.<br />
Feeding strategy: After optimizing the inoculum conditions, a fed-batch<br />
process was simulated in 125 mL shake flask due to a daily bolus feed with<br />
glutamine and glucose. The batch control ended in the death phase at day 3,<br />
whereas the fed-batch feed led to 6 day cultivation time. The feeding<br />
strategy with CB6 revealed a slightly improved cell growth. This result could<br />
be tremendously improved in a controlled 2L bioreactor leading to<br />
elongated process time (6 to 12 days), an increased viable cell concentration<br />
(from 1.6 × 10 6 cells/mL to 6.4 × 10 6 cells/mL) and higher antibody titer<br />
(450 mg/L compared to initial 110 mg/L) (Figure 1).<br />
Fed-batch was started with optimized medium (DMEM supplemented with<br />
6% FBS, 100 μg/L IGF and 0.2 g/L Pluronic). Glutamine was hold constant at<br />
1 mM and glucose at 2 g/L. Substrates and metabolites, cell concentration<br />
and antibody titer were measured with a chemical analyzer (Konelab,<br />
Thermo Scientific), an image-based cell counter (Cedex XS, Roche) and<br />
Protein A HPLC (Agilent) respectively each day.<br />
Conclusion: This data presents DoE as a powerful and efficient time<br />
saving tool in process optimization as well as a novel feeding strategy for<br />
fed-batch hybridoma process for increased IgG production. By employing<br />
DoE, FBS could be decreased from 10% to 6% by 100 μg/L human IGF and<br />
0.2 g/L Pluronic F68. For entirely serum-free hybridoma culture further<br />
critical ingredients like transferrin and albumin have to be replaced.<br />
However, serum-free media leads to higher production costs and can<br />
result in antibody yield reduction. Optimized medium was successfully<br />
used for subsequent bioreactor processes starting with a better cell<br />
performance. Fed-batch feeding with Hyclone Cell Boost 6 was beneficial<br />
for cell growth and antibody production compared to the conventional<br />
feed with glucose in medium. Both the optimized medium as well as the<br />
Cell Boost 6 feeding strategy led to a prolonged process time and<br />
increased antibody titer in the fermentation process.<br />
References<br />
1. Morris A, Schmid J: Effects of Insulin and LongR3 on Serum-Free Chinese<br />
Hamster Ovary Cell Cultures Expressing Two Recombinant Proteins.<br />
Biotechnology progress 2000.<br />
2. Eriksson L, Johansson E, Kettaneh-Wold N, Wikström C, Wold S: Design of<br />
Experiments: Principles and Applications Umea: UMETRICS ACADAEMY, 3<br />
2008, 425.<br />
P75<br />
High performance CHO cell line development platform for<br />
enhanced production of recombinant proteins including<br />
difficult-to-express proteins<br />
Pierre-Alain Girod 1* , Valérie Le Fourn 1 , David Calabrese 1 , Alexandre Regamey 1 ,<br />
Deborah Ley 2 , Nicolas Mermod 2<br />
1 Selexis SA, Plan-Les-Ouates, Switzerland;<br />
2 University of Lausanne,<br />
Switzerland<br />
E-mail: pierre-alain.girod@selexis.com<br />
BMC Proceedings 2013, 7(Suppl 6):P75<br />
Background: In an effort to improve product yield of mammalian cell<br />
lines, we have previously demonstrated that our proprietary DNA<br />
elements, Selexis Genetic Elements (SGEs), increase the transcription of a<br />
given transgene, thus boosting the over<strong>all</strong> expression of a therapeutic<br />
protein drug in mammalian cells [1]. However, there are additional cellular<br />
bottlenecks, notably in the molecular machineries of the secretory<br />
pathways. Most importantly, mammalian cells have some limitations in<br />
their intrinsic capacity to manage high level of protein synthesis as well as<br />
folding recombinant proteins. These bottlenecks often lead to increased<br />
cellular stress and, therefore, low production rates.<br />
Material and Methods: Our specific approach involves CHO cell line<br />
engineering. We constructed CHO-M libraries based upon the CHO-M<br />
genome and transcriptome and using unique proprietary transposon<br />
vectors harboring SGE DNA elements to compensate for rate-limiting<br />
factors [2]. Each CHO-Mplus library displays a diversity of auxiliary<br />
proteins involved in secretory pathway machineries and cellular<br />
metabolism. Collectively, the libraries address a broad range of expression<br />
issues.<br />
Figure 1(abstract P74) Fed-batch process with double feed - glutamine in medium and glucose (F1: with CB6; F2: without CB6).
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Page 99 of 151<br />
Figure 1(abstract P75) The iterative application of the CHO-Mplus libraries enabled >10 fold increase in productivity of ScFv:Fc without<br />
changes in gene copy number or transcription level of gene of interest.<br />
Results: Figure 1 shows that our CHO-Mplus libraries enabled the<br />
selection of a clonal cell line expressing 12 fold more product by<br />
comparison to the unmodified host cell [3].<br />
Conclusions: Our results demonstrate that components of the secretory<br />
and processing pathways can be limiting, and that engineering of the<br />
metabolic pathway (’omic’ profiling) improves the secretion efficiency of<br />
therapeutic proteins from CHO cells.<br />
References<br />
1. Girod PA, Nguyen DQ, Calabrese D, Puttini S, Grandjean M, Martinet D,<br />
Regamey A, Saugy D, Beckmann JS, Bucher P, Mermod N: Genome-wide<br />
prediction of matrix attachment regions that increase gene expression<br />
in mammalian cells. Nature Methods 2007, 4:747-753, Epub 2007 Aug 5.<br />
2. Ley D, Harraghy N, Le Fourn V, Bire S, Girod PA, Regamey A, Rouleux-<br />
Bonnin F, Bigot Y, Mermod N: MAR Elements and Transposons for<br />
Improved Transgene Integration and Expression. PLoS One 2013, 8:<br />
e62784.<br />
3. Le Fourn V, Girod PA, Buceta M, Regamey A, Mermod N: CHO cell<br />
engineering to prevent polypeptide aggregation and improve<br />
therapeutic protein secretion. Metab Eng 2013 [http://www.ncbi.nlm.nih.<br />
gov/pubmed/23380542], Feb 1. pii: S1096-7176(13)00002-5. doi: 10.1016/j.<br />
ymben.2012.12.003. [Epub ahead of print].<br />
P76<br />
Enhancement mechanism of antioxidant enzyme gene expression by<br />
hydrogen molecules<br />
Tomoya Kinjo 1 , Takeki Hamasaki 2 , Hanxu Yan 1 , Hidekazu Nakanishi 1 ,<br />
Tomohiro Yamakawa 1 , Kiichiro Teruya 1,2 , Shigeru Kabayama 3 ,<br />
Sanetaka Shirahata 1,2*<br />
1 Graduate School of Systems Life Sciences, Kyushu University, Fukuoka 812-<br />
8581, Japan;<br />
2 Department of Bioscience and Biotechnology, Faculty of<br />
Agriculture, Kyushu University, Fukuoka 812-8581, Japan;<br />
3 Nihon Trim Co.<br />
Ltd., Osaka 531-0076, Japan<br />
E-mail: sirahata@grt.kyushu-u.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):P76<br />
Background: Redox regulation system protects our body from oxidative<br />
stress-injury and keeps redox homeostasis. The hydrogen molecules (H 2 )<br />
exist as stable gas in the ordinal temperature and atmosphere. Recent study<br />
reports H 2 improve ischemia-reperfusion injury, glaucoma, Parkinson’s<br />
disease and atherosclerosis of animal models. It is supposed from these<br />
improvement results that H 2 participate in reduction of the oxidation stress,<br />
however, the reaction mechanism has not been clarified thoroughly. We<br />
surmised that intracellular redox regulation system is activated by H 2<br />
thereupon antioxidative activity is generated. Thus, we tried to find the<br />
effect of H 2 on the Nrf2 pathway, one of the redox regulation systems.<br />
Materials and methods: HT1080 cells, a human fibrosarcoma cell line,<br />
were incubated in a gas incubator at an atmosphere of 75%N 2 /20%O 2 /5%<br />
CO 2 or 75%H 2 /20%O 2 /5%CO 2 for24h.Then,afterthecellsweretreated<br />
with H 2 O 2 or fixative solution for 30 min or 15 min, the intracellular H 2 O 2<br />
and Nrf2 were determined by In cell analyzer and Confocal laser microscop<br />
using a BES-H 2 O 2 or anti-Nrf2 antibody, respectively. Furthermore, after<br />
extraction of mRNA from the treated HT1080 cells, the gene expressions<br />
were examined by using Real-time PCR.<br />
Results: The quantity of intracellular H 2 O 2 increased by hydrogen<br />
peroxide treatment was significantly decreased by pretreatment of H 2 .H 2<br />
enhanced the expression of catalase, glultathione peroxidase, Cu/Znsuperoxide<br />
dismutase, Nrf2 genes and Nrf2 protein.<br />
Conclusions: It was suggested that H 2 induced the expression level of<br />
antioxidant enzyme genes like catalase and glutathione peroxidase by<br />
increasing the expression level of the Nrf2 protein and decreased the<br />
amount of intracellular H 2 O 2 induced by the H 2 O 2 treatment in HT1080 cells.<br />
P77<br />
Evaluation of the impact of matrix stiffness on encapsulated HepaRG<br />
spheroids<br />
Sofia P Rebelo 1,2 , Marta Estrada 1,2 , Rita Costa 1,2 , Christophe Chesné 3 ,<br />
Catarina Brito 1,2 , Paula M Alves 1,2*<br />
1 iBET, Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras,<br />
Portugal; 2 Instituto de Tecnologia Química e Biológica, Universidade Nova de<br />
Lisboa, 2780-157 Oeiras, Portugal; 3 Biopredic International, Rennes, France (C.C.,<br />
R.L., S.C.)<br />
E-mail: marques@itqb.unl.pt<br />
BMC Proceedings 2013, 7(Suppl 6):P77<br />
Background: The drug development process is widely hampered by the<br />
lack of human models that recapitulate liver functionality and efficiently<br />
predict toxicity of new chemical compounds. Moreover, liver failure is a<br />
global medical problem, with transplantation being the only effective<br />
treatment currently available. The bipotent liver progenitor cell line HepaRG<br />
can be differentiated into cholangiocyte and hepatocyte-like cells that<br />
express major functions of mature hepatocytes, representing a valuable tool<br />
to model hepatic function [1]. Current two-dimensional (2D) protocols for<br />
the differentiation into mature hepatocyte-like cells fail to recapitulate the<br />
complex cell-cell interactions, which are crucial for maintaining polarity and<br />
inherent mature hepatic functionality. Herein, we present a threedimensional<br />
(3D) strategy for the culture of HepaRG cells based on the<br />
encapsulation of aggregates. The effect of matrix stiffness on expansion and<br />
differentiation was evaluated through encapsulation with different<br />
concentrations of alginate (1.1% and 2%). Further characterization of the<br />
hepatic features will reveal the extent of the hepatic functionality of the<br />
generated spheroids.<br />
Materials and methods: HepaRG cells were routinely propagated in static<br />
conditions as previously described [2]. Briefly, culture medium Williams E<br />
was supplemented with 1% (v/v) Glutamax, 1% (v/v) pen/strep, 5 μ g/ml<br />
insulin and 50 μ M hydrocortisone hemissuccinate and 10% (v/v) FBS and<br />
cultures were maintained at 37 ° C, 5% CO 2 . Spinner vessels with b<strong>all</strong> impeller<br />
(Wheaton) were inoculated with inoculums ranging from 5 to 8 × 10 5 cell/mL
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Page 100 of 151<br />
and an agitation ranging from 35 to 45 rpm to attain the desired aggregation<br />
conditions. Aggregate size was determined by measuring Ferret’s diameter<br />
using the Image J software (NIH). After 3 days of aggregation, spheroids were<br />
encapsulated in 1.1% and 2% (w/v) of Ultra Pure MVG alginate (UP MVG<br />
NovaMatrix, Pronova Biomedical) in NaCl 0.9% (w/v) solution. Encapsulation<br />
was performed in an electrostatic<strong>all</strong>y driven microencapsulation unit VarV1<br />
(Nisco) and cultures were maintained for 14 days in stirred culture conditions.<br />
Viability was determined by the double stain viability test - alginate beads<br />
were collected from stirred cultures, incubated with fluorescein diacetate<br />
(10 μg/mL) and TO-PRO3 ® (1 μM)andobservedonafluorescence<br />
microscope (Leica DMI6000) - and by the Trypan blue exclusion method -<br />
alginate beads were dissociated with a solution of Sodium citrate 50 mM,<br />
Sodium chloride 104 mM and spheroids were dissociated by incubation with<br />
Trypsin0.05%-EDTA(Gibco)andcountedtrypanblueexclusiondye.For<br />
characterization of the cultures, encapsulated spheroids were fixed as<br />
previously described [3] and incubated with ph<strong>all</strong>oidin and prolong gold with<br />
DAPI and images were acquired in a confocal microscope (Andor spinning<br />
disk).<br />
Results: In 2D cultures, HepaRG cells proliferate until confluence is reached<br />
and the cell-cell interactions established associated with the spatial<br />
constriction are postulated to trigger the differentiation program and<br />
maintain the differentiated state [1,4]. Moreover, the mechanochemical<br />
environment has been previously shown to strongly influence the liverspecific<br />
functions [5]. Thus, it was hypothesized that the microenvironment<br />
created by encapsulation of spheroids with an inert biomaterial with<br />
different stiffness levels, would promote differential behavior of the<br />
spheroids, towards differentiation or proliferation. Alginate concentrations<br />
of 1.1 and 2% (w/v) were used, given the 10 fold difference in stiffness,<br />
measured by the elastic modulus [6]. Both viability and the growth profile<br />
were monitored throughout culture time.<br />
In both culture conditions, the viability was maintained above 85%,<br />
showing that the alginate concentration does not affect diffusion of<br />
nutrients or oxygen to supply effectively the cell spheroids (Figure 1 A).<br />
Moreover, it was observed that the growth profile was comparable for the<br />
two cultures, with growth arrest after aggregation and no proliferation<br />
occurring either in both alginate concentrations (Figure 1 B). This suggests<br />
that the differentiation program is triggered either in softer and stiffer<br />
microenvironments, being 1.1% alginate concentration sufficient to initiate<br />
the process.<br />
The structural organization of the cell spheroids in both stiffness environments<br />
was characterized by the arrangement of actin filaments, which is<br />
associated to the tight junctions in highly polarized epithelial cells. As<br />
showninFigure1C,thecellsaredisposedinahighlypolarizedmanner,<br />
without necrotic centres.<br />
Conclusions: In the current work, the encapsulation of liver spheroids<br />
with different stiffness conditions was evaluated as a strategy to culture<br />
HepaRG cells. It was observed that the encapsulation with different<br />
alginate concentrations is compatible with maintenance of highly viable<br />
cultures of liver spheroids, with growth arrest and cell polarization<br />
promoted by spatial constriction and the enhanced cell-cell interactions<br />
in 3D.<br />
Acknowledgements: This work was supported by PTDC/EBB-BIO/112786/<br />
2009 and SFRH/BD/70264/2010 FCT, Portugal.<br />
References<br />
1. Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F, Guguen-Guillouzo C: The<br />
human hepatoma HepaRG cells: a highly differentiated model for<br />
studies of liver metabolism and toxicity of xenobiotics. Chem Biol Interact<br />
2007, 168:66-73.<br />
2. Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, Guyomard C,<br />
Lucas J, Trepo C, Guguen-Guillouzo C: Infection of a human hepatoma<br />
cell line by hepatitis B virus. Proc Natl Acad Sci USA 2002, 99:15655-15660.<br />
3. Tostoes RM, Leite SB, Serra M, Jensen J, Bjorquist P, Carrondo MJ, Brito C,<br />
Alves PM: Human liver cell spheroids in extended perfusion bioreactor<br />
culture for repeated-dose drug testing. Hepatology 2012, 55:1227-1236.<br />
4. Cerec V, Glaise D, Garnier D, Morosan S, Turlin B, Drenou B, Gripon P,<br />
Kremsdorf D, Guguen-Guillouzo C, Corlu A: Transdifferentiation of<br />
hepatocyte-like cells from the human hepatoma HepaRG cell line<br />
through bipotent progenitor. Hepatology 2007, 45:957-967.<br />
5. Semler EJ, Ranucci CS, Moghe PV: Mechanochemical manipulation of<br />
hepatocyte aggregation can selectively induce or repress liver-specific<br />
function. Biotechnol Bioeng 2000, 69:359-369.<br />
6. Martinsen A, Skjak-Braek G, Smidsrod O: Alginate as immobilization<br />
material: I. Correlation between chemical and physical properties of<br />
alginate gel beads. Biotechnol Bioeng 1989, 33:79-89.<br />
P78<br />
Feeding strategy optimization in interaction with target seeding<br />
density of a fed-batch process for monoclonal antibody production<br />
Marie-Françoise Clincke 1* , Grégory Mathy 1 , Laura Gimenez 1 ,<br />
Guillaume Le Révérend 1 , Boris Fessler 1 , Jimmy Stofferis 1 , Bassem Ben Yahia 1 ,<br />
Nicola Bonsu-Dartn<strong>all</strong> 2 , Laetitia Malphettes 1<br />
1 Cell Culture Process Sciences Group, BioTech Sciences, UCB Pharma S.A.,<br />
Braine L’Alleud, Belgium;<br />
2 In-Process Analytics Group, BioTech Sciences, UCB<br />
Celltech, Slough, UK<br />
E-mail: Marie-Francoise.Clincke@ucb.com<br />
BMC Proceedings 2013, 7(Suppl 6):P78<br />
Background: Current trend towards Quality by Design (QbD) leads the<br />
process development exercise towards systematic experimentation,<br />
rational development, process understanding, characterization and control.<br />
In this study, an example of the application of QbD approach is given.<br />
Optimization of the feeding strategy and the target seeding density was<br />
performed and interactions of the two parameters were assessed in order<br />
to enhance cell growth and MAb productivity. The feeding strategy was<br />
optimized to take into account daily process performance attributes and<br />
Figure 1(abstract P77) Characterization of encapsulated cultures of HepaRG spheroids (A) Viability assessed by staining the encapsulated spheroids<br />
with fluorescein diacetate (live, green) and TO-PRO3 ® (dead, red). Spheroids in 1.1 and 2% (w/v) of alginate after 14 days of culture are represented.<br />
Scale bar: 100 μm (B) Growth profile of encapsulated cultures of 1.1 and 2% (w/v) of alginate. (C) Immunofluorescence characterization of hepatic<br />
spheroids (1.1% alginate) after 14 days of culture. Actin filaments - green; Nuclei - blue. Scale bar: 10 μm.
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associated nutrient needs of the culture to maintain a balance between<br />
metabolism and MAb productivity. For scale up the feed strategy was<br />
simplified to become independent of daily process performance attributes.<br />
Feed ranging studies were performed to assess the robustness of the<br />
process.<br />
Materials and methods: 2L stirred tank bioreactors were run for 14 days<br />
in a fed-batch mode in a chemic<strong>all</strong>y defined medium. Feed was added<br />
daily from day 3 onwards. If required, antifoam C was added to the<br />
bioreactor by manual injections. DO, pH, and temperature were controlled<br />
at setpoint. DO was controlled using a multi-stage aeration cascade via a<br />
ring sparger. Viable cell concentration, cell viability, and average cell<br />
diameter were measured using a ViCell cell counter. The glucose, lactate,<br />
glutamine and ammonia concentrations were measured with a BioProfile<br />
Analyzer 400. On the day of harvest, the clarification was performed by<br />
centrifugation plus depth filtration. Monoclonal Antibody (MAb)<br />
concentration of the supernatant samples was quantified using Octet QK<br />
and Protein A high performance liquid chromatography.<br />
Results: Interaction study between feeding strategy and Target<br />
Seeding Density (TSD): Previous experiments performed with different<br />
daily fixed volume feed additions showed a correlation between feeding<br />
strategy and specific MAb productivity. It was observed that a significant<br />
decrease in the specific MAb productivity occurred if the feed ratio per the<br />
projection of a subset of process performance attributes was below a<br />
specific threshold (data not shown). A feed addition strategy based on the<br />
projected subset of process performance attributes was then developed.<br />
Based on previous screening study, feed ratio from 0.004 to 0.006 arbitrary<br />
units and and TSD from 0.30 to 0.40 arbitrary units were assessed. Custom<br />
DoE was performed with JMP SAS to study the interactions between both<br />
parameters. Number of interactions between the factors and the power of<br />
each factor were both fixed at 2. In total, 12 bioreactors were run. This<br />
Design of Experiment (DoE) was applied to the process development of a<br />
cell line 1 producing a monoclonal antibody and led to a 36% increase in<br />
the monoclonal antibody titer compared to control condition (Figure 1).<br />
The final feed ratio was based on (i) the improvement of MAb titer<br />
compared to the control condition, (ii) the scalability of the process<br />
(Culture start volume high enough to cover the impellers and low enough<br />
in order for Culture final volume to not exceed the maximum volume of<br />
the production bioreactor at large scale). TSD was fixed at 0.35 arbitrary<br />
units, so that a minimum dilution factor of 1:5 between the N-1 passage<br />
and the production bioreactor is achievable.<br />
Feeding strategy simplification, mode of feed addition, feeding<br />
ranging study: The design of the feeding strategy was simplified in order<br />
to facilitate the process transfer to large scale manufacture. Hence, based<br />
on the final feed ratio, the feed rates were fixed with a feed volume<br />
independent of the projected subset of process performance attributes.<br />
The pH of the feed is highly basic. In our 2L experiments, feed was added<br />
within less than 5 min, which generates pH excursions above 7.40.<br />
A strategy of slow bolus addition with a fixed minimum addition<br />
timeframe and with a fixed maximum flow rate was implemented, leading<br />
to minor pH-excursions during feeding with only minor CO 2 flows<br />
necessary to keep the pH within the pH deadband (data not shown). The<br />
robustness of the process was assessed by performing an experiment with<br />
over- and underfeeding cultures. Underfeeding at 20% below target had<br />
no impact on process performance (MAb titer) while feeding 20% above<br />
target led to a lower MAb titer (Table 1). No impact of underfeeding or<br />
overfeeding at ± 20% of the feed target was observed on the Acidic Peak<br />
Group (APG) and aggregate levels. Feeding 20% above target led to an<br />
increase in Mannose 5 species.<br />
Conclusions: DoE enabled us to study the impact of the feed addition<br />
strategy and the impact of the TSD on the Mab titer and PQAs at harvest<br />
in a time efficient manner. The feeding strategy was simplified to become<br />
independent of the projected subset of process performance attributes<br />
and to be scalable to large scale manufacture. The mode of feed addition<br />
was optimized to minimize pH-excursions during feeding. Feed ranging<br />
studies showed that underfeeding at 20% below target had no impact on<br />
MAbtiterandPQAswhilefeeding20%abovetargetledtoalowerMAb<br />
titer and an increase in Mannose 5 species (glycan). Fin<strong>all</strong>y, a 36% increase<br />
in the MAb titer was achieved in the feed optimized conditions compared<br />
to control condition at harvest with a feed strategy designed to be robust<br />
and scalable.<br />
P79<br />
Process development and optimization of fed-batch production<br />
processes for therapeutic proteins by CHO cells<br />
Marie-Françoise Clincke * , Mareike Harmsen, Laetitia Malphettes<br />
Cell Culture Process Sciences Group, BioTech Sciences, UCB Pharma S.A.,<br />
Braine L’Alleud, Belgium<br />
E-mail: Marie-Francoise.Clincke@ucb.com<br />
BMC Proceedings 2013, 7(Suppl 6):P79<br />
Figure 1(abstract P78) Impact of feed ratio and TSD on MAb titer at harvest day as well as one-way Anova study comparing the MAb titer at<br />
harvest (optimized process vs. baseline process in <strong>all</strong> runs)<br />
Table 1(abstract P78) MAb titers and Product Quality Attributes observed during the feed ranging study<br />
MAb titer (Normalized) APG (Normalized) Aggregate (Normalized) Mannose 5 (Normalized)<br />
Center point (n = 2) 1.00 1.00 1.00 1.00<br />
+20% Feed (n = 2) 0.54 0.97 0.95 1.78<br />
-20°% Feed (n = 2) 1.10 1.01 1.04 0.94
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Figure 1(abstract P79) Viable cell concentration and off-line pH, pCO 2 , osmolality, lactate and ammonia profiles during fed-batch culture (solid<br />
black line: cell line 2, process 1 strategy, short dash line: cell line 1, process 1, long dash line: cell line 2, process 2)<br />
Table 1(abstract P79) Comparison of MAb titers<br />
(normalized) obtained for both cell lines at 2L scale and<br />
80L scale<br />
Cell line 1, Process 1 Cell line 2, Process 2<br />
2L scale 1.00 1.00<br />
80L scale 0.99 1.09<br />
Background: In the biopharmaceutical industry, process development and<br />
optimization is key to produce high quality recombinant proteins at high<br />
yields. As technologies mature, pressure on cost and timelines becomes<br />
greater for delivering scalable and robust processes. Over<strong>all</strong>, process<br />
development should be viewed as a continuum from the early stages up to<br />
process validation. Here we outline a lean approach on upstream development<br />
during the initial phases to optimize yields while maintaining the desired<br />
product quality profiles. Early-stage process development was designed to
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lead to the establishment of a baseline process and to systematic<strong>all</strong>y include<br />
experiments with input parameters that have a high impact on performance<br />
and quality. At this stage, potential for pre-harvest titer and yield increases as<br />
well as product quality ch<strong>all</strong>enges were identified. Feed adjustments and<br />
systematic experiments with top, high, and medium impact parameters have<br />
then been performed to develop a robust and scalable process. This approach<br />
was applied to two early stage upstream processes.<br />
Materials and methods: 2L and 80L stirred tank bioreactors were run for<br />
14 days in a fed-batch mode in a chemic<strong>all</strong>y defined medium. Feed was<br />
added daily from day 3 onwards. If required, antifoam C was added to<br />
the bioreactor by manual injections. DO, pH, and temperature were<br />
controlled at setpoint. DO was controlled using a multi-stage aeration<br />
cascade via a ring sparger. Viable cell concentration, cell viability, and<br />
average cell diameter were measured using a ViCell cell counter. The<br />
glucose, lactate, glutamine and ammonia concentrations were measured<br />
with a BioProfile Analyzer 400. On the day of harvest, the clarification was<br />
performed by centrifugation plus depth filtration. Monoclonal Antibody<br />
(MAb) concentration of the supernatant samples was quantified using<br />
Protein A high performance liquid chromatography.<br />
Results: A lean and Quality by Design (QbD) approach on process<br />
development during the initial phases to optimize yields while maintaining<br />
the desired product quality profiles was adopted. In this approach, a<br />
workpackage including the expected high impact parameters (feeding<br />
strategy, seeding density, pH, temperature and the interaction studies) was<br />
defined. This workpackage was applied to the process development of a<br />
cell line 1 producing a monoclonal antibody and led to a 36% increase in<br />
the monoclonal antibody titer compared to control condition (data not<br />
shown). Then, the operational process parameters and feeding strategy<br />
developed for cell line 1 (process 1) were applied to a cell line 2 producing<br />
a monoclonal antibody fragment. The application of the process 1 strategy<br />
to a cell line 2 was not the best for cell line 2 and led to high pCO 2 level,<br />
high ammonia concentration, high osmolalities and low monoclonal<br />
antibody fragment titers (Figure 1). A feeding strategy was optimized for<br />
cell line 2 and pH set-point and deadband were also adjusted in order to<br />
decrease the pCO 2 level. This optimized process for cell line 2 led to higher<br />
performances (pCO 2 , ammonia concentration, and osmolalities values were<br />
maintained at a low level) with a 43% increase in the monoclonal antibody<br />
fragment titer (data not shown). Then both processes were scaled up to<br />
80L stirred tank bioreactors and comparable monoclonal antibody titers<br />
were obtained at 2L scale and 80L scale (Table 1). For the cell line 1,<br />
Product Quality Attributes such as Acidic Peak Group, aggregate and<br />
Mannose 5 were assessed and were maintained within the expected<br />
ranges with scale-up (data not shown).<br />
Conclusions: A similar process development approach was applied to<br />
both projects where identical high impact parameters were identified.<br />
Although process optimized for cell line 1 was not the best for cell line 2,<br />
we were able to use it as a starting point and were able to optimize within<br />
the tight timelines. For both projects, high titers were achieved following<br />
our lean approach on process development. The final process 1 optimized<br />
for a cell line 1 led to a 36% increase in monoclonal antibody titer. The<br />
final process 2 optimized for a cell line 2 led to a 43% increase in<br />
monoclonal antibody fragment titer. Comparable titers and product quality<br />
attributes were observed at 2L scale and 80L scale. Hence the adopted<br />
feeding strategy proved to be robust and scalable.<br />
P80<br />
Characterization of mAb aggregates in a mammalian cell culture<br />
production process<br />
Albert Paul * , Friedemann Hesse<br />
Institute of Applied Biotechnology, University of Applied Sciences Biberach,<br />
Biberach, 88400, Germany<br />
E-mail: paul@hochschule-bc.de<br />
BMC Proceedings 2013, 7(Suppl 6):P80<br />
Introduction: Protein aggregation is a major concern during monoclonal<br />
antibody (mAb) production [1,2]. The presence of aggregates can reduce the<br />
therapeutic efficacy of mAbs and trigger immunogenic responses upon<br />
administration [3]. Higher molecular weight (HMW) aggregates can be<br />
removed during downstream processing (DSP), but prevention of aggregate<br />
formation upstream could increase process yield [4,5]. Unfortunately,<br />
detection of aggregates upstream is ch<strong>all</strong>enging, since the size of aggregates<br />
ranges from sm<strong>all</strong> oligomers to visible particles and there is no single<br />
technique capable of measuring the broad range of aggregation phenomena<br />
[6,7]. For upstream detection of aggregates, <strong>all</strong> HMW species potenti<strong>all</strong>y<br />
present in the culture broth must be known. Therefore, we established<br />
methods to generate different types of aggregates and characterized the<br />
different HMW species using size exclusion high pressure liquid<br />
chromatography (SE-HPLC), dynamic light scattering (DLS) and UV<br />
spectroscopy. Furthermore, stability and traceability of the aggregates in cell<br />
culture medium and Chinese hamster ovary (CHO) DG44 supernatant were<br />
demonstrated. Fin<strong>all</strong>y, the established methods were used to monitor<br />
aggregate formation in a mAb producing CHO DG44 cell culture.<br />
Material and methods: Two mAbs produced in CHO DG44 cells and<br />
stored in 20 mM acetate at pH 3.5 were used for aggregation studies.<br />
Aggregation was induced using heat stress, pH-shift, high salt concentration<br />
and freeze-thawing. Heat stress was induced at 65 °C for different time<br />
periods. For the pH-shift, the antibody was diluted in citrate-phosphate<br />
buffer containing pH 3-8. NaCl concentrations for salt-induced aggregation<br />
varied from 50-1500 mM. A freeze-thawing cycle included incubation at<br />
-80 °C for 15 min followed by thawing at 25 °C for 15 min. The freeze-thaw<br />
cycle was repeated three times. The presence of sm<strong>all</strong> aggregates was<br />
evaluated using SE-HPLC equipped with a Yarra SEC4000 (Phenomenex)<br />
column. To identify the different HMW species the molecular weight was<br />
determined using SEC-MALS (multi-angle light scattering). Moreover, large<br />
aggregates were characterized using DLS (Zetasizer 3000HS, Malvern<br />
instruments) and UV spectroscopy (SpectraMax M5 e microplate reader,<br />
Molecular Devices). The size of large aggregates was displayed by<br />
the average diameter. The aggregation index (AI) was calculated from UV<br />
absorbance using the following equation: A 340 × 100/(A 280 -A 340 ).<br />
Furthermore, stability of induced aggregates in cell culture medium<br />
(SFM4CHO, Thermo Scientific) and CHO DG44 host cell supernatant was<br />
investigated. Therefore, freeze-thawed mAb2 was spiked into the culture<br />
medium as well as CHO DG44 host cell supernatant and analyzed via SE-<br />
HPLC. Fin<strong>all</strong>y, the supernatant of CHO DG44 mAb2 producer cells was<br />
analyzed directly after inoculation and at the end of cultivation. Based on<br />
results obtained from spiking aggregated mAb2 into CHO DG44 host cell<br />
supernatant, aggregate formation in a culture of a mAb producing CHO<br />
DG44 cell line was monitored.<br />
Results: All stress methods provoked aggregate formation. The mAbs<br />
showed formation of different aggregates using the different stress<br />
methods (Table 1). Heating the antibody only led to formation of large<br />
aggregates. Despite the loss of mAb2 monomer, no sm<strong>all</strong> aggregates were<br />
detected via SE-HPLC. However, heat induction provoked formation of<br />
large aggregates, whereby the average size (diameter > 1 μm) and AI<br />
increased over time at 65 °C. Hence, heat induction can only be used to<br />
generate large aggregates of the mAbs used in this study. The pH change<br />
provoked formation of sm<strong>all</strong> aggregates (dimer and oligomer) as well as<br />
large aggregates (diameter > 75 nm). With increasing pH dimer and<br />
oligomer levels also increased, whereas an increased diameter was only<br />
observed for pH 5 and 6. Thus, a shift to pH 6 can be used for induction of<br />
dimers, oligomers and large aggregates. The addition of NaCl provoked<br />
concentration-dependent formation of dimers and large aggregates<br />
(diameter>50nm)athigherNaClconcentrations(above500mM).In<br />
contrast to pH-induction, no oligomers larger than dimer were visible via<br />
SE-HPLC. Therefore, NaCl can be used for the fast generation of dimers and<br />
above a concentration of 500 mM for the induction of large aggregates.<br />
With increasing freeze thaw cycles formation of sm<strong>all</strong> aggregates occurred.<br />
Surprisingly, more aggregates were formed than with <strong>all</strong> other methods.<br />
Hence, freeze-thawing was used to study the stability of aggregates under<br />
culture conditions.<br />
Table 1(abstract P80) Formation of different HMW<br />
species using different induction methods<br />
Induction Method Sm<strong>all</strong> aggregates Large aggregates<br />
Dimer Oligomer<br />
Heat - - Up to 1 μm<br />
pH Increase with pH + At pH 5 and 6<br />
NaCl Increase with NaCl - Above 500 mM<br />
Freeze-thawing + + -
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Figure 1(abstract P80) Freeze-thawed mAb2 spiked into cell culture medium (A) and supernatant of CHO DG44 mAb producer cells (B).<br />
For this purpose, freeze-thawed mAb2 was spiked into culture medium<br />
and analyzed using SE-HPLC (Figure 1, A). Since the retention time of cell<br />
culture medium components differed from the freeze-thawed antibody,<br />
monomer and the aggregates were still detectable. Accordingly, freezethawing<br />
was preferred for the use in cell culture supernatant spiking<br />
experiments. The investigation of freeze-thawed (3x) mAb2 spiked into CHO<br />
DG44 host cell supernatant revealed that mAb2 monomers as well as the<br />
aggregates (22% dimer and 72% oligomer) were still detectable and<br />
quantifiable via SE-HPLC. Knowing the retention time of different aggregate<br />
species, the analysis of the supernatant of a CHO DG44 mAb producing cell<br />
line was performed at the beginning and after 144 h cultivation (Figure 1, B).<br />
Aggregates and monomer could successfully be detected after 144 h via<br />
SE-HPLC, whereas after inoculation neither monomer nor aggregates were<br />
visible. Therefore, the methods established in this work can be used to<br />
generate different types of aggregates as positive control to evaluate<br />
aggregate formation in cell culture supernatant.<br />
Summary: The stress methods used in this work induced different types of<br />
aggregates. Heating the antibody led to a loss of monomer and only<br />
formation of large aggregates. Dimers and oligomers were formed with<br />
increasing pH and large aggregates were formed at pH 5 and 6. A NaCl<br />
concentration dependent aggregate formation was observed, whereby<br />
only dimers were visible via SE-HPLC and large aggregates were only<br />
present at a NaCl concentration above 500 mM. Freeze-thawing induced<br />
more aggregates as with <strong>all</strong> other methods and was therefore used for the<br />
application under cell culture conditions. Spiking experiments of freezethawed<br />
mAb2 in culture medium and CHO DG44 host cell supernatant<br />
revealed that aggregates were still detectable and quantifiable under cell<br />
culture conditions. Fin<strong>all</strong>y, this work showed that aggregate formation<br />
directly in the supernatant of a CHO DG44 mAb producing cell line is<br />
possible.<br />
References<br />
1. Ishikawa T, Ito T, Endo R, Nakagawa K, Sawa E, Wakamatsu K: Influence of<br />
pH on heat-induced aggregation and degradation of therapeutic<br />
