大规模哺乳动物细胞培养中pCO_2的控制策略
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摘要
细胞培养工艺放大是生物抗体药物从实验室工艺走向临床和商业生产的必经之路,基于生物反应器的大规模细胞培养技术是制药企业的核心竞争力。哺乳动物细胞对环境变化高度敏感,大小规模反应器中工艺表现不一致成为放大的主要挑战,其中高二氧化碳分压(pCO_2)问题给大规模反应器中细胞生长、代谢和蛋白产量与质量带来严重影响。二氧化碳(CO_2)积累主要来自于细胞代谢产生CO_2与反应器传质水平的失衡。通过分析CO_2积累的原因,从培养工艺的优化和放大参数的合理设计两方面总结了降低大规模CO_2积累的方法,最后结合新型大规模反应器pCO_2预测模型,为工艺放大过程中pCO_2的有效控制提供参考。
Bioprocess scale-up is a fundamental component of process development in the biotechnology industry,which is the way to make a biologics from laboratory to commercial manufacturing. Large-scale cell culture technology based on bioreactor is the core competitiveness of pharmaceutical enterprises. Mammalian cells are highly sensitive to environmental changes,and the inconsistency in process performance has become a major challenge for scale-up,especially the carbon dioxide( CO_2) accumulation in large-scale bioreactors. CO_2 accumulation is a common feature of large scale mammalian cell culture due to the lower CO_2 stripping rate compared to small scale. When CO_2 partial pressure( pCO_2) is above a certain level( > 80 mm Hg),cell growth,metabolism and productivity can be impacted,as well as the product quality like N-glycans. By analyzing the causes of CO_2 accumulation,two strategies including the optimization of culture process and scale-up related parameters are discussed to reduce large-scale high pCO_2. In addition,a systematic pCO_2 control strategy by using the novel CO_2 stripping model is also discussed.
Bioprocess scale-up is a fundamental component of process development in the biotechnology industry,which is the way to make a biologics from laboratory to commercial manufacturing. Large-scale cell culture technology based on bioreactor is the core competitiveness of pharmaceutical enterprises. Mammalian cells are highly sensitive to environmental changes,and the inconsistency in process performance has become a major challenge for scale-up,especially the carbon dioxide( CO_2) accumulation in large-scale bioreactors. CO_2 accumulation is a common feature of large scale mammalian cell culture due to the lower CO_2 stripping rate compared to small scale. When CO_2 partial pressure( pCO_2) is above a certain level( > 80 mm Hg),cell growth,metabolism and productivity can be impacted,as well as the product quality like N-glycans. By analyzing the causes of CO_2 accumulation,two strategies including the optimization of culture process and scale-up related parameters are discussed to reduce large-scale high pCO_2. In addition,a systematic pCO_2 control strategy by using the novel CO_2 stripping model is also discussed.
引文
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[30] Xing Z,Kenty BM,Li ZJ,et al. Scale-up analysis for a CHO cell culture process in large-scale bioreactors[J]. Biotechnol Bioengin,2009,103(4):733-746.
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[32] Xing Z,Lewis AM,Borys MC,et al. A carbon dioxide stripping model for mammalian cell culture in manufacturing scale bioreactors[J]. Biotechnol Bioengin,2017,114(6):1184-1194.
[33] He C,Ye P,Wang H,et al. A systematic mass-transfer modeling approach for mammalian cell culture bioreactor scale-up[J].Biochem Engin J,2019,141:173-181.
[34] Sommeregger W,Sissolak B,Kandra K,et al. Quality by control:towards model predictive control of mammalian cell culture bioprocesses[J]. Biotechnol J,2017:1600546.
[2] Feng X,Cheng J,Li X,et al. Numerical simulation of turbulent flow in a baffled stirred tank with an explicit algebraic stress model[J]. Chem Engin Sci,2012,69(1):30-44.
[3] Kimura R,Miller WM. Effects of elevated p CO2and/or osmolality on the growth and recombinant t PA production of CHO cells[J].Biotechnol Bioengin,1996,52(1):152-160.
[4] Gray DR,Chen S,Howarth W,et al. CO2in large-scale and highdensity CHO cell perfusion culture[J]. Cytotechnol,1996,22:65-78.
[5] Dezengotita VM,Kimura R,Miller WM. Effects of CO2and osmolality on hybridoma cells:growth,metabolism and monoclonal antibody production[J]. Cytotechnol,1998,28(1-3):213-227.
[6] Zhu MM,Goyal A,Rank DL,et al. Effects of elevated p CO2and osmolality on growth of CHO cells and production of antibody-fusion protein B1:a case study[J]. Biotechnol Progress,2005,21(1):70.
[7] Mostafa SS,Gu X. Strategies for improved d CO2removal in largescale fed-batch cultures[J]. Biotechnol Progress,2003,19(1):45-51.
[8] Schmelzer AE,Miller WM. Hyperosmotic stress and elevated p CO2alter monoclonal antibody charge distribution and monosaccharide content[J]. Biotechnol Progress,2002,18(2):346-353.
[9] Zanghi JA,Schmelzer AE,Mendoza TP,et al. Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated p CO2or p H[J]. Biotechnol Bioengin,1999,65(2):182-191.
