A High Cell Density Approach to Fed-Batch Cell Culture for Production of Biopharmaceuticals Sampath Kumar,Kevin McCarthy,Linda Francullo,Kerstin Crowe,Robin Heller-Harrison,Wenge Wang,Gregory Hiller,and Mark Leonard
Abstract We have evaluated a more cell-intensive approach to both cell line adap-tation and production culture as a means of achieving high integrated viable cell density(IVCD)in order to improve cell culture process yields.Shakeflask bad Fed-batch(FB)production cultures were eded at multiple cell densities.Increas in ed density in the fed batch up to8x control ed density resulted in signif-icantly higher IVCD and titer with a2–3fold benefit relative to control eded cultures.Whereas the initial increments in production ed density resulted in a lin-ear increa in IVCD and titer,further increments showed diminishing returns.It appeared that the highest ed density production cultures were limited by nutrient supply and so richer medium and a higher feeding rate were then tested,resulting in further improvements in IVCD and titer.We also tested the effect of adapting the cells to high densities for over50generations prior to production cultures.For the production asssment,both the high-and standard-density adapted cells were eded at multiple cell densities.Effect of the adaptation was modest and vari-able.By combining high-ed adaptation,high production culture ed densities, enriched media and high feeding rate titer yields of up to9g/L were achieved.
1Introduction
An increasing number of antibodies and proteins produced in mammalian cells are approved as human therapeutics while the costs of production of the molecules remain high.The biopharmaceutical industry has been striving to improve the pro-ductivities of the production process as a means of reducing the cost of the drugs.
For recombinant protein production,volumetric productivity is a function of spe-cific cellular productivity(Qp)and integrated viable cell density(IVCD).Increas in either or both of the variables could potentially help improve the volumet-ric productivity of a cell culture process.While we are striving for productivity S.Kumar(B)
Bioprocessing R&D,Pfizer,Inc.,Andover,MA01810,USA
怎样折五角星e-mail:Sampath.kumar@Pfi
589 N.Jenkins et al.(eds.),Proceedings of the21st Annual Meeting of the European Society
for Animal Cell Technology(ESACT),Dublin,Ireland,June7–10,2009,ESACT Proceedings5, DOI10.1007/978-94-007-0884-6_97,C Springer Science+Business Media B.V.2012
590S.Kumar et al. improvements by both means,in the prent t of studies we focud on the lat-ter.We adopted a multi-pronged approach that involved cell line adaptation,high production culture ed densities,enriched media and high feeding rate.The study demonstrated significantly higher cell culture productivities.
2Materials and Methods
Two newly transfected cell lines,one stably expressing antibody X(Ab X),the other stably expressing small protein therapeutic Y(SPT Y),were adapted to grow at either standard or high ed densities during3-day/4-day passaging for50gener-ations or more.The rationale for the adaptations was(i)to acclimate the cells to a high-density environment which they would subquently experience under pro-duction cultures,and(ii)to achieve highfinal densities in N-1ed cultures that would enable eding of high-density production cultures.The relative performance of the cell lines was assd in FB using a pH-adjusted shakeflask model.Both the high-and standard-density adapted cell lines were eded at increasing cell den-sities in FB(control density and2x-4x-and8x-control density).Optimization of the ba medium as well as the feed strategy for the high ed pH-adjusted shakeflask FB was performed.The high-density-rich medium-FB cultures of the high-density adapted cells were also evaluated in bench-top bioreactors,and at160or500-L pilot scale,using the conditions
established in the shakeflask model.
3Results
During adaptation,the growth rate,cell viability and specific cellular productivity of the3-day/4-day cultures were comparable for the cells adapted under high-ed and standard-ed conditions(data not shown).
那些年一起追过的女孩In FB production cultures increas in ed density up to8x control resulted in significantly higher IVCD and titer with a maximum2-3-fold benefit relative to control-eded cultures(Fig.1).Whereas the initial increments produced nearly linear increas in IVCD and titer,further increments showed diminishing returns (Fig.2).Cultures eded at4and8x control ed densities showed early decline in cell viability(data not shown)suggesting limitation of nutrient supply.Hence, a richer ba medium and a higher feeding rate were then employed.Daily feeds totaling about3times the standard feed per FB resulted in further improvements in IVCD and titer(Fig.3).Subquently we employed the strategy of high-density adaptation,high ed density in FB,richer medium and higher feed rate to six more cell lines in both shakeflask and bench-top bioreactors and found similar results (data not shown).The effect of high-density adaption was modest and variable;three of the six cell lines showed5–50%increa in titer while the other three showed no improvement.
A High Cell Density Approach to Fed-Batch Cell Culture for Production of ...
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伤感的诗词Fig.1IVCD and titer of the two cell lines.Both standard-(dotted lines )and high-ed adapted (solid lines )lineages of each cell line were eded at control ed density (×)and 2x-( )4x-(o)and 8x-control density ( ).N =2
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Fig.2Increas in day-11IVCD and day-11titer in respon to increasing ed density for the high-ed density adapted lineages of the two cell lines.N =2
4Discussions
The past decade has witnesd major strides made in production of biopharmaceu-ticals in mammalian cell hosts both in terms of productivity and process robustness.The advances in productivity have been largely enabled by technologies that gener-ate and/or identify cell lines of high Qp,and media improvements that support high density cultures with high cell viabilities for extended durations.The prent study
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Fig.3IVCD and titer of the cell line expressing Ab X in the shake flask bad FB process employing richer ba medium and high feeding rate
一生只爱一个人demonstrates the potential for improvement in productivities through increasing production culture IVCD.
We employed a combination of strategies:(i)high-density cell adaptation,(ii)incread ed densities,and (iii)richer ba medium and high feeding rate.While each of the individual strategies parately produced improvements in outcome,the benefits from high ed adaptation were modest and variable.The combined strategy resulted in IVCDs up to 380E6cell-days/mL and titers of up to 9g/L in standard shake flask bad FB cultures of extended duration (up to 17days),a significant improvement over the control conditions.The process was further suc-cessfully demonstrated in bench-top bioreactors and subquently in 160–500L pilot scale bioreactors with titers up to 6g/L.