monoclonal antibodies. Biological & pharmaceutical bulletin 2010,<br />
33:1413-1417.<br />
2. Pease LF, Elliott JT, Tsai D-H, Zachariah MR, Tarlov MJ: Determination of<br />
protein aggregation with differential mobility analysis: application to IgG<br />
antibody. Biotechnology and bioengineering 2008, 101:1214-1222.<br />
3. Filipe V, Poole R, Oladunjoye O, Braeckmans K, Jiskoot W: Detection and<br />
characterization of subvisible aggregates of monoclonal IgG in serum.<br />
Pharmaceutical research 2012, 29:2202-12.<br />
4. Gomez N, Subramanian J, Ouyang J, Nguyen MDH, Hutchinson M,<br />
Sharma VK, Lin Aa, Yuk IH: Culture temperature modulates aggregation of<br />
recombinant antibody in cho cells. Biotechnology and bioengineering 2012,<br />
109:125-136.<br />
5. Jing Y, Borys M, Nayak S, Egan S, Qian Y, Pan S-H, Li ZJ: Identification of<br />
cell culture conditions to control protein aggregation of IgG fusion<br />
proteins expressed in Chinese hamster ovary cells. Process Biochemistry<br />
2012, 47:69-75.<br />
6. Philo JS: Is any measurement method optimal for <strong>all</strong> aggregate sizes and<br />
types? The AAPS journal 2006, 8:E564-E571.<br />
7. Arakawa T, Philo JS, Ejima D, Tsumoto K, Arisaka F: Aggregation Analysis of<br />
Therapeutic Proteins, Part 1: General Aspects and Techniques for<br />
Assessment. 2006, 4:42-43.<br />
P81<br />
Identification of process relevant miRNA in CHO cell lines - Process<br />
profiling reveals interesting targets for cell line engineering<br />
Fabian Stiefel 1* , Matthias Hackl 2 , Johannes Grilliari 2 , Friedemann Hesse 1<br />
1 Institute of Applied Biotechnology Biberach, Germany;<br />
2 University of Natural<br />
Resources and Life Sciences, Institute for Applied Microbiology, Vienna,<br />
Austria<br />
E-mail: stiefel@hochschule-bc.de<br />
BMC Proceedings 2013, 7(Suppl 6):P81<br />
Introduction: MicroRNAs (miRNAs) are sm<strong>all</strong> RNAs which function as<br />
regulators of posttranscriptional gene expression by binding to their mRNA<br />
targets [1]. MiRNAs are involved in crucial regulations of many signaling and<br />
metabolic pathways. In difference to other interfering RNAs (RNAi), miRNAs<br />
can target many mRNA, thus having an increased impact on regulation of<br />
gene expression. These properties of miRNAs makes them interesting and<br />
promising targets for biomarkers and cell line engineering [2,3]. Therefore,<br />
we studied miRNA profiles during different culture phases and process<br />
conditions and investigated the potential of differenti<strong>all</strong>y expressed miRNAs<br />
as targets for process optimization. This may help to pave the way to<br />
introduce a new layer of control for cell line engineering.<br />
Results: For miRNA target selection a strain from Chinese hamster ovary<br />
cells (CHO-DG44) was cultivated in a 2L bioreactor (Biostat B plus, Sartorius<br />
Stedim, Germany) in Batch mode and two different process conditions,<br />
control runs and temperature shift. For the control runs temperature was<br />
maintained at 37°C <strong>all</strong> time, while for the temperature shift the temperature<br />
was reduced to 30°C. Isolated RNA was analyzed using microarray<br />
technology (PowerScanner and HS 400 Pro Hybridisation station, Tecan,<br />
Germany and a cross-species chip containing miRNAs from human, mouse<br />
and rat, University of Graz) and the best differential expressed miRNA were<br />
cross-validated with qRT-PCR.<br />
The optimized bioreactor protocol, shown in Figure 1 A, each process<br />
condition included two independent biological replicates. The control<br />
runs show good growth behavior to a maximal viable cell concentration<br />
of 2.9 × 10 6 cells/ml. Reducing temperature from 37°C to 30 °C resulted<br />
in a clear inhibition of cell growth by sustained viability. From each time<br />
point of the different culture phases a sample was taken and total RNA<br />
was purified.<br />
Purified samples were labeled and then analyzed on a cross species<br />
miRNA microarray chip. Differential expressionwasalwayscalculated<br />
between the time points for the respective culture stage compared to<br />
day zero (shown in Table 1). The temperature shift from 37°C to 30°C
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Page 105 of 151<br />
Figure 1(abstract P81) A) Sample creation with final protocol of CHO-DG44 cells in 2L bioreactors. B) Summary of miRNA targets of microarray<br />
analysis for validation with qRT-PCR<br />
after 46 hours had a high impact on the miRNA profile with 22<br />
differenti<strong>all</strong>y expressed miRNAs for the early response from day three<br />
(D3-D0). In the late response (D5-D0) only three miRNAs and 18 miRNAs<br />
intheverylateresponse(D7-D0)weredifferenti<strong>all</strong>yexpressed.Two<br />
miRNAs were constantly expressed after shifting the temperature. In the<br />
control run at 37°C the number of differenti<strong>all</strong>y expressed miRNA was<br />
increasing during the course of the cell culture ranging from two miRNAs<br />
for the early to medium exponential phase, 12 miRNAs for the late<br />
exponential phase to 28 miRNA in the declining phase.<br />
To validate microarray normalization and results, differenti<strong>all</strong>y expressed<br />
miRNAs from the microarray analysis were cross-validated with qRT-PCR.<br />
This validation was conducted for respective miRNA of the time points<br />
before and after the temperature shift. Fold changes of mmu-miR-207<br />
(Log 2 FC of microarray was 2.0 and 2.9 for qRT-PCR) and mmu-471-5p<br />
207 (Log 2 FC of microarray was 4.4 and 5.0 for qRT-PCR) obtained from<br />
microarray and qRT-PCR technology were very comparable and showed<br />
same trends. This indicates that the microarray results can be used for a<br />
deeper analysis of the differenti<strong>all</strong>y expressed miRNAs.<br />
During a batch run, culture parameters are changing. In order to investigate<br />
the impact of these changes to miRNA profiles, time course of differential<br />
expression of miRNA during the different cell culture phases were analyzed.<br />
For the time courses of ten miRNAs in the temperature shift most of the<br />
miRNAs showed their highest differential expression shortly after the<br />
reduction of the temperature. Some miRNAs keep their level of differential<br />
expression, some return to normal levels three days after the temperature<br />
shift. One miRNA is differential expressed at the end of the observed culture<br />
phase. In the control run the number of differential expressed miRNAs and<br />
the fold change of the differential expression is increasing during<br />
progressing culture phase.<br />
Figure 1 B shows the differential expression of ten miRNA directly after the<br />
temperature shift and four miRNAs for the control runs between day zero<br />
and the declining phase at day seven. For the temperature shift differential<br />
expression ranges from log 2 FC 1.7 to 5.4 and 2.0 to 4.4 for the control<br />
Table 1(abstract P81) Summary of differenti<strong>all</strong>y<br />
expressed miRNAs in the control run and the<br />
temperature shift<br />
Amount of differenti<strong>all</strong>y expressed miRNA<br />
Control<br />
Temperature shift<br />
D2-D0 0 0<br />
D3-D0 2 22<br />
D5-D0 12 3<br />
D7-D0 28 18<br />
runs. This selection of miRNAs presented here may be interesting<br />
candidates for further investigation using miRNA overexpression/inhibition<br />
and phenotype studying.<br />
Conclusion: As a conclusion, with optimized bioreactor protocols it was<br />
possible to establish miRNA profiles of CHO-DG44 cells for different culture<br />
phases on cross species microarray chips. The number of differential<br />
expressed miRNAs was increasing by progressing of the culture phase.<br />
Addition<strong>all</strong>y, the impact of a temperature shift on the profiles revealed<br />
several highly differenti<strong>all</strong>y expressed miRNA. Some of these miRNAs were<br />
already cross-validated with qRT-PCR which confirmed the results from the<br />
microarray experiment. MiRNA targets of these two experimental<br />
approaches will help to increase the knowledge of the role of miRNAs<br />
during a bioreactor process and might pave the way for their use in cell line<br />
engineering.<br />
References<br />
1. Chen K, Rajewsky N: The evolution of gene regulation by transcription<br />
factors and microRNAs. Nature reviews Genetics 2007, 8:93-103.<br />
2. Barron N, Sanchez N, Kelly P, Clynes M: MicroRNAs: tiny targets for<br />
engineering CHO cell phenotypes? Biotechnology letters 2011, 33:11-21.<br />
3. Hackl M, Jadhav V, Jakobi T, Rupp O, Brinkrolf K, Goesmann A, Pühler A,<br />
Noll T, Borth N, Grillari J: Computational identification of microRNA gene<br />
loci and precursor microRNA sequences in CHO cell lines. J biotechnol<br />
2012, 158:151-155.<br />
P82<br />
Introducing a new chemic<strong>all</strong>y defined medium and feed for hybridoma<br />
cell lines<br />
Christoph Heinrich 1* , Tim F Beckmann 1 , Sandra Klausing 1 , Stefanie Maimann 2 ,<br />
Bernd Schröder 2 , Stefan Northoff 1<br />
1 TeutoCell AG, Bielefeld, 33613, Germany;<br />
2 Miltenyi Biotec GmbH, Teterow,<br />
17166, Germany<br />
E-mail: Christoph.Heinrich@teutocell.de<br />
BMC Proceedings 2013, 7(Suppl 6):P82<br />
Background: Hybridoma technology was established in the 2nd half of the<br />
20th century and in the view of current protein production it might seem<br />
old-fashioned. Despite, it is commonly used to produce monoclonal<br />
antibodies (mAbs) for R & D, clinical diagnostics or medical applications and<br />
the demand for mAbs produced by hybridomas is still high. However,<br />
compared to CHO, only a few serum-free hybridoma media are available<br />
and even less suppliers for chemic<strong>all</strong>y defined products are on the market.<br />
In this work, a new chemic<strong>all</strong>y defined medium and feed were developed to<br />
bring hybridoma processes to the next level and to target the existing gap<br />
in the market.<br />
Materials and methods: HybriMACS CD medium was developed using<br />
various research and production hybridoma cell lines from Bielefeld
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University (e.g. MF20, 187.1, HB8209) and industrial partners. HybriMACS<br />
CD medium was supplemented with 8 mM L-Glutamine for routine<br />
cultivation, batch and perfusion processes. For optimal performance of<br />
MF20 hybridoma cells (DSHB at the University of Iowa) the HybriMACS<br />
CD medium was supplemented with insulin (4 mg/L) or IGF (0.04 mg/L).<br />
All cultivations were carried out using standard conditions. Briefly,<br />
precultures and batch cultivations were performed in 125 mL and 250 mL<br />
Erlenmeyer flasks. Incubator conditions were set to 37 °C, 5% CO 2 and a<br />
relative humidity of 80%. For bioreactor cultivations closed-loop controlled<br />
2 L benchtop systems were used with parameters set to 37 °C, 40% DO and<br />
pH 7.1 +/- 0.05. Automated viable cell counting was performed using a<br />
Cedex (Innovatis). Monoclonal antibody (mAb) concentrations were<br />
determined with Protein A HPLC or ELISA (MF20 cell line).<br />
Results: Hybridoma cell growth in HybriMACS CD medium was compared<br />
to 12 competitor products in the time course of several passages and a<br />
final batch cultivation. For a mouse-mouse hybridoma cell line, maximum<br />
viable cell density (vcd) in HybriMACS CD was highest and for a ratmouse<br />
hybridoma cell line second-highest compared to growth in the 12<br />
competitor media, as shown in Figure 1 (A).<br />
Easy adaption from serum-containing medium was verified by direct<br />
thawing of five different hybridoma cell lines in HybriMACS CD (Figure 1 B).<br />
Furthermore, long-term stable growth of a hybridoma cell line in HybriMACS<br />
CD was also confirmed in cultivations for more than 80 days.<br />
The majority of tested cell lines reached a maximum cell density above<br />
2.5 to 5.0 × 10 6 cells/mL in uncontrolled and controlled batch processes<br />
using HybriMACS CD. For uncontrolled fed batch cultivations 1.0 × 10 7<br />
cells/mL were observed as maximum viable cell density, while controlled<br />
fed batch processes reached values above 1.5 × 10 7 cells/mL. The final<br />
antibody titer was increased at least by a factor of 5 in uncontrolled fed<br />
batches and up to 10 times in controlled fed batch cultivations using<br />
HybriMACS Feed Supplement. Exemplary results of controlled as well as<br />
uncontrolled batch and fed batch cultivations are shown in Table 1.<br />
Conclusions: HybriMACS CD is a chemic<strong>all</strong>y-defined, protein-free medium<br />
composition with no need for growth hormone supplementation. The<br />
speci<strong>all</strong>y designed formulation supports direct adaption of serumdependent<br />
hybridoma cells, even when starting from a serum-containing<br />
cell bank. In addition, the developed medium formulation enables stable<br />
long-term growth of hybridoma cell lines, supporting an unrestricted<br />
utilization in diverse processes. HybriMACS CD is suitable for bioreactor<br />
batch and perfusion processes reaching high cell densities and commonly<br />
accepted amounts of antibody. A speci<strong>all</strong>y tailored HybriMACS Feed<br />
Supplement increased final antibody titer at least by a factor of 5 to 10 for<br />
<strong>all</strong> tested hybridoma cell lines. This improvement can be further increased<br />
by customization of the generic feed regime, while maintaining suitable<br />
glucose and glutamine concentrations.<br />
P83<br />
Comparative study of bluetongue virus serotype 8 production on<br />
BHK-21 cells in a 50L Biostat® STR single-use bioreactors vs<br />
roller bottles<br />
Lídia Garcia * , Mercedes Mouriño, Alicia Urniza<br />
Zoetis Manufacturing & Research Spain, S.L Pfizer Olot S.L.U., Ctra.<br />
Camprodon s/n, La Riba, 17813 V<strong>all</strong> de Bianya (Girona), Spain<br />
E-mail: Lidia.garcia@zoetis.com<br />
BMC Proceedings 2013, 7(Suppl 6):P83<br />
Background: Bluetongue is a major disease of ruminant livestock that can<br />
have a substantial impact on income and animal welfare. Bluetongue virus<br />
serotype 8 (BTV-8) first emerged in the European Union in 2006, peaking at<br />
45,000 cases in 2008. Zoetis (formerly Pfizer Animal Health) licensed<br />
bluetongue vaccines (Zulvac 4 Ovis, Zulvac 1 Ovis, Zulvac 1 Bovis, Zulvac 8<br />
Ovis and Zulvac 8 Bovis and combinations) able to prevent viremia,<br />
stressing the role of the vaccine as an aid for the epidemiological control<br />
of the disease.<br />
One important issue to be taken into account in the development of<br />
vaccines is their cost, especi<strong>all</strong>y in veterinary use. Vaccine production<br />
requires high-yield, stable bioproduction systems and implementation of<br />
new technologies.<br />
Mammalian cells are the substrate for production of most of the veterinary<br />
vaccines. BHK-21 cells are commonly used to produce bluetongue<br />
vaccines.<br />
Figure 1(abstract P82) (A) Comparison of two different hybridoma cell lines in HybriMACS CD and 12 competitor media. (B) Growth behaviour<br />
of five serum-dependent hybridoma cell lines in HybriMACS CD directly after thawing.<br />
Table 1(abstract P82) Exemplary data of batch and fed batch cultivations under controlled (bioreactor) as well as<br />
uncontrolled (shaker) conditions<br />
Process<br />
Maximum vcd<br />
[10 6 cells/mL]<br />
IVCD<br />
[10 6 (cells*d)/mL]<br />
Shaker Batch 3.7 7.3 56.2<br />
Fed batch 10.5 39.5 447.5<br />
Bioreactor Batch 3.7 9.52 61.0<br />
Fed batch 17.3 107.6 1035.4<br />
Values represent mean from two biological replicates.<br />
Final mAb titer<br />
[mg/L]
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As an example, the use of the BTV-8 vaccine is routinely produced in roller<br />
bottles (RB). The aim of this study is to investigate Single-Use Bioreactor<br />
technology as an alternative to RB. This technology combines the basic<br />
concept of <strong>all</strong>owing the cells to attach to a surface (microcarriers) with the<br />
advantages of suspension, which <strong>all</strong>ows a better control of culture<br />
conditions and systematic and automatic culture process.<br />
Single use technology can also be an alternative to conventional production<br />
methods reducing facility complexity, possibility of the rapid expansion of<br />
the capacity of the production and to avoid the cleaning process and<br />
reduction of the risk of cross-contamination. Lower culture handling and<br />
more homogeneity can be achieved.<br />
Selection of appropriate culture conditions can be important to achieve<br />
consistent cell culture and virus production across sites and scales.<br />
Because characteristics like tank geometry and hardware (impellers,<br />
sparger) are not subject to change during scale-up, the scalability from<br />
50 L to 1000 L in the BIOSTAT® STR bioreactor can be an easy strategy for<br />
our production process.<br />
Materials and methods: Cell line: BHK-21. These cells were used<br />
because they are permissible to BTV replication. All cells were cultured at<br />
37°C in MEM-G medium supplemented with serum.<br />
Virus strain: BTV-8, strain BEL2006/02, supplied by “Veterinary and<br />
Agrochemical Research Centre” (VAR-CODA-CERVA), Ukkel, Belgium.<br />
Cultivation system: The growth of the BHK-21 cells and production of<br />
virus was performed in roller bottles and 50 L single-use bioreactor<br />
BIOSTAT® STR (Sartorius Stedim Biotech).<br />
BHK-21 cells were grown in microcarriers Cytodex-3 at 3g/L into the STR<br />
bioreactor and the cell production was optimized with respect to pH,<br />
temperature, stirring speed and aeration rate.<br />
Viable cell number was evaluated using the crystal violet dye nucleus<br />
staining method.<br />
Virus infection and titration<br />
The virus chosen to compare and prove the suitability of Single use<br />
technology for the production of viral vaccines was BTV-8.<br />
Confluent cells were infected at a constant MOI and harvesting was done<br />
at 100% CPE.<br />
Virus production was calculated according to the Spearman-Kärber method,<br />
expressing the result in tissue culture infectious doses (50%) (TCID 50 ).<br />
Cell growth and BTV-8 antigen production in the BIOSTAT® STR bioreactor<br />
was conducted at the optimal conditions determined previously on<br />
conventional bioreactors.<br />
Microcarriers elimination: Taking into account that for vaccine<br />
formulation microcarriers must be eliminated from the viral suspension,<br />
filtration through Sartopure PP2 cartridges (from Sartorius Stedim<br />
Biotech) was performed.<br />
Results: The final goal is to maximize productivity preserving its quality.<br />
How? By increasing cell concentration and cell productivity.<br />
To demonstrate the feasibility of bioreactors for microcarriers cell cultures,<br />
the growth of BHK-21 cells in roller bottles, and in the BIOSTAT® STR<br />
bioreactor was evaluated and compared.<br />
Results prove that when using the 50L BIOSTAT® STR bioreactor, BHK-21<br />
cells are attached and grow efficiently on microcarriers. Cell concentration<br />
yield in terms of average was higher than in roller bottles (Figure 1).<br />
The virus titers reached in the BIOSTAT® STR bioreactor were equal o<br />
higher than the levels obtained in roller bottles (Figure1).<br />
Conclusions: ▪ Comparable results between Roller bottles and 50 L<br />
BIOSTAT® STR bioreactor<br />
✓ cell density<br />
✓ productivity<br />
✓ product quality<br />
▪ BHK-21 cells grow efficiently on microcarriers. Conditions for cell<br />
attachment in terms of mixing conditions were optimized.<br />
▪ BTV-8 antigen with satisfactory yields can be obtained by culturing<br />
BHK-21 in a 50L BIOSTAT® STR bioreactor.<br />
▪ As expected, high density of BHK-21 cultures showed increased<br />
productivity<br />
▪ Microcarrier filtration causes no significant drop in virus titer.<br />
▪ With the conditions established with the 50 L BIOSTAT® STR<br />
bioreactor the reproducibility and the scale-up from 50 L to 1000 L<br />
can be easily performed.<br />
▪ Single-Use Bioreactor technology is a good alternative to Roller<br />
Bottles and is a suitable system for propagation of BTV-8 virus using<br />
adherent BHK cells on microcarriers. Involving reduction of costs,<br />
cleaning, sterilization etc.<br />
P84<br />
Golgi engineering of CHO cells by targeted integration of<br />
glycosyltransferases leads to the expression of novel Asn-linked<br />
oligosaccharide structures at secretory glycoproteins<br />
Tobias Reinl 1* , Nicolas Grammel 2 , Sebastian Kandzia 3 , Eckart Grabenhorst 1,2,3 ,<br />
Harald S Conradt 1,2,3<br />
1 Dept. Cell Engineering, Feodor-Lynen-Str. 35, 30625 Hannover, Germany;<br />
2 Dept. Mass Spectrometry, Feodor-Lynen-Str. 35, 30625 Hannover, Germany;<br />
3 Dept. Glycosylation Analysis GlycoThera GmbH, Feodor-Lynen-Str. 35, 30625<br />
Hannover, Germany<br />
E-mail: reinl@glycothera.de<br />
BMC Proceedings 2013, 7(Suppl 6):P84<br />
Background and novelty: N-glycans constitute an important information<br />
carrier in protein-driven signaling networks. Amongst others, N-glycans<br />
contribute to protein folding quality, adjust protein turnover and operate as<br />
address label for targeting proteins to specific cells and tissues [1]. Hence,<br />
the composition of N-glycans attached to recombinant glycoprotein<br />
therapeutics is vital for in-vivo therapeutic efficacy and strongly depends on<br />
the choice of the expression host [2,3]. Due to absence or silencing of<br />
glycosyltransferase genes homologue to human enzymes, biotechnologic<strong>all</strong>y<br />
used cell lines are limited by their intrinsic glycosylation machinery and<br />
produce host specific glycoforms.<br />
Cetuximab, a therapeutic chimeric mouse/human monoclonal antibody<br />
(IgG1), is N-glycosylated both at the CH2-domain (Asn299) and at the<br />
VH-domain (Asn88) (Figure 1A). Sold under the trade name Erbitux®,<br />
Cetuximab is expressed from a murine myeloma cell line and targets the<br />
human EGF receptor [4], which is overexpressed in about 1/3 of <strong>all</strong><br />
human cancers. The antibody is highly decorated with the aGal-epitope<br />
Figure 1(abstract P83) Comparison of cell growth and virus titer in roller bottles and in 50 liter BIOSTAT®CultiBagSTR single-use bioreactor
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Figure 1(abstract P84) (A) Non-reducing terminal oligosaccharide motifs attached to N-glycans of specific human glycoproteins (left side). Scheme of<br />
model glycoprotein Cetuximab with CH2- and VH-domain N-glycans (right side). (B) NP-HPLC-FLD elution profiles of 2-AB labeled oligosaccharides from<br />
VH-domain of Cetuximab after co-expression of the indicated glycosyltransferases.<br />
(Gala1-3Galb1-4GlcNAc) which has been shown to result in fatal <strong>all</strong>ergic/<br />
hypersensitivity response in several patients [5].<br />
The design of new quality-optimized and function<strong>all</strong>y improved biopharmaceuticals<br />
with properties conferred by host cell unrelated N-glycans<br />
requires a rational Golgi engineering strategy. Here, we apply GET, a system<br />
that enables the positioning of a desired catalytic glycosyltransferase<br />
activity into a favorable localization within the intracellular glycosylation<br />
machinery, to suspension CHO cells developed to secrete suitable amounts<br />
(200 μg/ml) of Cetuximab as a model glycoprotein. The presented Golgi<br />
engineering project aims in the extension of the intrinsic glycosylation<br />
repertoire enabling CHO cells to produce new human-type glycosylation<br />
motifs as indicated in Figure 1A: (i) GalNAcb1,4GlcNAc-R (LacdiNAc, LDN),(ii)<br />
GlcNAc in b1,4 linkage to central mannose residue (bisecting GlcNAc, bGN),<br />
(iii) Galb1,4(Fuca1,3)GlcNAc-R (Lewis X ,Le X )and(iv)NeuAca2,3Galb1,4<br />
(Fuca1,3)GlcNAc-R (Sialyl-LewisX, sLe X ). To assemble (ii) and (iv), we<br />
co-express GnT3 and FT7. As shown earlier, the latter enzyme catalyzes<br />
fucosylation exclusively of (iv). Therefore, we included in our study a variant<br />
of FT6 that is targeted to the early Golgi compartment with the aim to<br />
addition<strong>all</strong>y generate structure (iii) [6,7]. The uncommon LDN motif (i)<br />
which is e.g. detected on lutropin is assembled by human B4GalNT3 [8,9].<br />
We analyze oligosaccharides released from the products of genetic<strong>all</strong>y<br />
engineered CHO cells based on the resolution of single glycosylation sites<br />
of VH- and CH2- glycopeptides by quantitative NP-HPLC-FLD and use our<br />
comprehensive oligosaccharide standard library to identify novel<br />
oligosaccharide motifs.<br />
Experimental approach: Cloning of human glycosyltransferases and<br />
engineering of VAR FT6 [7] as well as construction of pGET expression<br />
plasmids encoding either the heavy and light chain of Cetuximab or the<br />
glycosyltransferase cDNAs was done acc. to standard DNA technologies.<br />
A stable clone with Cetuximab titers of 200 μg/ml and doubling times of
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25 hours was selected after transfection of pGET-Cetuximab in CHO cells.<br />
This clone was either mock- or co-transfected with pGET plasmids<br />
encoding the indicated glycosyltransferases. After shake flask<br />
subcultivation for 72 h Cetuximab was purified from supernatants,<br />
digested and applied to RP-HPLC peptide mapping. CH2- and VH-domain<br />
glycopeptides were separated and oligosaccharides were enzymatic<strong>all</strong>y<br />
released. After 2-AB labeling, the isolated oligosaccharides were subjected<br />
to quantitative NP-HPLC-FLD and ESI-TOF-MS and MS/MS analysis.<br />
Oligosaccharide structures were unambiguously identified in comparison<br />
to GlycoThera’s reference standard oligosaccharide library.<br />
Results and discussion: In combination with our site specific and<br />
quantitative micro glycan structure analysis we provide a modular system<br />
(GET) for the customized assembly of novel CHO unrelated oligosaccharide<br />
motifs. As exemplified for VH-domain, the NP-HPLC-FLD elution profiles of<br />
2-AB labeled oligosaccharides after heterologous co-expression of<br />
Cetuximab and the indicated glycosyltransferases are shown in Figure 1B.<br />
Quantitative results of <strong>all</strong> oligosaccharide structures are given in Figure 2.<br />
The Mock-transfected control approach reveals the intrinsic glycosylation<br />
repertoire of our stable CHO cell clone. Cetuximab is decorated with<br />
agalactosylated (35,5%), mono- (50,0%) and di-galactosylated (10,1%)<br />
diantennary complex-type N-glycans containing proximal a1,6-linked fucose<br />
at the CH2-domain. VH-domain N-glycans consist of neutral (13,8%), mono-<br />
(50,3%) and di-sialylated (35,8%) oligosaccharide structures. Whereas<br />
N-glycans from the market product Erbitux® produced in SP2/0 cells are<br />
extensively decorated with Gala1,3Gal and NeuGc (data not shown), those<br />
<strong>all</strong>ergenic structures are not detected in Cetuximab N-glycans from our CHO<br />
cell clone.<br />
The heterologous co-expression of wildtype B4GalNT3, GnT3 and FT7 and<br />
genetic<strong>all</strong>y modified FT6 results in the formation of the uncommon<br />
LacdiNAc motif (ca. 40%), the Lewis X and di-Lewis X structures (ca. 50%)<br />
and Sialyl-Lewis X (ca. 15%) almost exclusively on oligosaccharides from the<br />
VH-domain. Relevant modification of both VH-domain (ca. 40%) and CH2-<br />
domainglycans(ca.30%)isonlyachieved by GnT3 catalyzed attachment<br />
of bisecting GlcNAc. In addition, glycosyltransferase co-expression leads to<br />
charge state reduction of oligosaccharides by depletion of suitable<br />
acceptors for endogenous sialyltransferases. The strongest reduction in the<br />
content of neuraminic acid at VH-domain was observed by co-expression<br />
of VAR FT6 (ca. 55% reduction) and WT B4GalNT3 (ca. 50% reduction).<br />
Figure 2(abstract P84) Amount of oligosaccharide structures detected on CH2- and VH-domain of Cetuximab after heterologous glycosyltransferase<br />
co-expression (given in% peak area values after integration of NP-HPLC-FLD chromatograms)
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As a conclusion, Golgi engineering endows CHO cells to assemble<br />
significant amounts of LacdiNAc, bisecting GlcNAc, Lewis X and Sialyl-<br />
Lewis X to Cetuximab N-glycans (Figure 1B and Figure 2). Therefore, our<br />
glycosylation engineering strategy provides a tool to produce tailored<br />
N-glycosylation variants with defined structural motifs. As demonstrated,<br />
the tailored addition of bisecting GlcNAc to CH2-domain N-glycans<br />
increases ADCC of an aCD20 therapeutic mAB [10]. We therefore assume<br />
that the presented structural motifs exhibit novel therapeutic properties<br />
(ADCC, CDC, tissue specificity, serum half-life). Our strategy represents a<br />
relevant basis for the development of biotherapeutics and biobetters with<br />
potenti<strong>all</strong>y improved pharmacokinetics, pharmacodynamics, safety<br />
properties and in vivo therapeutic efficacy.<br />
References<br />
1. Varki A, Lowe JB: Biological Roles of Glycans. Essentials of Glycobiology<br />
Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press: Varki A,<br />
Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler<br />
ME, 2 2009, Chapter 6.<br />
2. Sinclair AM, Elliott S: Glycoengineering: the effect of glycosylation on the<br />
properties of therapeutic proteins. J Pharm Sci 2005, 94:1626-1635.<br />
3. Grabenhorst E, Schlenke P, Pohl S, Nimtz M, Conradt HS: Genetic<br />
engineering of recombinant glycoproteins and the glycosylation<br />
pathway in mammalian host cells. Glycoconj J 1999, 16:81-97.<br />
4. Erbitux® (Cetuximab): Prescribing Information. Bristol-Myers Squibb<br />
(1236886B3, Rev. March 2013).<br />
5. Commins SP, Platts-Mills TAE: Allergenicity of Carbohydrates and Their<br />
Role in Anaphylactic Events. Curr Allergy Asthma Rep 2010, 10:29-33.<br />
6. Grabenhorst E, Nimtz M, Costa J, Conradt HS: In Vivo Specificity of Human<br />
a1,3/4-Fucosyltransferases III-VII in the Biosynthesis of Lewis X and Sialyl<br />
Lewis X Motifs on Complex-type N-Glycans. J Biol Chem 1998,<br />
273:30985-30994.<br />
7. Grabenhorst E, Conradt HS: The cytoplasmic, transmembrane, and stem<br />
regions of glycosyltransferases specify their in vivo functional<br />
sublocalization and stability in the Golgi. J Biol Chem 1999,<br />
274:36107-36116.<br />
8. Sato T, Gotoh M, Kiyohara K, Kameyama A, Kubota T, Kikuchi N, Ishizuka Y,<br />
Iwasaki H, Togayachi A, Kudo T, Ohkura T, Nakanishi H, Narimatsu H:<br />
Molecular cloning and characterization of a novel human beta 1,4-Nacetylgalactosaminyltransferase,<br />
beta 4GalNAc-T3, responsible for the<br />
synthesis of N, N’-diacetyllactosediamine, galNAc beta 1-4GlcNAc. J Biol<br />
Chem 2003, 278:47534-47544.<br />
9. Fiete D, Srivastava V, Hindsgaul O, Baenziger JU: A hepatic<br />
reticuloendothelial cell receptor specific for SO4-4GalNAc beta<br />
1,4GlcNAc beta 1,2Man alpha that mediates rapid clearance of lutropin.<br />
Cell 1991, 67:1103-1110.<br />
10. Davies J, Jiang L, Pan LZ, LaBarre MJ, Anderson D, Reff M: Expression of<br />
GnTIII in a recombinant anti-CD20 CHO production cell line: Expression<br />
of antibodies with altered glycoforms leads to an increase in ADCC<br />
through higher affinity for FC gamma RIII. Biotechnol Bioeng 2001,<br />
74:288-294.<br />
P85<br />
Characterization of the influence of cultivation parameters on<br />
extracellular modifications of antibodies during fermentation<br />
Christian Hakemeyer * , Martin Pech, Gero Lipok, Alexander Herrmann<br />
Pharma Technical Development, Roche Diagnostics GmbH, Penzberg<br />
Germany<br />
E-mail: Christian.hakemeyer@roche.com<br />
BMC Proceedings 2013, 7(Suppl 6):P85<br />
Introduction: The production of protein-based medical agents, like<br />
monoclonal antibodies (Mabs), by biotechnological processes requires a<br />
comprehensive quality control. The pharmaceutical industry and national<br />
health authorities support the complete characterization of therapeutic<br />
proteins to increase the quality and safety. During numerous and<br />
different production steps like fermentation, purification and storage,<br />
various protein modifications on therapeutic products can occur, like<br />
deamidation of asparagine and glutamine, oxidation of methionine<br />
tryptophan residues, clipping of terminal amino acids, glycation and<br />
others.<br />
During the development of fermentation processes, good growth<br />
conditions for the cell culture are of primary importance to obtain<br />
maximal productivity [1]. Until now only few efforts have been made to<br />
investigate the development of extracellular antibody modifications and<br />
their sources during fermentation as the first phase of the productions<br />
process. Already known is the fact that pH-value and temperature can<br />
induce modifications on monoclonal antibodies [2].<br />
Aim of this work is to increase the knowledge about the development of<br />
extracellular modifications of monoclonal antibodies during the fermentation<br />
process. Therefore, parameters of fermentation were identified which<br />
influence modifications during cell-free incubation under common fermentation<br />
conditions (in shake flask and sm<strong>all</strong> scale bioreactor-systems).<br />
Results: The results from the shake flask experiments showed a different<br />
degree of changes of the charge isoform pattern (measured by IE-HPLC)<br />
for five analyzed antibodies during the approx. nine days of cell-free<br />
incubation. The respective increase of the amount of acidic regionwas<br />
strongly dependent on the specific protein. At the end of the incubation,<br />
the amount of the acidic region range from approx. 20 area-% to<br />
approx.75 area-% depending on the characteristics of the Mab. The<br />
increase in the acidic region correlated with a decrease of the main peak<br />
while the basic regionremained unchanged.<br />
The specific influence of the parameters pH, temperature and dissolved<br />
oxygen (DO) on the modification of antibodies was further characterized in<br />
full factorial DoE designed experiments for three Mabs. For this purpose,<br />
cell broth was taken at an early stage from standard 1.000 L fermentations<br />
with Chinese Hamster Ovary (CHO) cells and cells were removed by<br />
centrifugation. The cell-free supernatant was then transferred to sm<strong>all</strong><br />
scale bioreactors and incubated for approx. ten days under the conditions<br />
listed in table 1.<br />
In these experiments, elevated temperature conditions and higher pH<br />
values led to a faster modification (degradation) for <strong>all</strong> three investigated<br />
antibodies during the incubation compared to lower pH and temperature<br />
conditions, while dissolved oxygen level had no relevant impact on the<br />
kinetic of antibody degradation.<br />
The results of the cell-free incubation studies were used to develop a<br />
mathematical model was to predict the isoform pattern of the Mab during<br />
standard fermentations with CHO cells from inoculation to harvest. The<br />
amount of the acidic peak can be predicted, depending on the specific<br />
antibody characteristics as determined in the previous experiments, the<br />
concentration of the antibody during the cultivation, and the fermentation<br />
time and process conditions (pH, DO, temperature). Figure 1 shows an<br />
actual-by-predicted plot, comparing model predictions against measured<br />
values for several fermentations of one Mab. The model is well capable of<br />
predicting the amount of acidic isoform for this molecule.<br />
Conclusion: In this work, the influence of fermentation parameters (pH,<br />
DO, temperature) on the extracellular modification of Mabs (in the<br />
supernatant of cell broth) was examined. Higher temperature and higher<br />
pH values lead to a significant increase in the formation of the acid region<br />
species of Mabs compared to lower temperature and pH conditions. The<br />
impact of these process parameters on the modification kinetics of Mabs<br />
Table 1(abstract P85) Setup for the sm<strong>all</strong> scale<br />
fermentation experiments<br />
Experiment pH Temp. [°C] DO [%]<br />
1 6.7 33.0 45<br />
2 6.7 40.0 5<br />
3 7.0 36.5 25<br />
4 7.0 36.5 25<br />
5 7.3 33.0 5<br />
6 7.3 40.0 45<br />
7 6.7 40.0 45<br />
8 6.7 33.0 5<br />
9 7.0 36.5 25<br />
10 7.0 36.5 25<br />
11 7.3 33.0 45<br />
12 7.3 40.0 5
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Figure 1(abstract P85) Correlation of measured versus calculated amount of acidic isoforms<br />
during cell-free incubation was characterized. Furthermore, additional<br />
modifications were detected, as oxidation, deamidation, generation of<br />
pyro glutamic acid, separation of lysin (data not shown).<br />
The results of the incubation experiments in the sm<strong>all</strong> scale fermenter<br />
system lead to a mathematical prediction model for the increase of the<br />
acidic peak during a standard fermentation for the production of Mabs with<br />
CHO cells. This prediction model helps to develop robust fermentation<br />
processes.<br />
References<br />
1. Müthing J, Kemminer SE, Conradt HS, Sagi D, Nimtz M, Kärst U, Peter-<br />
Katalinic J: Effects of buffering conditions and culture pH on production<br />
ratesand glycosylation of clinical phase I anti-melanoma mouse IgG3<br />
