过量表达CHO细胞内源性糖基化酶对抗体五聚甘露糖修饰的影响

Effects of overexpressing endogenous glycosylases on the levels of mannose-5 of monoclonal antibody produced in CHO cells

目的研究CHO K1细胞内源性糖基化酶对细胞的生长代谢以及抗体中五聚甘露糖(Man5)水平的影响。方法构建含有抗体基因、糖基化酶基因共转染的CHO K1稳定细胞株以及空载细胞株,检测细胞群和克隆阶段的抗体滴度、Man5水平,使用RT-qPCR的方法检测过表达基因的转录水平,并分析单克隆细胞基因转录水平与Man5含量的相关性。结果研究显示,过表达糖基化酶基因后对细胞生长代谢的影响相对较小,糖基化酶基因单独过表达对降低蛋白中的Man5含量没有效果。将基因Man2a2和Mgat2组合过表达之后,可以将Man5的水平降低70%~80%,两个基因的转录水平都较高的情况下对降低Man5含量可以起到更加显著的作用。结论糖基化酶基因Man2a2和Mgat2组合过表达可以显著降低抗体蛋白中的Man5水平。

Objective This study explores the effects of the overexpression of endogenous glycosylases on cell growth,metabolism and mannose 5(Man5)level of monoclonal antibody in CHO K1 cells.Methods Multiple CHO K1 null cell lines and CHO K1 stable cell lines were generated by co-transfections of mock vector and target genes,respectively,in combination of selected glycosylase genes.The titer and Man5 level of target protein in stable pool and clone stage were detected.Then,RT-qPCR method was used to analyze the transcription levels of overexpressed genes.The correlation between gene transcription levels and Man5 levels of clones were analyzed.Results The overexpression of glycosylase genes exerted relatively weak effect on cell growth and metabolism.The overexpression of individual glycosylase genes also couldn’t reduce Man5 level.However,Man5 level could be reduced significantly by 70%-80%when the Man2a2 and Mgat2 genes were overexpressed together.Higher transcript levels of both glycosylase genes may play a more significant role on reducing the Man5 level.Conclusion Combined overexpression of glycosylase gene Man2a2 and Mgat2 can significantly reduce the Man5 level of antibody protein.