以CGRP为靶点的单抗药物的电荷变异体表征研究  

Characterization of charge variants in anti-CGRP monoclonal antibodies

作　　者：陈全姚 杜加亮[2] 刘万卉 王兰[2] CHEN Quanyao;DU Jialiang;LIU Wanhui;WANG Lan(Department of Pharmaceutical Analysis,School of Pharmacy,Yantai University,Yantai 264005,China;Division of Monoclonal Antibody Products,National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,State Key Laboratory of Drug Regulatory Science(SKLDRS))

机构地区：[1]烟台大学药学院药物分析教研室,烟台264005 [2]中国食品药品检定研究院单克隆抗体产品室,卫生部生物技术产品检定及标准化重点实验室,国家药品监督管理局生物制品质量研究与评价重点实验室,药品监管科学全国重点实验室,北京102629

出　　处：《山西医科大学学报》2024年第4期500-508,共9页Journal of Shanxi Medical University

基　　金：国家重点研发计划项目(2021YFF0600804)。

摘　　要：目的对抗降钙素基因相关肽(calcitonin gene-related peptide,CGRP)单抗及其电荷变异体进行充分表征分析。方法以抗CGRP单抗为研究对象,利用离子交换色谱对抗CGRP单抗电荷变异体进行分离收集,再利用液质联用技术(LC-MS/MS)从分子量、轻重链亚基以及翻译后修饰等方面对抗CGRP单抗和收集到的组分进行表征。结果利用离子交换色谱(IEC)仪器收集到抗CGRP单抗的电荷变异体分为主峰、酸性峰组(A1-A3)和碱性峰组(B1-B4)。质谱鉴定的抗CGRP单抗的相对分子质量为147103(主要糖型A2G1F,A2G0F);糖肽水平上的分析发现,含有N糖基化修饰的肽段序列为“TKPREEQFNSTYR”,糖基化位点为N296。酸碱变异体组分的N糖型修饰中A2G0F(>30%)和A2G1F(>35%)占比最大,并且酸性变异体中均存在唾液酸修饰;在所有组分中均检测到脱酰胺存在,酸性变异体脱酰胺比例较高;重链第109位,抗体骨架区FR区域上的色氨酸残基发生氧化的比例最高,并且酸性变异体发生氧化的比例总体高于碱性变异体;酸性变异体组分发生焦谷氨酸环化的比例远高于碱性变异体组分;抗CGRP单抗的赖氨酸(K)糖化位点比较丰富,轻链K188位点和重链K149、K245位点的糖化比例相对较高。结论通过表征分析发现,抗CGRP单抗酸性变异体的唾液酸修饰、氧化和焦谷氨酸环化发生比例高于碱性变异体,并且成功鉴定了各修饰发生的位点以及相对丰度。这些信息有助于评估是否需要进一步的药物强制降解研究和制定合理的的药物质量控制方法。Objective To perform full characterization of an anti-CGRP monoclonal antibody and its charge variants.Methods The charge variants of the anti-CGRP mAb were separated and collected through ion exchange chromatography(IEC),and then the anti-CGRP mAb and the components of collected charge variants were characterized in terms of molecular weight,light and heavy chain subunits,and post-translational modification by LC-MS/MS.Results The charge variants of the anti-CGRP mAb collected by IEC were divided into main peak,acidic peaks(A1-A3)and basic peaks(B1-B4).The relative molecular weight of the anti-CGRP mAb(mainly N-glycoform A2G1F and A2G0F)was 147103 by LC-MS.Detailed glycopeptide analysis revealed that the peptide containing the N-linked glycosylation site was TKPREEQFNSTYR,and the N-linked glycosylation site was located at N296.Additionally,A2G0F(>30%)and A2G1F(>35%)accounted for the largest proportion of N-glycans of the charge variants,and all the acidic charge variants exhibited sialic acid modifications.Deamidation was detected across all charge variants,with a higher incidence in the acidic variants.The oxidation level at Trp109 within the FR region of the heavy chain was the highest,and the acidic variants typically exhibited a higher oxidation proportion compared to the basic variants.Furthermore,the occurrence of pyroglutamate cyclization was significantly higher in acidic variants.The glycation sites of lysine of the anti-CGRP mAb were relatively abundant,with notable glycation proportions at light chain K188 and heavy chain K149 and K245.Conclusion The comprehensive characterization analysis shows that the acidic variants of the anti-CGRP mAb exhibit a higher proportion of sialic acid modification,oxidation,and pyroglutamate cyclization compared to the basic variants,and the sites and relative abundance of each modification are successfully identified.These information helps to assess the necessity for further forced drug degradation studies and develop the rational approach for drug quality control.

关 键 词：偏头痛 降钙素基因相关肽 电荷变异体 质谱表征 翻译后修饰 

分 类 号：R917[医药卫生—药物分析学]