高表达抗PD-1抗体CHO细胞株的筛选及代谢分析

Screening of Anti-PD-1 Antibody High-producing CHO Cell Line and Metabolism Analysis

目的筛选高表达抗PD-1抗体的工程细胞株和较优的基础及补料培养基,并进行代谢分析。方法分别在TPP摇管、摇瓶中通过流加培养的方式,从6株细胞株和5种商业培养基中筛选高表达抗PD-1抗体的GS-CHO细胞株和较优的基础及补料培养基。在流加培养过程中,检测葡萄糖、谷氨酰胺、谷氨酸、乳酸及氨等的浓度变化。同时为分析限制性营养因素,进一步对乳酸、氨及氨基酸代谢进行分析。结果通过TPP摇管初步筛选,显示122和126两株细胞在HyCell CHO培养基中表现最好,表达量分别为1.945和1.989g/L;在摇瓶中进一步验证显示122细胞在6株细胞中的最大活细胞密度图最高,达2.21×107cells/mL,表达量>2g/L。代谢分析发现,乳酸代谢切换和谷氨酸及丙氨酸代谢相关。氨基酸分析发现,蛋氨酸、异亮氨酸、亮氨酸等必需氨基酸可能为122细胞株生长和(或)表达限制性成分。结论筛选出高表达抗PD-1抗体的细胞株和较优培养基,同时代谢分析和氨基酸分析为后续细胞培养工艺开发及个性化培养基优化提供理性指导。

Objective To screen GS-CHO cell lines that have high expression of anti-PD-1antibody,their suitable basal and feed medium,and to analyze the metabolites.Methods We screened from6recombinant cell lines and5preferred commercial mediums by fed-batchin TPP tube and shake flask.At the same time,we measured glucose,glutamate,glutamine,lactate,and ammonia concentrations.To analyze restricted nutritional factors,we further explored amino acid metabolism in high-yield cell lines.Results With TPP tubes preliminary screening,122and126cell lines performed best in HyCell CHO medium with titer expression levels of1.945g/L and1.989g/L,respectively.Further verification in shake flasks showed that PVCD and titer of the122cell line in HyCell CHO medium were the highest,which could reached2.21×10^7cells/mL and titer greater than2g/L.Meanwhile,it was found that lactic acid metabolism switching was associated with glutamate and alanine metabolism.Amino acid metabolism revealed that essential amino acids such as methionine,isoleucine,and leucine may be the restrictive factors of growth and/or expression for122cell line.Conclusion We screened cell line with high expression of anti-PD1antibody and corresponding optimal medium.Furthermore,metabolism analysis of metabolites and amino acid may provide rational guidance for the subsequent cell culture process development and customized medium optimization.