铁离子和半胱氨酸对抗体电荷异构体分布的影响
Effect of Iron Ion and Cysteine on the Distribution of Antibody Charge Heterogeneity
刘学明 1蒋仁材 2韦梦娟 2唐鹏 2刘唯 2曹春来1
作者信息
- 1. 联邦生物科技(珠海横琴)有限公司,广东珠海 519060
- 2. 珠海联邦生物医药有限公司,广东珠海 519040
- 折叠
摘要
目的:研究Fe3+和Cys对CHO细胞表达免疫球蛋白G(IgG)电荷异构体比例的影响.方法:在补料分批培养外泌表达IgG的CHO细胞株中,以不同的策略单独或联合添加不同浓度的Fe3+或Cys;培养 14 d后离心得上清液,通过生化分析仪测定上清液中IgG的含量,利用Protein A MagBeads亲和纯化该抗体,通过糖基化修饰、阳离子亲和层析(CEX)、非还原十二烷基硫酸钠毛细管电泳(nrCE-SDS)、硼酸亲和层析(BAC)及疏水相互作用色谱(HIC)进行鉴定.结果:Fe3+的添加可提高IgG酸性异构体比例,并且Cys可增强Fe3+的提升效果,仅添加Cys对酸性异构体的比例没有影响;电荷异构体表征表明酸性异构体含量提高主要源于唾液酸修饰、片段、Fc段氨基酸位点氧化修饰以及糖化水平的增加.结论:Fe3+与Cys通过提高唾液酸修饰、片段的丰度、糖化水平以及某些氨基酸位点的氧化,导致IgG酸性异构体比例显著增加.
Abstract
Objective:To study the effects of Fe3+ and Cys on the immunoglobulin G(IgG)charge isomer ratio of CHO cells.Methods:Different concentrations of Fe3+ or Cys are added to CHO cell lines by different strategies.After 14 days of culture,the supernatant is centrifuged and the content of IgG in the supernatant is determined by biochemical analyzer,followed by affinity purification of ProteinA MagBeads,and finally IgG identification by glycoylation modification,cation exchange chromatography(CEX),non-reducing sodium dodecyl sulfate capillary electrophoresis(nrCE-SDS),boric acid affinity chromatography(BAC)andhydrophobic interaction chromatography(HIC).Results:The addition of Fe3+ could increase the proportion of acidic isomers in IgG,and Cys can enhance the lifting effect of Fe3+,while the addition of Cys alone does not increase the proportion of acidic isomers.The charge isomer characterization show that the increase of true acid isomer content is mainly due to the increase of sialic acid modification,the oxidative modification of amino acid sites in the Fc segment,and the increase of glycosylation level.Conclusion:Fe3+ and Cys can significantly increase the proportion of acid isomers by increasing sialic acid modification,fragment abundance,saccharification level and oxidation of some amino acid sites.
关键词
免疫球蛋白G/芬顿反应/氧化/Fe3+/Cys/电荷异构体Key words
IgG/Fenton reaction/oxidation/Fe3+/Cys/charge heterogeneity引用本文复制引用
出版年
2024
NETL
NSTL
万方数据