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Debottlenecking Manufacturing Capacity Using High Seed Density Production Processes
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
• 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
N-2 Step: 50L
N-1 Step: 200L
• Biopharmaceuticals have witnessed an exponential growth over
the past decade, with eight out ten best selling drugs in 2018 being
• 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-
• 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.
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
• With recent advancements, next-generation cell culture processes aim to
reduce process duration and increase flexibility in manufacturing
• Disruptive cell culture processes have shown to generate productivity and
product quality comparable with the conventional process while reducing
total process duration.
• 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
• 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.