抗结团剂对CHO细胞悬浮培养的影响

Effect of Anti-Clumping Agents on CHO Cell Suspension Culture

目的:本实验在摇瓶和3 L生物反应器内分别研究抗结团剂(Anti-Clumping Agent,ACA)对CHO-S和CHO-K1细胞种子链阶段和流加培养阶段细胞生长和代谢、蛋白表达以及关键质量的影响。方法:使用CHO-S和CHO-K1细胞在摇瓶和3 L生物反应器进行种子链扩增和流加培养,两个细胞在两个阶段均设计添加ACA的实验组和不添加ACA的对照组,以观察细胞结团情况,检测细胞生长密度及活率,并通过SEC-HPLC和CE-SDS分析抗体纯度,iCIEF法分析PI及电荷异构体。结果:CHO-S和CHO-K1细胞在种子链阶段添加ACA均能提高细胞的生长密度,缩短细胞倍增时间。在流加阶段添加ACA,CHO-S细胞出现代谢异常,乳酸累积,pH下降;CHO-K1细胞代谢稳定,生长代谢、蛋白产量及蛋白质量与不添加ACA的对照组没有明显区别。结论:结合实验数据、工艺复杂性以及放大成本考虑,在CHO-S和CHO-K1细胞培养中,只在种子链阶段添加ACA,以改善种子状态,缩短种子扩增周期。

Objective: This experiment is conducted to study the effects of anti-clumping agent(ACA) on cell growth and metabolism, protein expression and critical quality of CHO-S and CHO-K1 cells at the seed chain stage and fedbatch culture stage, respectively, in shake flasks and 3 L bioreactors. Methods: Experimental studies are performed using CHO-S and CHO-K1 cells on shake flasks and 3 L bioreactors in the seed chain expansion and fed-batch culture stage with and without the addition of ACA. Cell clumping is observed, cell growth density and viability are detected,and antibody purity is analyzed by SEC-HPLC and CE-SDS, and PI and charge isomers are analyzed by iCIEF.Results: Addition of ACA to CHO-S and CHO-K1 cells at seed chain stage can increase cell growth density and shorten cell doubling time. When ACA is added in the feeding phase, CHO-S cells showe abnormal metabolism, lactic acid accumulation, and pH decreased;CHO-K1 cell metabolism is stable, and there is no significant difference in growth metabolism, protein yield and protein quality between the control group and the control group without ACA. Conclusion:Considering the experimental data, process complexity and amplification cost, ACA is added only in the seed chain stage in CHO-S and CHO-K1 cell culture to improve the seed status and shorten the seed amplification cycle.