基于UPLC-Q-TOF-MS的双特异性抗体关键表征方法研究

Study on Key Characterizations of Bispecific Antibody Based on UPLC-Q-TOF-MS

关键词:  双特异性抗体  超高效液相色谱-三重四级杆/飞行时间质谱联用技术  相对分子质量  肽图分析

Key words:  bispecific antibody  UPLC-Q-TOF-MS  relative molecular mass  peptide mapping

摘要:

目的 建立Y-Body^®型双特异性抗体一级结构的超高效液相色谱-三重四级杆/飞行时间质谱联用技术(UPLC-Q-TOF-MS)研究方法。方法 以Y-Body^®型双特异性抗体M808为研究对象,基于UPLC-Q-TOF-MS检测完整抗体及各亚基去糖基化处理前后的相对分子质量、肽图(氨基酸序列),并通过Unify软件进行解析。结果 完整抗体去糖基化处理前后的相对分子质量分别为128417.6605,125040.7210;轻链去糖基化处理前后的相对分子质量分别为23447.2322,23445.5608;重链去糖基化处理前后的相对分子质量分别为50804.7732,49201.6051;单链去糖基化处理前后的相对分子质量分别为54189.9224,52415.7644;其氨基酸序列与理论序列基本一致,肽图的互补决定区覆盖率为100%;推测N-糖基化位点位于重链及单链Fc端,糖型以G1F和G2F为主。结论 通过UPLC-Q-TOF-MS及Unify软件可有效表征双特异性抗体的一级结构,为其质量标准的制定提供依据。

Abstract:

OBJECTIVE To study the primary structure of Y-Body^® bispecific antibody based on UPLC-Q-TOF-MS.METHODS Based on Y-Body^® bispecific antibody M808 as the object of study, the relative molecular mass of intact antibody and each subunits before and after deglycosylation, peptide mapping(amino acid sequence) was detected and analyzed by UPLC-Q-TOF-MS and Unify software.RESULTS The relative molecular mass of intact antibody and deglycosylation intact antibody were 128 417.660 5, 125 040.721 0. The relative molecular mass of light chain(LC) and deglycosylation LC were 23 447.232 2, 23 445.560 8. The relative molecular mass of heavy chain(HC) and deglycosylation HC were 50 804.773 2,49 201.605 1. The relative molecular mass of single chain(SC) and deglycosylation SC were 54 189.922 4, 52 415.764 4. The amino acid sequence was basically consistence with theoretical sequence, and the coverage of the complementary determining region attained 100%. N-glycosylation sites located at the Fc of HC and SC, mainly including G1F and G2F.CONCLUSION The primary structure of bispecific antibody can be effectively characterized by UPLC-Q-TOF-MS and Unify software, which provides a basis for ormulation of quality standards.