不同单克隆抗体聚集与断裂行为的多维分析  

Multi-Tiered Analysis of Aggregation and Fragmentation Behavior of Different Monoclonal Antibodies

作　　者：陆治锦 文玺凯 刘鲁杰 王宜冰 沈春雷 伍维 沈智勇 赵黎明[1] LU Zhijin;WEN Xikai;LIU Lujie;WANG Yibing;SHEN Chunlei;WU Wei;SHEN Zhiyong;ZHAO Liming(State Key Laboratory of Bioreactor Engineering,School of Biotechnology,East China University of Science and Technology,Shanghai 200237,China;Shanghai Zhenge Biotech Co.Ltd,Shanghai 201413,China;Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism,Shanghai 200237,China)

机构地区：[1]华东理工大学生物工程学院,生物反应器工程国家重点实验室,上海200237 [2]上海臻格生物技术有限公司,上海201413 [3]上海市细胞代谢光遗传学技术前沿科学研究基地,上海200237

出　　处：《华东理工大学学报(自然科学版)》2025年第1期42-49,共8页Journal of East China University of Science and Technology

基　　金：上海细胞代谢光遗传学前沿科学中心(上海市教委)资助。

摘　　要：采用尺寸排阻色谱法和分析型超速离心法对ZG154和ZG187抗体的分子大小变异体进行正交分析,系统解析了造成聚集和断裂的因素。结果表明,40℃时ZG154样品在天冬酰胺329位点发生脱酰胺修饰的比例增加了30.7%,通过增加分子间相互作用,相关修饰加速了聚集,并导致断裂的发生;40℃时ZG187样品在蛋氨酸430位点发生氧化修饰的比例增加了36.9%,相关修饰降低了蛋白的稳定性,导致了断裂的发生。研究结果为上述药物研发和生产过程中的聚集与断裂问题提供了检测和分析方法。This paper used size exclusion chromatography and analytical ultracentrifugation techniques to perform orthogonal analysis of the molecular size variants of ZG154 and ZG187 antibodies.Moreover,liquid chromatography-mass spectrometry,circular dichroism spectroscopy,dynamic light scattering and differential scanning fluorescence techniques were used to systematically analyze the factors causing aggregation and fragmentation.The results showed a 30.7%increase in the deamidation modification at the 329 site of asparagine in the ZG154 sample when stored at 40℃.The related modifications did not alter the secondary and tertiary structures,as well as the stability of the protein,but accelerated aggregation by increasing intermolecular interactions and led to fragmentation due to the production of succinimide.Moreover,there was a 36.9%increase in the oxidative modification at the lysine 430 site in the ZG187 sample when stored at 40℃,which reduced the stability of the protein and led to fragmentation.However,it did not change the secondary and tertiary structures of the protein,and the molecules always exhibited mutually exclusive interactions,thus not leading to aggregation.Furthermore,the changes in the sedimentation coefficient of ZG154 dimer and the non-covalent interactions between ZG187 molecular fragments were revealed through analytical ultracentrifugation sedimentation velocity analysis.These results describe suitable detection methods which can be combined to resolve the aggregation and fragmentation problems in the development and production of the above-mentioned drugs.

关 键 词：单克隆抗体 聚集 断裂 脱酰胺 氧化 

分 类 号：Q511[生物学—生物化学]