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

作　　者：崔新玲 胡志上[3] 孟晓光 钱小红[2] 朱涛[1,5] 应万涛 CUI Xin-ling;HU Zhi-shang;MENG Xiao-guang;QIAN Xiao-hong;ZHU Tao;YING Wan-tao(College of Biotechnology,Tianjin University of Science & Technology,Tianjin 300457,China;State Key Laboratory of Proteomics,Beijing Proteome Research Center,National Center for Protein Sciences (Beijing),Beijing 102206,China;National Institute of Metrology,Beijing 100013,China;Beijing C&N International Sci-techCo.,Ltd.,Beijing 102206,China;CANSINO BIOLOGICS INC. (CanSinoBIO ),Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]北京蛋白质组研究中心蛋白质组学国家重点实验室,国家蛋白质科学中心(北京),北京102206 [3]中国计量科学研究院,北京100013 [4]北京正旦国际科技有限责任公司,北京102206 [5]天津康希诺生物股份有限公司,天津300457

出　　处：《分析测试学报》2019年第8期939-945,共7页Journal of Instrumental Analysis

基　　金：国家重点研发计划(2017YFF0205400,2016YFA0501300);国家自然基金重点项目(81530021);创新基金课题(BWS14J052,16CXZ027)

摘　　要：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.

关 键 词：抗体对照品 分子量 等电点 电荷异质性 肽图 

分 类 号：O657[理学—分析化学]