CHO细胞高密度流加工艺参数优化  

Optimization of parameters for CHO cells high density fed batch

作　　者：纪海姣 于妍 杨金龙 王鹏银 JI Hai-jiao;YU Yan;YANG Jing-long;WANG Peng-yin(Upstream Technology Research Office,Tianshili Biomedical Co.,Ltd,Tianjin 300400,China)

机构地区：[1]天士力生物医药股份有限公司上游技术研究室,天津300400

出　　处：《生物技术》2023年第1期121-128,共8页Biotechnology

摘　　要：[目的]优化细胞接种密度、培养基、细胞培养温度等参数,提高生产细胞株PCSK9蛋白表达滴度。[方法]试验分四步进行,(1)探讨几款市售培养基优化组合后对CHO细胞株蛋白表达滴度的影响;(2)探讨10.0×10^(6)、15.0×10^(6)、50.0×10^(6)cells/mL高密度接种流加过程培养基、降温时间等对CHO细胞蛋白滴度的影响;(3)在(2)实验数据基础上继续优化,通过更换培养基继续探讨高密度流加工艺的可行性;(4)在反应器对实验数据进行工艺验证。[结果](1)CHO细胞PCSK9蛋白滴度提升至2.6 g/L,蛋白滴度成倍增长;(2)15.0×10^(6)cells/mL接种,30.0×10^(6)cells/mL降温至34℃,继续优化培养基1^(#)、2^(#)配比,蛋白滴度提升至4.5 g/L,反应器工艺验证,细胞生长状态稳定,蛋白滴度突破3.8 g/L;(3)继续筛选市售培养基,成功实现80.0×10^(6)cells/mL高密度流加接种,蛋白滴度提升至7.5 g/L。[结论]以贝谙基Eden-B600、Eden-400S培养基能够实现CHO细胞80.0×10^(6)cells/mL的高密度接种,流加培养D15蛋白滴度突破7.5 g/L。在细胞流加培养过程可以通过培养基成分优化、提高接种细胞密度等改善高密度流加工艺细胞株生长状态,提高目标蛋白产量。以国产培养基替换进口Sigma EX-CELL Advanced CHO Fed-Batch Medium极大地降低了生产成本。[Objective]To improve the titer of PCSK9 that processed by chinese hamster ovary cells(CHO) through optimization of cell density, medium, temperature and so on.[Method] Investigating the effect of several commercial medium or combination of several medium to the titer.Investigating the feasibility of high-density flow charging process, by improving the inoculation density.Based on the results of the second part experiment, continue screening commercial medium to realize high-density streaming progress and improve the titer.Verificating the results of the experiment optimization.[Result](1)The titer increased to 2.6 g/L in medium 4#,two times more than before.(2)The titer increased to 4.5 g/L through high density flow feeding process, three to four times more than before, the density of inoculation is 15.0×10^(6) cells/mL.(3) The titer increased to 7.5 g/L by changing new commercial medium and higher inoculation density, the density of inoculation as high as 80.0×10^(6) cells/mL.[Conclusion]By medim of Eden-B600,Eden-400S the density of CHO cells can be 80.0×10^(6) cells/mL,and the titer of D15 achived 7.5 g/L.The state and titer of cell in high-density fed-batch process can be optimized by optimizing the composition of medium and increasing the density of inoculation.The expression of the domestic medium may be higher than the foreign one, and the cost degraded greatly.

关 键 词：重组全人源PCSK9 高密度流加 CHO细胞 培养基优化 蛋白滴度 

分 类 号：Q784[生物学—分子生物学]