抗GD2单克隆抗体药物的质量控制  

Study on quality control of recombinant anti-GD2 monoclonal antibody

作　　者：杜加亮[1] 于传飞[1] 王文波[1] 武刚[1] 崔永霏 郭璐韵 杨雅岚[1] 俞小娟 李萌[1] 王兰[1] DU Jialiang;YU Chuanfei;WANG Wenbo;WU Gang;CUI Yongfei;GUO Luyun;YANG Yalan;YU Xiaojuan;LI Meng;WANG Lan(Division of Monoclonal Antibody Products,National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech 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,卫生部生物技术产品检定及标准化重点实验室,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：《山西医科大学学报》2022年第9期1176-1183,共8页Journal of Shanxi Medical University

基　　金：国家科技重大专项“重大新药创制”项目(2019ZX09732-002);中山市重大科技专项-战略性新兴产业技术公关专题(210204163866513)。

摘　　要：目的研究并建立针对抗双唾液酸神经节苷脂(disialoganglioside,GD2)单克隆抗体的关键质量属性质控策略。方法采用基于报告基因的抗体依赖性细胞介导的细胞吞噬(antibody-dependent cell-mediated phagocytosis,ADCP)效应以及基于细胞增殖抑制的补体依赖性细胞毒性(complement-dependent cytotoxicity,CDC)作用测定生物学活性。分别采用ELISA法和SPR-Biacore法测定GD2和CD16的结合活性。采用还原/非还原十二烷基硫酸钠毛细管凝胶电泳(capillary electrophoresis-sodium dodecyl sulfonate,CE-SDS)、分子排阻色谱(size exclusion-high performance liquid chromatography,SE-HPLC)测定单抗纯度。运用毛细管等电聚焦电泳(capillary isoelectric focusing electrophoresis,cIEF)测定电荷异质性。运用基于荧光检测器的超高效液相色谱法(UPLC)分析抗GD2单抗的糖基化(岩藻糖和半乳糖)。结果生物学活性中ADCP的半数最大效应浓度(concentration for 50%of maximal effect,EC_(50))值为(24.24±0.57)ng/ml,CDC的EC_(50)值为(0.49±0.003)ng/ml。GD2结合活性的EC_(50)值为(17.53±2.14)ng/ml,CD16结合活性的EC_(50)值为(99.40±1.43)nmol/L。非还原CE-SDS主峰面积百分比为(98.61±0.17)%,还原CE-SDS重链(HC)和轻链(LC)的峰面积百分比之和为(97.39±0.15)%,SE-HPLC主峰峰面积百分比为(98.32±0.01)%。cIEF分析中峰“l”、峰“m”和峰“n”的峰面积百分比分别为(14.88±0.15)%,(37.18±0.37)%和(38.67±0.50)%。糖基化分析中岩藻糖基化单糖合计所占比例为(97.41±0.04)%,半乳糖基化单糖合计所占比例为(18.93±0.07)%。结论针对抗GD2单抗的关键质量属性,研究并建立了相应的质量控制策略,以确保其安全、有效和质量可控,为该类单抗产品的质量控制提供参考依据。Objective To study and establish the quality control strategy for key quality attributes of anti-disialoganglioside(GD2)monoclonal antibodies(mAb).Methods Biological activity was measured using both the antibody-dependent cell-mediated phagocytosis(ADCP)effect based on reporter gene assay and the complement-dependent cytotoxicity(CDC)effect based on cell proliferation inhibition assay.The binding activities of GD2 and CD16 were determined by ELISA and SPR-Biacore,respectively.The antibody purity was controlled using the reduced and non-reduced capillary electrophoresis-sodium dodecyl sulfonate(CE-SDS),and the size exclusion-high performance liquid chromatography(SE-HPLC).Capillary isoelectric focusing electrophoresis(cIEF)was used to determine the charge heterogeneity.Ultra-HPLC(UPLC)based on fluorescence detector was used to analyze the glycosylation(fucosylation and galactosylation).Results The concentration for 50%of maximal effect(EC_(50))was(24.24±0.57)ng/ml for ADCP and(0.49±0.003)ng/ml for CDC.The EC_(50)was(17.53±2.14)ng/ml for GD2 binding activity and(99.40±1.43)nmol/L for CD16 binding activity.The percentage of the main peak area was(98.61±0.17)%in non-reduced CE-SDS,while the sum of the peak area percentages of heavy chain(HC)and light chain(LC)was(97.39±0.15)%in reduced CE-SDS.Meanwhile,the main peak area percentage was(98.32±0.01)%in SE-HPLC.In addition,the peak area percentages of peak“l”,“m”and“n”were(14.88±0.15)%,(37.18±0.37)%,and(38.67±0.50)%in cIEF,respectively.Finally,the total proportion of fucosylated monosaccharides was(97.41±0.04)%,and the total proportion of galactosylated monosaccharides was(18.93±0.07)%in glycosylation analysis.Conclusion The quality control strategies are established for the key quality attributes of anti-GD2 mAb to ensure its safety,effectiveness and quality,which provide a reference for the quality control of such mAb products.

关 键 词：双唾液酸神经节苷脂 单克隆抗体 药物质量控制 生物学活性 结合活性 

分 类 号：R927.11[医药卫生—药学]