monoclonal antibody r24. Biotechnol Bioeng 2003, 83:321-334.<br />
2. Usami A, Ohtsu A, Takahama S, Fujii T: The effect of pH,<br />
hydrogenperoxide and temperature on the stability of a human<br />
monoclonal antibody. J PharmBiomed Anal 1996, 14:1133-1140.<br />
P86<br />
Technology transfer and scale down model development strategy for<br />
biotherapeutics produced in mammalian cells<br />
Nadine Kochanowski * , Laetitia Malphettes<br />
Cell Culture Process Sciences Group, Biotech Sciences, UCB Pharma S.A.,<br />
Braine L’Alleud, 1420, Belgium<br />
E-mail: Nadine.Kochanowski@ucb.com<br />
BMC Proceedings 2013, 7(Suppl 6):P86<br />
Background: The goal of manufacturing process development for drug<br />
substance and drug product is to establish a commercial process capable of<br />
consistently producing drug substance/drug product of the intended<br />
quality. Based on regulatory requirements, the manufacturing process has to<br />
be characterized prior to process validation. Since performing the<br />
characterization study at the manufacturing scale is not practic<strong>all</strong>y feasible,<br />
development of a scale down model that represents the performance of the<br />
commercial process is essential to achieve reliable process characterization.<br />
The developed scale down model could also be applied for cell line<br />
selection, process and medium development, raw material evaluation, limit<br />
of cell age studies, process parameter excursions, etc... Process development<br />
and commercial production should not be on the critical path to market<br />
despite the compressed time-to-market expectations. That is why<br />
Technology Transfer (TT) is a vulnerable time for companies. According to<br />
World Health Organization, Transfer of technology is defined as “a logical<br />
procedure that controls the transfer of any process together with its<br />
documentation and professional expertise between development and<br />
manufacture or between manufacture sites”. In the pharmaceutical industry,<br />
Technology Transfer refers to the processes that are needed for successful<br />
progress from drug discovery to product development to clinical trials to<br />
full-scale commercialization or it is the process by which a developer of<br />
technology makes its technology available to commercial partner that will<br />
exploit the technology. This article describes the strategies and activities<br />
required to develop a scale down model. It also sketches a Technology<br />
Transfer approach for bioprocesses by focusing on the upstream part of a<br />
cell culture based process.<br />
Results: Scale down model development strategy: “Sm<strong>all</strong>-scale models<br />
can be developed and used to support process development studies. The<br />
development of a model should account for scale effects and be<br />
representative of the proposed commercial process. A scientific<strong>all</strong>y justified<br />
model can enable a prediction of quality, and can be used to support the<br />
extrapolation of operating conditions across multiple scales and equipment<br />
[2]. The key elements for designing a scale down model are inputs (raw<br />
materials and components, cell source, environmental conditions) and<br />
outputs (performance and product quality metrics, sample handling/<br />
storage, analytical methods). A scale down model can be equivalent for<br />
some outputs but not for <strong>all</strong> and still be a representative model. It should<br />
reproduceatsm<strong>all</strong>scaletheeffect/impactseenatlargescale.The<br />
acceptability of an observed offset has to be statistic<strong>all</strong>y evaluated and<br />
scientific<strong>all</strong>y understood.<br />
Technology Transfer strategy: “The goal of Technology Transfer<br />
activities is to transfer product and process knowledge from development<br />
to market, and within or between manufacturing sites to support product<br />
commercialization. This knowledge forms the basis for the manufacturing<br />
process, control strategy, process validation approach and ongoing<br />
continual improvement [1]. A dedicated Technology Transfer team has to<br />
be set up to facilitate and execute the process including experts in
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Figure 1(abstract P86) Technology Transfer (TT) process flow chart.<br />
Table 1(abstract P86) Technology transfer documentation<br />
Document<br />
Bill of materials<br />
Research and Development reports<br />
Risk assessment<br />
Process descriptions<br />
Content<br />
List of <strong>all</strong> components and their step of use (Supplier, grade)<br />
Historical data of pharmaceutical development of new drug substances and drug products at stage from<br />
early development to final application of approval - Quality profiles of manufacturing batches (including<br />
stability data) - Specifications and test methods of drug substances, intermediates, drug products, raw<br />
materials and components, and their rationale - Change histories of important processes and control<br />
parameters<br />
Process flow charts - Scale up - Equipment changes - Media and feed preparation<br />
Product information - Process step flow diagram - Cell culture steps description (cell line/inoculum/<br />
expansion/production bioreactor - Media and feed preparation - Harvest description - Raw materials/<br />
equipment)<br />
Technology transfer file Introduction - Manufacturing process description, process parameters - Equipment - Raw materials -<br />
Analyses - Safety, environment - Stability (conditions, results) - Packaging (cold chain requirements, etc...) -<br />
Cleaning - Shipment characteristics and proper validation if needed - Historical data available<br />
Technology transfer protocol Technology transfer description - Scope - Objective - Responsibilities - Process Description - Equipment list<br />
(receiving unit) - Raw material list - Reference of Master batch record/number of repetitions and status of<br />
batches/acceptance criteria/relevant specifications/description of coaching
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Table 1(abstract P86): Technology transfer documentation (Continued)<br />
Manufacturing and testing description<br />
of the process<br />
Routine and non-sampling plans<br />
Data recording list<br />
Deviation inventory<br />
Technology transfer report<br />
Product information - Process step flow diagram - Cell culture steps description (cell line/inoculum/cell<br />
expansion/production bioreactor) - Media and feed preparation - Harvest description (holding time/storage<br />
conditions) - Raw materials - Equipments<br />
List of <strong>all</strong> the samplings that should be taken and kept in addition to the in-process control samples listed<br />
in the manufacturing description<br />
Online and offline data to be monitored and recorded during the process<br />
Description in details of the deviations and reporting of the impact on the product titer and quality<br />
Technology transfer description - Objective - Scope - List of deviations and discussion - Process results and<br />
comparison to acceptance criteria -, Conclusions<br />
different fields (production, QA, QC, RA, MSAT, etc...). The whole<br />
Technology Transfer has to be coordinated by the technology transfer/<br />
project leader. Organization for Technology Transfer should be established<br />
and composed of both party members from both sites, roles and scope of<br />
responsibilities of each party should be clarified, and adequate<br />
communication and feedback of information should be ensured. Figure 1<br />
describes the main steps of the Technology Transfer. Technology Transfer<br />
can be considered successful if the Receiving Unit can routinely reproduce<br />
the transferred product, process, or method against a predefined set of<br />
specifications as agreed with the Sending Unit. The success of a Technology<br />
Transfer project will be largely dependent on the skill and performance of<br />
individuals assigned to the project from the Sending Unit and the Receiving<br />
Unit. The roles and responsibilities of the sending unit and the receiving unit<br />
have to be clearly defined. The documentation is a key element of<br />
Technology Transfer: it ensures consistent and controlled procedures for<br />
Technology Transfer and to run the process. Clear documentation should<br />
provide assurance of process and product knowledge (Table 1).<br />
Conclusions: A scale down models is a tool for developing and<br />
characterizing the process and should be designed and demonstrated as<br />
appropriate representations of the manufacturing process. The transfer of<br />
technology from R&D to the commercial production site is a critical<br />
process in the development and launch of a biotherapeutical product. The<br />
three primary considerations to be addressed during an effective<br />
technology transfer are the project plan, the people involved and the<br />
process.<br />
References<br />
1. ICHQ10 guideline: Pharmaceutical Quality System.<br />
2. ICHQ11 guideline: Development and manufacture of drug substances<br />
(chemical entities and biotechnological/biological entities).<br />
P87<br />
A modular flow-chamber bioreactor concept as a tool for continuous<br />
2D- and 3D-cell culture<br />
Christiane Goepfert 1 , Grit Blume 1 , Rebecca Faschian 1 , Stefanie Meyer 1 ,<br />
Cedric Schirmer 1 , Wiebke Müller-Wichards 2 , Jörg Müller 2 , Janine Fischer 3 ,<br />
Frank Feyerabend 3 , Ralf Pörtner 1*<br />
1 Institute of Bioprocess and Biosystems Engineering, Hamburg University of<br />
Technology Hamburg, D-21073, Germany;<br />
2 Institute of Micro System<br />
Technology, Hamburg University of Technology, Hamburg, D-21073,<br />
Germany;<br />
3 Department of Structural Research on Macromolecules, Institute<br />
of Materials Research, Helmholtz-Zentrum Geesthacht, Geesthacht, D-21502,<br />
Germany<br />
E-mail: poertner@tuhh.de<br />
BMC Proceedings 2013, 7(Suppl 6):P87<br />
Background: Advanced cell culture models, especi<strong>all</strong>y long-term 3D<br />
systems, require bioreactors <strong>all</strong>owing for cultivation under continuous flow<br />
conditions. Such culture models are for example tissue engineered implants,<br />
3D cultures for drug testing, in vitro models of cell growth and migration for<br />
wound healing studies, cell cultures for biomaterial testing. New ch<strong>all</strong>enges<br />
in drug testing and biomaterial development arise from regulatory<br />
requirements. Animal trials have to be replaced by cell culture assays,<br />
preferably by test systems with human material. Standard 2D monolayer<br />
cultures are often unsatisfactory and therefore tissue-like 3D cultures are<br />
suggested as an alternative. Here the design of a multi-well flow-chamber<br />
bioreactor as a tool for manufacturing advanced cell culture models is<br />
presented. Advantages of this reactor concept can be seen in constant flow<br />
conditions, removal of toxic reaction products, high cell densities, and<br />
improved metabolism [1]. The general design of the flow chamber<br />
bioreactor (FCBR) can easily be modified for different applications and<br />
analytical requirements.<br />
Concept: The concept of the flow-chamber bioreactor (FCBR) comprises<br />
the following features (Figure 1A): Simultaneous cultivation of multiple<br />
tissue constructs in special inserts; oxygen supply via surface aeration<br />
directly in the chamber; a uniform and thin medium layer which is created<br />
by a sm<strong>all</strong> barrier at the end of the flow channel to minimize the diffusion<br />
distance from the gas phase to the tissue constructs; medium supply from<br />
a reservoir bottle in a circulation loop via peristaltic pumps.<br />
Two designs are available: A closed system (single flow channel) with<br />
counter current flow of gas and medium for tissue-engineered constructs<br />
(Figure 1B), and a 24 well plate-based modular bioreactor (medorex, Nörten-<br />
Hardenberg, Germany) for miniaturized tissue constructs that permits the<br />
use of pipetting robots and standard plate readers (Figure 1C).<br />
For the latter one, the design of the 4 channels can be customized for<br />
various applications (Table 1). The lid of the plate is connected to tubings<br />
for medium recirculation. Medium is supplied via the first well and<br />
removed from the last well of each row (Figure 1C). Therefore 4 wells per<br />
row are available for construct cultivation.<br />
The closed system is aerated with humidified pre-mixed gas with optional<br />
composition. Therefore it can be handled independently from cell culture<br />
incubator. The 24 well-based system has to be placed in a humidified<br />
incubator for air supply from the incubator atmosphere.<br />
Fields of Application: For the above mentioned bioreactor designs, four<br />
applications are presented in the following.<br />
Example I: The single flow-channel bioreactor (Figure 1 (B)) was designed for<br />
the generation of three-dimensional cartilage-carrier constructs [2].<br />
The carriers consisting of a bone replacement material were covered with a<br />
1-2 mm cartilage layer. This reactor was used for long-term cultivation of<br />
cartilage-carrier-constructs with improved biochemical parameters (e.g.<br />
content of glycosaminoclycan, collagen type II) under constant conditions.<br />
Example II: The 24-well design was successfully applied to several cell culture<br />
models. Hepatocytes on porous 3D carriers were cultivated for 1-3 weeks and<br />
can be used as a model for drug testing [3]. After prolonged cultivation under<br />
continuous medium flow, the constructs are separated from each other for<br />
measurements in static operation mode to conduct viability and activity<br />
assays similar to procedures done in a standard multi well plate. Viability<br />
testing using Resazurin was performed repeatedly during cultivation.<br />
Furthermore, the EROD-assay for liver-specific cytochrome P450 activity was<br />
carried out at varying time points. Application for the resorption studies<br />
on magnesium implants is currently investigated by Prof. Willumeit,<br />
Dr. Feyerabend, HZ Geesthacht.<br />
Example III: A third layout of the MWFB was realized with four par<strong>all</strong>el<br />
flow channels instead of the separate wells. There is also the possibility to<br />
carry out material tests for cell expansion on specific materials (e.g.<br />
polymer films, collagen membranes, different coatings etc.).<br />
Example IV: Proliferation and migration of fibroblasts on collagen coated<br />
polymer foils integrated into the bioreactor was carried out using design IV<br />
(Figure 1 C). Electrical stimulation of NIH-3T3 fibroblasts resulted in the<br />
orientation of the cell cleavage plane perpendicular to the electric field<br />
vector. The electrodes were inserted into the chamber on a polymer foil<br />
clamped between the base plate and the 24 well plate equivalent top<br />
frame. The polymer foil can be removed and processed after the assays for<br />
staining and microscopic evaluation of the stimulated cells. The bottom
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Figure 1(abstract P87) Flow-chamber bioreactor (FCBR, medorex, Germany)(A) Concept (B) Closed system (single channel) with aeration for<br />
tissue-engineered constructs (C) 24 well plate-based modular bioreactor (medorex) for miniaturized constructs that permits the use of<br />
pipetting robots and standard plate readers (D) Flow chamber equipped with electrodes for stimulation.<br />
Table 1(abstract P87) Bioreactor configuration and applications<br />
Bioreactor design Potential applications Example<br />
I. Single channel, 6 variable culture inserts for 3D<br />
scaffolds transparent cover plate<br />
active aeration<br />
II.<br />
III.<br />
4 flow channels for perfusion 24 well plate layout<br />
inserts for 3D scaffolds surface aeration gas supply from<br />
humidified incubator<br />
As (II), transparent bottom plate for microscopy flow<br />
channels instead of separate wells<br />
IV. As (II) plus integrated of electrodes for electrical<br />
stimulation and impedance measurement<br />
Long term cultivation of 3D tissue constructs under flow<br />
conditions,<br />
tissue cultivation on implantable biomaterials<br />
Simultaneous cultivation of four 3D constructs per channel, 4<br />
channels available, separate functional tests can be carried out<br />
on single constructs<br />
Cultivation of shear-responsive cells, integration of biomaterials<br />
possible (e.g. a collagen membrane)<br />
Electrical stimulation of cell growth and orientation, impedance<br />
measurement of cell viability<br />
Cultivation of<br />
cartilage-carrier<br />
constructs [2]<br />
3D cultures of liver<br />
cells [3], biomaterial<br />
testing<br />
Cultivation of sweatgland<br />
associated cells<br />
(current)<br />
Orientation of mitotic<br />
axis [5]<br />
plate was realized in a transparent material for microscopy. The frequency<br />
of unipolar pulses can be varied between 16 Hz and 2 kHz, the voltage<br />
between 0 up to 600 mV and stimulation pulse to pause ratios between<br />
1:1, 1:10 and 1:100<br />
Conclusions: The flow chamber concept and its different modifications<br />
can be applied as an easily applicable and versatile tool for advanced cell<br />
culture models. The 24 well design issuitableforapplicationina<br />
standard cell culture lab without special bioreactor equipment: For<br />
medium supply, standard peristaltic pumps with 4 channels can be used.<br />
The bottom plate can be handled in a similar way as 24 well plates<br />
<strong>all</strong>owing for adaptation of standard assays to long-term 3D cultures,<br />
electric<strong>all</strong>y stimulated cells, or primary cells cultivated on membranes<br />
consisting of various biomaterials.<br />
References<br />
1. Pörtner R, Goepfert C, Wiegandt K, Janssen R, Ilinich E, Paetzhold H,<br />
Eisenbarth E, Morlock M: Technical Strategies to Improve Tissue<br />
Engineering of Cartilage Carrier Constructs - A Case Study. Adv Biochem<br />
Eng/Biotechnol 2009, 112:145-182.<br />
2. Nagel-Heyer S, Goepfert Ch, Adamietz P, Meenen NM, Petersen JP,<br />
Pörtner R: Flow-chamber bioreactor culture for generation of threedimensional<br />
cartilage-carrier-constructs. Bioproc Biosyst Eng 2005,<br />
27:273-280.<br />
3. Goepfert C, Scheurer W, Rohn S, Rathjen B, Meyer S, Dittmann A,<br />
Wiegandt K, Janßen R, Pörtner R: 3D-Bioreactor culture of human<br />
hepatoma cell line HepG2 as a promising tool for in vitro substance<br />
testing. BMC Proceedings 2011, 5:P61.
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4. Starbird R, Krautschneider W, Blume G, Bauhofer W: In Vitro<br />
Biocompatibility Study and Electrical Properties of the PEDOT, PEDOT<br />
Collagen-Coat, PEDOT Nanotubes and PEDOT Aerogels for Neural<br />
Electrodes. Biomedical Engineering (<strong>BioMed</strong> 2013) Proceedings Innsbruck,<br />
Austria 2013.<br />
5. Saß W, Blume G, Faschian R, Goepfert C, Müller J: Wachstumsstimulation<br />
von Fibroblasten mit Platin/PEDOT Elektroden auf hochflexiblen Folien.<br />
Mikrosystemtechnik Kongress VDE VERLAG BerlinBMBF; VDE; GMM; VDI/VDE-IT<br />
2012, ISBN 978-3-8007-3367-5.<br />
P88<br />
Platform process will give platform product - Can we afford it?<br />
Rohit Diwakar * , Sunaina Prabhu, Lavanya C Rao, Janani Kanakaraj, Kriti Shukla,<br />
Saravanan Desan, Dinesh Baskar, Ankur Bhatnagar, Anuj Goel<br />
Cell Culture Lab, Biocon Research Limited, Bangalore, India<br />
E-mail: rohit.diwakar@biocon.com<br />
BMC Proceedings 2013, 7(Suppl 6):P88<br />
Introduction: Manufacturing processes for therapeutic monoclonal<br />
antibodies (mAbs) have evolved immensely in the past two decades<br />
around two major thrust areas.<br />
1) Advancements in a) Cell line development-breakthrough and<br />
incremental knowledge gain in technology b) Media and feed formulation<br />
strategies c) Advent of Disposables and Instrumentation technologies thus<br />
offering significant improvements to Process Development (PD).<br />
2) Establishment of platform processes to leverage faster PD [1,2].<br />
A platform process gener<strong>all</strong>y consists of a standard i) Cell line development<br />
technique, ii) Basal medium and feeds, iii) Process parameters and scale-up<br />
approach. The biggest advantage of using the platform process for the PD<br />
group is in expediting the project timelines. The platform approach also<br />
benefits from well-established and validated work flows in Manufacturing,<br />
QA, QC and Supply-chain groups.<br />
Certain disadvantages have also been cited for the platform approach. For<br />
example, modifications in the platform process are gener<strong>all</strong>y discouraged<br />
due to time, cost and efforts required in accommodating such changes.<br />
Also, as process conditions can substanti<strong>all</strong>y impact the product quality<br />
(PQ) attributes, a platform approach does not <strong>all</strong>ow any significant<br />
changes in the PQ attributes, if desired.<br />
Materials and methods: In this study, CHO cell lines were cultured in<br />
chemic<strong>all</strong>y defined medium. Experiments were carried out in 2L stirred<br />
tank bioreactors and 125mL shake flasks running at 140 rpm in 5% CO 2<br />
controlled incubator shaker. Cell count and viability were determined<br />
using haemocytometer. Lactate, glucose, osmolality and IgG concentration<br />
was also estimated along with glycosylation profiling.<br />
Results and discussion: Case 1: Multiple cell lines developed using<br />
same technology expressing different mAbs: Using the same cloning<br />
technology, cell lines expressing mAbs 1-4 were developed. These cell<br />
lines when run with the platform process showed very similar growth, titer<br />
and glycosylation profiles. Glycan profilethusproducedisrepresentedas<br />
three species; type I, II and III.<br />
The advantage of platform process was evident from the similarity of glycan<br />
profiles achieved in <strong>all</strong> the mAbs run with this process. However, for mAbs 3<br />
and 4, the target glycan profile was significantly different. The platform<br />
process gave 20-30% higher glycan type 1 than the respective targets. In<br />
order to match the targeted glycan profile, a few changes were made:<br />
i) mAb 3: New feed introduced to reduce glycan type 1; feeding<br />
strategy was optimized during PD.<br />
Figure 1(abstract P88) (Clockwise direction) a) Viability comparison between control (mAb1-4) and mAb5 and 6. b) Viability comparison between<br />
platform and modified process for mAb5 and 6. c) Cell count comparison and d) Lactate comparison between cell line technology 1 and 2. As expected,<br />
PQ profiles between these two clones were very different.
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ii) mAb 4: In addition to feeding strategy used for mAb3, changes in<br />
process parameter (pH and DO) set-points were done to achieve<br />
desired glycosylation profiles.<br />
Case 2: Difference in lead clone selection criteria - growth vs.<br />
specific productivity: Clone selection is done by ranking the clones<br />
based on parameters such as cell growth, titer, specific productivity (PCD)<br />
and PQ. In this study, the lead clones were shortlisted based on different<br />
strategies. For mAbs 1-4, the lead clone was shortlisted based on cell<br />
growth and titer as dominant selection criteria. For mAbs 5 and 6, PCD was<br />
the dominant selection criterion. The other aspects of the cloning<br />
technique were same in <strong>all</strong> cell lines.<br />
When lead clones for mAb 5 and 6 were run in platform process they<br />
showed poor growth characteristics (Figure 1a). The early drop in viability<br />
made these clones unfit for a manufacturing process. Changes in the<br />
platform process were attempted to overcome this manufacturing concern:<br />
i) mAb 5: Culture longevity was increased by restricting cell growth. This<br />
was achieved by reducing nutrient levels in the production medium.<br />
ii) mAb 6: Lactate and ammonia accumulation was reduced by<br />
optimizing medium/feed composition and pH, DO control ranges.<br />
The modified processes significantly improved the culture longevity and<br />
viability profiles, making them suitable for manufacturing (Figure 1b).<br />
Case 3: Cell lines expressing the same mAb developed using<br />
different technology: Two cloning technologies, 1 and 2 were used to<br />
develop clones expressing the same mAb. The major differences in the<br />
technologies were i) host cell lines ii) design of vector and its mechanism<br />
in the genome. Both cell lines were run with the same platform process<br />
and a two-fold difference in cell count was observed between them<br />
(Figure 1c). The lactate levels were also markedly different (Figure 1d),<br />
possibly indicating differences in nutrient metabolism. The lactate<br />
differences also reflected in the pH profiles.<br />
Summary: Case 1: The use of platform process enabled accelerated PD<br />
from cell culture perspective. However, accommodating the specific PQ<br />
requirements resulted in extended process development, affecting timelines.<br />
Case 2: Change in clone selection criteria was observed to significantly<br />
impact culture performance while applying platform process. This almost<br />
resulted in rejection of these clones, thus extending PD timelines. This<br />
was prevented by modifying the platform process.<br />
Case 3: Clones developed using different cloning technologies when run<br />
with the platform process resulted in different cell culture and PQ<br />
profiles. Therefore, the type of cloning technique forms an integral part<br />
of the platform process.<br />
Though platform process was not suitable in most of the cases discussed<br />
here, it still offers advantages like expedited project timelines and<br />
established work flows. These benefits were achieved by establishing four<br />
versions of the platform process to meet the varied cell culture and PQ<br />
requirements. Based on the cell line characteristics and target PQ profiles,<br />
the appropriate version is chosen to initiate PD. These versions retained the<br />
major advantages of the platform process such as having common media<br />
and feeds with only changes in their concentrations and set point of main<br />
process parameters to achieve desired PQ.<br />
Acknowledgements: Cell Culture Lab - Ruchika Srivastava, Vana Raja S,<br />
Chandrashekhar K.N<br />
Characterization Lab - Varshini Priya, Laxmi Adhikari<br />
Purification Lab - Shashank Sharma<br />
References<br />
1. Kelley B: Industrialization of mAb production technology. Landes<br />
Biosciences 2009, 5:443-452, mAbs 1.<br />
2. Li F, Vijayasankaran N, Shen A, Kiss R, Amanullah A: Cell culture processes for<br />
monoclonal antibody production. Landes Biosciences 2010, 5:466-477, mAbs 2.<br />
P89<br />
Applications of biomass probe in PAT<br />
Chandrashekhar K Nanjegowda, Nirmala K Ramappa, Pradeep V Ravichandran,<br />
Deepak Vengovan, Saravanan Desan, Dinesh Baskar * , Ankur Bhatnagar, Anuj Goel<br />
Cell Culture Lab, Biocon Research Limited, Bangalore, India<br />
E-mail: dinesh.baskar@biocon.com<br />
BMC Proceedings 2013, 7(Suppl 6):P89<br />
Introduction: In biologics manufacturing, process consistency is essential to<br />
produce the desired product quality over the product life cycle. Process<br />
monitoring is an important tool to achieve consistency and robustness.<br />
Typical process parameters monitored at upstream are viable cell<br />
concentration (VCC), viability, titer, nutrient levels, waste metabolites,<br />
osmolality, pH, DO and pCO 2 . Tradition<strong>all</strong>y pH, DO and pCO 2 are monitored<br />
using online sensors while others are measured by offline sampling<br />
methods. With recent advances in sensor technology, probes are now<br />
available to reliably estimate some of these parameters online. One such<br />
tool is biomass probe which estimates VCC by measuring capacitance in the<br />
bioreactor. In this work two cases are presented where biomass probe has<br />
advantages over traditional offline sampling and can be used as an effective<br />
PAT tool to monitor and improve process consistency and robustness.<br />
Experimental Approach: CHO and NS0 cell lines were used to run fed<br />
batch (70L) and perfusion (1KL) runs. The perfusion bioreactor used two<br />
Spin filters (SF) as cell retention device that could be switched when<br />
required. Biomass probe readings were compared to the VCC estimated<br />
by offline sampling.<br />
Results and discussions: In Fed Batch runs, offline and online VCC values<br />
were very comparable during the initial days of the run and deviated with<br />
increased process duration and drop in cell viability. In the Perfusion Batch,<br />
the offline and online VCC values were comparable throughout the run.<br />
The current work focusses on the phases where online biomass probe<br />
can be reliably used to improve efficiencies of both Fed Batch and<br />
Perfusion processes.<br />
Case 1: Improving process efficiency in Fed batch: Inoculum<br />
propagation and transfer: Inoculum plays a critical role in the process<br />
performance; therefore inoculum consistency is very important. Inoculum<br />
development step requires cells to be transferred to the next stage while<br />
they are in the exponential phase. This is norm<strong>all</strong>y done by sampling the<br />
seed bioreactors, measuring the cell counts and transferring cells to the<br />
next stage.<br />
As this requires sampling for cell counting, due to rapid cell growth in this<br />
phase, gener<strong>all</strong>y a wide range of acceptable cell concentration is given for<br />
practical reasons. Although during this broad range of acceptable cell<br />
concentration, cells are in their exponential phase, the volume of inoculum<br />
added into the bioreactor changes the spent media ratio inside the<br />
production bioreactor considerably.<br />
By measuring VCC online using a biomass probe, it was possible to transfer<br />
the inoculum at much precise cell concentration thus achieving consistent<br />
volumetric inoculum ratios in production bioreactor (Figure 1a).This resulted<br />
in an improved consistency in the cell culture profiles of the production run.<br />
Feeding based on online VCCmeasurements:AFedBatchprocess<br />
requires frequent additions of nutrient feeds to the bioreactor. These feeds<br />
are gener<strong>all</strong>y added either by sampling and measuring concentrations of<br />
residual nutrients or based on predefined culture time intervals. By feeding<br />
based on fixed culture duration, nutrients are added at same age but at<br />
different cell concentration. Feeding based on biomass probe readings<br />
helped in maintaining the nutrients as per VCC, thus preventing<br />
accumulation or depletion of nutrients in the process and eliminating batchto-batch<br />
variations (Figure 1b).<br />
Case 2: Improving process efficiency in Perfusion: In our process, loss<br />
in cell-retention in the perfusion device led to decrease in cell conc. and<br />
productivity. By monitoring retention continuously, corrective actions<br />
could be taken to reduce these losses. Introducing a biomass probe in the<br />
perfusate line overcame operational constraints of frequent sampling to<br />
monitor retention efficiency.<br />
Effective switching of the retention filters: As the SF clogs, there is a<br />
drop in perfusate volume being drawn from the filter, which results in<br />
pressure drop in the harvest line. Whenever the line pressure drops<br />
significantly, the perfusion is switched to the other filter. Calculations show<br />
reduction in retention efficiency of the filters from about 90% to 50%<br />
(Figure 1c). This reduction indicates cell loss through the filter resulting in<br />
significant drop in bioreactor VCC (Figure 1d).<br />
To prevent a significant loss of cells from the bioreactor, it was decided to<br />
switch the filter by monitoring the retention by biomass probe in the<br />
perfusate line. Two biomass probes were inserted in the bioreactor and<br />
the perfusion outlet to measure the bioreactor cell concentration and the<br />
cells lost through the filter during perfusion. The filter was switched when<br />
the retention efficiency drop below 70%. This helped in preventing<br />
significant loss of viable cells from the bioreactor due to cell leakage<br />
through filters.
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Figure 1(abstract P89) (In clockwise direction) a) Inoculum transfer range using offline (±15%) and online (±5%) VCC measurements. b) Feeding<br />
strategy comparison based on time and online probe readings c) Profiles of batches comparing N. VCC (Normalized VCC) and N. VVD. d) Drop in cell<br />
retention leading to increased cell leakage through the filter.<br />
Effective control of perfusion rates: The perfusion rate in a perfusion<br />
run is gener<strong>all</strong>y reported as VVD (volume of medium perfused per<br />
bioreactor volume per day). As the VCC in the bioreactor increases, VVD is<br />
increased to provide additional nutrients for the cells. Although increase in<br />
VVD favours higher cell concentration, a drop in bioreactor VCC is also<br />
seen occasion<strong>all</strong>y at higher VVD (Figure 1d, batch 1). Upon investigation it<br />
was evident that in these cases when VVD was increased, cell<br />
concentration in the bioreactor decreased due to increased cell leakage<br />
through the filters. Hence it was decided to control the VVD based on cell<br />
retention values. The VVD in the batch 2 was gradu<strong>all</strong>y increased<br />
considering the retention efficiency of the filter. A higher VCC was<br />
obtained in this batch compared to batch 1 even at lower VVD, due to<br />
lower cell loss through the filters.<br />
Summary: In the current study, effective use of biomass probe was<br />
demonstrated in applications ranging from direct measurement of VCC to<br />
indirect applications during perfusion. The probe can be used for these and<br />
similar applications as an effective PAT tool to improve process consistency<br />
and robustness.<br />
Acknowledgements: Manufacturing team: Jiju Kumar, Raghu S,<br />
Kathiravan N, Santoshkumar Guddad<br />
Cell culture lab: Rohit Diwakar, Kriti Shukla, Vana Raja S, Abdul Waheed,<br />
Janani Kanakaraj.<br />
P90<br />
Understanding cell behavior in cultivation processes - A metabolic<br />
approach<br />
Jonas Aretz 1 , Tobias Thüte 1 , Sebastian Scholz 1 , Klaudia Kersting 1 ,<br />
Thomas Noll 1,2 , Heino Büntemeyer 1*<br />
1 Institute of Cell Culture Technology, Bielefeld University, Bielefeld, Germany;<br />
2 Center for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, Germany<br />
E-mail: heino.buentemeyer@uni-bielefeld.de<br />
BMC Proceedings 2013, 7(Suppl 6):P90<br />
Background: During cultivation cells undergo a tremendous change in<br />
their metabolism when shifting from one state to another or when<br />
parameters are changed. To understand the changes in intracellular<br />
metabolite concentrations and their impact on cell performance we used a<br />
systematic approach. By employing the chemostat mode at different<br />
steady state conditions we investigated the alterations of the<br />
concentrations of key metabolites during cultivations of a human<br />
production cell line.<br />
Methods: Chemostat cultivations were performed with the AGE1.hn AAT<br />
cell line (Probiogen AG, Berlin, Germany) and TC-42 medium (Teutocell AG,<br />
Bielefeld, Germany) in a fully controlled 2 litre benchtop bioreactor<br />
(Sartorius, Göttingen, Germany). Different dilution rates of 0.24 d -1 , 0.33 d -1 ,<br />
and 0.40 d -1 and pH values of pH 6.9, pH 7.15, and pH 7.3 were performed<br />
using the same bioreactor setup. For stopping the cell metabolism an<br />
established fast filtration method [1] was used for rapid quenching.<br />
Metabolites were extracted from cells using liquid/liquid extraction. Extracts<br />
were analyzed by using hydrophilic interaction chromatography (HILIC) and<br />
ESI-MS/MS mass spectometry. Extracellular amino acids and pyruvate were<br />
analyzed by pre-column derivatization and RP-HPLC [2], glucose and lactate<br />
using a YSI 2700 bioanalyser.<br />
Results: The comparative analysis of the three steady state dilution rates<br />
shows the great impact of changing extracellular conditions on the<br />
intracellular metabolite pools which may also lead to an altered<br />
productivity. For example, as been shown in Figure 1A the specific<br />
pyruvate consumption rate, qPyr, as well as the intracellular pyruvate pools<br />
decrease with increasing dilution rates, while qGlc and qGln increase at<br />
the same time. While some metabolite pools show great differences<br />
between different dilution rates others remain more or less constant.<br />
A malonate inhibition of the TCA cycle (Figure 1B) appears mainly at low<br />
dilution rates, which might be an effect of glucose and/or glutamine<br />
limitation at those steady states.<br />
Although qGlc, qPyr as well as qGln decrease with increasing pH values<br />
(data not shown), the intracellular TCA pools remain constant due to a<br />
catabolism of further amino acids (Table 1). This may have led to a lower<br />
waste of ammonia, lactate and glycine at higher pH values.<br />
The analysis of the intracellular nucleotide pools show that while the<br />
concentrations of almost <strong>all</strong> nucleotides dropped with increasing dilution<br />
rates, they were more or less stable at changing pH values (data not<br />
shown).
