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Debottlenecking Manufacturing Capacity Using High Seed Density Production Processes

Authored and presented by: Richard Ottman, Yogender Gowtham, S. Rameez, K. C. Zhang, S. S. Mostafa.

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Debottlenecking Manufacturing Capacity Using High Seed Density Production Processes

  1. 1. Debottlenecking Manufacturing Capacity Using High Seed Density Production Processes Richard Ottman, Yogender Gowtham, S. Rameez, K. C. Zhang, S. S. Mostafa. Process Development and Manufacturing, KBI Biopharma, Durham, NC A key bottleneck for mammalian cell culture productivity is the extended duration of the process with inoculum seed train and production culture stretching between 4-6 weeks in duration. Introducing flexibility in scheduling and execution of cell culture manufacturing campaigns via a reduction in process duration can be one of the key strategy for maximizing facility utilization and facilitating the progression of multiple therapeutics to clinical trials. In this work, we investigated the initiation of CHO cell culture production runs using cultures utilizing Selexis’ CHO-M cell line paired with higher seeding density which allowed for process time to be reduced to 10 days from 14 - 15 days while maintaining titers and product quality. To achieve this result optimal media and feed combinations were first identified that supported higher peak cell densities. Fed- bath culture timeline was shifted to align with peak cell densities and process parameters refined to maintain culture health. This improved process was identified in ambr15 and ambr250 scale down models and successfully transitioned to 3L and 15L bioreactors to demonstrate successful scale up and optimization of process parameters. Overall, this approach offers a realistic possibility of decreasing the cell culture manufacturing timeline and introduces flexibility in scheduling and executing manufacturing. Advancing Manufacturing Timelines • Conventional CHO cell biomanufacturing processes are typically conducted over an extended period, lasting anywhere between 4-6 weeks. • The manufacturing run usually comprises of vial thaw followed by inoculum expansion across scales (shake flasks, Wave® bioreactor and Xcellerex bioreactor) followed by the production run (mostly fed-batch) to generate desired product. High Seed Density Mammalian Cell Culture Processes Summary Vial thaw (3 days) Seed Train Expansion (15-21 days) N-2 Step: 50L Wave Bioreactor (3 days) N-1 Step: 200L XDR Bioreactor (3 days) Fed-batch Production: 2000L XDR Bioreactor (14-16 days) • Biopharmaceuticals have witnessed an exponential growth over the past decade, with eight out ten best selling drugs in 2018 being biologics. • More than a third of biopharmaceutical manufacturing processes uses mammalian cells, particularly CHO cells, as the host for producing the biologics. Biopharmaceuticals 1982 to 20141 Annual Biopharmaceutical Sales2 • The extended duration of the CHO cell manufacturing process is a key bottleneck in the biopharmaceutical industry. • The recent advancements in CHO cell line productivity (>5g/L titer) and efficient media and feed systems can be utilized towards developing next-gen manufacturing processes. • Next-generation cell culture processes should aim at reducing process duration and increase flexibility in scheduling and execution of manufacturing campaigns. • Such a change is necessary to maximize facility utilization and to accommodate the increasing portfolio of multiple therapeutics. • Next-generation manufacturing cell culture processes should aim to disrupt the conventional workflow by accelerating the inoculum expansion step and/or the production bioreactor step. • The proprietary expression vectors (SUREtech VectorsTM that contain Selexis SGE® Genetic Elements), combined with high-throughput screening of clones provides cell lines capable of achieving higher cell growth and productivities even for difficult-to- express proteins. • Optimization of the cell culture medium is important in order to support higher cell densities. Chemically defined media were screened in shake flask batch cultures. Clear differences were noted between formulations, half of the tested formulations were able to promote fast growth up to days 3-4, fewer were able to sustain growth past day 4. • Using the media that supported both growth and higher densities, blends were developed that supported higher peak cell densities and ultimately higher titers compared against the parental media. Conventional Processes Medium Optimization Process High Seed Density Seed Train Passage 1 Passage 2 2-3 day passages (Shake Flasks) 10-14 day culture Passage 3 Passage 4 Abstract • With recent advancements, next-generation cell culture processes aim to reduce process duration and increase flexibility in manufacturing campaigns. • Disruptive cell culture processes have shown to generate productivity and product quality comparable with the conventional process while reducing total process duration. Next Steps • Developing media and feeds specific for high density cultures. • Scale-up of high cell density cell culture processes • Evaluate more molecules/cell lines especially non-mAbs Acknowledgments • KBI Process Development Team • KBI Analytical Development Team • Selexis Team Shukla A.A., Rameez S., Wolfe L.S., Oien N. (2017) High-Throughput Process Development for Biopharmaceuticals. In: Advances in Biochemical Engineering/Biotechnology. Springer, Berlin, Heidelberg Rameez S. Direct inoculum of bioreactors with CHO cells from frozen seed bags to eliminate continual seed trains and improve facility utilization. (2017) The Cell Culture Dish • The intrinsic higher cell growth of Selexis’ CHO-M cell lines combined with optimized media and feed systems can be utilized towards developing high density seed trains. • Facility utilization increases by 14 - 27% for two or three days decrease in cell culture duration. • A CHO-M cell line expressing an Fc-fusion protein was adapted to 6 commercially available media and 4 different blends of media for 4 passages. To reduce seed train expansion timelines, the target medium should promote low population doubling times. • Following media adaptation a shake flask batch study was conducted to identify media and blends that supported the highest growth with out addition of feeds. • High density seed trains were optimized with a small feed addition on the second day of the n-2 and n-1 passages. This maintains growth of the high density cultures which has a carry over effect maintaining low PDTs through to the production bioreactor. High Density Production Bioreactor Process • Production cultures inoculated at up to 8x seeding density display similar growth curves and do not display any significant differences in cell viability through 10 days in culture. • Production cultures of 2 different CHO-M cell lines expressing either an Fc-fusion or bi-specific Mab. Cultures were inoculated at 4x, 5x, and 10x seeding density. Optimized medium and higher cell densities consistently produce equivalent titers in fewer production days.

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