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IgG1型单克隆抗体对照品的一级结构解析
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作者单位
崔新玲,胡志上,孟晓光,钱小红,朱涛,应万涛 1.天津科技大学生物工程学院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)3.中国计量科学研究院4.北京正旦国际科技有限责任公司5.天津康希诺生物股份有限公司 
基金项目: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)
中文摘要: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Exactive plus EMR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148 285.0~149 020.8,完整去糖后分子量为145 810.4,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。
中文关键词:抗体对照品  分子量  等电点  电荷异质性  肽图
 
Primary Structural Ananlysis of Reference IgG1 Monoclonal Antibody
Abstract: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(MW) before and after PNGaseF digestion,then calculating the N glycan content.The MW of the reference antibody ranged from 148 285.0 to 149 020.8,which was 145 810.4 after PNGaseF digestion,and the N glycan content ranged from 1.67% to 2.15%.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.
Key Words:reference antibody  molecular weight  isoelectric point(pI)  charge heterogeneity  peptide mapping
引用本文:崔新玲,胡志上,孟晓光,钱小红,朱涛,应万涛.IgG1型单克隆抗体对照品的一级结构解析[J].分析测试学报,2019,(8):939-945.
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