重组嵌合抗CD20IgG1型单克隆抗体的结构验证Structure-药学学报.PDF

752 药学学报 Acta Pharmaceutica Sinica 2010, 45 (6) : 752755 重组嵌合抗 CD20 IgG1 型单克隆抗体的结构验证 磊, 饶春明, 高 凯, 史新昌, 赵 阳, 王军志* ( 中国药品生物制品检定所, 北京 100050) 摘要: 本文选择一种重组嵌合抗CD20 IgG1 型单抗, 应用液质联用仪及N -末端测序仪对其进行结构验证。 对该单抗进行还原、烷基化、酶解等处理后, 对其氨基酸序列、二硫键配对方式、糖链类型及糖基化位点进行 分析测定。结果显示, 该单抗轻、重链氨基酸序列与理论一致。通过液质肽图的解析, 对单抗 10 条二硫键的配 对方式进行了验证; 通过比较单抗重链切糖前、后的相对分 质量, 预测单抗所含糖链类型为岩藻糖化的双触角 复杂型N -糖, 糖基化位点位于重链的Asn301 上。本方法可为该类重组单抗制品的质量控制及其参考品的结构确 证提供参考。 关键词: 抗CD20 IgG1 单克隆抗体; 结构验证; 高效液相色谱-质谱联用 中图分类号: R917 文献标识码:A 文章编号: 05 13-4870 (2010) 06-0752-04 Structure verification of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody * TAO Lei, RAO Chun-ming, GAO Kai, SHI Xin-chang, ZHAO Yang, WANG Jun-zhi (National Institute for the Control of Pharmaceutical and Biological Products, Beij ing 100050, China) Abstract: Structure of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody was verified by the application of high-performance liquid chromatography-mass spectrometry (HPLC-MS) and N -terminal sequencer. Molecular masses, N -terminal sequences and peptide maps of the antibody treated with different reagents and enzymes were measured. Results indicate that the amino acid sequences of light and heavy chains and 10 disulfide bonds were consistent with theoretical structure. By comparison of molecular masses and peptide maps for the fully glycosylated and deglycosylated samples, the N-linked glycosylation site was identified. The method is simple, rapid, precise, and could be referred to the quality control an