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Figure 1(abstract P90) Metabolite pool sizes in Glycolysis (A) and TCA (B) at different dilution rates. The metabolism at the three different<br />
dilution rates 0,24 d -1 (left), 0,33 d -1 (middle), 0,4 d -1 (right) is shown. Specific rates are illustrated with filled bars and given in nmol cell -1 d -1 .<br />
Stripped bars illustrate pool sizes which are given in mM (extracellular) and μM (intracellular), respectively.<br />
Conclusions: Although more data have to be raised to get a comprehensive<br />
insight into cell metabolism it could be shown that chemostat cultures<br />
performed at steady state conditions are a valuable tool for investigating cell<br />
behaviour on an intracellular basis. A much better data stability can be<br />
obtained than in batch or fed-batch cultures.<br />
Acknowledgements: Funding by the BMBF, Germany, Grand Nr.<br />
0315275A is gratefully acknowledged.<br />
References<br />
1. Volmer M, Northoff S, Scholz S, Thüte T, Büntemeyer H, Noll T: Fast<br />
filtration for metabolome sampling of suspended animal cells. Appl<br />
Microbiol Biotechnol 2011, 94:659-671.<br />
2. Büntemeyer H: Methods for off-line analysis in animal cell culture.<br />
Encyclopedia of Industrial Biotechnology. Bioprocess, Bioseparation, and Cell<br />
Technology New York: Wiley: Flickinger M 2010.
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Table 1(abstract P90) Correlation of specific rates q xxx<br />
with the adjusted pH values during steady state<br />
pH 6,9 pH 7,15 pH 7,3<br />
q NH3 430 ± 27 243 ± 9 207 ± 19<br />
q Lac 4751 ± 298 3766 ± 143 3548 ± 325<br />
q Glc - 3660 ± 230 - 3302 ± 126 - 3301 ± 302<br />
q Pyr - 155 ± 10 - 121 ± 5 -84 ± 8<br />
q Gln - 527 ± 33 - 488 ± 19 - 484 ± 44<br />
q Asp - 63 ± 4 - 123 ± 5 -153 ± 14<br />
q Glu 66 ± 4 29 ± 1 - 16 ±2<br />
q Asn - 17 ±1 - 42 ± 2 -45 ± 4<br />
q Ser -91 ± 6 -198 ± 8 - 191 ± 17<br />
q His - 13 ± 1 - 23 ± 1 -5 ± 1<br />
q Gly 32±2 9±0 7±1<br />
q Thr -26±2 61±2 67±6<br />
q Arg - 39 ±2 - 97 ± 4 - 109 ± 10<br />
q Ala 101 ± 6 48 ± 2 99 ± 9<br />
q Tyr - 10 ± 1 -29 ± 1 29 ± 2<br />
q Met -20 ± 1 -39 ± 2 - 40 ± 4<br />
q Val -37 ± 2 -79 ± 3 - 88 ± 8<br />
q Trp -5±0 -8±0 -9±1<br />
q Phe - 10 ± 1 -36 ± 1 -36 ± 3<br />
q Ile -35±2 -68±3 -72±7<br />
q Leu - 63 ± 4 -111 ± 4 -122 ± 11<br />
q Lys - 21 ± 1 - 89 ± 3 - 100 ± 9<br />
The specific rates are given in pmol cell -1 d -1 . (Negative values indicate<br />
consumed metabolites.)<br />
P91<br />
Engineering characterisation of single-use bioreactor technology for<br />
mammalian cell culture applications<br />
Akinlolu Odeleye * , Gary J Lye, Martina Micheletti<br />
Department of Biochemical Engineering, University College London, London,<br />
WC1E 7JE, UK<br />
E-mail: akinlolu.odeleye.09@ucl.ac.uk<br />
BMC Proceedings 2013, 7(Suppl 6):P91<br />
Background: The commercial success of mammalian cell-derived<br />
recombinant proteins has fostered an increase in demand for novel<br />
single-use bioreactor (SUB) systems, that facilitate greater productivity,<br />
increased flexibility and reduced costs. Whilst maintaining auspicious<br />
mixing parameters, these systems exhibit fluid flow regimes unlike those<br />
encountered in traditional glass/stainless steel bioreactors. With such<br />
disparate mixing environments between SUBs currently on the market,<br />
the traditional scale-up procedures applied to stirred tank reactors (STRs)<br />
are not adequate. The aim of this work is to conduct a fundamental<br />
investigation into the hydrodynamics of single-use bioreactors at<br />
laboratory scale to understand its impact upon the growth, metabolic<br />
activity and protein productivity of an antibody-producing mammalian<br />
cell culture.<br />
Materials and methods: This work presents a study characterising the<br />
macro-mixing, fluid flow pattern, turbulent kinetic energy (TKE), energy<br />
dissipation rates (EDRs), and shear stresses within these bioreactor<br />
systems carried out using 2-dimensional Particle Image Velocimetry (PIV).<br />
PIV enables acquisition of whole-field flow characteristics through<br />
instantaneous velocity measurements. The SUBs employed in the PIV<br />
measurements include the 3L CellReady (Merck Millipore), PBS Biotech’s<br />
PBS 3 bioreactor and the Sartorius 2L BIOSTAT Cultibag RM.<br />
The CellReady is a stirred tank bioreactor (3 litre volume), housing a 3-bladed<br />
upward-pumping marine scoping impeller. The PIV study was conducted<br />
using the actual vessel which has an internal diameter (D T ) of 137 mm and<br />
height (H T ) of 249 mm. The marine scoping impeller (D I ) is 76.2 mm in<br />
diameter and is located near the bottom with a clearance of 30mm from the<br />
base. Measurements were obtained at varying impeller rates from 80 to<br />
350rpm (corresponding to Re = 8699 to 38057). The PBS 3 is a pneumatic<strong>all</strong>y<br />
driven bioreactor (3 litre volume) whose mixing is induced through the<br />
buoyancy of bubbles. PIV measurements were again obtained utilising the<br />
actual PBS 3 vessel in the central vertical plane of the bioreactor at wheel<br />
speeds of 20, 27, 33 and 38rpm. The Sartorius Cultibag RM is a rocked bag<br />
bioreactor with a 2 litre total volume. A custom-made Sartorius Cultibag<br />
mimic and rocking platform was manufactured to enable the required<br />
optical access for PIV investigations. Measurements were taken at a rocking<br />
speed of 25 rpm, in the vertical plane 8.5cm from the outer edge of the<br />
bioreactor. Fluid working volume (wv) was varied at 30, 40, 50 and 60% wv.<br />
A biological study into the impact of these fluid dynamic characteristics<br />
on mammalian cell culture performance and behaviour is presented.<br />
CellReady and Cultibag cell cultures were conducted using the GS-CHO<br />
cell-line (Lonza) producing an IgG 4 (B72.3) antibody. The impeller speed<br />
and working volume are used to vary the hydrodynamic environment<br />
within the CellReady, whilst the rocker speed is the varied parameter in<br />
the Cultibag RM.<br />
Results and discussion: The upward-pumping 3-bladed impeller within<br />
the CellReady engenders compartmentalisation of the fluid flow. This in<br />
turn contributes to the wide range of turbulence levels conveyed between<br />
the lower quarter and upper three quarters of the fluid. The maximum<br />
fluid velocity of 0.25U tip is achieved in the impeller discharge stream (at<br />
approximately r/R = 0.65 and z/H = 0.15) as shown in Figure 1, whilst the<br />
peak axial and radial turbulent velocities (ũ) are 0.15U tip and 0.11U tip<br />
respectively.<br />
Disparity in cellular growth and viability throughout a range of CellReady<br />
operating conditions (80 rpm-2.4L, 200rpm-2.4L and 350 rpm-1L) was not<br />
substantial, although a significant reduction in cell specific productivity<br />
was found at 350 rpm and 1L working volume. This is considered to be the<br />
most stressful hydrodynamic environment tested. Cells grown at these<br />
conditions displayed a metabolic shift from lactate production to net<br />
lactate consumption, without a reduction in glucose uptake. A possible<br />
reason for these observations is increased oxidative stress resulting from<br />
the higher agitation rate and gas entrainment [1,2].<br />
The PBS exhibits a greater degree of fluid dynamic homogeneity when<br />
compared to the CellReady. Although, TKE is more than 10 times lower<br />
than values observed in the CellReady’s impeller zone (which ranges from<br />
0.0026 to 0.0455 m 2 /s 2 at the varying impeller rates tested). Whilst TKE in<br />
the PBS peaks at approximately 0.0022 m 2 /m 2 with a wheel speed of<br />
38 rpm, the fluid attains velocities of up to 50% of the PBS wheel speed.<br />
This corresponds to velocities of up to 15 cm/s, which is within a similar<br />
range to the values observed in the CellReady.<br />
The Sartorius RM induces fluid velocities of up to 37 cm/s at 25 rpm,<br />
although fluid velocity and turbulence is dominated by the radial<br />
component. EDR and TKE remain relatively low at 25 rpm, with mean wholefield<br />
ensemble-averaged values of up to 0.0044 m 2 /s 3 and 0.0020 m 2 /s 2<br />
respectively. These measurements are significantly lower than the mean EDR<br />
values of 0.0052 to 0.14 m 2 /s 3 (over the RPM range of N = 80 to 350 rpm)<br />
determined in the upper three quarters of the CellReady alone. Cellular<br />
response to an increase in turbulence within the rocked bag bioreactor<br />
(25 to 42rpm), results in an increase in stationary phase viable cell<br />
concentration (VCC) of 20%. In addition, cell metabolic activity and cell<br />
specific protein productivity remains relatively unchanged. The augmented<br />
homogeneity and consistency in reference to turbulence and shear stresses<br />
within the Sartorius RM may enable the cells to adapt to the more rigorous<br />
mixing, thus maintaining cell specific productivity as well as enhancing VCC.<br />
Also,cellsgrownintheSartoriusRMexhibitmorethan60%greatercell<br />
specific productivity levels and up to 37% greater IgG 4 titres compared to<br />
those grown in the CellReady. Even though IgG 4 productivity increases<br />
within the Cultibag, investigations into product quality are necessary.<br />
Given the shifts seen in metabolic behaviour and cell specific productivity, it<br />
can be concluded that the fluid dynamic environment will impact upon<br />
cellular performance. Clearly, the range of EDRs and TKEs experienced by<br />
the culture is just as pertinent as the peak turbulence levels. Therefore,<br />
determining the critical hydrodynamic parameters applicable to the<br />
different flow regimes found in SUBs, will enable greater cross-compatibility<br />
and scalability across the range of SUBs.
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Figure 1(abstract P91) a) Time-resolved mean normalized velocity contour plot obtained at N = 200rpm, Re = 21747, V L =2.4L.b) Time-resolved<br />
turbulent velocity ( ũij) contour plot obtained at N = 200rpm, Re = 21747, V L = 2.4 L. Resolution of 0.815mm.<br />
References<br />
1. Mckenna T: Oxidative stress on mammalian cell cultures during<br />
recombinant protein expression. Linkoping University Institute of<br />
Technology 2009, 10.<br />
2. Sengupta N, Rose ST, Morgan J: Metabolic flux analysis of CHO cell<br />
metabolism in the late non-growth phase. Biotechnol Bioeng 2011,<br />
108:82-92.<br />
P92<br />
Enhancing cell growth and antibody production in CHO cells by siRNA<br />
knockdown of novel target genes<br />
Sandra Klausing 1* , Oliver Krämer 1 , Thomas Noll 1,2<br />
1 Institute of Cell Culture Technology, Bielefeld University, Bielefeld, Germany;<br />
2 Center for Biotechnology (CeBiTec), Bielefeld, Germany<br />
E-mail: Sandra.klausing2@uni-bielefeld.de<br />
BMC Proceedings 2013, 7(Suppl 6):P92<br />
Background: Seven out of the ten top-selling biopharmaceuticals in 2011<br />
are produced in Chinese Hamster Ovary (CHO) cells [1]. This tremendous<br />
commercial interest makes the development and application of strategies<br />
for cell line optimization, like gene overexpression or knockdown to<br />
enhance cell specific productivity and cellular growth, highly interesting. In<br />
this work, we investigated the knockdown effect of novel target genes by<br />
siRNA as a powerful tool for CHO cell line engineering.<br />
Materials and methods: CHO DP-12 cells (clone #1934, ATCC CRL-12445)<br />
were used as a model cell line, producing an anti IL-8 antibody. Cultivations<br />
were performed in 125 mL shaking flasks at 37 °C, 5% CO 2 ,185rpmand<br />
5 cm shaker orbit. For fed-batch processes, TCx2D feed supplement<br />
(TeutoCell AG) and a predefined feeding regime were applied identic<strong>all</strong>y for<br />
<strong>all</strong> cultures. Viable cell densities (vcd) and cell viability were measured by a<br />
Cedex Sytem (Innovatis). Monoclonal antibody (mAb) concentrations were<br />
determined via HPLC and a protein A column (Life Technologies).<br />
Target genes were chosen based on well-known signaling pathways (e.g.<br />
apoptosis, cell cycle or histone modification) as well as from previous results<br />
of a CHO cDNA microarray [2]. Mediators of apoptosis Bad and JNK were<br />
chosen as target genes for evaluation after knockdown, as well as Set, a<br />
protein involved in histone modification. Mcm5 is involved in DNA<br />
replication but its regulative role is not completely understood. Fin<strong>all</strong>y,<br />
knockdown of target gene P (patent pending) was investigated. Short<br />
hairpin RNA (shRNA) sequences were designed and cloned into a shRNA<br />
expression vector which was stably introduced into CHO DP-12 cells via<br />
lentiviral gene delivery. After selection with 5 μg/mL puromycin, successful<br />
siRNA-mediated mRNA knockdown (kd) of the target gene was verified by<br />
quantitative real-time PCR (qPCR). Transduced cell pools were evaluated in<br />
batch and fed-batch shaker cultivations with regard to growth performance<br />
and antibody productivity.<br />
Results: Through siRNA-mediated RNA interference, a high stable gene<br />
knockdown in the cell pools was achieved for target gene Set, JNK, Bad and<br />
P. Transcript levels were reduced by 57% (knockdown of JNK) up to 93%<br />
(knockdown of P), as shown in Figure 1A. Due to the procedure of lentiviral<br />
infection and puromycin selection, a slight variation in transcript levels of<br />
some target genes was observed even for an empty vector control cell pool<br />
in comparison to untreated CHO DP-12 cells. Unexpectedly, despite<br />
genomic integration of Mcm5-targeting shRNA, Mcm5 transcription was<br />
found to be up-regulated in two separate measurements of the respective<br />
cell pool.<br />
In batch shaker cultivations, <strong>all</strong> cells with a stable vector integration<br />
exhibited higher maximum vcds, compared to the untreated CHO DP-12<br />
culture. Cells with a stable knockdown of apoptosis mediator Bad reached<br />
the highest vcd with 121·10 5 cells/mL. However, final antibody titers did<br />
not exceed the titer of the empty vector control cell pool (data not shown).<br />
Fed-batch shaker cultivation increased maximum cell densities as well as<br />
process duration and revealed a strong influence of siRNA mediated gene<br />
knockdown (Figure 1B and C). The maximum vcd was increased for cells<br />
with stable expression of a shRNA targeting JNK (by 23%), Bad (by 44%),<br />
Mcm5 (by 45%) and P (by 74%) compared to empty vector control cells. In<br />
comparison to this control cell pool, maximum mAb titer was higher for cell<br />
pools JNK-kd, Mcm5-kd and P-kd. Mean cell specific productivity between<br />
day 4 and day 8 of the cultivation was increased in cell pools Set-kd as well<br />
as P-kd. The highest mAb titer of 456 mg/L was detected for cells with a<br />
stable knockdown of gene P.<br />
Conclusions: siRNA knockdown of target genes is an effective tool for<br />
CHO cell engineering in order to achieve higher viable cell densities and<br />
mAb titers. The stable transduction of shRNA targeting Mcm5 resulted in a<br />
slight increase of the transcript level, nevertheless, vcd and product titer<br />
were enhanced. This effect will be further analyzed. Knockdown of target<br />
gene P led to increased vcd in fed-batch cultivation (by 123%), higher
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Figure 1(abstract P92) (A) Relative mRNA ratio of target genes in cell pools with stable shRNA expression and the empty vector control cell pool<br />
compared to untreated CHO DP-12 cells. (B) Viable cell density and viability during fed-batch shaker cultivation of cell pools and untreated cells. (C)<br />
Maximum mAb titer and mean cell specific productivity (csp) between day 4 and 8 for <strong>all</strong> cultures in fed-batch cultivation.<br />
maximummAbtiter(by159%)andhighercspbetweenday4and8(by<br />
70%), compared to untreated CHO DP-12 cells, which makes this target<br />
gene a highly interesting candidate for cell line engineering. Stable<br />
transduction with an empty vector also influenced cellular behavior of the<br />
control cell pool compared to untreated CHO DP-12 cells. This is likely due<br />
to the random integration of the transfer vector and a selection for more<br />
robust and faster growing cells during the procedure of lentiviral infection<br />
and puromycin selection. Further reasons are under investigation. Single<br />
cell clone isolation for the presented cell pools will most likely result in<br />
further improvements of viable cell density and product titer.<br />
References<br />
1. Huggett B, Lähteenmaki R: Public biotech 2011 - the numbers. Nature<br />
Biotechnology 2012, 30:751-757.<br />
2. Klausing S, Krämer O, Noll T: Bioreactor cultivation of CHO DP-12 cells<br />
under sodium butyrate treatment - comparative transcriptome analysis<br />
with CHO cDNA microarrays. BMC Proceedings 2011, 5(Suppl 8):P98.<br />
P93<br />
Skin and hair-on-a-chip: Hair and skin assembly versus native skin<br />
maintenance in a chip-based perfusion system<br />
Ilka Wagner 1* , Beren Atac 1 , Gerd Lindner 1 , Reyk Horland 1 , Matthias Busek 1 ,<br />
Frank Sonntag 2 , Udo Klotzbach 2 , Alexander Thomas 1 , Roland Lauster 1 ,<br />
Uwe Marx 1<br />
1 Technische Universität Berlin - Berlin, Germany;<br />
2 Fraunhofer IWS - Dresden,<br />
Germany<br />
E-mail: ilka.wagner@tu-berlin.de<br />
BMC Proceedings 2013, 7(Suppl 6):P93<br />
Background and novelty: In recent decades, substantial progress to<br />
mimic structures and complex functions of human skin in the form of skin<br />
equivalents has been achieved. Different approaches to generate functional<br />
skin models were made possible by the use of improved bioreactor<br />
technologies and advanced tissue engineering. Although various forms of<br />
skin models are successfully being used in clinical applications, in basic<br />
research, current systems still lack essential physiological properties for<br />
toxicity testing and compound screening (such as for the REACH program)<br />
and are not suitable for high-throughput processes.<br />
Experimental approach: In particular, further bioengineering is necessary<br />
for the implementation of adipose tissue, hair follicles and a functional<br />
vascular network into these models. In addition, miniaturization, nutrient<br />
and oxygen supply, and online monitoring systems have to be implemented<br />
in sophisticated culture systems. To become one step closer to the in vivo<br />
situation, we produced microfollicles as in vitro hair equivalents and<br />
integrated them into skin models. These microfollicles containing skin<br />
tissues were cultured under static and dynamic<strong>all</strong>y perfused conditions and<br />
were compared to ex vivo scalp and foreskin skin organ cultures. Dynamic<br />
cultivation was performed in our Multi-Organ-Chip system (Figure 1 A).<br />
Results and discussion: The formation of functional neopapillae needs<br />
more than 48 hours. After the addition of keratinocytes and melanocytes,<br />
the self-organizing microorganoids follow a stringent pattern of follicularlike<br />
formation by generating polarized segments, sheath formations and<br />
the production of a hair shaft-like fiber. We show that the de novo<br />
formation of human microfollicles in vitro is accompanied by basic hair<br />
follicle like characteristics. The microfollicles can be used to study<br />
mesenchymal-epithelial-neuroectodermal interactions and for the in vitro<br />
testing of hair growth-modulating substances and pigmentary effects. As<br />
the hair follicle is highly vascularized, it supports penetration of substances<br />
into the skin and further into the bloodstream. Testing of topic<strong>all</strong>y applied<br />
substances might therefore be performed with significantly enhanced<br />
validity by the incorporation of a microfollicle into a dynamic chip-based<br />
bioreactor containing a skin equivalent which mimics a physiological<br />
penetration route. Commerci<strong>all</strong>y available skin equivalent EpiDermFT were<br />
cultured in the Multi-Organ-Chip for 7 days with subcuteaneous tissue and<br />
showed better viability and comparable histological results to native skin<br />
(Figure 1 C-J). Cellular and nutritional effect of the subcueaneous tissue is<br />
visible even under static conditions. Presence of subcuteaneous tissue<br />
decreased the expression of Tenascin C in dermis which is a marker for<br />
inflamation and fibrosis. Integritiy of the epidermis and proliferating cells<br />
in epidermis kept prominently in combined tissues. Figure 1 B showes the<br />
staining of a skin equivalent with an successfully inserted microfollicle.<br />
Conclusion: Perfusion of the combined tissue provides better integration<br />
and associated to viability of the subcuteaneous tissue. In general, presence<br />
of subcutaneous tissue increased the longevity of the in vitro skin equivalent<br />
in both static and especi<strong>all</strong>y in Multi-Organ-Chip cultures with improved<br />
tissue architecture. A skin equivalent with integrated microfollicles and
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Figure 1(abstract P93) Microfluidic device for perfused skin equivalent culture and integrated Microfollicle (A) Dynamic chip-based bioreactor<br />
for continuous perfusion culture of skin equivalents with integrated microfollicles. (B) PanCytokeratin immunoflourescent staining of a skin<br />
equivalent with an inserted microfollicle. (C-J) In vitro skin equivalents (MatTek) cultured for 7 days in MOC or static conditions with and without<br />
subcutaneous tissue (SCT) and compared to ex vivo foreskin. (C-F) H&E staining and (G-J) immunofluorescence staining for epidermal markers Cytokeratin<br />
10 and 15. Dashed lines mark the border between the skin equivalent and the subecuteaneous tissue. Scale bars indicate 100 μm.<br />
subcutaneous tissue under dynamic perfusion will be the most suitable<br />
model for long-term cultivation and more efficient drug studies and one<br />
step closer to mimic in vivo skin.<br />
Acknowledgements: The work has been funded by the German Federal<br />
Ministry for Education and Research, GO-Bio Grand No. 0315569.<br />
P94<br />
2D fluorescence spectroscopy for real-time aggregation monitoring in<br />
upstream processing<br />
Karen Schwab * , Friedemann Hesse<br />
Institute of Applied Biotechnology, University of Applied Science Biberach,<br />
88400 Germany<br />
E-mail: schwab@hochschule-bc.de<br />
BMC Proceedings 2013, 7(Suppl 6):P94<br />
Introduction: Product aggregation is one side effect of rising yields due to<br />
process improvement and therefore accompanied with massive product<br />
loss during downstream processing (DSP). But it is already in literature<br />
described, that product aggregation also occurs during the fermentation<br />
process and is caused by various process operations [1]. Real-time<br />
bioprocess monitoring and thus on-line product quality control during<br />
upstream processing (USP) enables to address this issue during process<br />
development. For bioprocess control, 2D fluorescence spectroscopy in<br />
combination with chemometric modeling based on fluorescence signals<br />
derived from cells and medium components is a promising tool and<br />
described in literature [2]. Furthermore extrinsic fluorescence dyes are<br />
widely used to detect and quantify aggregated protein [3]. In this study,<br />
2D fluorescence spectroscopy in combination with three different extrinsic<br />
fluorescence dyes were evaluated, in order to establish a process control<br />
tool which enables real-time product control during USP.<br />
Materials and methods: A CHO DG44 cell line producing a monoclonal<br />
antibody (mAb) was cultivated in a 2 liter bioreactor (Sartorius AG) in<br />
fed-batch mode. Metabolites and substrate concentrations were determined<br />
using Konelab 20XT (Thermo Scientific) and cell concentration and<br />
viability via CEDEX XS system (Innovartis-Roche AG). The product titer was<br />
determined with protein-A HPLC. Furthermore, culture supernatant samples<br />
were applied to the size exclusion column Yarra S4000 (Phenomenex) after<br />
filtration. The intrinsic fluorescence signal at 355nm was recorded with a<br />
fluorescence detector (Gynkotek), in order to determine the monomer to<br />
aggregate ratio in the sample. Samples were taken twice a day and<br />
incubated with ANS, bis-ANS and Thioflavin T at 3 different concentrations<br />
respectively. Full 2D scans from 270nm to 590nm of these samples were<br />
taken with the DELTA BioView® sensor. These scans were used as data<br />
input for chemometric modeling, where the target data was the mAb<br />
aggregate concentration.<br />
Results: A common approach to analyze aggregated mAb in cell culture<br />
comprises the isolation of the mAb by protein A HPLC subsequently<br />
followed by size exclusion chromatography [1,4]. However, the capture step<br />
itself may have an influence on product aggregation. Therefore, in this study<br />
we tried to avoid the capture step by directly applying cell culture<br />
supernatant onto the size exclusion column after a filtration step. The signal<br />
derived from the cell culture medium and host cell proteins could be<br />
separated from mAb monomer and aggregate signal (Figure 1D). This<br />
<strong>all</strong>owed direct quantification of mAb aggregates in culture broth via size<br />
exclusion chromatography (SEC). Fluorescent dyes such as ANS, and its<br />
dimeric analogon 4,4’-bis-1-anilinonaphthalene-8-sulfonate (Bis-ANS) as well<br />
as thioflavin T interact noncovalently with hydrophobic regions of the<br />
aggregated protein [3]. To our knowledge, up to now these dyes were not<br />
used as additives in mammalian cell cultures. Therefore, a major concern<br />
was their toxicity towards the CHO production cell line. Toxicity screens in<br />
microtiter plates (data not shown) revealed that already 4μM bis-ANS as well<br />
as 4μM thioflavin T reduced the specific growth rate strongly. The in<br />
literature reported concentrations for these dyes in DSP approaches [3] were<br />
considerably higher hence their sensitivity limits in cell culture had to be<br />
evaluated. In order to enable a direct comparison of fluorescence intensity
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Figure 1(abstract P94) PCA score plots for <strong>all</strong> Bis-ANS (A), thiovlavin T (B) and ANS (C) concentrations, where PC2 is displayed over PC1. T=0<br />
indicates data of 2D scans taken directly after inoculation. (D) SEC chromatogram of the intrinsic fluorescence emission signal at 355nm. Monomer, dimer<br />
and oligomer fractions of mAb were detectable; furthermore a separation from the medium and host cell protein signal was possible.<br />
Table 1(abstract P94) PLS results for selected dye concentrations used in the fed-batch fermentation experiment<br />
Dye PC’s R-Square RMSE Offset Slope<br />
w/o dye 3 calibration data set 0.96 1.27 0.43 0.96<br />
validation data set 0.72 3.62 2.75 0.70<br />
2μM Bis-ANS 4 calibration data set 0.98 10.9 2.28 0.98<br />
validation data set 0.93 19.36 -4.20 0.97<br />
80μM ANS 4 calibration data set 0.98 0.82 0.18 0.98<br />
validation data set 0.85 2.08 1.44 0.85<br />
25μM Th T 2 calibration data set 0.99 5.18 0.52 0.99<br />
validation data set 0.96 14.54 6.23 0.93<br />
2D fluorescence scans were taken as x-data and the mAb aggregate concentration was used as target data for chemometric modeling. Validation data sets were<br />
generated with cross validation.
