抗CD3/CD20双特异性抗体生物学活性测定方法的建立  

Establishment of biological activity assay method of anti-CD3/CD20 bispecific antibody

作　　者：俞小娟 刘丽 白海娇 韩晓捷 刘春雨[1] 付志浩[1] 李萌[1] 杜加亮[1] 徐刚领 段茂芹 杨雅岚[1] 崔春博 陈浩彬 于传飞[1] 王兰[1] YU Xiaojuan;LIU Li;BAI Haijiao;HAN Xiaojie;LIU Chunyu;FU Zhihao;LI Meng;DU Jialiang;XU Gangling;DUAN Maoqin;YANG Yalan;CUI Chunbo;CHEN Haobin;YU Chuanfei;WANG Lan(National Institutes for Food and Drug Control,State Key Laboratory of Drug Regulatory Science,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,Beijing 102629,China;Tianjin Institute for Drug Control)

机构地区：[1]中国食品药品检定研究院,药品监管科学全国重点实验室,国家卫生健康委生物技术产品检定方法及其标准化重点实验室,国家药品监督管理局生物制品质量研究与评价重点实验室,北京102629 [2]天津市药品检验研究院

出　　处：《山西医科大学学报》2024年第7期922-928,共7页Journal of Shanxi Medical University

基　　金：国家自然科学基金项目(21702007);国家重点研发计划项目(2021YFF0600804)。

摘　　要：目的建立基于报告基因的抗CD3/CD20双特异性抗体生物学活性检测方法。方法以WIL2-S细胞系作为靶细胞,以Jurkat-NF-κB-Luc转基因细胞系作为效应细胞,对抗体的量效范围、孵育时间、诱导时间、效靶比及细胞接种密度进行优化,构建可用于测定抗CD3/CD20双特异性抗体生物学活性的报告基因法。依据ICHQ2的指导原则对方法进行全面验证,包括专属性、准确性、精密度、线性、稳定性。结果成功建立了具有量效关系的抗CD3/CD20双特异性抗体生物学活性检测方法,该方法的抗体起始浓度为500 ng/mL,稀释倍数为1∶4,共计8个浓度点(500,125,31.25,7.81,1.95,0.49,0.12,0.031 ng/mL),效靶比为4∶1,诱导时间为4 h。本方法具有良好的专属性;5个不同稀释组回收率样品经测定,相对效价分别为59.63%±4.57%,75.54%±4.05%,99.98%±9.63%,123.90%±5.54%和142.51%±12.82%;对应的回收率分别为93.17%±7.15%,94.42%±5.06%,99.98%±9.63%,99.12%±4.43%以及91.21%±8.21%,CV分别为7.67%,5.35%,9.63%,4.47%和9.00%,上述结果的变异系数CV值均<10%;实测效价与理论效价线性回归分析相关系数R^(2)=0.9823,线性良好;本研究建立的方法可以敏感检测出抗体结构变化对生物活性的影响,对双特异性抗体的稳定性检测有一定的指导意义。结论利用转基因细胞法成功建立了抗CD3/CD20双特异性抗体生物学活性检测方法,该方法专属性强、准确性高、重复性好,可用于评价抗CD3/CD20双特异性抗体生物学活性。Objective To establish a cell-based reporter gene assay(RGA)for bioactivity determination of anti-CD3/CD20 bispecific antibodies.Methods With WIL2-S cell line as target cells and Jurkat-NF-κB-Luc transgenic cell line as effector cells,the RGA for determining the biological activity of anti-CD3/CD20 bispecific antibodies was established by optimizing antibody concentration range,balance time,induction time,effector-to-target ratio,and cell seeding density.The specificity,the accuracy,the precision,the linearity,and the stability of RGA were comprehensively validated according to the guidelines of ICH Q2.Results A dose-response relationship-based assay method for determining the biological activity of anti-CD3/CD20 bispecific antibody was successfully established.The optimized conditions were as follows:the initial antibody concentration of anti-CD3/CD20 bispecific antibody of 500 ng/mL,the dilution rate of 1∶4,eight detected concentrations(500,125,31.25,7.81,1.95,0.49,0.12,0.031 ng/mL),the effector-to-target ratio of 4∶1,and the induction time of 4 h.The method possessed good specificity.The relative potency of bispecific anti-CD3/CD20 antibody was 59.63%±4.57%,75.54%±4.05%,99.98%±9.63%,123.90%±5.54%and 142.51%±12.82%for recovery samples under five dilute concentrations,and the corresponding recovery was 93.17%±7.15%,94.42%±5.06%,99.98%±9.63%,99.12%±4.43%and 91.21%±8.21%,respectively.The CV value was 7.67%,5.35%,9.63%,4.47%and 9.00%,respectively,and CV values of the above results were all lower than 10%.The measured potency to expected potency demonstrated good linearity(R^(2)=0.9823).The method established in this study was sensitive to detect the effect of structural changes of antibodies on biological activity,and had certain guiding significance for the stability test of bispecific antibody.Conclusion A biological activity assay method for anti-CD3/CD20 bispecific antibodies is successfully established using a transgenic cell-based approach.The method demonstrates strong specificity,high accurac

关 键 词：抗CD3/CD20双特异性抗体 双特异性抗体 生物学活性 报告基因 优化 验证 

分 类 号：R92[医药卫生—药学]