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、N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 147 992;去糖完整相对分子质量为145 103;轻链相对分子质量为23 450,重链相对分子质量(主要糖型,A2G0F)为 50 548;重链Fd段相对分子质量为25 355,重链sFc段(A2G0F, 1xK_Loss) 25 189。使用Trypsin & Chymotrypsin分别进行蛋白酶解,单抗氨基酸序列覆盖率为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种主要糖型,包括A2G0F(59.8%)、A2G1Fa(18.66%)、A2G1Fb(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。
Abstract
OBJECTIVE To establish a mass spectrometry method for the qualitative analysis of anti-CTLA4 monoclonal antibody and its N-linked glycosylation. METHODS The anti-CTLA4 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 147 992; the deglycosylation molecular weight of anti-CTLA4 mAb is 145 103; the molecular weight of light chain is 23 450; the molecular weight of heavy chain (A2G0F) is 50 548; the heavy chain Fd segment has a molecular weight of 25 355 and the heavy chain sFc segment (A2G0F,1xK_Loss) has a molecular weight of 25 189. 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%), A2G1Fb(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
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Key words
anti-CTLA4 monoclonal antibody /
high resolution liquid mass spectrometry /
molecular weight /
amino acid sequence coverage /
disulfide bond /
N-glycosylation
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中图分类号：
R915
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参考文献
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脚注
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基金
科技重大专项项目资助(2018ZX09101001-003-001)
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