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increase generated by dye aggregate interaction, the DELTA BioView® sensor<br />
was used at-line during the fed-batch fermentation. For chemometric<br />
modeling, fluorescence maps were preprocessed by principal component<br />
analysis (PCA), in order to capture the data input with the highest variance<br />
over the cultivation time. PCA results indicated that the sensitivity of Bis-ANS<br />
and ANS was very high towards aggregated mAb. Furthermore, increasing<br />
Bis-ANS concentrations increased the score values of PC1 in general (Figure<br />
1A), contrary to ANS where score values of PC2 increased (Figure 1C). For<br />
thioflavin T score values differed greatly when low and high dye<br />
concentrations were compared, starting at one point (Figure 1B).<br />
Furthermore, the mAb aggregate titer was used as target for partial least<br />
square regression (PLS) (Table 1) and resulting calibration and validation<br />
models showed low root square mean error (RMSE) values as well as good<br />
slopes and R-squares for ANS and Bis-ANS. Besides that, the chemometric<br />
model computed with 2D scans taken from cell culture without additional<br />
dye showed a slope of 0.7 and R-square value of 0.72 for the validation data<br />
set. This indicated that the quality of the chemometic models seemed to be<br />
improved when an additional fluorescence signal based on dye mAb<br />
aggregate interaction was generated in the 2D scans. Moreover, only 25μM<br />
thioflavin T enabled a solid calibration model (Table 1). This raised the<br />
suspicion, that there might be only weak interactions of dye and aggregated<br />
mAb. In consequence these preliminary results indicated, that thioflavin T<br />
which is norm<strong>all</strong>y used for detection of fibrils seemed to be less favorable<br />
for the detection of mAb aggregates.<br />
Conclusions: Suitable fluorescence dye candidates were selected and<br />
based on sensitivity and toxicity, ANS and Bis-ANS proved to be<br />
promising candidates for further work. Direct quantification of mAb<br />
aggregates in cell culture broth was possible with SE-HPLC based on the<br />
intrinsic fluorescence of mAb. The fed-batch fermentation experiment,<br />
where the DELTA BioView® sensor was used at-line, enabled a direct<br />
comparison of different dye concentrations. Therefore, this experiment<br />
demonstrated that for bis-ANS even lower concentrations than already<br />
used might be applicable due to its high sensitivity towards mAb<br />
aggregates. Moreover, the results indicated that product aggregation is<br />
not only a side effect of rising titers, because mAb aggregates were also<br />
present at early fermentations time points.<br />
References<br />
1. Gomez N, Subramanian J, Ouyang J, Nguyen M, Hutchinson M, Sharma V,<br />
Lin A, Yu I: Culture temperature modulates aggregation of recombinant<br />
antibody in CHO cells. Process Biochem 2012, 47:69-75.<br />
2. Teixeira A, Portugal C, Carinhas N, Dias J, Crespo J, Alves P, Carrondo M,<br />
Oliveira R: In situ 2D fluorometry and chemometric monitoring of<br />
mammalian cell cultures. Biotechnol Bioeng 2009, 102:1098-1106.<br />
3. Hawe A, Sutter M, Jiskoot W: Extrinsic fluorescent dyes as tools for<br />
protein characterization. Pharm Res 2008, 25:1487-1499.<br />
4. Jing Y, Borysa M, Nayakb S, Egana S, Qiana Y, Pana S, Li Z: Identification of<br />
cell culture conditions to control protein aggregation of IgG fusion<br />
proteins expressed in Chinese hamster ovary cells. Biotechnol Bioeng<br />
2012, 109:125-136.<br />
P95<br />
Use of microcarriers in Mobius® CellReady bioreactors to support<br />
growth of adherent cells<br />
Michael McGlothlen * , Donghui Jing, Christopher Martin, Michael Phillips,<br />
Robert Shaw<br />
EMD Millipore Corporation, 80 Ashby Rd, Bedford MA 01730, USA<br />
E-mail: Michael.mcglothlen@emdmillipore.com<br />
BMC Proceedings 2013, 7(Suppl 6):P95<br />
Mixing: Manufacturer specifications show Cytodex 3 ® and Solohill®<br />
microcarriers to be similar in density and size. Working with this assumption,<br />
mixing studies where performed using the Cytodex 3 ® microcarriers in 3L<br />
Mobius® CellReady and Solohill® Collagen coated in 50L single use<br />
bioreactor to determine the slowest agitation speed or the just suspended<br />
mixing power inputs (P/V) js , required to fully suspend the microcarriers so<br />
that the beads are equ<strong>all</strong>y distributed in the bioreactor.<br />
Microcarrier distribution was assessed by sampling the bioreactor at varying<br />
depths. Then the dry weight of the microcarrier was used to determine the<br />
% relative sample weight to the target weight.<br />
Mixing Results: Data show the (P/V) js to be ~0.6W/m 3 in both the 3L and<br />
50L single use bioreactors<br />
100% distribution corresponds to the theoretical concentration of<br />
microcarriers, which is 3g/L Cytodex 3 ® in 3L bioreactor and 15g/L Solohill®<br />
Collagen microcarriers in 50L bioreactor<br />
Cell Growth: Initial cell culture runs were performed with MDCK and<br />
Human Mesenchymal Stem Cells (hMSCs) to evaluate the bioreactor<br />
agitation to support cell growth in the 3L Mobius® CellReady single use<br />
bioreactor. The conditions that showed the best performance could then<br />
scaled to the 50L Mobius® bioreactor.<br />
1. Cultured MDCK cells on Cytodex 3 ® microcarriers grew to a peak cell<br />
density of ~1e6cells/mL using a power input of 0.6W/m 3 with a 2L<br />
working volume after 3 days.<br />
2. Cultured hMSCs on Solohill® microcarriers grew to a maximum<br />
total cell number of 6e6 cells using power input of 0.6-0.8W/m 3 with<br />
a 2.4L working volume after 12 days.<br />
Conclusions: 1. Data from the mixing experiments demonstrate the just<br />
suspended mixing power input was determined to be ~0.6W/m 3 .<br />
2. Cell growth experiments with hMSCs demonstrate comparable cell<br />
growth in the 3L and 50L Mobius® CellReady bioreactor with total<br />
number of hMSCs reaching 4e8 and 9e9 cells after 12 days at a<br />
agitation power input of 0.6-0.8W/m 3<br />
3. Initial cell growth experiments with adherent MDCK cells<br />
demonstrate an agitation power/volume input of 0.6W/m 3 may<br />
provide the best performance for cell growth with peak cell densities<br />
~1.0e6 cells/mL after 3 days<br />
4. Comparable MDCK cell growth is observed:<br />
Mobius® CellReady Bioreactor 3L<br />
Mobius® CellReady Bioreactor 50L<br />
Rocking Bioreactor 20L<br />
P96<br />
CHO starter cell lines for manufacturing of proteins with pre-defined<br />
glycoprofiles<br />
Karsten Winkler 1* , Michael Thiele 1,2* , Rita Berthold 1 , Nicole Kirschenbaum 1 ,<br />
Marco Sczepanski 1 , Henning von Horsten 1,3 , Susanne Seitz 1 , Norbert Arnold 2 ,<br />
Axel J Scheidig 2 , Volker Sandig 1<br />
1 ProBioGen AG, D-13086 Berlin, Germany;<br />
2 Christian-Albrechts-Universität zu<br />
Kiel, D-24118 Kiel, Germany;<br />
3 Hochschule für Technik und Wirtschaft Berlin,<br />
D-10138 Berlin, Germany<br />
E-mail: michael.thiele@probiogen.de<br />
BMC Proceedings 2013, 7(Suppl 6):P96<br />
Backround: Glycosylation of protein therapeutics is influenced by a<br />
multifaceted mix of product intrinsic properties, host cell genetics and<br />
upstream process parameters. Industrial CHO cell lines may have several<br />
deficits in their glycosylation pattern for some applications, like high fucose<br />
content (corresponding to a low ADCC profile) and low galactosylation and<br />
sialylation levels (proposed to decrease activity and/or pharmacokinetics).<br />
We have successfully applied the GlymaxX® technology [1] abolishing fucose<br />
synthesis in well-established CHO DG44 and K1 platforms and pre-existing<br />
producer cell lines (glycan modulator GM1). Here we extend this strategy by<br />
other engineering approaches to enable production of protein therapeutics<br />
with desired glycosylation features. Through stable integration of other<br />
Table 1(abstract P95)<br />
Physical Characteristics of Microcarriers<br />
Microcarrier Cytodex 3® Solohill® Collagen Coated<br />
Density (g/ml) 1.04 1.03<br />
Hydrated Size (μm) 141-211 125-212<br />
Concentration (g/ml) 3 15
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Table 2(abstract P95)<br />
MDCK/Cytodex 3® Microcarriers Process Table<br />
Variable<br />
Value<br />
Cells MDCK hMSCs<br />
Inoculation 4e5 cells/mL<br />
5e3 cells/mL<br />
Density<br />
Substrate Cytodex 3® Solohill®<br />
Growth Media DMEM w/4.5g/L Glucose, 2% FBS, 1% NEAA and 2mM L-Glutamine DMEM low glucose, 10% FBS, 8ng/ml bFGF, 2mM<br />
Glutamine, 1X Pen/Strep<br />
pH 7 NA<br />
DO (% 45 NA<br />
Saturation)<br />
Feed 1 Day 1: 100% Growth Media Day 6: 1000ml low glucose fresh medium<br />
Feed 2 Day 3: Drain 50% of the working volume and reefed with equal volume Day 9: 400ml high glucose fresh medium<br />
of Growth Media<br />
Batch Duration 7 days 12 days<br />
Figure 1(abstract P95) Illustrates the attachment of MDCK and hMSCs to Cytodex 3 ® and Solohill® microcarriers<br />
Figure 2(abstract P95) compares the viable cell density of MDCK cells at increasing power/volume impeller inputs and different bioreactors<br />
genes for glycosylation enzymes we are able to tune galactosylation (glycan<br />
modulator GM2) and sialylation (glycan modulator GM3). These glycan<br />
modulators can specific<strong>all</strong>y be combined to address certain desired<br />
oligosaccharide patterns.<br />
We postulate that modulating effects of GM2 and GM3 require a specific<br />
expression level. In this case the combination of high level target protein<br />
expression and defined levels of glycan modulators becomes extremely rare.<br />
Therefore, the characterization of clones with individual stable levels of<br />
glycanmodulator expression is a prerequisite for industrial application.<br />
Materials and methods: Two vectors expressing either GM2 alone or GM2<br />
and GM3 in combination were constructed to evaluate modulator effects.<br />
This technology was applied to both, CHO-DG44 and K1 cells to generate<br />
modified host cell pools. Modulator host cell clones were generated out of<br />
appropriate DG44 pools and characterized for growth and modulator gene<br />
expression using a 7-day shaker batch culture and RT-qPCR respectively.<br />
A human IgG and a Fc-Fusion protein carrying a single N-glycosylation side<br />
in the CH2 domain were chosen as model proteins. After stable transfection<br />
of human IgG into GM2 and Fc-fusion protein into GM2/3 clones, the
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Figure 3(abstract P95) shows the viable cell density of hMSCs in the 3L and 50L Mobius® CellReady Bioreactor<br />
resulting test modulator clone pools were analyzed in fed batch shaker<br />
assays. Harvested culture supernatants were purified and subjected to N-<br />
Glycan profile analysis performed by Hydrophilic-Interaction-Chromatography<br />
(HILIC).<br />
Results: Characterization of modulator host cell clones for proliferation<br />
and modulator mRNA expression indicated that growth behavior is not<br />
influenced by modulator expression level. Therefore only GMx-mRNA<br />
level were used to select five to six clones expressing a broad range of<br />
either GM2 alone or GM2 and GM3 in combination. Each selected<br />
modulator host cell clone was transfected with the corresponding model<br />
protein in duplicates (indicated by A or B).<br />
Final fed batch assays gave typical clone pool results with growth profiles<br />
showing high comparability between clone pools expressing the same<br />
model protein (Table 1). Peak viable cell densities (VCD) of about 3E7 vc/mL<br />
were reached with maximum titers of 1.2 g/L hum IgG and 2.4 g/L Fc-Fusion<br />
protein within 12 days, while final viabilities were in most cases above 80%.<br />
Up to 3 fold different titers between pools A and B of the same starter clone<br />
were observed depending on selection schemes and process management.<br />
As it is given by the conveyer like nature of the glycosylation machinery the<br />
content of a certain glycan structure cannot be increased without<br />
decreasing the output of the preliminary structures. Therefore the<br />
hypergalactosylation effect of GM2 should result in a shift towards more<br />
G2F structures and for the combination of GM2 and GM3 a shift towards<br />
more G2FS1 structures is anticipated, while even the G2F content could be<br />
decreased. As shown in Figure 1 the expected shifts were observed,<br />
demonstrating that the glycan modulators are working in the intended way.<br />
Addition<strong>all</strong>y, we found a positive correlation between the level of modulator<br />
gene expression and the degree of glycan modifying effect. Clone pools<br />
with highest modulator expression levels displayed the highest content of<br />
the desired structures e.g. G2F for GM2 clones and G2FS1 for GM2/3 clones.<br />
This reflects a 15 - 20-fold increase of these target structures compared to<br />
clone pools with low or moderate modulator expression (Table 1).<br />
Despite substantial differences in productivity and process between A and<br />
B clone pool duplicates (2 - 3 fold difference in titers) in most cases only<br />
slight shifts of certain oligosaccharide structures were observed (e.g. clone<br />
pool 3 - 5 and 8, 9). This indicates that the glycan pattern is more<br />
Table 1(abstract P96) Data of selected clone pools shown in Figure 1<br />
Model protein: human IgG<br />
Model protein: Fc-Fusion protein<br />
Clone pool no. 1 2 4 6 7 10<br />
Index A B A B A B A B A B A B<br />
relative modulator mRNA expression<br />
GM2 5.8 5.8 2.5 2.5 0.4 0.4 1.5 1.5 3.7 3.7 0.6 0.6<br />
GM3 3.2 3.2 1.4 1.4 0.3 0.3<br />
Key process parameter<br />
Peak VCD (cell/mL) 20 20 31 24 28 24 31 24 29 25 27 25<br />
Final-vitality (%) 73 82 82 88 87 91 87 87 86 84 89 93<br />
Titer (g/L) 0.6 0.4 0.9 0.7 1.0 0.6 2.1 1.0 1.8 1.0 2.3 1.1<br />
N-Glycan analysis<br />
G0F (%) 2 1 62 55 71 68 37 24 19 19 46 41<br />
G1F (%) 19 13 23 29 18 21 27 32 35 34 33 38<br />
G2 (%) 3 4 1 1 1 1 1 1 1 1 1 1<br />
G2F (%) 61 67 4 6 2 3 3 7 11 10 9 12<br />
G1FS1 (%) 2 3
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Figure 1(abstract P96) HILIC chromatograms of clone pools with distinct modulator expression levels. A: GM2 clone pools, B: GM2/3 clone pools.<br />
With increasing GM2 activity a clear shift towards G2F structures can be observed. While the increasing activities of GM2 and GM3 correlates positively<br />
with the G2FS1 content.<br />
depended on clone specific modulator gene expression than on<br />
glycoprotein expression level.<br />
Conclusions: Expression of GM2 and GM3 in CHO cell lines can effectively<br />
change the glycosylation pattern of target proteins in a dose dependent<br />
manner. Growth and productivity characteristics are similar to unmodified<br />
host cells and maintain their suitability for clinical and commercial<br />
production.<br />
The degree of glycomodulation is reproducible and relatively independent<br />
of target glycoprotein expression level. This <strong>all</strong>ows a prediction of<br />
glycosylation patterns of glyco-proteins produced in certain host cell<br />
clones in relation to modulator expression level.<br />
Fin<strong>all</strong>y, a comprehensive set of engineered, biopharmaceutical CHO<br />
production cell lines were generated and characterized, individu<strong>all</strong>y<br />
optimized for enhanced ADCC activity, adjusted galactosylation or sialylation<br />
levels of the target proteins. This elaborate cellular toolbox <strong>all</strong>ows the rapid<br />
and targeted creation of antibody and glycoprotein molecules with specific<br />
pre-defined glycan profiles.<br />
Reference<br />
1. von Horsten HH, Ogorek C, Blanchard V, Demmler C, Giese C, Winkler K,<br />
Kaup M, Berger M, Jordan I, Sandig V: Production of non-fucosylated<br />
antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-<br />
hexulose reductase. Glycob 2010, 20:1607-1618.<br />
P97<br />
Dynamic profiling of amino acid transport and metabolism in Chinese<br />
hamster ovary cell culture<br />
Sarantos Kyriakopoulos 1 , Karen M Polizzi 2,3 , Cleo Kontoravdi 1*<br />
1 Centre for Process Systems Engineering, Department of Chemical<br />
Engineering and Chemical Technology, Imperial College London, UK;<br />
2 Division of Molecular Biosciences, Imperial College London, UK;<br />
3 Centre for<br />
Synthetic Biology and Innovation, Imperial College London, UK<br />
E-mail: cleo.kontoravdi98@imperial.ac.uk<br />
BMC Proceedings 2013, 7(Suppl 6):P97<br />
Introduction: Chinese Hamster Ovary (CHO) cells are the most widely used<br />
industrial hosts for the production of recombinant DNA technology drugs<br />
[1]. In such processes amino acids (a.a.) are vital nutrients for growth, but<br />
also building blocks of the recombinant protein (rprotein). Our research aims<br />
to establish a better understanding of a.a. transport in and out of cells, since<br />
this could have significant impact on increasing productivity and designing<br />
feeding strategies during bioprocessing.<br />
There are about 46 a.a. transporter proteins in mammalian cells, the genes of<br />
which are presented in Table 1 along with their substrates and <strong>all</strong> are<br />
members of the Solute Carriers (SLC) database [2]. A.a. transporters are
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Table 1(abstract P97) Amino acid transporter genes based on the SLC database [2]<br />
System GENES Substrates Expresion/Type of<br />
regulation<br />
A SLC38a1 Ala, Asn, Cys, Gln, His, Ser below detection<br />
limits<br />
SLC38a2 Ala, Asn, Cys, Gln, Gly,<br />
His, Met, Pro, Ser<br />
System GENES Substrates Expresion/Type<br />
of regulation<br />
PAT SLC36a1 Gly, Ala, Pro, b- Ala,<br />
Tau<br />
remains stable<br />
between cell lines b SLC36a2 Gly, Ala, Pro low a<br />
SLC38a4 Ala, Asn, Cys, Gly, Ser, Thr within cell culture c SLC36a3 putative low a<br />
ASC SLC1a4 Ala, Ser, Cys, Thr within cell culture c SLC36a4 Ala, Pro, Trp remains stable<br />
SLC1a5 Ala, Ser, Cys, Thr, Gln,<br />
Asn<br />
both d T SLC16a10 Phe, Tyr, Trp low a<br />
asc<br />
SLC7a10/<br />
SLC3a2<br />
Ala, Cys, Gly, Ser, Thr low a X - AG SLC1a1 Asp, Glu low a<br />
B 0 SLC6a19 Pro, Leu, Val, Ile, Met low a SLC1a2 Asp, Glu both d<br />
SLC6a15 Pro, Leu, Val, Ile, Met remains stable SLC1a3 Asp, Glu between cell lines b<br />
B 0,+ SLC6a14 basic & neutral a.a. not checked SLC1a6 Asp, Glu below detection<br />
limits<br />
b 0,+<br />
SLC7a9/<br />
SLC3a1<br />
Arg, Lys, Cystine low a SLC1a7 Asp, Glu below detection<br />
limits<br />
b SLC6a6 Tau, b-Ala both d x - c SLC7a11/<br />
SLC3a2<br />
Glu, Cystine<br />
Gly SLC6a9 Gly within cell culture c y + SLC7a1 Arg, Lys, His both d<br />
SLC6a5 Gly low a SLC7a2 Arg, Lys, His low a<br />
SLC6a18 Gly below detection<br />
limits<br />
IMINO SLC6a20 Pro low a y + L SLC7a7/<br />
SLC3a2<br />
L<br />
SLC7a5/<br />
SLC3a2<br />
SLC7a8/<br />
SLC3a2<br />
Cys, Leu, Phe, Trp, Val,<br />
Tyr, Ile, His, Met<br />
both d<br />
neutral a.a., except Pro low a His & sm<strong>all</strong><br />
peptides<br />
SLC7a3 Arg, Lys, His low a<br />
SLC7a6/<br />
SLC3a2<br />
Lys, Arg, Gln, His, Leu,<br />
Met<br />
Lys, Arg, Gln, His, Leu,<br />
Met, Ala, Cys<br />
within cell culture c<br />
both d<br />
remains stable<br />
SLC15a3 His between cell lines b<br />
SLC43a1 Leu, Ile, Met, Phe low a SLC15a4 His between cell lines b<br />
SLC43a2 Leu, Ile, Met, Phe between cell lines b Heavy subunits of<br />
hetero-meric<br />
SLC3a1<br />
various based on<br />
“partner”<br />
SLC43a3 putative between cell lines b SLC3a2 various based on<br />
“partner”<br />
N SLC38a3 Ala, Asn, Gln, His not checked Not in a system SLC6a7 Pro not checked<br />
SLC38a5 Gln, Asn, His, Ser both d SLC6a17 neutral a.a. not checked<br />
SLC7a13 Asp, Glu not checked<br />
SLC12A8 putative not checked<br />
The “Expression/Type of regulation” column refers to our results for the CHO cell lines described in the materials & methods section: a low levels-refers to<br />
fractional copies per cell; b regulation between cell lines-refers to regulation significantly higher than two fold at least at a time point between the different cell<br />
lines presented; c regulation within cell culture-refers to differential expression (significantly higher than two fold) at least at a time point within cell culture of a<br />
given cell line; d both types of regulation-refers to a gene presenting both b and c as discussed previously.<br />
low a<br />
both d<br />
subject to different expression profiles among mammalian cells and are<br />
grouped into more than 18 systems, based on sequence homology and<br />
function.<br />
To our knowledge, there is no comprehensive study of a.a. transporters in<br />
industri<strong>all</strong>y relevant CHO cells in the literature. To that direction, a.a.<br />
transporter genes were profiled during batch culture of three CHO cell lines<br />
with varying levels of productivity. In par<strong>all</strong>el, the intra- and extracellular<br />
levels of a.a. were quantified.<br />
Materials and methods: Three cell lines were kindly donated by Lonza<br />
Biologics. GSn8 cell line was transfected with an empty glutamine synthetase<br />
(GS) vector. GS35 and GS46 cell lines were both transfected with a GS vector<br />
that also carries the heavy and light chains of a chimeric IgG4 antibody. The<br />
specific productivity of cell line GS46, quantified by a commercial ELISA kit<br />
(Bethyl laboratories, US), is approximately double that of GS35 one.<br />
Batch cultures were performed in triplicate in 1L Erlenmeyer flasks with a<br />
working volume of 300mL in CD-CHO medium (Invitrogen, UK) supplemented<br />
with 25 μM MSX (Sigma, UK). Viable cell concentration was<br />
determined daily using the trypan blue dye exclusion method.<br />
40 a.a. transporters were studied in <strong>all</strong> cell lines using real time<br />
quantitative reverse transcription polymerasechainreactiononsamples<br />
from different phases of batch culture. Samples were collected at day 4<br />
(exponential phase) and day 6 & day 7 (stationary phase) of the growth<br />
curve for <strong>all</strong> cell lines (samples were also taken at day 3 for IgG4 producers<br />
only and day 9 for the null cell line only). Results are reported against the<br />
housekeeping gene “actb”. Housekeeping genes “vezt” and “hirip3” were<br />
also well correlated.<br />
The extracellular and intracellular a.a. profiles were monitored daily using<br />
high performance liquid chromatography (PicoTag, Waters, UK). Intracellular
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samples were quenched with 0.9% w/v NaCl and extracted with a 50%<br />
aqueous acetonitrile solution, as described in [3].<br />
Results: The results (Table 1) reveal that ~30% of transporters are lowly<br />
expressed (fractional copies per cell), 9% are below levels of detection,<br />
whereas 40% are significantly differenti<strong>all</strong>y expressed either during batch<br />
cell culture, or between cell lines, or both. The remaining transporters<br />
appear to remain stable.<br />
Regulation within culture: The majority of the transporters are found to<br />
be upregulated at stationary phase for <strong>all</strong> cell lines, as also presented in<br />
Figure 1, where a mapping of a.a. metabolism and transport has been<br />
illustrated for the null cell line. Specific<strong>all</strong>y, five genes encoding for<br />
transporters of a.a. relating to the glutathione (GSH) pathway were found<br />
to be upregulated significantly higher than 2 fold at stationary phase,<br />
when compared to exponential phase for <strong>all</strong> cell lines. These genes were:<br />
slc1a4 (Ala and Cys), slc6a9 (Gly), slc1a2 (Glu and Asp), slc7a11 (Cystine and<br />
Glu), and heteromeric transporter slc3a2 which partners with slc7a11. GSH<br />
is a well-known marker of oxidative stress [4], high levels of which have<br />
been associated with high productivity [5].<br />
Regulation between cell lines: In their majority, genes were found to be<br />
upregulated for protein producing cell lines at <strong>all</strong> time points. Genes whose<br />
expression is upregulated significantly (two-fold or higher) in the proteinproducers<br />
at <strong>all</strong> time points analyzed were: slc43a2 (system L, leucine and<br />
branched-chain a.a.) and slc1a2 (system X - AG, glutamate and aspartate).<br />
However, no genes, apart from slc6a6 (taurine and b-Ala), were found to be<br />
differenti<strong>all</strong>y expressed between high (GS46) and low producer (GS35). We<br />
find slc6a6 gene differenti<strong>all</strong>y expressed early in cell culture (day 3), which<br />
makesushypothesizethatthegenecould be a candidate for selection<br />
purposes. The overexpression of this gene in CHO cells has been found to<br />
significantly enhance growth and productivity [6].<br />
Feeding strategy based on order of feeding: The a.a. transporters gene<br />
expression findings correlate well with the extracellular and intracellular<br />
concentration profiles of their respective substrates (Figure 1). By analysing<br />
the differenti<strong>all</strong>y expressed genes for a specific cell line a feeding strategy<br />
can be designed. For example, we find transporter slc7a5, of system L,<br />
highly upregulated at stationary phase for the null cell line (Figure 1). This<br />
transporter exchanges an intracellular neutral a.a. with an extracellular<br />
Figure 1(abstract P97) A map associating the differenti<strong>all</strong>y expressed amino acid transporters for the null cell line, their amino acid substrates,<br />
and the intracellular concentrations (femtomol/cell, in the area designated by the “IN” tag) and extracellular concentrations (mM, in the area<br />
designated by the “OUT” tag) of the latter. A.a. transport is highlighted by the black box. The expression of the mRNA levels of the differenti<strong>all</strong>y<br />
expressed a.a. transporters (in mRNA copies per cell) at different phases of cell culture, exponential (day 4), stationary (days 6 & 7), and decline (day 9) is<br />
displayed at the bottom, where stationary phase samples are averaged, since not statistic<strong>all</strong>y different (for ease of statistical analysis visualization). The<br />
relevant energy utilisation mechanisms of each system are also depicted (top). Genes: slc6a9 (glycine), slc1a2 (acidic a.a.), slc7a7 (basic and branched chain<br />
a.a.) and its heteromeric transporter slc3a2 were also found to be differenti<strong>all</strong>y expressed, but are not presented in this figure. Our chosen a.a. analysis<br />
method was not able to quantify cysteine (L-Cys) levels.
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branched chain one (isoleucine, leucine, valine). Branched chain amino acids<br />
are associated with the mTor sign<strong>all</strong>ing pathway, essential regulator for<br />
many physiological roles in mammalian cells [7]. Hence, a feeding strategy<br />
can be proposed, where neutral amino acids are fed first and followed by<br />
branched chain amino acids, in order for them to be more effectively<br />
uptaken. A similar type of pre-conditioning was found to significantly<br />
enhance cellular protein production in another type of mammalian cells [7].<br />
Conclusions: Glutathione pathway associated a.a. transporters (slc1a2,<br />
slc1a4, slc6a9, slc7a11/slc3a2) can be targeted as genetic engineering<br />
targets, since are <strong>all</strong> found highly upregulated at stationary phase of cell<br />
culture. Addition<strong>all</strong>y, transporters slc1a2, slc43a2 are associated with rprotein<br />
productivity, since <strong>all</strong> of them are found to be upregulated for producing<br />
cell lines vs the null. Gene slc6a6, carrying taurine and b-alanine, can be<br />
associated with high productivity (as also suggested in [6]), as was also<br />
found to be differenti<strong>all</strong>y expressed in the high vs the low producer early in<br />
cell culture. A feeding strategy can be proposed, based on our results that<br />
remains to be tested experiment<strong>all</strong>y. Fin<strong>all</strong>y, extending this integrative<br />
approach to the proteome level would help link regulation at the<br />
transcriptomic level to actual differences in transport capability.<br />
Acknowledgements: S.K. would like to thank EPSRC & iChemE for<br />
financialsupport.K.P.wouldliketothankRCUKandC.K.thanksRCUK&<br />
Lonza Biologics for their Fellowships.<br />
References<br />
1. Kyriakopoulos S, Kontoravdi C: Analysis of the landscape of biologic<strong>all</strong>yderived<br />
pharmaceuticals in Europe: Dominant production systems,<br />
molecule types on the rise and approval trends. European journal of<br />
pharmaceutical sciences: official journal of the European Federation for<br />
Pharmaceutical Sciences 2012, 48:428-441.<br />
2. Hediger MA, Romero MF, Peng JB, Rolfs A, Takanaga H, Bruford EA: The<br />
ABCs of solute carriers: physiological, pathological and therapeutic<br />
implications of human membrane transport proteins - Introduction. Pflug<br />
Arch Eur J Phy 2004, 447:465-468.<br />
3. Dietmair S, Timmins NE, Gray PP, Nielsen LK, Kromer JO: Towards<br />
quantitative metabolomics of mammalian cells: Development of a<br />
metabolite extraction protocol. Anal Biochem 2010, 404:155-164.<br />
4. Selvarasu S, Ho YS, Chong WPK, Wong NSC, Yusufi FNK, Lee YY, Yap MGS,<br />
Lee DY: Combined in silico modeling and metabolomics analysis to<br />
characterize fed-batch CHO cell culture. Biotechnol Bioeng 2012,<br />
109:1415-1429.<br />
5. Chong WP, Thng SH, Hiu AP, Lee DY, Chan EC, Ho YS: LC-MS-based<br />
metabolic characterization of high monoclonal antibody-producing<br />
Chinese hamster ovary cells. Biotechnol Bioeng 2012, 109:3103-3111.<br />
6. Tabuchi H, Sugiyama T, Tanaka S, Tainaka S: Overexpression of Taurine<br />
Transporter in Chinese Hamster Ovary Cells Can Enhance Cell Viability<br />
and Product Yield, While Promoting Glutamine Consumption. Biotechnol<br />
Bioeng 2010, 107:998-1003.<br />
7. Nicklin P, Bergman P, Zhang BL, Triantafellow E, Wang H, Nyfeler B,<br />
Yang HD, Hild M, Kung C, Wilson C, et al: Bidirectional Transport of Amino<br />
Acids Regulates mTOR and Autophagy. Cell 2009, 136:521-534.<br />
P98<br />
Optimized platform medium and feed for rCHO cell lines using the<br />
CHEF1® expression system<br />
William Paul 1* , Raymond Davis 2 , Andrew Campbell 1 , Sarah Terkildsen 2 ,<br />
Vann Brasher 2 , James Powell 2 , Blake Engelbert 2 , Howard Clarke 2<br />
1 Life Technologies Corporation (PD-Direct® Bioprocess Services), 3175 Staley<br />
Road, Grand Island, NY, 14072 USA;<br />
2 CMC Biologics, 22021 20th Avenue SE,<br />
Bothell, WA, 98021 USA<br />
E-mail: william.paul@lifetech.com<br />
BMC Proceedings 2013, 7(Suppl 6):P98<br />
Chinese Hamster Ovary (CHO) cells are widely used in biomanufacturing and<br />
biomedical research to produce proteins of clinical significance. The<br />
environment the cells grow in to produce these proteins is complex and<br />
varies across the industry. One key variable in production processes is the<br />
cell culture medium used. Media can include chemic<strong>all</strong>y-defined components<br />
such as amino acids, vitamins, lipids, metal salts, and buffers. In addition,<br />
undefined components such as proteins, serum, or hydrolysates may be<br />
added. To reduce complexity, increase consistency, and comply with<br />
increasing demands from regulatory entities, chemic<strong>all</strong>y-defined formulations<br />
are preferred and can be developed and optimized for a given cell line.<br />
While a medium and feed can be optimized for every cell line/clone,<br />
developing a platform system provides a cost-effective option while ensuring<br />
a high level of growth and productivity.<br />
In this collaboration, between Life Technologies PD-Direct® and CMC<br />
Biologics, a single animal origin-free, hydrolysate-free base platform medium<br />
and three synergistic feed media were developed for use with recombinant<br />
CHO cell lines engineered using the CHEF1® expression system to produce<br />
monoclonal antibodies. The CHEF1 expression system utilizes regulatory<br />
domains from the Chinese hamster elongation factor 1 (EF1a) gene to drive<br />
production of heterologous proteins [1]. Serum-free, suspension adapted<br />
CHO DG44 cells were transfected with CHEF1 plasmids harboring 2 different<br />
IgG1 MAb genes and used as test cell lines to develop a platform feed<br />
system. A cell culture production platform system (CHEF1, base medium,<br />
feed media) was developed and optimized using two cell lines that were<br />
previously grown in an undefined culture system. The new platform growth<br />
system developed here, showed an average 1.6 fold improvement in titer<br />
for the two cell lines compared to the performance using the undefined<br />
culture system.<br />
Using Design of Experiment (DOE) methods, we performed a Feed<br />
Mixtures experiment and a 2-Level Categoric experiment in shake flasks<br />
(culture parameters are shown in Table 1). Cell counts and viabilities were<br />
determined using a Cedex AS20 automated cell counter (Innovatis Inc.).<br />
Product titer was measured by Protein A HPLC. Performance data from<br />
the Feed Mixtures experiment were analyzed using Design Expert® (Stat-<br />
Ease®). Select spent media samples from the best performing Feed<br />
Mixtures conditions were analyzed for glucose, amino acids and select<br />
water-soluble vitamins using immobilized enzyme (YSI Life Sciences),<br />
UPLC (Waters AccQ-Tag - reverse phase with UV detection) and HPLC<br />
(ion-pair reverse phase using a UV detection), respectively. The Feed<br />
mixtures data were used to calculate nutrient consumption rates, which<br />
in turn were used to develop 3 balanced feeds (at neutral pH). A separate<br />
Feed Supplement (at high pH) was designed to facilitate delivery of<br />
components that were needed at levels above solubility limits in a<br />
neutral solution. These feeds and the Feed Supplement were then tested<br />
in a 2-Level Categoric experiment, evaluating feed volume, feed schedule,<br />
and the feed supplement. Performance data from this experiment were<br />
analyzed using Design Expert. Select spent media samples from the best<br />
performing conditions were analyzed for glucose, amino acids and select<br />
water-soluble vitamins. These data demonstrated that the three feeds<br />
were balanced and, when the feed supplement was included, provided<br />
nutrients at levels sufficient for continued growth/productivity. The best<br />
performing feed system (balanced feed [BF1] and feed supplement [FS])<br />
was used in a bioreactor confirmatory experiment (culture parameters<br />
shown in Table 1). In addition, a day 0 feed was designed (BF5 - included<br />
recombinant growth factors) and tested in the bioreactor.<br />
Supplementing BF5 at 3% (v/v) prior to inoculation and feeding 4%BF1 on<br />
day4,5%onday6,3%onday8,2%onday10,and1%(v/v)ondays12<br />
and 14 and FS at 0.2% (v/v) on alternate days starting on day 3 provided<br />
an environment for both cell lines that resulted in productivity superior to<br />
the control condition; cell line #1 reached 1.1 gm/L (control = 0.5 gm/L)<br />
and cell line #2 reached 2.0 gm/L (control = 1.6 gm/L).<br />
Since the cost of dry format media is more economical than liquid media at<br />
GMP scale, the feeds were converted from liquid format to dry formats; BF1<br />
was converted to Advanced Granulated Technology (AGT) format and the<br />
Feed Supplement was converted to a dry powder media (DPM). Once<br />
hydrated, these feeds were tested to confirm equivalency, achieving similar<br />
growth and productivity patterns as their liquid counterparts. Addition<strong>all</strong>y,<br />
these feeds have been concentrated to reduce the dilution effect that many<br />
commercial feeds produce, resulting in an approximate 76% reduction in<br />
feed volume added over the life of the culture (Figure 1). This is a significant<br />
reduction in the volume of fluid requiring in-process handling and<br />
downstream processing; saving time, equipment, and money. This feed<br />
system development collaboration yielded a 112% improvement (over the<br />
control condition) in product titer for cell line #1 and a 25% improvement in<br />
product titer for cell line #2 (Figure 1). The base medium and the newly<br />
developed final feed system provide an animal origin-free, hydrolysate-free<br />
growth environment. For the purposes of many commercial cGMP<br />
processes, this culture system provides an economical solution. Addition of<br />
a proprietary undefined feed (CMC Biologics), prior to inoculation, on top of<br />
this balanced feed system has been shown to boost productivity by about<br />
15% over using just the balanced feed system. Next steps include evaluating<br />
protein quality and validating at production scale.
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Table 1(abstract P98) Culture Parameter Conditions and Set-Points<br />
Parameters<br />
Shake Flask/Culture Volume 125 mL vented Erlenmeyer 30 mL working volume<br />
Bioreactor/Culture Volume 3 L single-use CellReady bioreactor 2 L working volume<br />
Seeding Density<br />
5x10 5 viable cells/mL<br />
Temperature 37°C ± 0.5°C (days 0 - 4) 34°C ± 0.5°C (day 5 - end)<br />
CO 2 Level (Shake Flask) 6% ± 1% (days 0 - 4) 2% ± 1% (day 5 - end)<br />
pH (Bioreactor) 7.0 ± 0.2<br />
RPM (Shake Flask) 120 ± 5<br />
RPM (Bioreactor) 200 ± 10<br />
Dissolved Oxygen (Bioreactor) 60% ± 5%<br />
Reference<br />
1. Running Deer J, Allison DS: High-Level Expression of Proteins in<br />
Mammalian Cells Using Transcription Regulatory Sequences from the<br />
Chinese Hamster EF-1a Gene. Biotechnol 2004, 20:880-889, Prog.<br />
P99<br />
Profiling of glycosylation gene expression in CHO fed-batch cultures in<br />
response to glycosylation-enhancing medium components<br />
Ryan Boniface 1* , Jeoffrey Schageman 2 , Brian Sanderson 2 , Michael Gillmeister 1 ,<br />
Angel Varela-Rohena 1 , John Yan 3 , Yolanda Tennico 3 , Shawn Barrett 1 ,<br />
Robert Setterquist 2 , Stephen Gorfien 1<br />
1 Life Technologies Corporation, 3175 Staley Road, Grand Island, New York,<br />
USA, 14072;<br />
2 Life Technologies Corporation, 2130 Woodward, Austin, Texas,<br />
USA, 78744;<br />
3 Life Technologies Corporation, 29851 Willow Creek, Eugene,<br />
Oregon, USA, 97402<br />
E-mail: Ryan.Boniface@lifetech.com<br />
BMC Proceedings 2013, 7(Suppl 6):P99<br />
Introduction: Characterization of the glycosylation profile of a recombinant<br />
protein product is an important part of defining product quality in the<br />
bioproduction industry. Development of a protein with desired characteristics<br />
would require the capacity to modify and target specific glycosylation<br />
patterns as well as an understanding of the implications of changes to these<br />
glycosylation profiles. Previous cell culture studies have demonstrated the<br />
ability to modulate glycan profiles without negative impact to culture growth<br />
and product titer through the addition of glycosylation-enhancing medium<br />
components. With new methods, including increased measurement<br />
sensitivity and new capabilities in RNA-Seq technology, it is possible to<br />
develop a glycosylation gene expression profile for CHO cells. Specific<br />
glycosylation genes can then be tracked to ensure that the addition of these<br />
compounds will not negatively impact gene expression. Analyses comparing<br />
growth and titer, glycan distribution, and transcriptome differences can<br />
present us with potential insight into what changes are taking place on a<br />
genetic level in the cell in response to changes in medium and culture<br />
conditions.<br />
Materials and methods: (All Materials were from Life Technologies unless<br />
otherwise indicated)<br />
Cell culture: CHO-S® and DG44 derived recombinant cells expressing the<br />
same IgG molecule were grown in CD FortiCHO medium supplemented<br />
with 4mM L-glutamine and 1:100 Anti-Clumping Agent.<br />
Fed-batch bioreactor: DASGIP bioreactor with 500mL initial working<br />
volume seeded at 0.3x10 5 viable cells/ml in CD FortiCHO medium. 10% CD<br />
EfficientFeed C (EFC) feeding on days 3, 5 and 7 for CHO-S® cultures, and<br />
feedingondays4,6and8forDG44cultures. Glucose concentration was<br />
maintained above 3g/L. Component A and/or component B were added on<br />
thefirstdayoffeeding(day3forCHO-S®andday4forDG44cultures).<br />
Culture conditions were maintained as follows; pH 7.0 +/- 0.05, 50% DO, 37°C,<br />
110 rpm. Cell densities and viabilities were measured using a Vi-CELL®<br />
counter (Beckman Coulter). Metabolites (glucose, ammonia, lactate) and IgG<br />
were measured using a Cedex® Bio HT Instrument (Roche).<br />
Glycan analysis: Protein supernatant samples were collected and purified<br />
using POROS® MabCapture® A resin. Samples glycan profiles were analyzed<br />
on an Applied Biosystems® 3500 Series Genetic Analyzer.<br />
Transcriptome analysis: RNA was extracted at several time points during<br />
the culture. A total of 174 potential glycosylation specific gene targets were<br />
Figure 1(abstract P98) Summary of Optimization Collaboration - Improved titer by 112% (cell line #1), by 25% (cell line #2), and reduced volume<br />
fed by 76%.
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identified and primers designed to these using reference sequences from<br />
Chinese hamster ovary, mouse, rat and human. A total of 34 samples were<br />
multiplexed on a Proton PI chip on the Ion Torrent PGM.<br />
Results and discussion: The use of components A and B with CHO-S® cells<br />
in CD FortiCHO medium causes a considerable increase in the level of<br />
galactosylation of the recombinant IgG (Figure 1) as shown by the shift in<br />
the glycosylation profile from G0F to G1F and G2F. The use of targeted<br />
transcriptome analysis revealed that the changes observed in the<br />
glycosylation profile do not translate to noticeable differences in the<br />
expression levels of the glycosylation genes. There are changes in gene<br />
expression levels with culture age but they are not altered by the additions<br />
of components A and/or B. It was origin<strong>all</strong>y theorized that components A<br />
and B could act as cofactors or substrates within the glycosylation enzymatic<br />
pathways but this could not be confirmed without an understanding of the<br />
glycosylation gene profile. The changes in the glycosylation patterns<br />
combined with the absence of changes in the gene expression data lend<br />
support to this theory. With this information it is apparent that the additions<br />
of the glycosylation-enhancing components A and B can increase<br />
galactosylation of recombinant proteins with no negative effect on growth,<br />
titer or glycosylation gene expression.<br />
The comparison between CHO-S® and DG44 cultures without supplementation<br />
with components A or B revealed the DG44 culture had better<br />
galactosylation with increased proportions of G1F and G2F. Both cell lines<br />
express high levels of DDOST, RPN1, DAD1 and SST3A which are <strong>all</strong> part of<br />
the oligosaccharyltransferase complex which catalyzes the transfer of high<br />
mannose oligosaccharides from lipid-linked oligosaccharide donors to the<br />
asparagines on the Asn-X-Ser/Thr of the polypeptide chain. The DG44 cells<br />
differ from the CHO-S® cells with increases in: ALG2, ALG3, ALG9 and ALG12<br />
Figure 1(abstract P99) Glycan analysis data measured as the percentage of total glycans. (A) The glycan profile for the CHO-S® culture with no<br />
addition of components A and/or B. (B) These data indicate that the addition of component A to the culture results in very little change to the glycan<br />
profile, only slight increase in the percent of G1F on days 5 and 7. (C) The addition of component B to the culture shifts the glycan profile from primarily<br />
non-galactosylated G0F to increased G1F (single galactose) and G2F (two galactose) glycoforms. (D) The addition of both components A and B results in<br />
a change in galactosylation indicated by the increase in both G1F and G2F and an over<strong>all</strong> reduction of G0F. In every condition, G0F increases with time<br />
but this is minimized with the addition of both components A and B. The majority of protein glycoforms within this experiment are fucosylated and the<br />
addition of components A and/or B does not appear to alter this.
