离子交换色谱对抗VEGFR2单抗的电荷异质性分析

Analysis of charge heterogeneity for anti-VEGFR2 monoclonal antibody by CEX-HPLC

目的采用离子交换高效液相色谱(CEX-HPLC)分析抗VEGFR2(抗血管内皮生长因子受体2)单抗制品的电荷异质性。方法应用CEX-HPLC技术,对VEGFR2单抗进行电荷异质性分析,并结合羧肽酶B(CpB)和N-糖酰胺酶F(EndoF2)酶切,初步研究其电荷异质性的成因。结果用CEX-HPLC分析CpB酶切前后单抗,证明其碱性变异体主要由C末端赖氨酸不均一性引起;分析EndoF2酶切前后处理的单抗,证明其酸性变异体部分由N-糖末端上的唾液酸修饰所引起。通过2种酶的顺序酶切可相对准确地分析含C-末端赖氨酸以及含唾液酸单抗的比例。结论采用CEX-HPLC可较好地分析单抗的电荷异质性;并结合合适的酶切处理,可判断单抗主要电荷异质性的来源,为保证单抗制品生产工艺的稳定性及质量控制提供了有效手段。

Objective To analyze the charge heterogeneity for anti-VEGFR2 monoclonal antibody with CEX-HPLC.Meth-ods The charge heterogeneity for anti-VEGFR2 monoclonal antibody was analyzed ,and the original cause of the charge heterogeneity was studied by CEX-HPLC,and in combination with the consecutive digestion treatments of CpB and EndoF enzymes.Results The comparison between untreated and CpB treated antibody revealed that the basic variants were main -ly caused by non-homogeneity of C-terminal lysine on antibody ,and the comparison between untreated and EndoF treated antibody showed that the acidic variants were partially caused by sialic acid modification at the N-glycan terminal on anti-body.Furthermore,the relatively accurate ratio of the antibody with C-terminal lysines and sialic acid at N-glycan terminus could be quantified separately ,it is by using of consecutive treatments of the antibody with the two enzymes .Conclusion The charge heterogeneity for the monoclonal antibody could be well analyzed and the origin cause of main charge heteroge -neity for the monoclonal antibody decided too .This study provides an effective tool for ensuring the stability of production process and quality control for anti -VEGFR2 monoclonal antibody .