[10] Nahler G. International conference on harmonisation(ICH)[J].Diction Pharmac Med,2009,69(3-4):96-96.
[11] Xu S,Hoshan L,Jiang R,et al. A practical approach in bioreactor scale-up and process transfer using a combination of constant P/V and VVM as the criterion[J]. Biotechnol Progress,2017,33(4):1146-1159.
[12] Sieblist C,Jenzsch M,Pohlscheidt M. Equipment characterization to mitigate risks during transfers of cell culture manufacturing processes[J]. Cytotechnol,2016,68(4):1381-1401.
[13] Goudar CT,Matanguihan R,Long E,et al. Decreased p CO2accumulation by eliminating bicarbonate addition to high cell-density cultures[J]. Biotechnol Bioengin,2007,96(6):1107-1117.
[14] Takuma S,Hirashima C,Piret JM. Dependence on glucose limitation of the p CO2influences on CHO cell growth,metabolism and Ig G production[J]. Biotechnol Bioengin,2007,97(6):1479-1488.
[15] Gagnon M,Hiller G,Luan YT,et al. High-end p H-controlled delivery of glucose effectively suppresses lactate accumulation in CHO fed-batch cultures[J]. Biotechnol Bioengin,2011,108(6):1328-1337.
[16] Xu S,Jiang R,Mueller R,et al. Probing lactate metabolism variations in large-scale bioreactors[J]. Biotechnol Progress,2018,34(3):756-766.
[17] Brunner M,Doppler P,Klein T,et al. Elevated p CO2affects the lactate metabolic shift in CHO cell culture processes[J]. Engin Life Sci,2018,18(3):204-214.
[18] Darja O,Stanislav M,Sasa S,et al. Responses of CHO cell lines to increased p CO2at normal(37°C)and reduced(33°C)culture temperatures[J]. J Biotechnol,2016,219:98-109.
[19] Brunner M,Fricke J,Kroll P,et al. Investigation of the interactions of critical scale-up parameters(p H,p O2and p CO2)on CHO batch performance and critical quality attributes[J]. Bioprocess Biosystems Engin,2017,40(2):251-263.
[20] Matanguihan R,Sajan E,Zachariou M,et al. Solution to the high dissolved CO2problem in high-density perfusion culture of mammalian cells. Animal Cell Technology:From Target to Market[M]. Springer Netherlands,2001.
[21] Yang WC,Minkler DF,Kshirsagar R,et al. Concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity[J]. J Biotechnol,2016,217:1-11.
[22] Brunner M,Braun P,Doppler P,et al. The impact of p H inhomogeneities on CHO cell physiology and fed-batch process performance-two-compartment scale-down modelling and intracellular p H excursion[J]. Biotechnol J,2017,12(7):1600633.
[23] Hoshan L,Jiang R,Moroney J,et al. Effective bioreactor p H control using only sparging gases[J]. Biotechnol Progress,2018.
[24] Li J,Wong CL,Vijayasankaran N,et al. Feeding lactate for CHO cell culture processes:impact on culture metabolism and performance[J]. Biotechnol Bioengin,2012,109(5):1173-1186.
[25] Nienow AW. Reactor engineering in large scale animal cell culture[J]. Cytotechnol,2006,50(1-3):9-33.
[26] Sieblist C,Hageholz O,Aehle M,et al. Insights into large-scale cell-culture reactors:II. Gas-phase mixing and CO2stripping[J].Biotechnol J,2011,6(12):1547-1556.
[27]郑兵兵.动物细胞培养用生物反应器内流体混合及气液传质特性研究[D].上海:华东理工大学,2013.
[28] Dreher T,Husemann U,Ruhl S,et al. Design space definition for a stirred single-use bioreactor family from 50 to 2000 L scale[J].BMC Proceed,2013,14(3):304-310.
[29] Matsunaga N,Kano K,Maki Y,et al. Culture scale-up studies as seen from the viewpoint of oxygen supply and dissolved carbon dioxide stripping[J]. J Biosci Bioengin,2009,107(4):412-418.
[30] Xing Z,Kenty BM,Li ZJ,et al. Scale-up analysis for a CHO cell culture process in large-scale bioreactors[J]. Biotechnol Bioengin,2009,103(4):733-746.
[31] Zhu Y,Cuenca JV,Zhou W,et al. NS0 cell damage by high gas velocity sparging in protein-free and cholesterol-free cultures[J].Bioengin Biotechnol,2008,101(4):751-760.
[32] Xing Z,Lewis AM,Borys MC,et al. A carbon dioxide stripping model for mammalian cell culture in manufacturing scale bioreactors[J]. Biotechnol Bioengin,2017,114(6):1184-1194.
[33] He C,Ye P,Wang H,et al. A systematic mass-transfer modeling approach for mammalian cell culture bioreactor scale-up[J].Biochem Engin J,2019,141:173-181.
[34] Sommeregger W,Sissolak B,Kandra K,et al. Quality by control:towards model predictive control of mammalian cell culture bioprocesses[J]. Biotechnol J,2017:1600546.
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