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(mannosyltransferases), ALG8 and ALG10 (glucosyltransferases), ALG14<br />
(acetylglucosaminyltransferase), and B4GALT5 (galactosyltransferase). These<br />
increases in gene expression in DG44 cells seem to coincide with the higher<br />
galactosylation profiles observed in the glycan analysis.<br />
Conclusions: Differences in growth, titer and glycoform distribution were<br />
observed between CHO-S® and CHO DG44 cells. DG44 cells had higher<br />
expression of glycosylation transferase genes compared to CHO-S® cells.<br />
Components A and B had synergistic effects on terminal galactosylation<br />
(Figure 1), showed no changes in gene expression and could be acting as<br />
cofactors/substrates with glycosylation enzymes.<br />
Acknowledgements: The Austin team (Natalie Hernandez, Laura<br />
Chapman, Angie Cheng, Lea Kristi and Daniel Williams) for library<br />
preparation and transcriptome analysis.<br />
P100<br />
How to assess chemic<strong>all</strong>y defined media and feeds from 9 suppliers on<br />
CHO cells producing mAb<br />
Aurore Polès-Lahille * , Margaux Paillet, Aurélie Da Silva, Nora Kadi, Eric Basque,<br />
Flavien Thuet, David Balbuena, Sébastien Ribault<br />
Merck Biodevelopment, Martillac, France, 33650<br />
E-mail: aurore.lahille@merckgroup.com<br />
BMC Proceedings 2013, 7(Suppl 6):P100<br />
Introduction: Mammalian cell culture medium development has widely<br />
evolved in recent years. The use of hydrolysates as serum replacement<br />
has led to process variability due to lot-to-lot variations. The undefined<br />
composition of these media could also increase the process optimization<br />
timelines, sometimes with limited impact on process performances. With<br />
the reduction of process development activities for preclinical and Phase I<br />
studies, medium and feed platforms raised. The objective of the media<br />
was to ensure cell growth only in order to go as fast as possible to<br />
production bioreactors while the feeds were responsible for productivity<br />
and production length. Either companies spent several months if not<br />
years to develop their own generic medium and feed platforms or they<br />
used commercial ones, sometimes under licenses. The medium and feed<br />
platform assessment also started earlier in the product development<br />
process. Clone screening was performed more and more in fed-batch<br />
conditions rather than batch ones. Thus screening tools, scale-down<br />
models of bioreactors, with lower and lower working volumes were<br />
designed. Another cell culture process evolution was the development of<br />
new expression systems without any selection agents. In order to assess<br />
our screening scale-down model, between 20 to 35 chemic<strong>all</strong>y defined<br />
platforms from 9 suppliers were screened with 3 CHO host cell lines/<br />
expression systems.<br />
Methods: The following protocol was followed for 3 different CHO cell<br />
lines producing mAb:<br />
- CHO host cell 1 - expression system n°1 : mAb I<br />
- CHO host cell 1 - expression system n°2 : mAb II<br />
- CHO host cell 2 - expression system n°3 : mAb III<br />
Each medium was prepared, supplemented according to cell requirements,<br />
0.2 μm PVDF filtrated and stored into at least 2 separated bottles. A sterility<br />
test was performed on each bottle before use. Each cell line was thawed<br />
and amplified during at least one week in its usual medium. Then the cells<br />
were adapted to each medium for at least 8 passages in either 125 mL<br />
shake flasks or 50 mL spin tubes in duplicate. Each media was preheated at<br />
37°C before use and one bottle was used per duplicate in order to reduce<br />
contamination risk. After cell adaptation, fed-batch platform assessment was<br />
performed in 50 mL spin tubes at 37°C with a seeding density around 0.25 *<br />
10 6 viable cells/mL. Every 2 to 3 days, samples were taken to measure pH,<br />
pO 2 ,pCO 2 , viable cell density, viability, glucose and lactate levels. The<br />
feeding strategy applied was the same for each cell line and agreed with<br />
each supplier. The cultures were stopped when the viability was below 60%<br />
or after 16-17 days.<br />
Results: The objective of cell culture media is to sustain cell growth in order<br />
to quickly seed the production bioreactor. Here are the doubling times<br />
measured on the 3 cell lines (Table 1).<br />
Despite having the same host cell, cell growth was different between<br />
mAb I and mAb II. The expression system could have a significant impact<br />
Table 1(abstract P100) Doubling time of each cell line in<br />
each medium after adaptation<br />
mAb I mAb II mAb III<br />
Cellvento CHO-200 medium 20 h 32 h 17 h<br />
Supplier A medium 1 23 h 35 h 23 h<br />
Supplier A medium 2 20 h 63 h 22 h<br />
Supplier A medium 3 20 h 24 h 18 h<br />
Supplier B medium 1 21 h 20 h 18 h<br />
Supplier B medium 2 24 h 66 h 24 h<br />
Supplier C medium 1 26 h 18 h 20 h<br />
Supplier C medium 2 22 h 18 h 18 h<br />
Supplier D medium 1 21 h 19 h 18 h<br />
Supplier E medium 1 26 h 35 h 28 h<br />
Supplier E medium 2 23 h 20 h 19 h<br />
Supplier F medium 1 21 h 26 h 17 h<br />
Supplier G medium 1 21 h 20 h 18 h<br />
Supplier G medium 2<br />
19 h<br />
Supplier G medium 3<br />
21 h<br />
Supplier G medium 4<br />
19 h<br />
Supplier H medium 1<br />
21 h<br />
Supplier H medium 2<br />
21 h<br />
on cell growth behavior. In order to separate the different platform<br />
results, a color was assigned to each supplier and platform assessed<br />
(Figure 1).<br />
Depending on the CHO host cell and the expression system, each platform<br />
had different performances. Some platforms seemed to be more robust<br />
than others in terms of final titer. The lactate metabolism was also<br />
compared between the different platforms. Most of the platforms had a<br />
maximum lactate concentration measured around 1 - 1.5 g/L. Some<br />
platforms went above 2 g/L of lactate, which could be difficult to scale-up<br />
in bioreactors. The practical aspect was also studied as it can facilitate the<br />
implementation and the tech transfer. Some platforms assessed had 2<br />
feeds added everyday while others only had 1 feed added 3 times.<br />
Molecule quality was also compared between platforms in terms of High<br />
Molecule Weight and cIEF.<br />
Conclusions: We have implemented a strong protocol for medium and<br />
feed screening with up to 70 spin tubes manipulated in par<strong>all</strong>el. More than<br />
3000 sterile manipulations were performed under a laminar flow without<br />
any contaminations. These experiments <strong>all</strong>ow us to define robust platforms<br />
in terms of cell growth, productivity and metabolism on different CHO host<br />
cell lines and expression systems.<br />
P101<br />
Evaluation of single-use bioreactors for perfusion processes<br />
Aurore Polès-Lahille * , Flavien Thuet, David Balbuena, Sébastien Ribault<br />
Merck Biodevelopment, Martillac, France, 33650<br />
E-mail: aurore.lahille@merckgroup.com<br />
BMC Proceedings 2013, 7(Suppl 6):P101<br />
Introduction: Single-Use Bioreactors are now commonly used for Process<br />
Development activities, as seeding bioreactors or to produce Drug<br />
Substances. The advantages of this equipment have been well demonstrated<br />
over the last years on batch/fed-batch processes. Continuous<br />
processes were widely applied in the past to increase the over<strong>all</strong><br />
productivity of sm<strong>all</strong> bioreactors or for sensitive molecule production. The<br />
process control, contamination risk and complexity were the main concerns<br />
of this operation mode. However, the bioprocessing trends and technology<br />
evolution led to reconsidering the perfusion processes. The aim of this study
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Figure 1(abstract P100) Colors assigned to each supplier. Final mAb I (top right side), mAb II (bottom left side) and mab III (bottom right side) titers<br />
for <strong>all</strong> platforms compared to Cellvento CHO-200 medium.<br />
was to combine standard single-use bioreactors with different perfusion<br />
technologies and to compare productivity and molecule quality.<br />
Methods: A CHO cell line producing a mAb was thawed and amplified in<br />
shake flasks using Cellvento CHO-100 medium. When a sufficient<br />
amount of cells was reached, 2 Mobius® CellReady 3L bioreactors were<br />
launched in par<strong>all</strong>el: one in batch mode and one in perfusion mode<br />
using Cellvento CHO-100 medium. Two perfusion technologies were<br />
assessed: the Fibra-Cel® Disks (Eppendorf) and the Alternative Tangential<br />
Flow (Refine) ones. The Mobius® CellReady 3L bioreactor was not<br />
modified to perform perfusion processes aseptic<strong>all</strong>y transferred into a<br />
Mobius® CellReady 3L bioreactor through the probe port. Regarding the<br />
ATF technology, an ATF-2 system was first washed with water then<br />
autoclaved and welded to the harvest line of a Mobius® CellReady 3L<br />
bioreactor. The bioreactor conditions were 37°C with pH maintained<br />
between 6.80- and 7.10. The Dissolved Oxygen set point was 50% and<br />
stirrer speed 104 rpm. The viable cell density, viability, metabolism and<br />
titers were measured at least daily. The perfusion was initiated at 0.5 vvm<br />
when the lactate was above 0.5 g/L and increased daily based on glucose<br />
and lactate levels up to 1 vvm for the Fibra-Cel ® technology and up to<br />
2 vvm for the ATF one. In order to increase the oxygen transfer at high<br />
cell density, a decision tree was applied. For the Fibra-Cel® technology,<br />
the mAb was collected in harvest bags welded to a side port while for<br />
the ATF, the molecule remained inside the Mobius® CellReady 3L<br />
bioreactor with the use of a 50 kDa hollow fiber. In order to measure the<br />
quality of the mAb produced, samples were collected on day 7, day<br />
10 and the last bioreactor day. Titers and HCP levels were directly<br />
measured on harvest while SE-HPLC and cIEF were performed on ProSep®<br />
Ultra Plus eluates.<br />
Results: As expected, the cells grew on Fibra-Cel® Disks after 2 days. Thus<br />
only a few cells were in suspension from day 3 to day 14 (end of the<br />
bioreactor). Regarding the ATF technology, a maximum cell density of<br />
33 millions cells/mL was reached (Figure 1).<br />
The glucose concentration was well maintained between 5 and 6,5 g/L<br />
while the lactate was not above 1.5 g/L in perfusion bioreactors. A steady<br />
state was maintained over several days. The global productivity of each<br />
process mode was calculated and compared to the batch one. The<br />
perfusion technologies increased the mAb quantity obtained compared to<br />
abatchmode.TheATF technology increased the final mAb titer by 2.9<br />
fold and the Fibra-Cel® technology increased the mAb quantity by 1.2 fold<br />
(Table 1).<br />
The quality attributes of the mAb obtained in batch and perfusion modes<br />
were also compared. The molecule produced during the perfusion processes<br />
was more acid than the ones produced in batch and fed-batch modes.<br />
Therefore the mAb produced with Fibra-Cel® and ATF technologies in<br />
Mobius® CellReady 3L bioreactor could have a higher half-life than the<br />
molecule produced in batch and fed-batch modes. Regarding the Host Cell<br />
Proteins, Low Molecular Weight and High Molecular Weight over<strong>all</strong> contents,<br />
the ATF technology generates more contaminants while the Fibra-Cel®<br />
reduces them compared to a batch process (Table 1). Fin<strong>all</strong>y, the upstream<br />
cost to reach the ATF quantity was compared between batch and<br />
perfusion processes at different scales. The ATF technology can reduce<br />
process cost in disposable bioreactors whatever the scale compared to the<br />
batch mode while the Fibra-Cel® process cost is higher due to higher<br />
medium quantity necessary (Table 1).<br />
Conclusions: Without any modification of the Mobius® CellReady 3L<br />
bioreactor, we were able to demonstrate the compatibility of this single use<br />
bioreactor to a mAb perfusion process. Using two different technologies, the<br />
over<strong>all</strong> performances, molecule quality, contaminant level and cost were<br />
compared. This study demonstrates the flexibility of existing disposable<br />
bioreactors to new bioprocessing technologies.
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Figure 1(abstract P101) Viable cell density in suspension in batch and perfusion processes measured in Mobius® CellReady 3L.<br />
P102<br />
Profiling and engineering of microRNAs for enhancing recombinant<br />
protein productivity in Chinese hamster ovary cells<br />
Wan Ping Loh 1* , Bernard Loo 1 , Lihan Zhou 2 , Peiqing Zhang 1 , Dong Yup Lee 1,2<br />
, Yuan Sheng Yang 1 , Kong Peng Lam 1<br />
1 Bioprocessing Technology Institute, 20 Biopolis Way, #06-01 Centros,<br />
Singapore 138668;<br />
2 Department of Biochemistry, National University of<br />
Singapore, 8 Medical Drive 4, Blk MD7 #05-04, Singapore 117597<br />
BMC Proceedings 2013, 7(Suppl 6):P102<br />
Background: Chinese hamster ovary (CHO) cells have become dominant<br />
host cells in the biopharmaceutical industry due to their capacity for<br />
proper protein folding, assembly and post-translational modifications.<br />
However, low specific productivity (qp) places limitations on yields<br />
obtained from mammalian host cells. MicroRNAs (miRNAs), a novel class of<br />
short, non-coding RNAs which negatively regulate target gene expression<br />
at post-transcriptional levels, have emerged as promising targets for<br />
engineering of CHO cell factories to enhance recombinant protein<br />
production. While engineering of miRNAs for enhanced cell growth and<br />
delayed cell death have been reported, miRNA targets which can enhance<br />
qp have not been identified to date.<br />
Materials and methods: To understand the role of miRNAs in conferring<br />
high qp phenotype in CHO cells, we carried out high throughput<br />
sequencing of 4 in-house generated IgG-expressing CHO sub-clones of<br />
varying qps. Reads were mapped to miRBase and 22 miRNAs were found to<br />
be differenti<strong>all</strong>y expressed between the high and low producers. These<br />
miRNAs were stably transfected into an IgG-expressing sub-clone to assess<br />
their effects on growth, titer, qp and product quality attributes.<br />
Results: Over-expression of miRs-17, 19b, 20a and 92a individu<strong>all</strong>y and in<br />
combination resulted in 13-27% increases in titer and 14-24% increases in<br />
qp in stably transfected pools. No significant alterations in proliferation rates<br />
were observed. 20-45 single cell clones were randomly selected from each<br />
of the 5 transfected pools for characterization. Statistical analyses showed<br />
significant differences in titer/qp between the high- and low-miRNA<br />
expressing single cell clones. The highest producing single cell clones<br />
exhibited ~100% increases in titer and qp compared to non-transfected<br />
cells. A correlation was found between increased miR-19b levels (>1.3-fold)<br />
and enhanced qp and titer. Over-expression of miR-19b does not appear to<br />
impact IgG aggregation significantly.<br />
Table 1(abstract P101) Global productivity, Host cell Proteins, High Molecular Weight and Low Molecular Weight contents<br />
in perfusion processes compared to batch ones reached in Mobius® CellReady 3L bioreactor in addition to upstream cost<br />
to reach ATF mAb quantity, in perfusion processes compared to batch one in Mobius® CellReady Family<br />
Batch mode Fibra-Cel® technology ATF technology<br />
Final Titer 100% 121% 290%<br />
Host Cell Proteins 100% 28% 144%<br />
High Molecular Weight 100% 87% 198%<br />
Low Molecular Weight 100% 68% 107%<br />
Upstream cost at 3L GLP Scale 100% 108% 47%<br />
Upstream cost at 50L GLP Scale 100% 175% 84%<br />
Upstream cost at 200L GMP Scale 100% 134% 47%
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Conclusions: To our knowledge, this is the first report of enhancement<br />
of recombinant protein productivity by stable miRNA over-expression.<br />
The genes and cellular pathways targeted by these miRNAs specific to<br />
enhancing protein productivity are under investigation and will be<br />
reported.<br />
Acknowledgements: This work was supported by the Biomedical<br />
Research Council/Science and Engineering Research Council of A*STAR<br />
(Agency for Science, Technology and Research), Singapore. The authors<br />
would like to thank Faraaz Noor Khan Yusufi, Ju Xin Chin for their<br />
assistance in processing of next-generation sequencing data, and Corrine<br />
Wan, Gavin Teo, Daniel Chew, Lyn Chiin Sim, Ce Huang Poo and Kong<br />
Meng Hoi for their technical assistance in IgG purification, aggregation<br />
and glycosylation analyses.<br />
P103<br />
Designing clinical-grade integrated strategies for the downstream<br />
processing of human mesenchymal stem cells<br />
Bárbara Cunha 1,2 , Margarida Serra 1,2 , Cristina Peixoto 1,2 , Marta Silva 1,2 ,<br />
Manuel Carrondo 2,3 , Paula Alves 1,2*<br />
1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa,<br />
Av. da República, 2780-157 Oeiras, Portugal;<br />
2 iBET, Instituto de Biologia<br />
Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal;<br />
3 Departamento de Química, Faculdade de Ciências e Tecnologia,<br />
Universidade Nova de Lisboa, 2829-516 Monte da Caparica, Portugal<br />
E-mail: marques@itqb.unl.pt<br />
BMC Proceedings 2013, 7(Suppl 6):P103<br />
Background: During the past decade, human stem cells have been the<br />
focus of an increased interest due to their potential in clinical applications,<br />
as a therapeutic alternative for several diseases. Within this context, human<br />
mesenchymal stem cells (hMSCs) have gained special attention due to<br />
their immune-modulatory characteristics, as well as in secreting bioactive<br />
molecules with anti-inflammatory and regenerative features [1].<br />
In order to face the high demands of hMSCs (from 10 5 to 10 9 cells<br />
per patient) [2] to be used in therapies, the establishment of robust<br />
manufacturing platforms that can ensure the efficient production,<br />
purification and formulation of stem cell-based products is still a ch<strong>all</strong>enge.<br />
Although substantial efforts have been performed on the development of<br />
clinical-grade bioprocesses for the expansion of hMSCs in microcarrier-based<br />
stirred culture systems, the incorporation of downstream strategies that<br />
assure efficient cell-bead separation and consequent hMSC concentration<br />
(i.e. volume reduction) and washing is required to deliver safe hMSCs to the<br />
clinic [3,4].<br />
Therefore, the main aim of this work was the design of integrated<br />
methodologies (filtration and membrane technology approaches) [5] for the<br />
robust and clinical-grade downstream processing of hMSC.<br />
Materials and methods: Cell culture: hMSCs (STEMCELL Technologies)<br />
were cultivated in IMDM supplemented with 10% of fetal bovine serum<br />
(FBS) or in MesenCult®-XF Medium (STEMCELL Technologies) supplemented<br />
with 2 mM L-glutamine (Life Sciences) at 37°C in a humidified<br />
atmosphere of 5% CO 2 , according to manufacture recommendations. These<br />
cells were routinely propagated in static conditions (T-flasks) or on<br />
microcarriers (SoloHill Engineering, Inc) using stirred culture systems<br />
(spinner vessels and bioreactors). Cell concentration and viability were<br />
determined by counting the cells in a hemacytometer using the standard<br />
trypan blue exclusion method.<br />
Cell characterization and quality control tests: Standard procedures for the<br />
analysis of cell surface markers (CD90, CD73, CD45, CD34) using flow<br />
cytometry tools, as well as cell-based assays for the evaluation of cell<br />
proliferation capacity (CFU assay - colony-forming unit) and differentiation<br />
potential (differentiation into osteoblasts and adipocytes) were performed,<br />
following the manufacturer’s recommendations.<br />
Downstream processing: After harvesting, the microcarriers were removed<br />
from the cell suspension using nylon filters (Millipore) with different pore<br />
sizes (100, 80 and 30 μm). The clarified cell-based materials were<br />
concentrated by tangential flow filtration (TFF) using polysulfone hollowfiber<br />
cartridges with 0.45 μm pore size.<br />
Results: Over the past years, as scale-up platforms for the biomanufacturing<br />
of hMSCs become robust enough to yield high cell quantities to support<br />
cell-based therapies, culture media supplemented with FBS are becoming<br />
less used. This requirement is in line with what is advised by regulatory<br />
agencies, due to the main drawbacks associated to the use of FBS, such as<br />
the variability between different lots and suppliers and the risk of<br />
contamination with animal pathogens, which may trigger an immune<br />
response upon MSC therapy [5]. Within this context, large efforts have been<br />
made towards the development of serum- and xeno- free culture medium<br />
formulations for the expansion of hMSC. Thus, on a first approach, we<br />
evaluated the feasibility of propagating hMSCs in a serum- and xeno-free<br />
culture medium, the MesenCult®-XF medium, and further compared cell<br />
growth profile with standard medium formulation (e.g. IMDM + 10% FBS).<br />
Our results showed that, hMSC can be successfully expanded in MesenCult®-<br />
XF medium, presenting a constant population doubling length (PDL) of<br />
approximately 2 in each cell passage and a cumulative PDL of 12.5 in a total<br />
of 42 days (Figure 1A). Moreover, hMSCs showed an accelerated cell growth<br />
and increased lifespan when compared to the hMSC cultivated in standard<br />
culture medium supplemented with FBS where hMSCs presented limited<br />
proliferation capacity (Figure 1A). This was an expected outcome since<br />
MesenCult®-XF medium was design to enhance hMSC expansion from<br />
primary human bone marrow and cultured-expanded cells, leading to longterm<br />
cultures. It is important to mention that hMSCs maintained<br />
their characteristics after expansion in MesenCult®-XF medium, namely<br />
immunophenotype, proliferation capacity and multipotency (results not<br />
shown).<br />
After the expansion of hMSCs in microcarrier-based stirred culture systems,<br />
different downstream strategies were evaluated for the purification of<br />
hMSCs. First, the clarification step was carried out to remove the<br />
microcarriers from the cell suspension. For this purpose, nylon filters were<br />
used and the effect of the mesh pore size on cell recovery yields and<br />
viability was evaluated. Our results showed that nylon is a suitable material<br />
for the clarification step since it ensured efficient removal of microcarriers<br />
(no beads were detected after filtration processing) without compromising<br />
cell viability (Figure 1B). Moreover, we demonstrated that higher mesh<br />
pore sizes yielded higher cell recoveries (Figure 1B).<br />
For the cell concentration and volume reduction step, preliminary<br />
experiments were performed with human foreskin fibroblasts (Figure 1C).<br />
With this cellular system, a concentration factor (in volume) of 10 times was<br />
successfully achieved using TFF processes, yielding 70-80% of recovered<br />
cells with high viabilities (Figure 1C). Process validation with hMSCs is<br />
ongoing but first results were encouraging since we were able to<br />
concentrate 2 times hMSCs while ensuring high cell recovery yields (96%)<br />
and viabilities (98%) (Figure 1C). In addition, hMSCs maintained their<br />
immunophenotype, as well as their proliferation capacity and multilineage<br />
differentiation potential at the end of <strong>all</strong> steps of the downstream process<br />
(results not shown).<br />
Conclusions: While upstream technologies mature to meet the increasing<br />
demand of hMSCs, biomanufacturing bottlenecks are now shifting towards<br />
the downstream processing of stem cells. This work shows our first<br />
approach to tackle such bottlenecks. More specific<strong>all</strong>y, we demonstrate that<br />
standard filtration techniques and TFF systems are suitable and robust<br />
approaches for the downstream processing of hMSCs. Using these strategies<br />
we were able to ensure efficient removal of the major impurities of the<br />
cellular suspension (microcarriers) and further concentrate cell-based<br />
products up to 10 times without compromising their viability and quality.<br />
However, further improvements in cell concentration and polishing steps<br />
are still required. Nonetheless, this work provides important insights towards<br />
the establishment of robust and clinical-grade bioprocesses for the<br />
purification of hMSCs to be integrated and applied in the biomanufacturing<br />
of cell-based therapies.<br />
Acknowledgements: The authors acknowledge the NanoGene project<br />
(EuroNanoMed ERA-Net initiative) and the project EXPL/BBB-EBI/1003/2012 -<br />
“Development of a scalable strategy for stem cells purification” funded by<br />
Fundação para a Ciência e Tecnologia (FCT) for financial support, as well as<br />
MIT-Portugal program and FCT for the grant SFRH/BD/51940/2012.<br />
References<br />
1. Patel A, Genovese J: Potential clinical applications of adult human<br />
mesenchymal stem cell (Prochymal®) therapy. Stem Cells and Cloning:<br />
Advances and Applications 2011, 4:61-72.<br />
2. Chen A, Reuveny S, Oh S: Application of human mesenchymal and<br />
pluripotent stem cell microcarrier cultures in cellular therapy:<br />
Achievements and future direction. Biotechnology Advances 2013 in press.<br />
3. Serra M, Brito C, Correia C, Alves PM: Process engineering of human<br />
pluripotent stem cells for clinical application. Trends in Biotechnol 2012,<br />
30:350-359.
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Page 137 of 151<br />
Figure 1(abstract P103) Up- and down- stream processing of hMSCs. A) Growth profile of hMSC culture; profile of cumulative PDLs of hMSC cultured<br />
in MesenCult®-XF (blue line) or in IMDM + 10% FBS (purple line) medium along culture time. B) Microcarriers’ removal using nylon filters with different<br />
pore sizes (100, 80 and 30 μm). The (blue) bars represent the cell recovery yields, while the (green) line represents cell viability. C) Major outcomes<br />
achieved after TFF processing of human foreskin fibroblasts and hMSCs.<br />
4. Pattasseril J, Varadaraju H, Lock L, Rowley J: Downstream Technology<br />
Landscape for Large-Scale Therapeutic Cell Processing. BioProcess<br />
International 2013, 11:46-52.<br />
5. Peixoto C, Ferreira T, Sousa M, Carrondo MJT, Alves PM: Towards<br />
purification of adenoviral vectors based on membrane technology.<br />
Biotechnol Prog 2008, 24:1290-1296.<br />
6. Spees J, Gregory C, Singh H, Tucker H, Peister A, Lynch P, Hsu S, Smith J,<br />
Prockop D: Internalized antigens must be removed to prepare<br />
hypoimmunogenic mesenchymal stem cells for cell and gene therapy.<br />
Mol Ther 2004, 9:747-756.<br />
P104<br />
Culture supplement extracted from rice bran for better serum-free<br />
culture<br />
Satoshi Terada 1* , Satoko Moriyama 1 , Ken Fukumoto 1 , Yui Okada 1 ,<br />
Rinaka Yamauchi 1 , Yoko Suzuki 1 , Masayuki Taniguchi 2 , Shigeru Moriyama 3 ,<br />
Takuo Tsuno 3<br />
1 Department of Applied Chemistry and Biotechnology, University of Fukui,<br />
Fukui, 910-8507, Japan;<br />
2 Niigata University, Niigata, 950-2102, Japan;<br />
3 Tsuno<br />
Food Industrial Co., Ltd, Katsuragi-cho, Wakayama, 649-7122, Japan<br />
E-mail: terada@u-fukui.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):P104<br />
Introduction: In mammalian cell culture, fetal bovine serum (FBS) and<br />
proteins including albumin (BSA) have been extensively added to culture<br />
media as growth factor. But mammal-derived factors are potent source of<br />
various infections such as abnormal prion and viruses, and so alternative<br />
supplement is eagerly required. The alternative must be chemic<strong>all</strong>y defined<br />
or obtained from plant, as well as should be produced in commercial<br />
quantities and stably supplied.<br />
As an alternative supplement, we focused on rice bran extract (RBE), byproduct<br />
of milling in the production of refined white rice, because rice bran<br />
contains abundant nutrients and proteins [1] as well as antioxidants [2] and<br />
because rice is cultivated plant, indicative of huge and stable supply.<br />
Materials and methods: Preparation of RBE: RBE was extracted in<br />
alkaline solution and then precipitated with acid. The precipitate was freezedried.<br />
Effect of RBE on the culture of various cell lines: Mitogenic activity of<br />
RBE was evaluated using cell lines. Cells were cultured in ASF104 medium<br />
with or without RBE for several days. Then viable cell densities were counted<br />
by trypan-blue method and concentration of MoAb was measured by ELISA.<br />
Effect of RBE on the culture of MSC: Mesenchymal stem cells (MSCs)<br />
were isolated from male Wistar rats and expanded in purchased serum-free<br />
medium or conventional medium containing FBS. The expanded cells were<br />
transferred to differentiation medium into bone. The differentiated cells to<br />
bone were readily stained. Triplicated culture.<br />
Effect of RBE on the culture of pancreatic islets: Pancreatic islets were<br />
obtained from male Lewis rats and cultured in RPMI medium supplemented<br />
with RBE or FBS for eight days.<br />
Results and discussion: Effect of RBE on the culture of various cell<br />
lines: On growth and MoAb production of hybridoma in serum-free<br />
medium, desired effects of RBE were observed and the effect was superior<br />
to BSA.<br />
Similarly, serum-free culture of CHO-DP12 added with RBE exhibited<br />
increased cell growth and production.<br />
Growth of HepG2 and HeLa cells in the serum-free medium was also<br />
improved.
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Together <strong>all</strong>, RBE had mitogenic activity on various cell lines.<br />
Effect of RBE on the culture of MSC: As primary cells, MSCs from Wistar<br />
rat were expanded in serum-free medium with RBE or without and then the<br />
medium was changed into osteoblast-inducing medium. While MSCs<br />
expanded in the serum-free medium lost it, the cells expanded in the<br />
presence of RBE retained the potency, suggesting that RBE contains<br />
physiologic<strong>all</strong>y active substances maintaining potency of differentiation<br />
during ex vivo serum-free culture.<br />
Effect of RBE on the culture of pancreatic islets: Pancreatic islets, isolated<br />
from Lewis rats, were also tested in the presence of RBE. While islets died<br />
out by one week in basal medium, islets successfully survived in the<br />
presence of RB. This result supports that RBE could alternate FBS in islets<br />
culture.<br />
Conclusion: RBE successfully improved the serum-free culture of four cell<br />
lines including hybridoma, CHO, HepG2 and HeLa, as well as primary<br />
culture of MSCs and pancreatic islets. These results indicate that RBE<br />
would be useful as culture supplement in serum-free media.<br />
References<br />
1. Adebiyi AP, Adebiyi AO, Hasegawa Y, Ogawa T, Muramoto K: Isolation and<br />
characterization of protein fractions from deoiled rice bran. European<br />
Food Research and Technology 2008, 10.<br />
2. Adebiyi AP, Adebiyi AO, Yamashita J, Ogawa T, Muramoto K: Purification and<br />
characterization of antioxidative peptides derived from rice bran protein<br />
hydrolysates. European Food Research and Technology 2008, 10.<br />
P105<br />
Cryopreservative solution using rakkyo fructan as cryoprotectant<br />
Satoshi Terada 1 , Shinya Mizui 1 , Yasuhito Chida 1 , Masafumi Shimizu 1 ,<br />
Akiko Ogawa 2* , Takeshi Ohura 3 , Kyo-ichi Kobayashi 3 , Saori Yasukawa 4 ,<br />
Nobuyuki Moriyama 4<br />
1 Department of Applied Chemistry and Biotechnology, University of Fukui,<br />
3-9-1 Bunkyo, Fukui, 910-8507, Japan;<br />
2 Department of Chemistry and<br />
Biochemistry, Suzuka National College of Technology, Shiroko-cho, Suzuka,<br />
510-0294, Japan;<br />
3 Fukui Prefectural Food Process, 1-1-1 Maruoka-chotubonouchi,<br />
Sakai, 910-0343, Japan;<br />
4 ELLE ROSE CO., Ltd., 4-200<br />
Saburoumaru, Fukui, 910-0033, Japan<br />
E-mail: ogawa@chem.suzuka-ct.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):P105<br />
Introduction: Cryopreservation of the cells <strong>all</strong>ows great flexible application<br />
for cell therapy, as well as industrial production of biologics such as<br />
antibody therapeutics. Convention<strong>all</strong>y, cryopreservative solution contains<br />
both of fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) as a<br />
cryoprotectant [1]. However, both of them have problems. FBS frequently<br />
induces differentiation of stem cells and so it should not be used for cell<br />
therapy. Addition<strong>all</strong>y, FBS has serious concern about zoonotic infections<br />
such as abnormal prions, pathogen of bovine spongiform encephalopathy<br />
(BSE) [2,3], indicating necessity of FBS-free cryopreservative solution. DMSO<br />
has cytotoxicity and often induces stem cells to differentiate [3]. Therefore, it<br />
is necessary to reduce the concentration of DMSO in cryoprotectant<br />
solution. In this study, we report that rakkyo fructan, plant-derived<br />
polysaccharide, significantly improved the viability of the cells frozen in<br />
DMSO-free solution.<br />
Materials and methods: Cell line and culture condition: A mouse<br />
hybridoma 2E3-O [4] was used for this study. 2E3-O was cultured in ASF104<br />
(Ajinomoto, Tokyo, Japan) with 1 g/L bovine serum albumin (BSA, Wako<br />
pure chemical industries, Osaka, Japan).<br />
Polysaccharides and cryopreservative solution: Rakkyo fructan was<br />
purified by the method in previous study [5]. Low molecular weight inulin<br />
and high one were produced by Fuji Nihon Seito Co. (Tokyo, Japan). Levan<br />
was purchased from Wako pure chemical industries. Each polysaccharide<br />
was solved in phosphate buffer saline (PBS). FBS containing 10% DMSO was<br />
used as positive control.<br />
Cryopreservative procedure: 2E3-O cells were pre-cultured until 60-70%<br />
confluent before cryopreservation. They were collected by centrifugation,<br />
removed the culture supernatant and then suspended in the cryopreservative<br />
solution. They were transferred to freezing tubes, placed in a BIOCELL<br />
container (Nihon freezer, Tokyo, Japan), frozen and stored at -80°C for several<br />
days.<br />
Thawing procedure and re-culture: Stored cells were defrosted at 37°C<br />
rapidly then transferred to the culture medium. The defrosted cells were<br />
centrifuged in order to the cryopreservative solution. Collected cells were<br />
suspended by the culture medium again. A part of them was stained with<br />
trypan blue exclusion method and counted with hemocytometer. The other<br />
one was re-cultured in a multi well plate for several days. After that, grown<br />
cells were stained with trypan blue exclusion method and counted with<br />
hemocytometer.<br />
Results and discussion: 2E3-O cells stored in 3 w/v%, 10 w/v% or 30 w/v%<br />
rakkyo fructan solution. After frozen and thawed in 10 w/v% or 30 w/v%<br />
rakkyo fructan solution, 2E3-O cells successfully survived and proliferated<br />
(Figure 1). On the other hand, <strong>all</strong> 2E3-O cells stored in 3 w/v% rakkyo fructan<br />
solution were dead (data not shown). This result shows that using rakkyo<br />
fructan will be effective for serum-free cryopreservation without DMSO.<br />
To compare the effect of rakkyo fructan on cellular protection, other fructans<br />
such as inulin and levan were also used for cryopreservation. Four fructans<br />
Figure 1(abstract P104) Effect of RBE on Serum-free Culture of<br />
Islets. Islets were cultured in RPMI 1640 medium in the presence of RBE<br />
or FBS as positive control.<br />
Figure 1(abstract P105) The time curse of viable cell number after<br />
thawing of frozen cells. 2E3-O cells were stored for three days in<br />
10 w/v% rakkyo fructan (triangles) or 30 w/v% rakkyo fructan (circles).<br />
The experiment was four trials.
