IgG1型单克隆抗体对照品的一级结构解析

Primary Structural Ananlysis of Reference IgG1 Monoclonal Antibody

对单克隆抗体药物对照品(IgG1型)进行了全面的结构表征。采用ExactiveplusEMR质谱测定了去N糖前后抗体的精确分子量,并通过计算N糖含量得出其完整分子量为148285.0~149020.8,完整去糖后分子量为145810.4,N糖含量为1.67%~2.15%。优化了全柱成像实时等电聚焦毛细管电泳(WCID-cIEF)检测方法,结果显示该抗体等电点分布在8.21~8.74之间,其中主成分等电点为8.61,相对含量为61.43%。继而使用离子交换色谱检测了电荷异质性,发现碱性峰含量为4.1%,酸性峰含量为23.9%,主峰含量为72.0%,与WCID-cIEF结果吻合,并证明了方法间良好的重现性。通过切糖前后的肽图分析,获得糖肽分布信息,识别出糖修饰位点及重轻链的互补决定区(CDR),甲硫氨酸(M)的氧化位点及氧化率,并通过抗体还原前后的肽图解析出二硫键连接。研究结果同时提示该抗体对照品无C末端糖化修饰现象。

A comprehensive structural analysis on reference monoclonal antibody (IgG1 ) was conducted,which provides a reference for further in-depth research of quality and future quality controling.Exactive plus EMR mass spectrometry was used to detect the precise molecular weight ( M W ) before and after PNGaseF digestion,then calculating the N-glycan content.The M W of the reference antibody ranged from148285.0 to149 020.8,which was145 810.4 after PNGaseF digestion,and the N-glycan content ranged from1.67 % to2.15 %.The WCID-cIEF results showed that the isoelectric point (pI ) of the antibody ranged from 8.21 to 8.74,in which the pI of the main component was 8.61 and the relative content was 61.43 %.Then,the charge heterogeneity was detected by ion exchange chromatography.The contents for basic peak,acid peak and main peak were 4.1 %,23.9 % and 72.0 %,respectively,which were consistent with the pI distribution by WCID-cIEF analysis.Furthermore,the glycopeptide distribution and the glycosylated sites were analyzed by peptide mapping before and after PNGaseF digestion,as well as the complementary determinant region (CDR ) of the heavy and light chains,and the oxidation sites and oxidation ratio of methionine.The disulfide bonds were also analyzed by peptide mapping before and after antibody reduction.Finally,the glycation of the C-terminal of the antibody was detected.Results showed that there was no glycation occured in the C-terminal of the reference antibody.