Assessment of process performance and product quality in high-performing fedbatch

Transcription

1 Assessment of process performance and product quality in high-performing fedbatch Thomas Falkman, Anita Vitina, Annika Morrison, Tomas Björkman, and Andreas Castan GE Healthcare Life Sciences, Björkgatan 30, Uppsala, Sweden First presented at the 23 rd ESACT meeting, Lille, France June 23-26, 2013

2 Abstract A stable, robust and well-characterized process that generates a product with the defined critical quality attributes is of great importance for the biomanufacturer. In this study, we investigated the performance of a CHO fed-batch in five fed-batch using ActiCHO Media System; in WAVE Bioreactor system at 10 L and 25 L scale, and in a conventional, stainless steel, stirred-tank (SS-STR) bioreactor at 100 L scale. The CHO cell line was of DG44 origin and stably transfected to produce a human IgG1 monoclonal antibody. Consistent process performance was demonstrated with respect to cell growth, cell viability, IgG1 production, metabolite patterns, osmolality, glycan distribution, and aggregate levels, independent of bioreactor system or scale. 2

3 Materials and Methods WAVE Bioreactor 20/50 system (GE Healthcare). WAVEPOD II (GE Healthcare). Cellbag 20 L and 50 L with optical DO and ph sensor (GE Healthcare). 100 L SS-STR Bioreactor (Belach Bioteknik AB). CHO DG44 cell line expressing monoclonal antibody IgG1 (licensed by Cellca GmbH). ActiCHO P medium (GE Healthcare) supplemented with 6 mm L-glutamine. ActiCHO Feed A and Feed B medium (GE Healthcare). Nutrients and physical parameters were measured off-line using a Bioprofile Flex (Nova Biomedical). Cell density and viability were measured using a CASY Cell Counter and Analyzer (Roche). IgG titers were determined with Biacore system (GE Healthcare). IgG was purified using HiTrap MabSelect SuRe Protein A columns (GE Healthcare). Aggregate and fragments were quantified using Tosoh TSK G3000SWxl Size Exclusion Chromatography (Tosoh). Feeding of glucose started at day 6 post-inoculation and was carried out to maintain a glucose level of 4 g/l. Glycosylation profile was obtained by enzymatic cleavage of the purified IgG followed by liquid chromatography mass spectrometry. 3

4 Materials and Methods Bioreactor settings are listed in Table 1. Cells were inoculated at e6 cells/ml in production medium (ActiCHO P). Feeding with Feed A and Feed B started at day 3 post-inoculation with 43.2 g/l/d and 4.32 g/l/d, respectively. 4

5 Results and Discussions Cell growth and productivity were comparable among the five, demonstrating a similar process performance in the different cultivation vessels and scales. The maximum cell density was in the range of 20 million cells/ml and the peak IgG1 production level was 5 g/l (Fig 1). 5

6 Results and Discussions The glucose concentrations were maintained at 4 g/l (Fig 2) and the lactate concentrations were around 0.5 g/l in the end, after an initial stage with lactate production of up to 2 g/l (Fig 3). The observed metabolic switch is the combined result of the characteristics of the CHO DG44 clone, with a balanced nutrient supply from the media and feeds. 6

7 Results and Discussions The feeds are highly concentrated but the osmolality was consistent at a level of 340 in all cell (Fig 3-4), indicating a correct tailoring of the total nutrient amount fed. 7 product quality in high-performing fed-batch

8 Results and Discussions The studied product quality attributes aligned well with the robust growth performance and was consistent between the. The GOF, G1F and G2F glycan distribution was even through-out the course of the culture showing reliable process performance in different vessel formats and scales (Fig. 5-7). 8

9 Results and Discussions The aggregate level was low, around 1 % (Fig. 9) and the percentage of monomers was high (Fig 10). 9 product quality in high-performing fed-batch

10 Conclusion Comparable process performance in all cell culture runs was demonstrated with respect to: cell growth and cell viability IgG production metabolite patterns (glucose and lactate concentrations) osmolality A comparable product quality profile was demonstrated with respect to glycan distribution aggregate levels In conclusion, by using the ActiCHO Media System, a consistent performance could be demonstrated among all, independent of bioreactor system or scale. 10

11 Acknowledgments Glycan analysis data was provided by Cobra Biologics Ltd. GE, imagination at work, and GE monogram are trademarks of General Electric Company. ActiCHO, Biacore, HiTrap, Cellbag, MabSelect SuRe, WAVE Bioreactor, WAVEPOD are trademarks of GE Healthcare companies. All third party trademarks are the property of their respective owners. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information General Electric Company All rights reserved. First published June 2013 GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden 11