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Table 1(abstract P105) Viable cell number of 2E3-O cells after frozen-thawing process<br />
Cryopreservative solution Mean degree of polymerization Viable cell number (×10 6 )<br />
30 w/v% rakkyo fructan 390 99.5<br />
30 w/v% inulin (low molecular weight) 16 64.5<br />
10 w/v% inulin (high molecular weight) 19 5.0<br />
1 w/v% levan 1000 0.2<br />
Positive control - 111<br />
2E3-O cells were stored for three days. 1.18 × 10 6 cells were frozen.<br />
were different in molecular weight and solubility. Rakkyo fructan and low<br />
molecular weight inulin solved in water very much but high molecular<br />
weight inulin solved in water up to 10 w/v% and levan dissolved in water.<br />
Rakkyo fructan was the highest viable cell number among fructans (Table 1).<br />
This result indicates that rakkyo fructan can protect animal cells more<br />
effectively than other fructans. Using rakkyo fructan has some advantages:<br />
1) using rakkyo fructan can avoid pathogenic contamination, 2) using rakkyo<br />
fructan will not be occurred osmotic change of stored cells because<br />
molecular weight of rakkyo fructan is over 10,000 (i.e. 30 w/v% rakkyo<br />
fructan is about 0.03 M), and 3) rakkyo fructan is high water soluble, which is<br />
easy to use.<br />
Conclusion: In conclusion, the freezing media using rakkyo fructan will be<br />
extensively used to protect animal cells against freezing stress without<br />
DMSO.<br />
References<br />
1. Seth G: Freezing mammalian cells for production of biopharmaceuticals.<br />
Methods 2012, 56:424-431.<br />
2. Tonti GA, Mannello F: From bone marrow to therapeutic applications:<br />
different behavior and genetic/epigenetic stability during mesenchymal<br />
stem cell expansion in autologous and foetal bovine sera? Int J Dev Biol<br />
2008, 52:1023-1032.<br />
3. Santos NC, Figueira-Coelho J, Martines-Silva J, Saldanha C: Multidisciplinary<br />
utilization of dimethyl sulfoxide: pharmacological, cellular, and<br />
molecular aspects. Biochem Pharmacol 2003, 65:1035-1041.<br />
4. Makishima F, Terada S, Mikami T, Suzuki E: Interleukin-6 is antiproliferative<br />
to a mouse hybridoma cell line and promotive for its antibody<br />
productivity. Cytotechnology 1992, 10:15-23.<br />
5. Kobayashi K, Futigami S, Nishikawa K, Inaki Y, Tsuji Y: Japanese patent<br />
application H10-158306 1998.<br />
P106<br />
Rice bran extract (RBE) as supplement for cell culture<br />
Satoko Moriyama 1 , Ken Fukumoto 1 , Masayuki Taniguchi 2 , Shigeru Moriyama 3 ,<br />
Takuo Tsuno 3 , Satoshi Terada 1*<br />
1 University of Fukui, Fukui, 910-8507, Japan;<br />
2 Niigata University, Niigata,<br />
950-2102, Japan;<br />
3 Tsuno Food Industrial Co., Ltd, Katsuragi-cho, Wakayama,<br />
649-7122, Japan<br />
E-mail: terada@u-fukui.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):P106<br />
Introduction: In mammalian cell culture, fetal bovine serum (FBS) or<br />
proteins obtained from mammals are usu<strong>all</strong>y supplemented to culture<br />
media. Since the use of animal-derived components may cause an infection<br />
of virus and other pathogens, alternative supplement derived from nonmammals<br />
is eagerly required in cell culture for producing biotherapeutics<br />
and for cell therapy [1]. As an alternative supplement, we focused on rice<br />
bran extract (RBE), because several studies have been done and reported<br />
that RBE has some biological effects such as enhancement of NK cell activity<br />
and anti-inflammatory effect on mice [2] and antioxidant effect [3].<br />
Rice bran, by-product of milling in the production of refined white rice,<br />
contains abundant nutrients and proteins. In this study, the effect of RBE<br />
was examined in the serum-free culture.<br />
Materials and methods: Effect of RBE on several cell lines: RBE was<br />
extracted from rice bran in an alkaline solution, precipitated with acid, and<br />
subsequently freeze-dried. The proceeding was performed by Tsuno Food<br />
Infdustrial Co., Ltd. To test the effect, RBE was supplemented to the culture<br />
of hybridoma cells, Chinese hamster ovary cells (CHO-DP12), hepatoma<br />
HepG2 and HeLa. The cells were cultured in 24 well plate (Sumitomo<br />
Bakelite, Japan) with 1 ml ASF104 medium (Ajinomoto, Japan) containing<br />
RBE or BSA (Wako, Japan) as positive control. The cell density was estimated<br />
using a hemacytometer. Viable cells were distinguished from dead cells by<br />
trypan blue dye exclusion method. The production of antibodies from<br />
hybridoma and CHO-DP12 cell was measured by ELISA method.<br />
Fractionation of RBE with UF membrane: In order to identify the<br />
growth factor(s) in RBE, fractionations were performed using UF<br />
membranes. RBE was fractionated into the permeable and residual fraction<br />
with ultrafiltration membrane Amicon Ultra-15 (Merck Millipore, Germany)<br />
at 4,000 rpm, 40 min and 4°C. The fractions and whole RBE were added to<br />
the culture of hybridoma and CHO-DP12 cells.<br />
Results and discussion: Enhanced cell growth and productivity<br />
using RBE: Figure 1 shows an enhanced proliferation by RBE. On growth<br />
and monoclonal antibody production of hybridoma cells, RBE had desired<br />
effect and the effect of RBE was superior to that of BSA. Similarly, to CHO-<br />
DP12 cells, addition of RBE exhibited increased cell growth and improved<br />
the productivity of humanized antibody. Growth of HepG2 and HeLa cells<br />
were also enhanced in the presence of RBE.<br />
Improvement of fractionated RBE by UF membrane: Fractionated<br />
RBEs by UF membranes were also tested. The fraction of RBE more than<br />
60 kDa improved the proliferation of hybridoma cells and the level was<br />
superior to that of whole RBE, while the fraction less than 60 kDa inhibited<br />
the proliferation. This results suggest that in RBE, some lower molecular<br />
inhibitor(s) and higher molecular growth factor(s) would be contained.<br />
Conclusion: We provide the first evidence that RBE is an attractive culture<br />
supplement to improve the proliferation and the production of mammalian<br />
cells.<br />
References<br />
1. Leopold G, Thomas RK, Sonia N, Manfred R: Emerging trends in plasmafree<br />
manufacturing of recombinant protein therapeutics expressed in<br />
mammalian cells. Biotechnology journal 2009, 4:186-201.<br />
2. Kim HY, Kim JH, Yang SB, Hong SG, Lee SA, Hwang SJ, Shin KS, Suh HJ,<br />
Park MH: A polysaccharide extracted from rice bran fermented with<br />
Lentinus edodes enhances natural killer cell activity and exhibits<br />
anticancer effects. Journal of medicinal food 2007, 10:25-31.<br />
3. Elisa R, Consuelo SM, Miramontes E, Juan B, Ana GM, Olga C, Rosa C,<br />
Juan P: Nutraceutical composition, antioxidant activity and<br />
hypocholesterolemic effect of water-soluble enzymatic extract from rice<br />
bran. Food Research International 2009, 42:387-393.<br />
P107<br />
Identification of mitogenic factor in rice bran for better mammalian cell<br />
culture<br />
Yoko Suzuki 1 , Satoko Moriyama 1 , Masayuki Taniguchi 2 , Shigeru Moriyama 3 ,<br />
Takuo Tsuno 3 , Satoshi Terada 1*<br />
1 University of Fukui, Fukui, 910-8507, Japan;<br />
2 Niigata University, Niigata,<br />
950-2102, Japan;<br />
3 Tsuno Food Industrial Co., Ltd, Katsuragi-cho, Wakayama,<br />
649-7122, Japan<br />
E-mail: terada@u-fukui.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):P107<br />
Introduction: In cell culture for biopharmaceutical production, serum-free<br />
culture is required in order to avoid the risks associated with components of<br />
mammal origin such as BSE. Although many serum-free medium have been<br />
developed, there is yet room for improvement and protein hydrolysates<br />
from crops are widely used as additives to improve the culture.
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Figure 1(abstract P106) Cell growth in serum-free medium containing RBE. a mouse hybridoma cell, b HeLa.<br />
We found that rice bran extract (RBE), not hydrolysate, successfully<br />
improved the proliferation of various cells as well as recombinant protein<br />
production of CHO cells when RBE was added into serum-free culture.<br />
Several studies have been done and reported that rice bran has antioxidant<br />
potential [1,2] and a rice bran 57-kDa protein showed cell adhesion activity<br />
for murine Lewis lung carcinoma cells [3].<br />
RBE contains various components such as proteins and the factors activating<br />
mammalian cells are not identified yet. In this study, we aim to identify the<br />
effective factor in RBE.<br />
Our colleague reports that heavier molecular weight fraction of RBE<br />
improves the proliferation of various cells. Addition<strong>all</strong>y, protein is the most<br />
abundant component in RBE. Together with them, some of the proteins in<br />
RBE would be the effective factor or the mitogen. We first determined<br />
whether some of the proteins in RBE are the bio-active factor or not, and<br />
then tried to identify which protein in RBE is the bio-active factor.<br />
Materials and methods: Effect of heat treatment on RBE: RBE was<br />
autoclaved at 121°C for 20 minutes. The heated RBE was supplemented<br />
into the culture of murine hybridoma cell line 2E3-O. Hybridoma cells<br />
were cultured in 24-well plate (Sumitomo Bakelite, Japan) with 1 ml<br />
ASF104 medium (Ajinomoto, Japan) in the presence of heated RBE. On<br />
day 3, viable cell number was determined by trypan blue dye exclusion<br />
with hemocytometer.<br />
Effect of trypsin treatment on RBE: RBE was digested with trypsin at 37°C<br />
for 24 hours. The treated RBE was SDS electrophoresed to confirm RBE was<br />
digested and to decide the condition. The trypsinized RBE was supplemented<br />
to the culture of hybridoma cells. On day three, viable cell number was<br />
determined.<br />
Proteins in Rice Bran: Two kinds of oryzacystatins are known in rice bran;<br />
oryzacystatin I and II. Antiserum against both oryzacystatin I and II was<br />
prepared, and mobilized in HiTrap Protein A column (GE Healthcare, USA).<br />
Using affinity chromatography, oryzacystatin I and II were eluted with 100<br />
mM Glycine-HCL (pH 2.9) containing 2 M Urea. All purification steps were<br />
done at 4°C.<br />
The purified oryzacystatin was supplemented to the culture of hybridoma<br />
cells. On day three, viable cell number was determined.<br />
Results and discussion: Autoclaved RBE lost mitogenic activity: While<br />
un-heated RBE successfully improved the proliferation, autoclaved RBE<br />
failed, suggesting that mitogenic factors in RBE would be heat-sensitive.<br />
Trypsinized RBE lost mitogenic activity: Most of proteins including 31<br />
kDa protein of RBE were successfully digested with trypsin. Although the<br />
proliferation of the cells treated with undigested RBE was stimulated, that<br />
of the cells treated with trypsinized RBE was not, suggesting that<br />
effective factors in RBE would be some proteins. Effect of trypsinized RBE<br />
on hybridoma cell growth is shown in Figure 1.<br />
Purified Oryzacistatin from RBE did not improve the cellular<br />
proliferation: Oryzacystatin obtained from RBE did not improve the<br />
culture of hybridoma, suggesting that oryzacystatin would not be mitogen.<br />
Other proteins in RBE would have mitogenic effects on mammalian cells.<br />
Conclusions: RBE improves the culture of various cells. Both of autoclaved<br />
and trypsinized RBE had lost the mitogenic effect, suggesting that bioactive<br />
factors in RBE would be heat-sensitive ingredients, probably<br />
proteins.<br />
Among abundant proteins in rice bran, oryzacystatin was purified from<br />
RBE and supplemented into the culture, but it failed to improve the<br />
culture. Other proteins in RBE will be tested to identify bio-active factor<br />
in RBE.<br />
References<br />
1. Adebiyi AP, Adebiyi AO, Hasegawa Y, Ogawa T, Muramoto K: Isolation and<br />
characterization of protein fractions from deoiled rice bran. European<br />
Food Research and Technology 2008, 10.<br />
2. Adebiyi AP, Adebiyi AO, Yamashita J, Ogawa T, Muramoto K: Purification<br />
and characterization of antioxidative peptides derived from rice bran<br />
protein hydrolysates. European Food Research and Technology 2008, 10.<br />
3. Shoji Y, Mita T, Isemura M, Mega T, Hase S, Isemura S, Aoyagi Y: A<br />
Fibronectin-binding Protein from Rice Bran with Cell Adhesion Activity<br />
for Animal Tumor Cells. Biosci Biotechnol Biochem 2001, 65:1181-1186.<br />
Figure 1(abstract P107) Effect of trypsinized RBE on hybridoma cell<br />
growth. RBE was digested with trypsin at 37°C for 24 hours.
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P108<br />
Protein folding and glycosylation process are influenced by mild<br />
hypothermia in batch culture and by specific growth rate in continuous<br />
cultures of CHO cells producing rht-PA<br />
Mauricio Vergara 1 , Silvana Becerra 2 , Julio Berrios 1 , Juan Reyes 3 ,<br />
Cristian Acevedo 4 , Ramon Gonzalez 5 , Nelson Osses 3 , Claudia Altamirano 1,2*<br />
1 Escuela Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso,<br />
Valparaíso, 2362806, Chile;<br />
2 Centro Regional En Alimentos Saludables<br />
(CREAS), Valparaíso, 2340025, Chile;<br />
3 Instituto Química, Pontificia Universidad<br />
Católica de Valparaíso, Valparaíso, 2340025, Chile;<br />
4 Centro de Biotecnología,<br />
Universidad Técnica Federico Santa María, Valparaíso, 2390123, Chile;<br />
5 Department of Chemical and Biomolecular engineering, RICE University,<br />
Houston, 77055, USA<br />
E-mail: Claudia.altamirano@ucv.cl<br />
BMC Proceedings 2013, 7(Suppl 6):P108<br />
Background: CHO cells are the primary host for the production of<br />
different biopharmaceuticals, including recombinant proteins, monoclonal<br />
antibodies, vaccines, etc. Primarily due to their ability to perform properly<br />
folding and glycosylation processes required for these proteins acquire<br />
adequate biological functionality.<br />
However, culturing of these cells in the bioreactor still presents a number of<br />
disadvantages, among which can be mention: nutrient depletion, toxic<br />
byproducts accumulation, limited oxygen transfer, etc. These issues limit the<br />
cell growth and early onset of programmed cell death, which restricts the<br />
longevity of cultures and jointly specific productivity of recombinant protein.<br />
To overcome these limitations, different approaches have been made to<br />
maximize the productivity of these cultures. One of these approaches, that<br />
has gained importance during the last 20 years is the use of mild<br />
hypothermic temperatures, within a range of 33°C to 30°C. This strategy has<br />
been demonstrated to reduce the rate of growth and metabolism of cells<br />
but in turn increases the longevity of cultures and increase in specific<br />
productivity of a wide range of recombinant proteins in batch cultures [1,2].<br />
One possible cause involved in the increase of specific productivity of<br />
recombinant proteins, is the increase in folding capacity and expression of<br />
chaperones from endoplasmic reticulum [3,4]. However, the intracellular<br />
mechanisms underlying the effect of temperature on the stages of posttranslational<br />
protein synthesis are still poorly understood.<br />
In this regard, the study of endoplasmic reticulum processes (folding,<br />
assembly and glycosylation of proteins, and degradation of misfolded<br />
proteins through ERAD pathway) has reached a high interest in recent<br />
years [4,5]. Reports show that the expression of several proteins associated<br />
with the various processes that take place in the ER, are affected under<br />
conditions of mild hypothermia. However, this phenomenon has not been<br />
analyzed from a process perspective.<br />
Thus, this study investigated the effect of mild hypothermic temperatures<br />
(33°C) on the process of protein folding of rht-PA expressed in CHO cells.<br />
For this, inhibitors of protein translation, glycosylation and endoplasmic<br />
reticulum associated degradation pathways (ERAD I: via the ubiquitin/<br />
proteasome and ERAD II: autophagosome/Lysosome) were used. Two<br />
experimental approaches were evaluated: batch culture and continuous<br />
culture.<br />
Materials and methods: Batch Culture: CHO cells were cultured in<br />
HyClone SFM4CHO medium with out glucose, supplemented with 20 mM<br />
glucose, at 95% relative humidity in an atmosphere of 5% CO2, at<br />
temperatures of 37°C or 33°C. The inhibitors used to block processes in<br />
the endoplasmic reticulum were: cycloheximide (Sigma, C4859)-protein<br />
translation; tunicamycin (Sigma, T7765)-N-glycosylation of proteins,<br />
MG132 (Merck, 474790)-ERAD I pathway; Pepstatin A (Merck, 516485),<br />
Leupeptin (Merck, 108976) and E64d (Sigma, E8640)-ERAD II pathway.<br />
Continuous culture: The bioreactor was inoculated and operated in<br />
batch-mode during 48 h and it was then supplied with sterile feed<br />
throughout the period of operation. A series of four experiments was<br />
performed, in duplicate, at 37°C or 33°C, keeping D, at 0.014 and 0.012 h -1 .<br />
Cultures were considered to reach steady-state (SS) when, after at least<br />
four residence times, both, the number of viable cells and lactate<br />
concentration, were constant in two consecutive samples.<br />
Cell growth was measured by counting cells by trypan blue method;<br />
consumption and production of metabolites were measured by biochemical<br />
analyzer (YSI 2700); protein rht-PA was measured by ELISA (Trinilize tPA<br />
antigen) and enzymatic activity of the protein was measured by amidolytic<br />
assay (S-2288 peptide, Chromogenix Italy). The results were analyzed by the<br />
mathematical technique of PCA (Principal Component Analysis).<br />
Results: The results of the batch cultures may indicate that the process of<br />
protein folding is sensitive to mild hypothermia. Inhibition of glycosylation<br />
process and ERAD pathways (ERAD I or II), under conditions of low<br />
temperature, promotes the accumulation of intracellular deglycosylated rht-PA<br />
as shown in Table 1. This response may indicate that the protein folding<br />
process is attenuated under conditions of mild hypothermia, promoting<br />
unfolded protein degradation by both ERAD pathways in CHO cells.<br />
Recent reports [6,7] show that the effect of mild hypothermia condition in<br />
batch culture is associated predominant with a decrease on specific cell<br />
growth rate rather a decrease on culture temperature. To evaluate this fact,<br />
we carried out continuous cultures at different dilution rates.<br />
These results show that the degradation of the protein would be more<br />
related to the decrease in specific growth rate than the temperature<br />
decrease. Also show that the temperature decrease would promote an<br />
increase in protein folding capacity of the endoplasmic reticulum. This fact is<br />
clearly observed at low specific growth rate (Table 1).<br />
The cell behavior was evaluated using the technique of principal component<br />
analysis (PCA) in both, batch and continuous culture Figure 1.<br />
Table 1(abstract P108) Intracellular rht-PA content (% of control) on CHO cells by inhibition of translation and<br />
glycosylation prosesses and ERAD I and II pathways at 37°C and 33°C<br />
Dilution rate (h -1 )<br />
0.014 0.012<br />
Temperature Temperature Temperature<br />
Batch Cultures 37°C 33°C Continuous Cultures 37°C 33°C 37°C 33°C<br />
CC 100 1 100 2 SS 100 3 100 4 100 5 100 6<br />
TM* 107 140 CHX/ERAD I** 120 117 185 139<br />
TM/ERAD I* 87 201 CHX/ERAD II** 107 115 242 150<br />
TM/ERAD II* 79 176<br />
CC: Control Culture; TM: Culture inhibited glycosylation; TM/ERAD I or II: Culture inhibited glycosylation and ERAD I or II; SS: Steady State; CHX/ERAD I or II:<br />
Culture inhibited translation and ERAD I or II; *Values at 24 hours after perturbation with inhibitors respect to CC value at 0 h. **At 48 hours after perturbation<br />
with inhibitors respect to value at SS.<br />
1 Concentration (8,8 ng/10 6 cells).<br />
2 Concentration (8,6 ng/10 6 cells).<br />
3 Concentration (7,9 ng/10 6 cells).<br />
4 Concentration (6,5 ng/10 6 cells).<br />
5 Concentration (4,2 ng/10 6 cells).<br />
6 Concentration (6,1 ng/10 6 cells).
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Figure 1(abstract P108) First principal plane and Loads of first and second principal component of Batch and Continuous cultures.<br />
A: First principal plane of Batch culture B: First principal plane of continuous culture; C: Loads of first and second principal component of Batch cultures.<br />
D: Loads of first and second principal component of Continuous cultures.<br />
The first principal plane (PC1 axis and PC2 axis) of batch cultures (Figure<br />
1A) shows that there are only two values whose behavior is significantly<br />
away from the origin (P < 0,05). These correspond to the behavior of the<br />
tested batch cultures at 24 h at 37°C and 33°C, respectively. This indicates<br />
the great influence of culture temperature on cell behavior. The first<br />
principal plane of continuous culture (Figure 1B) shows the behavior of<br />
cells organized into two major groups, which are correlated with both<br />
dilution rates tested.<br />
PC1 loads of batch cultures (Figure 1C) suggest that low temperature<br />
reduces the ability of the protein folding; this would explain the<br />
accumulation of intracellular deglycosylated rht-PA. However, loads of PC1<br />
from continuous cultures (Figure 1D) shows that increasing of intracellular<br />
rht-PA content is associated with the reduction in the rate of dilution and is<br />
not associated with a lower temperature.<br />
Conclusions: Experimental approach of continuous culture revealed that<br />
reduction on specific growth rate is associated to an increase ERAD<br />
activity on rht-PA while the temperature reduction may have a positive<br />
effect on protein folding. Moreover, PCA analysis indicated that specific<br />
growth rate is also responsible for general behavior exposed by CHO<br />
cells.<br />
References<br />
1. Yoon SK, Song JY, Lee GM: Effect of low culture temperature on specific<br />
productivity, transcription level, and heterogeneity of erythropoietin in<br />
Chinese hamster ovary cells. Biotechnol Bioeng 2003, 82:289-298.<br />
2. Bollati-Fogolín M, Forno G, Nimtz M, Conradt HS, Etcheverrigaray M,<br />
Kratje R: Temperature reduction in cultures of hGM-CSF-expressing CHO<br />
cells: effect on productivity and product quality. Biotechnology Progress<br />
2005, 21:17-21.
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3. Baik JY, Lee MS, An SR, Yoon SK, Joo EJ, Kim YH, Park HW, Lee GM: Initial<br />
Transcriptome and Proteome Analyses of Low Culture Temperature-<br />
Induced Expression in CHO Cells Producing Erythropoietin. Biotechnol<br />
Bioeng 2006, 93:361-371.<br />
4. Masterton RJ, Roobol A, Al-Fageeh M, Carden M, Smales CM: Post-<br />
Translational Events of a Model Reporter Protein Proceed With Higher<br />
Fidelity and Accuracy Upon Mild Hypothermic Culturing of Chinese<br />
Hamster Ovary Cells. Biotechnol Bioeng 2010, 105:215-220.<br />
5. Gomez N, Subramanian J, Ouyang J, Nguyen M, Hutchinson M, Sharma VK,<br />
Lin AA, Yuk IH: Culture Temperature Modulates Aggregation of<br />
Recombinant Antibody in CHO Cells. Biotechnol Bioeng 2012, 109:125-136.<br />
6. Becerra S, Berrios J, Osses N, Altamirano C: Exploring the effect of mild<br />
hypothermia on CHO cell productivity. Biochem Eng J 2012, 60:1-8.<br />
7. Vergara M, Becerra S, Berrios J, Osses N, Reyes J, Rodríguez-Moyá M,<br />
Gonzalez R, Altamirano C: Differential effect of culture temperature and<br />
specific growth rate on CHO cell behavior in continuous culture. Bioch<br />
Eng J 2013, submitted.<br />
P109<br />
The combined use of platinum nanoparticles and hydrogen molecules<br />
induces caspase-dependent apoptosis<br />
Takeki Hamasaki 1 , Tomoya Kinjyo 2 , Hidekazu Nakanishi 2 , Kiichiro Teruya 1,2 ,<br />
Sigeru Kabayama 3 , Sanetaka Shirahata 1,2*<br />
1 Department of Bioscience and Bioengineering, Faculty of Agriculture,<br />
Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan;<br />
2 Graduate School of Systems Life Sciences, Kyushu University, 6-10-1<br />
Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan;<br />
3 Nihon Trim Co. LTD., 34-8-1<br />
Ooyodonaka, Kita-ku, Osaka 531-0076, Japan<br />
E-mail: sanetaka@grt.kyushu-u.ac.jp<br />
BMC Proceedings 2013, 7(Suppl 6):P109<br />
We previously reported electrochemic<strong>all</strong>y reduced water (ERW), produced<br />
near the cathode by electrolysis, exhibits reductive activity. We also revealed<br />
that ERW contains Pt nanoparticles (Pt nps) derived from Pt-coated titanium<br />
electrodes in addition to high concentration of dissolved molecular<br />
hydrogen (H 2 ) by in vitro assay, and Pt nps exhibit powerful ROS scavenger<br />
activity and catalysis activity converting H 2 to active hydrogen. Our study<br />
investigates apoptosis inducibility of H 2 and synthesized Pt nps on human<br />
promyelocytic leukaemia HL60 cells. Human promyelocytic leukaemia cells<br />
(HL60) were cultured in RPMI 1640 medium supplemented with 10% FBS,<br />
2.0 mM l-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin. Cultures<br />
were incubated in an atmosphere of 75%(v/v) H 2 /20%(v/v) O 2 /5%(v/v) CO 2 ,<br />
75%(v/v) He/20%(v/v) O 2 /5%(v/v) CO 2 atmosphere or 75%(v/v) N2/20%(v/v)<br />
O 2 /5%(v/v) CO 2 atmosphere for 12-48 hr after incubated with Pt nps for 2 h.<br />
Untreated cultures were included as controls. Cytotoxicity was determined<br />
by cell-counter. Apoptosis pathway of HL60 cells was investigated by Sub<br />
G-1 assay.<br />
Growth suppression was not observed when cells were treated with Pt nps<br />
or H 2 only. Analysis of cell cycle and activity of caspase-3 suggested that<br />
combination use of both Pt nps and H 2 induced apoptosis in HL60 cells. Our<br />
caspase activity experimentation suggests that apoptosis was caused via<br />
caspase-8 activation. These results suggested that atomic hydrogen from H 2<br />
induces caspase-8 dependent apoptosis. The cytotoxicity was not detected<br />
in Pt nps or H 2 separately treated cells. Apoptosis was determined only<br />
when cells were treated with both Pt nps and H 2 , suggesaspase-8<br />
dependent apoptosis was caused by atomic hydrogen produced from H 2 by<br />
catalyst activity of Pt nps.<br />
P110<br />
Rec. ST6Gal-I variants to control enzymatic activity in processes of<br />
in vitro glycoengineering<br />
Alfred M Engel 1* , Harald Sobek 1 , Michael Greif 1 , Sebastian Malik 2 ,<br />
Marco Thomann 2 , Christine Jung 2 , Dietmar Reusch 2 , Doris Ribitsch 4 ,<br />
Sabine Zitzenbacher 4 , Christiane Luley 4 , Katharina Schmoelzer 4 ,<br />
Tibor Czabany 5 , Bernd Nidetzky 4,5 , Helmut Schwab 4,6 , Rainer Mueller 3<br />
1 Professional Diagnostics, Roche Diagnostics GmbH, 82372 Penzberg,<br />
Germany;<br />
2 Pharma Biotech Development, Roche Diagnostics GmbH, 82372<br />
Penzberg, Germany;<br />
3 Applied Science, Roche Diagnostics GmbH, 82372<br />
Penzberg, Germany;<br />
4 ACIB GmbH, 8010 Graz, Austria;<br />
5 Institute of<br />
Biotechnology and Biochemical Engineering, Graz University of Technology,<br />
8010 Graz, Austria;<br />
6 Institute of Molecular Biotechnology, Graz University of<br />
Technology, 8010 Graz, Austria<br />
E-mail: alfred.engel@roche.com<br />
BMC Proceedings 2013, 7(Suppl 6):P110<br />
Background: Glycosylation is an important posttranslational modification<br />
of proteins influencing protein folding, stability and regulation of the<br />
biological activity. The sialyl mojety (sialic acid, 5-N-acetylneuramic acid) is<br />
usu<strong>all</strong>y exposed at the terminal position of N-glycosylation and therefore, a<br />
major contributor to biological recognition and ligand function, e.g. IgG<br />
featuring terminal sialic acids were shown to induce less inflammatory<br />
response and increased serum half-life.<br />
The biosynthesis of sialyl conjugates is controlled by a set of sugar-active<br />
enzymes including sialyltransferases which are classified as ST3, ST6 and<br />
ST8 based on the hydroxyl position of the glycosyl acceptor the Neu5Ac is<br />
transferred to [1]. The ST6 family consists of 2 subfamilies, ST6Gal and<br />
ST6GalNAc. ST6Gal catalyzes the transfer of Neu5Ac residues to the<br />
hydroxyl group in C6 of a terminal galactose residue of type 2 disaccharide<br />
(Galb1-4GlcNAc).<br />
To our knowledge, the access to recombinant ST6Gal-I for therapeutic<br />
applications is still limited due to low expression and/or poor activity in<br />
various hosts (Pichia pastoris, Spodoptera frugiperda and E. coli).<br />
The present study describes the high-yield expression of two variants of<br />
human beta-galactoside alpha-2,6 sialyltransferase 1 (ST6Gal-I, EC 2.4.99.1;<br />
data base entry P15907) by transient gene expression in HEK293 cells with<br />
yields >100 mg/L featuring distinct mono- (G2+1SA) as well as bi- (G2+2SA)<br />
sialylation activity.<br />
Materials and methods: Two N-termin<strong>all</strong>y truncated fragments of human<br />
ST6Gal-I (delta89, residues 89-406, and delta108, residues 109-406) were<br />
designed for transient gene expression (TGE): Instead of the natural leader<br />
sequence and N-terminal residues, both ST6Gal-I coding regions harbor<br />
the Erythropoietin (EPO) signal sequence in order to ensure correct<br />
processing of the polypeptides by the secretion machinery. Following<br />
cloning into pM1MT, expression of the ST6Gal-I coding sequences is under<br />
control of a hCMV promoter followed by an intron A.<br />
Sialyltransferase assays: 1. Asialofetuin was used as acceptor and CMP-9F-<br />
NANA as donor substrate. Enzymatic activity was determined by measuring<br />
the transfer of 9F-NANA to asialofetuin. 2. Recombinant humanized IgG1<br />
and IgG4 monoclonal antibodies (mabs), characterized as G2+0SA, as well as<br />
desialylated EPO were used as targets in sialylation experiments (30 μg<br />
enzyme/300 μg target protein). Both enzyme variants of ST6Gal-I (delta89<br />
and delta108) were used under identical reaction conditions and the<br />
sialylation status was analyzed by mass spectrometry.<br />
Results: In using the suspension-adapted human embryonic kidney (HEK)<br />
293-F cell line, a modified serum-free FreeStyle medium platform plus<br />
transfection by the 293-Free reagent, we were able to inst<strong>all</strong> a TGE shaker<br />
fermentation process with product yields of up to 200 mg/L culture<br />
supernatant. Both variants delta89 and delta108 could be isolated to >98%<br />
purity by a simple 2-step purification protocol.<br />
To our surprise, both variants show a distinct and different sialylation<br />
activity as shown by sialylation kinetics of a IgG4 molecule (Figure 1).<br />
Recently, the crystal structure of the delta89 variant could be determined<br />
as first human ST6Gal-I by SIRAS phasing using an iodide soak as<br />
derivative I [2]: An elongated glycan from a cryst<strong>all</strong>ographic neighbour<br />
binds to the active site, mimicking a substrate complex. An analysis of<br />
substrate interactions and comparison to other sialyltransferases <strong>all</strong>ows<br />
modelling of a Michaelis complex and conclusions on the catalytic<br />
mechanism.<br />
Due to their high expression rates and easy purification, both recombinant<br />
variants (delta89 and delta108) of human ST6Gal-I are available in large<br />
quantities and high purity. Both variants are active with high molecular<br />
weight substrates like monoclonal antibodies. To our surprise, they show<br />
different performance in sialylation experiments using with bi-antennary<br />
glycans such as mabs as well as tetra-antennary glycans (data not shown)<br />
as substrate. Under identical reaction conditions, bi-sialylated glycans are<br />
obtained in using variant delta89, whereas delta108 yields mono-sialylated<br />
glycans.<br />
Our findings on variant delta108 are in contradiction to previous studies [3]<br />
claiming that the conserved QVWxKDS sequence, residues 94-100 of<br />
human ST6Gal-I, being essential for its catalytic activity.
