聚乙二醇化重组人源化抗肿瘤坏死因子α单克隆抗体的质量控制  

Quality control study of PEGylated recombinant anti-TNFαmonoclonal antibody

作　　者：徐刚领 杜加亮[1] 武刚[1] 段茂芹 崔永霏 王文波[1] 付志浩[1] 于传飞[1] 王兰[1] XU Gangling;DU Jialiang;WU Gang;DUAN Maoqin;CUI Yongfei;WANG Wenbo;FU Zhihao;YU Chuanfei;WANG Lan(Division of Monoclonal Antibodies,National Institutes for Food and Drug Control,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)

机构地区：[1]中国食品药品检定研究院单克隆抗体产品室国家卫生健康委员会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

出　　处：《中国生物制品学杂志》2024年第8期964-969,共6页Chinese Journal of Biologicals

基　　金：国家重点研发计划蛋白类生物制品主成分及其相关成分精准定量检测技术研究(2021YFF0600804).

摘　　要：目的建立针对聚乙二醇(polyethylene glycol,PEG)化重组人源化抗肿瘤坏死因子α(tumor necrosis factorα,TNFα)抗体(PEG-抗TNFα单抗)的关键质量属性质控方法。方法采用肽图谱鉴别PEG-抗TNFα单抗,分子排阻高效液相色谱(size exclusion-high performance liquid chromatography,SEC-HPLC)法检测分子大小异质性,阳离子交换高效液相色谱(cation exchange-high performance liquid chromatography,CEX-HPLC)法检测电荷异质性,反相超高效液相色谱(reversed-phase-ultra performance liquid chromatography,RP-UPLC)法检测PEG残留量及错配异构体含量;利用稳定转染TNFα相关受体下游反应元件驱动的报告基因的HEK293细胞测定生物学活性。结果PEG-抗TNFα单抗具有相应的特征图谱,可用于鉴别;SEC-HPLC单体峰相对百分含量为(99.66±0.01)%,高分子量物质峰相对百分含量为(0.31±0.01)%,低分子量物质均低于定量限;CEX-HPLC主峰区峰相对百分含量为(98.31±0.10)%,酸性峰区峰相对百分含量为(1.31±0.04)%,碱性峰区峰相对百分含量为(0.38±0.07)%;PEG含量低于定量限,错配异构体含量为(0.76±0.007)%;生物学活性的半数最大效应浓度(concentration for 50%of maximal effect,EC50)值为(2.22±0.11)ng/mL。结论针对PEG-抗TNFα单抗的关键质量属性建立了相应的质量控制方法,可确保其安全、有效及质量可控,为该类单抗产品的质量控制方法和策略提供了参考。Objective To establish quality control methods for PEGylated recombinant anti-tumor necrosis factorα(TNFα)monoclonal antibody(PEG-anti-TNFαmAb).Methods Peptide mapping was used for identification of the PEG-anti-TNFαmAb.Size exclusion-high performance liquid chromatography(SEC-HPLC)methods were used to measure the molecular size heterogeneity,and cation exchange-high performance liquid chromatography(CEX-HPLC)was used to test the charge heterogeneity.The residual polyethylene glycol(PEG)and mismatched isomers were determined by reversed-phase-ultra performance liquid chromatography(RP-UPLC).The potency was measured by HEK293 cells stably transfected with reporter gene driven by downstream reaction elements of TNFαassociated receptors.Results Peptide mapping detection of PEG-anti-TNFαmAb had the corresponding characteristic map,which played a good role in the identification.The peak area percentages of monomer and high molecular weight(HMW)determined by SEC-HPLC were(99.66±0.01)%and(0.31±0.01)%,respectively,and that of low molecular weight(LMW)was lower than the limit of quantification.The peak area percentages of the main subtypes,the acid subtype,and the basic subtype analyzed by CEX-HPLC were(98.31±0.10)%,(1.31±0.04)%and(0.38±0.07)%,respectively.The residual PEG was lower than the limit of quantification and the content of mismatched isomers was(0.76±0.007)%.The concentration for 50%of maximal effect(ECso)of PEG-anti-TNFαmAb in bioassay was(2.22±O.11)ng/mL.Conclusion According to the key quality attributes of PEG-anti-TNFαmAb,the corresponding quality control methods were established,which can ensure its safety,effectiveness and controllable quality,and provide reference for the quality control methods and strategies of PEG-anti-TNFαmAb products.

关 键 词：聚乙二醇 肿瘤坏死因子Α 单克隆抗体 质量控制 

分 类 号：R91[医药卫生—药学]