利用高分辨液相质谱综合表征抗CTLA4单克隆抗体的研究  

Comprehensive Characterization of Anti-CTLA4 Monoclonal Antibody by Liquid Chromatography High Resolution Mass Spectrometry

作　　者：武刚[1] 王文波[1] 于传飞[1] 崔永菲 张荣建[1] 王兰[1] WU Gang;WANG Wen-bo;YU Chuan-fei;CUI Yong-fei;ZHANG Rong-jian;WANG Lan(Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,National Institutes for Food and Drug Control,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院,卫生部生物技术产品检定及标准化重点实验室,北京102629

出　　处：《中国药学杂志》2020年第6期457-464,共8页Chinese Pharmaceutical Journal

基　　金：科技重大专项项目资助(2018ZX09101001-003-001)。

摘　　要：目的研究建立抗CTLA4单抗质谱表征方法。方法以抗CTLA4单抗为研究对象,利用液相色谱-串联质谱联用技术(LC-MS/MS),通过优化液相条件与质谱参数,应用液质联用技术从完整蛋白相对分子质量、亚基相对分子质量、氨基酸序列覆盖率、二硫键、糖基化位点、W糖基化修饰等方面对抗CTLA4单抗进行全面的表征。结果抗CTLA4单抗完整相对分子质量(主要糖型,A2G0F/A2G0F)约为147992;去糖完整相对分子质量为145103;轻链相对分子质量为23450,重链相对分子质量(主要糖塑,A2G0F)为50548;重链Fd段相对分子质量为25355,重链sFc段(A2G0F,lxK_Loss)25189。使用Trypsin&Chymotrypsin分别进行蛋白酶解,单抗氛基酸序列覆盖率为100%;16对二硫键全部得到鉴定,与理论二硫键连接方式一致;N糖基化修饰所在的肽段序列为EEQYNSTYR,N-糖基化修饰位点位于重链的第298位天冬酰胺残基上,有13种主要糖塑,包括A2G0F(59.8%)、A2GlFa(18.66%)、A2GlFb(7.28%)、A2G2F(3.2%)、M5(1.66%)、A2S1G0F(1.17%)、A1G0F(1.16%)、A3G1F(0.95%)、A2G0(0.94%)、A2S2F(0.78%)、A2S1G1F(0.67%)、A3G0F(0.53%)和A1G1F(0.51%)。结论建立了基于质谱技术对CTLA4单抗系统的表征分析方法。OBJECTIVE To establish a mass spectrometry method for the qualitative analysis of anti-GTLA4 monoclonal antibody and its N-linked glycosylation.METHODS The anti-CTIA4 monoclonal antibody was characterized by liquid-mass technique from the aspects of intact molecular weight,subunit molecular weight,amino acid sequence coverage,disulfide bond,N-linked glycosylation site and glycoform.RESULTS The molecular weight of anti-CTLA4 mAb(A2G0F/A2G0F)is 147992;the deglycosylation molecular weight of anti-CTLA4 mAb is 145103;the molecular weight of light chain is 23450;the molecular weight of heavy chain(A2G0F)is 50548;the heavy chain Fd segment has a molecular weight of 25355 and the heavy chain sFc segment(A2G0F,lxK_Loss)has a molecular weight of 25189.Peptide mapping was performed with Trypsin&Chymotrypsin,and the coverage of the amino acid sequence was 100%.The peptide containing the N-linked glycosylations site is EEQYNSTYR,and the N-glycosylation site is located at Asn298 of the heavy chains.Thirteen glycoform were characterized including A2G0F(59.8%),A2G1Fa(18.66%),A2GlFb(7.28%),A2G2F(3.2%),M5(1.66%),A2S1G0F(1.17%),A1G0F(1.16%),A3G1F(0.95%),A2G0(0.94%),A2S2F(0.78%),A2S1G1F(0.67%),A3G0F(0.53%)and A1G1F(0.51%).CONCLUSION A method for qualitative and quantitative analysis of monoclonal antibody is established.

关 键 词：抗CTLA4单抗 高分辨液相质谱表征 相对分子质量 氨基酸序列覆盖率 二硫键 N糖谱 

分 类 号：R915[医药卫生—微生物与生化药学]