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Figure 1(abstract P110) Left panel: Variant delta89 yields 88% G2+2SA sialylation. However, after prolonged incubation (24 hrs), the bi-sialylation is<br />
reduced to a stable mono-sialylation product, presumably by a sialydase activity. Right panel: Variant delta108 yields 70% G2+1SA and 7% G2+2SA<br />
sialylation.<br />
To our knowledge, these human ST6Gal-I variants are the first enzymes<br />
available in large quantities and currently, recombinant alpha-2,3<br />
sialyltransferase 1 (ST3Gal-I) and beta-1,4 galactosyltransferase 2 (B4Gal-T2)<br />
are developed in order to strengthen this enzyme portfolio. Together with<br />
the already available donor substrates (activated sugars), a complete set of<br />
reagents will be soon available for the commercial glycoengineering of<br />
proteins.<br />
References<br />
1. Weijers CA, Franssen MC, Visser GM: Glycosyltransferase-catalyzed<br />
synthesis of bioactive oligosaccharides. Biotechnol Adv 2008, 26:436-456.<br />
2. Kuhn B, Benz J, Greif M, Engel AM, Sobek H, Rudolph MG: Crystal structure<br />
of human 2,6 sialyltransferase reveals mode of binding of complex<br />
glycans. Acta Cryst<strong>all</strong>ographica 2013, D69:1826-1838.<br />
3. Donadio S, Dubois C, Fichant G, Roybon L, Guillemot JC, Breton C, Ronin C:<br />
Recognition of cell surface acceptors by two human alpha-2,6-<br />
sialyltransferases produced in CHO cells. Biochimie 2003, 85:311-321.<br />
P111<br />
Accelerating stable recombinant cell line development by targeted<br />
integration<br />
Bernd Rehberger * , Claas Wodarczyk, Britta Reichenbächer, Janet Köhler,<br />
Renée Weber, Dethardt Müller<br />
Rentschler Biotechnologie GmbH, 88471 Laupheim, Germany<br />
E-mail: bernd.rehberger@rentschler.de<br />
BMC Proceedings 2013, 7(Suppl 6):P111<br />
Introduction: Targeted integration (TI) <strong>all</strong>ows fast and reproducible genetic<br />
modification of well characterized previously tagged host cells thus<br />
generating producer cells with predictable qualities. Concurrently, timelines<br />
are cut by 50% compared to random integration (RI) based cell line<br />
development. In contrast to commonly low productivities of cell lines<br />
generated by TI, we developed a system for CHO cells leading to<br />
productivities of more than 1 g/L within weeks using the TurboCell platform.<br />
The system is based on CHO K1 cells that have been tagged with a GFP<br />
expression cassette flanked by recombinase recognition sites. Following<br />
GFP based FACS enrichment and cloning of the tagged cells, over 4000<br />
clones were screened for growth, productivity, GFP expression stability<br />
and integration status of the GFP expression cassette. The best clones<br />
were selected to be used as “Master TurboCell” (MTC) host cell lines for<br />
recombinant cell line development.<br />
Generation of producer TurboCell lines: A selected MTC host cell line<br />
is co-transfected using a TurboCell expression plasmid containing the gene<br />
of interest (GOI) expression cassette flanked by matching recombinase<br />
recognition sites together with a plasmid encoding the recombinase enzyme<br />
required for RMCE. Upon transfection both plasmids enter the MTC’s nucleus<br />
initiating transient expression of the recombinase which further mediates the<br />
stable exchange of the GFP expression cassette against the GOI expression<br />
cassette. Thus, the GOI is stably introduced into the tagged genomic spot<br />
shortly after the transfection. Cells are cultivated for a few days to recover<br />
from the transfection procedure and to <strong>all</strong>ow GFP to fade out of RMCE<br />
positive cells. The transfected pools are thereupon sorted by FACS in order to<br />
remove the majority of GFP positive cells. The remaining producer<br />
TurboCell(PTC) pools in general comprise of 90-99% GFP negative, GOI<br />
expressing cells that are genetic<strong>all</strong>y identical due to the conserved locus of<br />
GOI integration. This <strong>all</strong>ows the production of recombinant protein from PTC<br />
enriched pools at a very early stage of 3 weeks upon transfection. Due to<br />
their genetic homogeneity the physiological diversity of the clones within<br />
the pool is limited thus leading to only sm<strong>all</strong> variations in the recombinant<br />
protein produced. Therefore, material drug candidate screening prepared on<br />
the parental PTC pool level should only differ slightly from material produced<br />
from clones thereof. Following FACS sorting, the PTC pools can be cloned, if<br />
required. Due to the high degree of similarity of <strong>all</strong> clones, the screening<br />
effort to find the best clone can be limited to about 10 clones. Recombinant<br />
protein material from clones can be produced 9 weeks upon transfection.<br />
Molecular biological analysis of producer TurboCell lines: In order<br />
to prove successful RMCE reproducibly taking place without additional<br />
random integration of the remaining plasmid, genomic DNA was prepared<br />
from clonal PTC for Southern Blot analysis. The genomic DNA was digested<br />
with a restriction enzyme cutting the correctly integrated targeting vector<br />
into two pieces, one fragment only comprising internal vector sequences, as<br />
well as a second fragment also comprising CHO derived sequences of the<br />
specific integration locus. As both fragments carry sequences of the CMV<br />
promotor, both can be visualized using one single CMV promoter-specific<br />
probe. As only two bands occur in case of successful RMCE, cell lines<br />
showing more than two bands indicate clones with randomly integrated<br />
targeting vector molecules in addition to RMCE. Statistics of several cell line<br />
generation projects show that in about 90% of <strong>all</strong> analyzed clones a correct<br />
RMCE without additional random integration events takes place. This <strong>all</strong>ows<br />
for a significant reduction in clone screening efforts to a level of 10 clones<br />
per project.<br />
Process characteristics: To show the feasibility of the TurboCell system<br />
for recombinant protein production in fed batch cultivations, a PTC clone<br />
producing IgG1 was cultivated in a stirred tank bioreactor. The data of this<br />
bioreactor were compared to two shake flask fed batch runs performed with<br />
the same PTC and the same media system (Rentschler’s proprietary media +<br />
GE Healthcare’s ActiCHO feed) in par<strong>all</strong>el. Figure 1a shows a comparison of<br />
viable cell density and product concentration. The better performance in the<br />
bioreactor indicates that the PTC can be easily transferred from shake flask<br />
to bioreactor settings. The maximum cell density of 13*10 6 cells/mL, as well<br />
as the integral of viable cells over the cultivation time, and the maximum<br />
product titer of more than 1 g/L IgG1 proved the feasibility of the<br />
TurboCell system for the production of recombinant proteins even in<br />
larger amounts at a very early stage of a biopharmaceutical development<br />
project.<br />
Analysis of Protein Quality: Both amount of glycosylation and glycosylation<br />
pattern in different Turbo Cell subsets were analyzed<br />
Figure 1b shows that clones derived from the same parental pool differ<br />
only slightly regarding their glyco pattern and they are very similar to their<br />
parental pool. Even if different antibodies are expressed from pools<br />
derived from the same MTC the variation between the pools is within the<br />
range of clones compared to each other. Significant variations in the glyco
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Figure 1a(abstract P111) Comparison of cell growth and recombinant protein production in a bioreactor versus shake flask. Figure 1b:<br />
Comparison of glycopatterns. The first row compares three clones derived from one parental pool of one defined MTC with each other and with the<br />
relevant parental pool. The second row compares mAb material derived from two PTC pools derived from different, not related MTCs. The third row<br />
compares two types of IgG1 antibodies expressed from PTC pools derived from the same MTC.<br />
pattern can only be detected, if antibody material derived from pools<br />
descendent from different, unrelated MTCs is compared (indicated by<br />
yellow arrows).<br />
Conclusions: Within 3 weeks upon transfection and targeted integration,<br />
producer cell pools were FACS sorted to purities of >95%. These cells were<br />
suited for high quality recombinant protein material production in fed batch<br />
runs exceeding 1 g/L IgG1. Clones generated thereof behaved similar to the<br />
pools in terms of productivity and product quality, cell growth and<br />
metabolism. From those clones analyzed a mean of about 90% showed<br />
successful RMCE without unintended random integration. Cellular properties<br />
and productivities of the clones were as expected and variations between<br />
different clones were marginal. Thus, the TurboCell system reduces clone<br />
screening efforts to a minimum <strong>all</strong>owing the simultaneous production of<br />
multiple recombinant proteins in stable CHO cells with optimal use of<br />
resources. This makes the TurboCell system an interesting tool for<br />
candidatescreeningandearlyphasesmaterialproductioneveninlarge<br />
scale setups.<br />
P112<br />
Differential affects of low glucose on the macroheterogeneity and<br />
microheterogeneity of glycosylation in CHO-EG2 camelid monoclonal<br />
antibodies<br />
Bo Liu 1* , Carina Villacres-Barragan 1 , Erika Lattova 2 , Maureen Spearman 1 ,<br />
Michael Butler 1<br />
1 Dept of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2,<br />
CA, USA;<br />
2 Dept of Chemistry, University of Manitoba, Winnipeg, Manitoba,<br />
R3T 2N2, CA, USA<br />
E-mail: bo.liu422@gmail.com<br />
BMC Proceedings 2013, 7(Suppl 6):P112<br />
Background: The demand for high yield recombinant protein production<br />
systems has focused industry on culture media and feed strategies that<br />
optimize productivity, yet maintain product quality attributes such as<br />
glycosylation. Minimizing media components such as glucose, reduces the
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production of lactate, but may also affect glycosylation. The first steps in the<br />
glycosylation pathway involve the synthesis of lipid-linked oligosaccharides<br />
(LLOs). Glycan macroheterogeneity is introduced by variation in site-specific<br />
glycosylation with the transfer of the oligo-saccharide to the protein. Further<br />
modification of the oligosaccharide can occur through processing reactions,<br />
where some sugars are removed and additional sugars added. This<br />
produces microheterogeneity of the glycan pool. Both macroheterogeneity<br />
and microheterogeneity may be affected by fermentation conditions. The<br />
objective of this study has been to investigate the effect of variable<br />
concentrations of glucose on the glycosylation patterns of a camelid<br />
monoclonal antibody produced in Chinese hamster ovary (CHO) cells and to<br />
further evaluate their effect on components of the N-glycosylation pathway.<br />
Materials and methods: A CHO cell line recombinantly expressing<br />
chimeric antibodies EG2 with a camelid single domain fused to human Fc<br />
regions was used in this study. Cells were inoculated at 2.6 x 10 6 cells/ml<br />
into 7 shake flasks (250 ml) each containing 80 ml of media with a different<br />
initial glucose concentration varying from 0 to 25 mM. The cultures were<br />
maintained and monitored under standard shaking conditions in an<br />
incubator over a 24 hr period.<br />
Cells were harvested and quenched to stop any subsequent metabolic<br />
activities [1]. LLOs were extracted from the cells using a previously<br />
established method [2]. Mild acid cleaved glycans were labeled with 2-<br />
aminobenzamide and analyzed by high performance liquid chromatography<br />
(HPLC) using the technique of hydrophilic interaction liquid chromatography<br />
(HILIC). The structures were assigned using standard GU values from the<br />
GlycoBase database (NIBRT.ie) [3] and confirmed by Mass spectrometric<br />
analysis.<br />
Antibodies were purified from culture supernatants with a Protein A affinity<br />
column and run under denaturing conditions on 8-16% SDS-PAGE gels and<br />
stained with Coomassie Brilliant Blue (CBB). The density ratio between<br />
upper and lower bands was determined by densitometry. The protein<br />
bands were removed by scalpel, washed, and treated with Peptide-N-<br />
Glycosidase F for 18 h to remove the attached glycans. MS analysis was<br />
carried out on the MALDI-TOF/TOF mass spectrometer to confirm<br />
aglycosylated Mabs in the lower band, and glycosylated proteins present<br />
in the upper band. The isolated N-linked glycans were labeled with 2-AB<br />
[4]. Glycan structures were assigned using standard GU values from HILIC<br />
analysis in GlycoBase. Structures were confirmed by exoglycosidase<br />
enzymatic digestion arrays according to method of Royle et al (2010).<br />
Results: Peaks corresponding to the LLOs from each of the previously<br />
described cultures with varying glucose concentration cultures were<br />
compared (Figure 1.A.). Samples from cultures containing 25mM glucose<br />
displayed a prominent large peak with a GU value of 11.7 representing<br />
63% of the total LLOs and designated as the Glc3Man9GlcNAc2 a structure<br />
(Figure 1.A.). Sm<strong>all</strong> peaks were designated as Glc2Man9GlcNAc2,<br />
Glc1Man9GlcNAc2, Man9GlcNAc2, Man5GlcNAc2 and Man2GlcNAc2<br />
structures. For cells grown at an initial glucose concentration of less than<br />
15 mM the predominant peak was Man2GlcNAc2 with a significant level of<br />
the Man5GlcNAc2 structure but the percentage of the Glc3Man9GlcNAc2<br />
structure was reduced significantly to 2.9% of the over<strong>all</strong> LLOs. It is<br />
important to note that these cultures (≤15mM glucose) were under<br />
conditions of glucose depletion for at least 4 h prior to harvest.<br />
LLO with a completed glycan structure Glc3Man9GlcNAc2 is an essential<br />
precursor for N-glycosylation. Thus, the effect of glucose concentration on<br />
the macroheterogeneity and microheterogeneity of the fully formed<br />
glycoprotein were examined next. Protein A purified antibodies from cultures<br />
after 24 h were analyzed on reduced SDS-PAGE gels 1. B. The antibodies<br />
produced by cells grown in 17.5-25 mM glucose displayed one single strong<br />
band corresponding to the glycosylated heavy chain. Proteins isolated from<br />
cell culture with 15 mM initial glucose concentration (Lane 5) showed a faint<br />
band underneath the predominant gel band. The proportional density of the<br />
lower band in the 12.5 mM glucose sample was 26% which increased<br />
gradu<strong>all</strong>y to 52% for samples taken from cultures with no added glucose<br />
(Table 1). The lower protein bands were suspected to be deglycosylated<br />
proteins due to an estimated 2% weight loss, which corresponds to the<br />
typical mass of glycan found on IgGs [5]. Samples of antibody showing two<br />
gel bands were analyzed by MALDI-MS. This showed m/z values of 82,670<br />
and 79,350 which are the expected masses of the glycosylated and nonglycosylated<br />
forms, respectively of the complete antibodies.<br />
To compare the difference in glycosylation profiles of EG2 antibodies<br />
induced by various glucose concentrations, the glycans were released from<br />
the Protein A-purified Mabs with PNGase F, and analyzed by HILIC HPLC.<br />
The glycan pool was separated into six major peaks which eluted between<br />
33 and 43 minutes with corresponding GU values between 5 and 9<br />
(Figure 1. C.). Structures were provision<strong>all</strong>y assigned from GU values with<br />
reference to the Glycobase and confirmed by a series of exoglycosidase<br />
enzyme array digestions. This <strong>all</strong>owed the identification of biantennary<br />
glycan structures with variable galactosylation, fucosylation and sialylation.<br />
The predominant glycan structure of antibodies isolated from the 25 mM<br />
glucose culture was the fully galactosylated biantennary and fucosylated<br />
structure, Fuc(6)GlcNAc2Gal2 , which comprised 60% of the over<strong>all</strong> glycans.<br />
Fuc(6)GlcNAc2Gal0 and Fuc(6)GlcNAc2Gal2 structures were determined at<br />
6% and 34%, respectively. The structures were found in samples from <strong>all</strong><br />
cultures analyzed but there was a significant shift to lower galactosylation<br />
and sialylation in samples derived from cultures with lower glucose.<br />
a Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine.<br />
b Fuc, fucose; Gal, galactose.<br />
Each glycan pool was assigned a galactosylation index (GI) and a sialylation<br />
index (SI) based upon the relative peak areas on the HPLC profile. In this<br />
experiment the GI value changed from 0.35 to 0.72 as the availability of<br />
glucose increased for the cells. Sialylation is dependent upon prior<br />
galactosylation of a glycan and consequently shows lower values with<br />
corresponding SI values from 0.019 to 0.058. There was a strong positive<br />
correlation between the GI and SI value determined for each sample and<br />
the time spent by the corresponding cells in glucose deprived media over<br />
the 24 h experimental period (R2 = 0.965 and 0.936 for the GI and SI<br />
values respectively; Figure 1.D.).<br />
Conclusion: N-glycosylation is an important post-translation modification in<br />
mammalian cells, which is known to impact the quality and efficacy of<br />
therapeutic recombinant proteins. In this study, we focused on the effect of<br />
glucose concentration on several aspects in N-glycosylation pathways in<br />
CHO-EG2 cells. The depletion of glucose as the main carbohydrate source<br />
during cell culture, can reduce the capacity for N-glycosylation. Reduced<br />
availability of the full-length LLO precursor occurred by glucose deprivation<br />
and resulted in the accumulation of truncated dolichol linked glycans. This<br />
led to reduced glycosylation in the EG2 antibodies. Glucose deprivation also<br />
led to changes in microheterogeneity with a decrease in galactosylation and<br />
sialylation. It is concluded that low glucose concentrations in culture altered<br />
LLO synthesis and N-glycan profiles of the antibody.<br />
Acknowledgements: This work is supported by the Natural Sciences and<br />
Engineering Research Council of Canada (NSERC) and MabNet. Author<br />
would like to thank Dr. Michael Butler at University of Manitoba for<br />
instructing and <strong>all</strong> members in Butler’s lab.<br />
References<br />
1. Sellick CA, Hansen R, Maqsood AR, Dunn WB, Stephens GM, Goodacre R,<br />
Dickson AJ: Effective quenching processes for physiologic<strong>all</strong>y valid<br />
metabolite profiling of suspension cultured Mammalian cells. Anal Chem<br />
2009, 81:174-183.<br />
2. Gao N, Lehrman MA: Non-radioactive analysis of lipid-linked<br />
oligosaccharide compositions by fluorophore-assisted carbohydrate<br />
electrophoresis. Meth Enzymol 2006, 415:3-20.<br />
3. Royle LL, Campbell MPM, Radcliffe CMC, Rudd PMP, Dwek RAR: GlycoBase<br />
and autoGU: tools for HPLC-based glycan analysis. Bioinformatics 2008,<br />
24:1214-1216.<br />
4. Detailed Structural Analysis of N-Glycans Released From Glycoproteins<br />
in SDS-PAGE Gel Bands Using HPLC Combined With Exoglycosidase<br />
Array Digestions. Methods in Molecular Biology 2010, 347:125-143.<br />
5. Deisenhofer J: Cryst<strong>all</strong>ographic refinement and atomic models of a human<br />
Fc fragment and its complex with fragment B of protein A from Staphylococcus<br />
aureus at 2.9- and 2.8-A resolution. Biochemistry 1981,<br />
20:2361-2370.<br />
P113<br />
Development and application of an automated, multiwell plate based<br />
screening system for suspension cell culture<br />
Sven Markert * , Carsten Musmann, Klaus Joeris<br />
Roche Diagnostics GmbH, Pharma Biotech Production and Development,<br />
Penzberg, Germany<br />
E-mail: Sven.Markert@roche.com<br />
BMC Proceedings 2013, 7(Suppl 6):P113<br />
Introduction: The already presented automated, multiwell plate (MWP)<br />
based screening system for suspension cell culture is now routinely used in
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Figure 1(abstract P112) The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy<br />
and the N-glycosylation profile of a monoclonal antibody. A. Lipid-linked oligosaccharide (LLO) profiles. The glycans from each sample were acid<br />
hydrolyzed from the lipid carriers, 2-AB labeled and detected by HILIC. (Glc Δ Man Ο and GlcNAc ?). B. Separation of EG2 antibodies on reduced 8-16%<br />
SDS-PAGE gel. The purified antibody in lane 8 was isolated from the culture prior to the 24 h incubation. Upper bands in lanes 1 to 4 correspond to<br />
glycosylated antibodies, and the lower bands were determined to be non-glycosylated antibodies. C. HPLC profiles of N-glycans isolated from EG2<br />
antibodies produced by CHO cells with various initial glucose concentrations during a 24 h incubation. D. The effect of exposure time of cells to media<br />
depleted of glucose on the galactosylation (GI; |) and the sialylation (SI; ?) indices of EG2 antibodies produced by CHO cells.<br />
process development. It is characterized by a fully automated workflow with<br />
integrated analytical instrumentation and uses shaken 6-24 well plates as<br />
bioreactors which can be run in batch and fed-batch mode with a capacity<br />
of up to 384 reactors in par<strong>all</strong>el [1].<br />
A wide ranging analytical portfolio is available to monitor cell culture<br />
processes and to characterize product quality. Assays running on the<br />
screening system comprise the determination of cell concentration and<br />
viability, quantification of nutrients and metabolites as well as detection of<br />
apoptosis level and staining of organelles. Addition<strong>all</strong>y a RT-qPCR method<br />
has been setup to measure gene expression level in a high throughput<br />
manner. Having a large network in-house to high throughput groups of<br />
the analytical department a lot of advanced methods can easily be<br />
Table 1(abstract P112) Quantitative densitometry of Protein A purified EG2 antibodies stained with coomassie blue (n =5)<br />
Initial glucose concentration (mM) % Glycosylated protein % Non-glycosylation protein<br />
0 48±1 52±1<br />
5 60±4 40±4<br />
10 69 ± 2 31 ± 2<br />
12.5 74 ± 2 26 ± 2<br />
15 100 0<br />
17.5 100 0<br />
25 100 0<br />
Control 100 0
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performed like chromatographic and mass spectrometry to characterize<br />
product quality.<br />
Current work focuses on expanding the analytical portfolio to develop<br />
control strategies for automated cell culture processes. Besides setting up<br />
a robust method for pH measurement we evaluate different spectroscopic<br />
techniques like Raman, infrared or 2D fluorescence as fast and powerful<br />
analytical tools.<br />
Results: Scale-up prediction: The comparability of results obtained with<br />
multiwell plates and bioreactors had to be verified to develop a screening<br />
system for the predictive scale-up.<br />
Using several late stage project cell lines growing in suspension the<br />
comparability of results obtained with automated, shaken multiwell plates<br />
and bioreactors with a volume of up to 1.000 L could be verified. The<br />
effects of process optimization steps on cell culture performance and<br />
product quality were shown in multiwell plates and bioreactors. Thus, the<br />
automated cell culture screening system can be used for scale up<br />
prediction.<br />
Application of pH measurement and pH control: A fully automated,<br />
multiwell plate based pH measurement assay and a pH control strategy<br />
was developed for the screening system. The established assay is based<br />
on the use of pH sensitive absorption and fluorescent dyes which are<br />
added to a cell culture sample. The advantages of this method comprise<br />
a short analytical time and the low sample volume per sample. The assay<br />
is characterized by a high precision and robustness without any probe<br />
drift during a cultivation time of up to two weeks.<br />
The successful application of the developed pH measurement and pH<br />
control could be confirmed by getting comparable pH profiles from MWP<br />
and bioreactor under the same conditions and can be kept equal by<br />
controlling the pH (Figure 1A).<br />
In a second experiment a pH shift of 0.4 pH values after 72 hours was<br />
performed (Figure 1B). The target pH was reached exactly and it could be<br />
controlled at a stable level using the developed pH measurement assay.<br />
Feasibility of Raman spectroscopy as high throughput analytical<br />
tool: Raman spectroscopy is a powerful tool for the detection and<br />
quantification of several components in cell culture processes at once. Using<br />
this fast and non-invasive analytical technique there will be no reagent costs<br />
and no sample consumption what this technique makes ideal for sm<strong>all</strong> scale<br />
high throughput systems.<br />
The feasibility of Raman spectroscopy was shown for the quantification of<br />
different metabolites and nutrients, i.e.glucose,lactateandglutamine.<br />
For the quantification of glucose (0 g/L to 20g/L), lactate (0 g/L to 10 g/L)<br />
and glutamine (0 g/L to 20 g/L) a good correlation with a high prediction<br />
accuracy could be shown.<br />
Conclusions and outlook: The developed robotic screening system is<br />
capable of performing a fully automated workflow consisting of incubation,<br />
sampling, feeding and near real-time analytics. In the performed experiments<br />
the scalability from mL scale up to 1000 L scale could be shown.<br />
Expanding the analytical portfolio a robust and fast pH measurement assay<br />
was developed to enable pH control in multiwell plates. This assay as well<br />
as pH control was tested during the cultivation of two late stage project<br />
cell lines resulting in comparable pH profiles and cell culture performance.<br />
These results enable the routinely use of the developed pH measurement<br />
and control strategy. Addition<strong>all</strong>y the proof of concept for Raman<br />
spectroscopy as a powerful tool for the quantification of metabolites and<br />
nutrients for the automated screening system could be shown. Further<br />
spectroscopic techniques using infrared or fluorescence will be evaluated.<br />
Acknowledgements: The authors would like to thank <strong>all</strong> internship and<br />
diploma students (R. Wetzel, K. Moeser, P. Linke, S. Spielmann, K. Müller,<br />
B. Frommeyer, J. Wisbauer), the Roche Penzberg pilot plant and GMP facility<br />
team, <strong>all</strong> Roche Penzberg portfolio project teams and the University of<br />
Hannover (Prof. Dr. Thomas Scheper, Dr. D. Solle).<br />
Reference<br />
1. Markert S, Joeris K: Development of an automated, multiwell plate based<br />
screening system for suspension cell culture. BMC Proc 2011, 5(Suppl 8):<br />
O9, Nov 22.<br />
P114<br />
Characterization of recombinant IgA producing CHO cell lines by qPCR<br />
David Reinhart 1 , Wolfgang Sommeregger 1 , Monika Debreczeny 2 ,<br />
Elisabeth Gludovacz 1 , Renate Kunert 1*<br />
1 Vienna Institute of BioTechnology, Department of Biotechnology, University<br />
of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria;<br />
2 Vienna Institute of BioTechnology, Imaging Center, University of Natural<br />
Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria<br />
E-mail: kunert@boku.ac.at<br />
BMC Proceedings 2013, 7(Suppl 6):P114<br />
Materials and methods: CHO host (ATCC CRL-9096) and recombinant cell<br />
lines [1] were cultivated in spinner vessels (Techne, UK) with 50 mL medium<br />
(ProCHO5, Switzerland), at 37°C and 50 rpm.<br />
Genomic DNA (gDNA) was isolated from 2 × 10 6 cells using the DNA Blood<br />
Mini Kit (Qiagen, Netherlands) according to the manufacturers’ instructions.<br />
Quantification was performed spectrophotometric<strong>all</strong>y at an absorbance of<br />
260 nm and the purity was determined by measuring the ratio at 260 nm<br />
and 280 nm. gDNA samples were stored at 4°C. Cellular RNA was isolated<br />
from 5 × 10 6 cells using the Ambion Tri Reagent Solution (Life Technologies,<br />
CA) according to the manufacturers’ instructions. To remove DNA<br />
contaminations from extracted RNA the preparation was digested with 3 U<br />
DNase I (Qiagen, Netherlands) for 30 min at RT together with 160 U RNase<br />
inhibitor (Life Technologies, CA) and then inactivated for 10 min at 75°C<br />
before another RNA precipitation step. Purified total RNA was dissolved in<br />
25 μl RNase free water containing 60 U RNase inhibitor. cDNA was obtained<br />
Figure 1(abstract P113) (A) Comparability of the pH profile between the MWP reference process and the 2L bioreactor reference process.<br />
Addition<strong>all</strong>y further pH profile and product concentration under different media compositions. (B) pH sensitive process with pH shift. The target pH,<br />
before and after the shift, was achieved by pH control.
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by reverse transcription. 1.5 μg RNA,1μg random primers (Promega, WI)<br />
and12.5nmoldNTPs(NewEnglandBiolabs,MA)wereincubatedina<br />
reaction volume of 14 μl for 5 min at 70°C and 2 min at room temperature.<br />
Then, 40 U RNase inhibitor, 200 U M-MLV reverse transcriptase and buffer<br />
(both Promega, WI) were added to a reaction volume of 20 μl and<br />
incubated for 30 min at 37°C before denaturation for 5 min at 95°C.<br />
Real-time PCR (qPCR) analysis was performed on a MiniOpticon qPCR<br />
device (Biorad, CA). Primers and the fluorogenic hydrolysis probes were<br />
synthesized by Sigma (MO). Same primers and probes were used for the<br />
analysis of gDNA and cDNA. The reaction mix included iQ Supermix<br />
(Biorad, CA), 6 pmol primer and 4 pmol hydrolysis probe for HC, JC and<br />
ß-actin quantification or 12 pmol primer and 8 pmol hydrolysis probe for<br />
LC determination in 20 μl reaction volume. 3 ng pre-denatured (99°C, 10<br />
min) gDNA or 3 μL cDNA from a 1:50 dilution of the reverse transcription<br />
reaction was used directly for qPCR. Negative controls (NC), no template<br />
controls (NTC) and no reverse transcriptase controls (NRT) for transcript<br />
analysis were included in each run. The quantification cycle (Cq) was<br />
determined by linear regression and baseline subtraction using the CFX<br />
Manager (Biorad, CA). The mean qPCR efficiencies for HC, LC, JC and<br />
ß-actin were calculated from raw fluorescence data using the LinRegPCR<br />
software application, V12.17 [2]. Quantification was done by relative<br />
quantification with efficiency correction [3] using ß-actin as internal<br />
reference and expressed as ratios.<br />
Results and discussion: qPCR was performed in six technical replicates.<br />
The Cq values and calculated efficiencies were well reproducible<br />
(Table 1). gDNA analysis revealed an over<strong>all</strong> higher exogenic GCN for the<br />
low producer 4B3-IgA than for 3D6-IgA (Figure 1). On the genomic level<br />
clone 4B3-IgA contained two times more HC, three times more JC and<br />
four times more LC than 3D6-IgA. Both clones incorporated more HC<br />
genes than JC than LC. This could be due to the presence of the dhfr<br />
amplification gene on the HC plasmid, whereas the neomycin resistance<br />
gene was located on the JC plasmid. No selection marker was included<br />
on the LC plasmid.<br />
mRNA levels were addition<strong>all</strong>y quantified by qPCR to exclude any<br />
misinterpretation of our analysis due to incompletely transfected<br />
expression cassettes, chromosomal position effects or transgene silencing.<br />
Despite higher gene copy numbers 4B3-IgA contained only half of HC and<br />
JC transcripts as compared to 3D6-IgA. LC was transcribed with the same<br />
range of efficiency and resulted in three times more LC mRNA copies. In<br />
contrast to gDNA results, LC mRNA content greatly exceeded that of HC<br />
and JC in both clones (Figure 1). Hence, LC content, which has been<br />
proposed to be critical for high antibody productivities [4], should not<br />
have been limited by mRNA. Summarized, the respective mRNA levels<br />
differed slightly between the two recombinant cell lines, but were<br />
presumably not sufficient for the low specific productivity of clone 4B3-IgA.<br />
Conclusions: An over<strong>all</strong> higher exogenic GCN was determined for the<br />
low producer 4B3-IgA as compared to 3D6-IgA. Both clones incorporated<br />
more HC genes than JC than LC. Despite higher GCNs 4B3-IgA contained<br />
only half of HC and JC mRNA transcripts as compared to 3D6-IgA. LC was<br />
transcribed with similar efficiencies whereas LC mRNA content greatly<br />
exceeded that of HC and JC in both clones. All in <strong>all</strong>, differences in<br />
specific productivity, intracellular antibody chain content and volumetric<br />
titers of the cell lines could not sufficiently be explained by qPCR data of<br />
GCN and mRNA levels. Therefore, bottlenecks are believed to occur<br />
further upstream in the translational and/or protein processing machinery.<br />
Acknowledgements: This study was funded by the European Community’s<br />
Seventh Framework Programme (FP7/2002-2013) under grant agreement N°<br />
201038, EuroNeut-41 and sponsored by Polymun Scientific Immunbiologische<br />
Forschung GmbH, Donaustraße 99, 3400 Klosterneuburg, Austria.<br />
References<br />
1. Reinhart D, Weik R, Kunert R: Recombinant IgA production: single step<br />
affinity purification using camelid ligands and product characterization.<br />
J Immunol Methods 2012, 378:95-101.<br />
2. Ramakers C, Ruijter JM, Deprez RH, Moorman AF: Assumption-free analysis<br />
of quantitative real-time polymerase chain reaction (PCR) data. Neurosci<br />
Lett 2003, 339:62-66.<br />
3. Pfaffl MW: A new mathematical model for relative quantification in realtime<br />
RT-PCR. Nucl Acids Res 2001, 29:e45.<br />
4. Borth N, Strutzenberger K, Kunert R, Steinfellner W, Katinger H: Analysis of<br />
changes during subclone development and ageing of human antibodyproducing<br />
heterohybridoma cells by northern blot and flow cytometry.<br />
J Biotechnol 1999, 67:57-66.<br />
P115<br />
Data integration methodology that couples novel bioreactor<br />
monitoring tools, automated sampling, and applied mathematics to<br />
redefine bioproduction processes<br />
Lisa J Graham * , Jeffrey F Breit, Lynn A Davis, Corey C Dow-Hygeland,<br />
Brandon J Downey<br />
Bend Research Inc, Bend, OR, USA<br />
E-mail: lisa.graham@bendresearch.com<br />
BMC Proceedings 2013, 7(Suppl 6):P115<br />
Cell physiology dynamic<strong>all</strong>y affects the nutrient requirements of a culture.<br />
It is critical to obtain data over appropriate time intervals to assess the<br />
Table 1(abstract P114) Calculated efficiencies (E), Cq and ΔCq values and copies relative to ß-actin for gDNA and<br />
cDNA derived from clones 3D6-IgA and 4B3-IgA<br />
GOI Target Clone Cq max. SD [%] E SD (%) ΔCq ß-actin Copies relative to ß-actin<br />
ß-actin gDNA 3D6-IgA 24.60 0.20 2.07 2.22 n/a n/a<br />
4B3-IgA 24.21 0.14 2.07 2.22 n/a n/a<br />
cDNA 3D6-IgA 18.52 0.13 2.03 0.43 n/a n/a<br />
4B3-IgA 16.25 0.63 2.04 1.33 n/a n/a<br />
HC gDNA 3D6-IgA 23.56 0.16 1.95 3.32 -1.03 8.28<br />
4B3-IgA 22.11 0.14 1.95 3.32 -2.11 16.44<br />
cDNA 3D6-IgA 21.78 0.17 1.91 1.35 3.26 0.38<br />
4B3-IgA 19.50 0.68 1.97 1.53 3.25 0.20<br />
JC gDNA 3D6-IgA 24.81 0.03 1.95 0.94 0.22 3.80<br />
4B3-IgA 22.77 0.10 1.95 0.94 -1.44 11.20<br />
cDNA 3D6-IgA 24.52 0.23 1.82 0.87 5.97 0.22<br />
4B3-IgA 20.81 1.54 1.96 0.27 4.56 0.10<br />
LC gDNA 3D6-IgA 24.90 0.14 2.05 0.59 0.31 0.98<br />
4B3-IgA 21.50 0.21 2.11 1.21 -2.71 4.40<br />
cDNA 3D6-IgA 20.26 0.20 1.88 0.75 1.73 1.30<br />
4B3-IgA 15.02 2.36 1.98 1.30 -1.22 3.93
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Figure 1(abstract P114) Gene copy number and transcript level of recombinant clones expressing 3D6-IgA or 4B3-IgA. The abundance of LC<br />
( ), JC ( ) and HC ( ) genes was calculated relative to ß-actin.<br />
impact of process conditions on the cell population. By optimizing<br />
bioreactor operation, feed strategies and media composition, we can limit<br />
the number of experiments to obtain the empirical data sets.<br />
For this poster, we present an emerging process-development methodology<br />
that is based on applying novel and existing bioreactor monitoring<br />
technologies, coupled with applied mathematics, to bioreactor processes. This<br />
approach employs tools like dielectric spectroscopy, aseptic autosamplers,<br />
and cell-based bioreactor models. We will illustrate how information gained<br />
from these tools can be coupled through utilization of the proper data<br />
integration and applied mathematics techniques.<br />
The knowledge gained using this improved process development<br />
methodology also supports a less-invasive monitoring and feedback<br />
system, and can be implemented using a customized bioreactor control<br />
code.<br />
P116<br />
Multicellular tumor spheroids in microcapsules as a novel 3D in vitro<br />
model in tumor biology<br />
Elena Markvicheva 1* , Daria Zaytseva-Zotova 1 , Roman Akasov 1 , Sergey Burov 2 ,<br />
Isabelle Chevalot 3 , Annie Marc 3<br />
1 Shemyakin-Ovchinnikov Inst Bioorg Chem Rus Acad Sci, 117997 Moscow,<br />
Russia;<br />
2 Institute of Macromolecular Compounds Rus Acad Sci, 199004 St-<br />
Petersburg, Russia;<br />
3 CNRS, Laboratoire Réactions et Génie des Procédés, UMR<br />
7274, Université de Lorraine, Vandoeuvre-lès-Nancy Cedex, 54505, France<br />
E-mail: lemarkv@hotmail.com<br />
BMC Proceedings 2013, 7(Suppl 6):P116<br />
Background: Advantages of microencapsulation as a 3D growth system<br />
are chemic<strong>all</strong>y and spati<strong>all</strong>y defined 3D network of extracellular matrix<br />
components, cell-to-cell and cell-to-matrix interactions governing<br />
differentiation, proliferation and cell function in vivo. The study is aimed at<br />
i) optimization of techniques for preparing microcapsules; ii) generation of<br />
multicellular tumor spheroids (MTS) by culturing tumor cells in the<br />
microcapsules; iii) study of anticancer treatment effects for both<br />
photodynamic therapy (PDT) and anti-cancer drug screening. The model<br />
<strong>all</strong>ows to estimate drug doses or parameters for PDT in vitro before<br />
carrying out preclinical tests, and thereby to reduce a number and costs of<br />
experiments with animals commonly used.<br />
Materials and methods: To form MTS, tumor cell lines (mouse melanoma<br />
cells M3, human breast adenocarcinoma cells MCF-7, mouse myeloma Sp2/<br />
0 cells, human CCRF-CEM and CEM/Cl cell lines, HeLa) were encapsulated<br />
in polyelectrolyte microcapsules (200-600 μm), and cultivated for 3-4<br />
weeks [1]. Microcapsules were fabricated from alginate (polyanion) and<br />
various polycations, namely natural polymers (modified chitosan, DEAEdextran<br />
etc) and novel smart co-polymers (e.g. chitosan-graft-polyvinyl<br />
alcohol copolymers) synthesized by a Solid-State Reactive Blending<br />
technique [2]. The copolymers were characterized by FTIR, GPC and<br />
elemental analysis.<br />
Results: MTS based MCF-7 cells were prepared and used to study<br />
effects of PDT. To study the effect of irradiation parameters on cell<br />
viability, 2 photosensitizers (PS), namely photosense and chlorine e6<br />
were used. Phototoxicity of PS depended on PS concentration and light<br />
energy density in both monolayer culture (MLC) and MTS. Study of cell<br />
morphology in MLC and MTS before and after PDT revealed that light
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energy density increase within the range of 30-70 J/cm2 resulted in cell<br />
apoptosis. However, cell survival in MTS was much higher than this in<br />
the MLC. MTS were also used to test some antitumor therapeutics<br />
(methotrexate, doxorubicin and their derivatives). An enhanced cell<br />
resistance in MTS compared to MLC both for normal and Dox-resistant<br />
cells (MCF-7, MCF-7/DXR, respectively) were observed. MTS were also<br />
proposed to evaluate cytotoxicity not only of novel therapeutics but<br />
also nanosized drug delivery systems (liposomes, micelles, nanoparticles<br />
and nanoemulsions).<br />
Acknowledgements: The authors are greatful to Dr. T. Akopova<br />
(Moscow) for synthesis of chitosan-graft-polyvinyl alcohol copolymers<br />
used in this study. The authors also thank CNRS and Russian foundation<br />
for basic research for support of the research (PICS-Russia project N°<br />
5598 - 2010-2012).<br />
References<br />
1. Zaytseva-Zotova D, Marc A, Chevalot I, Markvicheva E: Biocompatible<br />
Smart Microcapsules Based on Chitosan-Poly(Vinyl Alcohol) Copolymers<br />
for Cultivation of Animal Cells. Adv Eng Mater 2011, 13:B493-B503.<br />
2. Akopova TA, Moguilevskaia EL, Ozerin AN, Zelenetskii AN, Vladimirov LV,<br />
Zhorin VA: Proc Int Conf Mechanochemical Synthesis and Sintering Novosibirsk<br />
2004, 199.<br />
Cite <strong>abstracts</strong> in this supplement using the relevant abstract number,<br />
e.g.: Markvicheva et al.: Multicellular tumor spheroids in microcapsules<br />
as a novel 3D in vitro model in tumor biology. BMC Proceedings 2013, 7<br />
(Suppl 6):P116