CHO细胞表达重组人生长激素-Fc融合蛋白糖基化类型的优化  

Optimization of glycosylation type of recombinant human growth hormone-Fc fusion protein expressed in CHO cells

作　　者：张凯宁 刘涵 朱秋媚 富锐丽 刘玉林 孙瑞欣 刘景会 赵竞男 韩雪容 ZHANG Kai-ning;LIU Han;ZHU Qiu-mei;FU Rui-li;LIU Yu-lin;SUN Rui-xin;LIU Jing-hui;ZHAO Jing-nan;HAN Xue-rong(不详;School of Life Science and Technology,Changchun University of Science and Technology,Changchun130000,Jilin Province,China)

机构地区：[1]长春理工大学生命科学技术学院,吉林长春130000 [2]长春生物制品研究所有限责任公司细胞因子室,吉林长春130000

出　　处：《中国生物制品学杂志》2023年第2期200-206,共7页Chinese Journal of Biologicals

基　　金：吉林省科技厅发展计划项目(2018414056GH);吉林省发改委项目(2020C028-3)。

摘　　要：目的优化CHO细胞表达重组人生长激素-Fc(recombinant human growth hormone-Fc,rhGH-Fc)融合蛋白,以获得更优的糖基化比例及更低含量的高甘露糖。方法在7 L生物反应器中培养表达rhGH-Fc的CHO细胞,通过改善补料培养基成分(使用3种商业化培养基:Gly-1:EX-CELL Glycosylation Adjust、Gly-2:SHEFF-CHO PLUS PG ACF、Gly-3:EfficientFeed C+AGT Supplement&GlycanTune C+Total Feed),对rhGH-Fc糖基化修饰进行调整,并通过质谱分析目的蛋白的糖基化类型及比例。结果Gly-1、Gly-2、Gly-3的G0F(主要糖型一般为G0、G1和G2,F为岩藻糖)分别为32.89%、58.66%、33.28%,G1F分别为31.39%、18.03%、34.90%,G2F分别为31.39%、18.03%、34.90%,Gly-1与Gly-3使目的蛋白含有更少的G0F及更多的G2F;Gly-3补料方案在高甘露糖修饰方面较其他两种方案更少。结论Gly-1培养基使糖基化修饰从G0F向G1F、G2F转变,Gly-2培养基使糖基化修饰从G2F、G1F向G0F转变,Gly-3培养基使糖基化修饰从G0F向G1F、G2F转变,且高甘露糖含量在5%以下。Gly-3培养基可能具有更好的修改目的蛋白糖基化类型及比例的效果。Objective To optimize the expression of recombinant human growth hormone-Fc(rhGH-Fc)fusion protein in CHO cells in order to obtain better glycosylation ratio and lower content of highmannose.Methods CHO cells expressing rhGH-Fc were cultured in a 7 L bioreactor.The glycosylation modifications of rhGH-Fc were adjusted by improving the composition of feeding media(using three commercial media:Gly-1:EX-CELL Glycosylation Adjust,Gly-2:SHEFF-CHO PLUS PG ACF and Gly-3:EfficientFeed C+AGT Supplement&GlycanTune C+Total Feed),and the glycosylation type and proportion of the target proteins were analyzed by mass spectrometry.Results The G0F(main glycosylation types:G0,G1 and G2;F:fucose)of Gly-1,Gly-2 and Gly-3 were 32.89%,58.66%and 33.28%,the G1F were 31.39%,18.03%and 34.90%,and the G2F were 31.39%,18.03%and 34.90%,respectively.Gly-1 and Gly-3 made the target protein contain less G0F while more G2F;Gly-3 feeding scheme-showed less high mannose modification than the other two schemes.Conclusion Gly-1 medium changed the glycosylation modification from G0F to G1F and G2F,while Gly-2 medium changed that from G2F and G1F to G0F.However,Gly-3 medium changed the glycosylation modification from G0F to G1F and G2F,and the contentof high mannose was less than 5%,which may have a better effect on modifying glycosylation type and proportion of the target protein.

关 键 词：重组人生长激素-Fc融合蛋白 CHO-K1细胞 糖基化修饰 

分 类 号：Q251[生物学—细胞生物学]