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BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 54 of 151 Samples of a model antibody produced in commercially available CHO reference medium and TeutoCell’s platform medium using two different producer clones were desalted, denaturated and treated with PNGaseF. Glycanswereconcentratedviasolid-phaseextractionandanalyzedby MALDI-TOF mass spectrometry. Signal-to-noise ratios of specific masses were used for calculations of relative amounts. Results: The performance of the platform medium was evaluated using a set of eleven different CHO cell lines in comparison to an industrially relevant, protein-free and chemically defined medium. For all tested cell lines, the maximum viable cell density (vcd) as well as the integrated viable cell density (ivcd) and the product titer were higher compared to the reference. In numbers, the improvement in vcd ranged between a factor of 1.7 and 2.5, in ivcd between a factor of 1.2 and 4.2 and in product titer between a factor of 1.4 and 2.7 in batch cultures. By this improvement viable cell densities of up to 17.53·10 6 cells/mL and product titer of 1015 mg/L were reached. An overview of these results is illustrated in Figure 1. Furthermore, the potential influence of the utilized medium on product glycosylation was examined. For this, antibody harvest from two different clones cultivated in reference and platform medium was analyzed. The results of relative quantification of glycan structures by mass spectrometry showed highly comparable profiles for the reference and platform medium. An overview of the glycoanalysis is given in Table 1. As an additional application, the platform medium was successfully utilized as a basis for a chemically defined cloning medium in limited dilution experiments. For different cell lines single cell growth was achieved and cells were effectively expanded from 96-well plate format up to shaking flask cultures. Conclusions: Within this work a chemically defined and animal-component free media platform was successfully implemented, which supports high performance growth and productivity without supplementation of proteins or growth hormones. In addition, its streamlined formulation of less than 50 components increases the design space for the efficient development of custom formulations. The suitability as a platform medium was verified by the successful cultivation of a wide range of cell lines including CHO-DG44, CHO-GS and CHO-K1 clones and the feature of easy adaption from serum containing and commercially available formulations. For all tested cell lines stable high performance cultivations with high product yields were achieved, with consistent glycosylation profiles. As a further field of application, the platform medium provides the basis for single cell growth following limited dilution. Acknowledgements: Parts of this work were financially supported by the German Federal Ministry of Education and Research - BMBF (#031A106). Responsibility for the content lies with the author. P36 Streamlined process development using the Micro24 Bioreactor system Steve RC Warr * , John PJ Betts, Shahina Ahmad, Katy V Newell, Gary B Finka Upstream Process Research, GlaxoSmithKline, Stevenage, SG1 2NY, UK E-mail: steve.r.warr@gsk.com BMC Proceedings 2013, 7(Suppl 6):P36 Introduction: The Pall Micro24 Bioreactor system is one of several microbioreactor systems that have been commercialised in recent years in response to the demand to reduce costs and shorten process development time lines. We have previously demonstrated that the Micro24 Bioreactor system can be integrated successfully into the later stages of cell line screening programmes and that the results correlate well with those from more conventional methods [1]. Further process development for these selected cell lines traditionally utilises bench top bioreactors to define appropriate process conditions giving the desired process outcomes Figure 1(abstract P35) Comparison of growth performance (A) and product titer (B) using the reference and platform medium. To illustrate the improvement in relation to the reference medium, the mean of 5 producer- and the corresponding parental cell line were normalized to the results obtained in the reference medium (C and D). The error bars show the deviation between the different cell lines. The development progress of the platform medium is represented by one major interstage.
BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 55 of 151 Table 1(abstract P35) Comparison of the glycosylation pattern of a model antibody produced in two different cell lines using the reference medium and the platform medium Structure Relative Amount of Glycan Structure [%] High Producer 1 High Producer 2 Reference Shaker Platform Shaker Platform Bioreactor Reference Shaker Platform Shaker Platform Bioreactor Man3 - - - 4 ± 2 1 ± 2 - Man5 7 ± 2 3 ± 2 5 ± 2 12 ± 3 9 ± 2 4 ± 1 G0F-GlcNAc 2 ± 2 2 ± 1 4 ± 2 10 ± 4 5 ± 0 2 ± 2 G0F 46 ± 2 50 ± 5 47 ± 5 47 ± 5 47 ± 2 47 ± 5 G1 6±3 6±2 8±2 3±1 9±1 5±1 G1F 30 ± 3 32 ± 5 29 ± 7 19 ± 2 23 ± 3 35 ± 2 G2F 9±2 7±2 7±2 5±1 6±1 7±2 The relative amounts of detected structures are given in % with the standard deviation of four independent analyses. although this approach can be time consuming and resource intensive. However the Micro24 Bioreactor system allows up to 24 different process conditions to be run concurrently thereby facilitating efficient process development. This work describes the use of the Micro24 Bioreactor system to identify improved process conditions for different cell lines and their subsequent validation in bioreactors. Micro-24 bioreactor system (Pall): This system comprises 24 bioreactors (7 ml working volume) each capable of independent temperature, dissolved oxygen and pH control. The main limitation of the system is the lack of automation meaning that any feed additions or sample removal must be made manually and similarly, for mammalian cultures, upwards pH control is achieved by the manual addition of NaHCO 3 . Engineering characterisation studies carried out at UCL (data not shown) have shown how conditions within the individual Micro24 chambers compare with those in bioreactors and recent results also indicate that the selection of the Micro24 plate type is critical in ensuring good correlation with performance in traditional bioreactors. Within the Micro24 Bioreactor system cell cultures are carried out in presterilised polycarbonate mammalian cell culture cassettes which are inoculated manually in a laminar flow cabinet before sealing with Type A single use closures and incubation under experimental conditions. Methods: Chemically defined medium and feeds were used throughout this work. Unless otherwise stated standard experimental conditions were used. (35°C, pH 6.95, 30% Dissolved Oxygen (DO)). Viable cell numbers and viability were determined using a ViCell Cell Viability Analyser (Beckman Coulter) and antibody titres were determined using an Immage Immunochemistry System (Beckman Coulter). Process optimisation: Typical process relevant factors that can be tested in the Micro24 include feed regime, pH, DO and temperature. The effects of these types of factors are best tested using a Design of Experiments (DoE) approach to assess the effects not only of different factors but also of the interactions between them. Such data can then be used to build predictive models of process performance to specify the appropriate operating conditions in larger scale bioreactors. We have already developed and are using a similar approach for microbial dAb processes. The data below shows examples of how we have used this system to identify improvements to platform processes for specific cell lines. Case Study 1 - process conditions: In this experiment the effects of changes to the platform process pH and DO set points on the performance of a mAb producing cell line were assessed in the Micro24 using a DoE approach with different operating conditions in each well. This data demonstrated that although the dissolved oxygen level had little effect on viable cell numbers, titres and specific productivity, operating at a higher pH than the standard platform set point resulted in an increase in titre and in specific productivity. There was no significant interaction between the factors. Bioreactor validation (1) - 2 litre scale: The high pH process identified from the Micro24 was run in 2 litre bioreactors and compared to the standard platform process. At the high pH set point cell numbers during the later stages of the process were slightly reduced compared to the control and as in the Micro24 higher titres were produced under higher pH conditions. However, as in the Micro24 the greatest effect of increased pH was on specific productivity which in the bioreactors was increased by approximately 35% compared to the control. Bioreactor validation (2) - 50 litre scale: Similar results were achieved at the50litrescaleforadifferentcelllinerunninginthesameplatform process but producing a different molecule (Figure 1). There was little effect on the cell numbers but the higher pH condition resulted in increased titre, culture duration, volumetric productivity and specific productivity. Case study 2 - feeding regime: The Micro24 can be used to investigate the effect of different feeding regimes on culture performance. We have already demonstrated that the effect of feed addition on culture performance in the Micro24 is similar to that in shake flasks [1]; the data below (Table 1) shows that for a chemically defined process multiple feed additions have a similar effect in 2 litre bioreactors to the Micro24. In both systems the addition of the feed results in significant increases in cell numbers and titre. Culture duration is increased and the overall specific activity is increased by 63% in the Micro24 and 79% in the bioreactors. Discussion: Our previous work has demonstrated how the Micro24 system can be used for mammalian cell line selection [1] and the data presented here extends the application of the Micro24 into mammalian process development. The parallel nature of the Micro24 enables process relevant factors to be tested in DoE experiments and these data show that improved process conditions such as increased pH and feed additions identified in the Micro24 can be used to achieve process improvements in bioreactors. The validation of the Micro24 results in bioreactors suggests that the integration of this technology into mammalian process development could reduce significantly the numbers of bioreactors required to achieve process improvements which could result in reduced resource requirements and improved timelines. Reference 1. Warr S, Patel J, Ho R, Newell K: Use of Micro Bioreactor systems to streamline cell line evaluation and upstream process development for monoclonal antibody production. BMC Proceedings 2011, 5(Suppl 8):P14. P37 Temperature dependency of immunoglobulin production in novel human partner cell line Galina Kaseko * , Marjorie Liu, Edwin Hoe, Qiong Li, Mercedes Ballesteros, Tohsak Mahaworasilpa The Stephen Sanig Research Institute, Sydney, NSW, 2015 Australia E-mail: g.kaseko@ssri.org.au BMC Proceedings 2013, 7(Suppl 6):P37 Introduction: A number of immunoglobulin (Ig) secreting human hybrid cell lines were created using one-on-one somatic cell hybridization of a rare human tumor infiltrating B lymphocyte and a cell of a novel human cell line (WTM), developed in house and described earlier [1]. These hybrid cell lines secret various amounts of tumor-derived immunoglobulins (Igs) of different specificities. Current investigative efforts are directed towards
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