以CD79b为靶点抗体偶联药物的质量研究  

Quality control of anti-CD79b antibody-vc-MMAE

作　　者：李萌[1] 赵雪羽 武刚[1] 杜加亮[1] 王文波[1] 郭璐韵 龙彩凤 杨雅岚[1] 付志浩[1] 俞小娟 刘春雨[1] 段茂芹 徐刚领 于传飞[1] 王兰[1] LI Meng;ZHAO Xue-yu;WU Gang;DU Jia-liang;WANG Wen-bo;GUO Lu-yun;LONG Cai-feng;YANG Ya-lan;FU Zhi-hao;YU Xiao-juan;LIU Chun-yu;DUAN Mao-qin;XU Gang-ling;YU Chuan-fei;WANG Lan(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;China Pharmaceutical University,Nanjing 211198,China)

机构地区：[1]中国食品药品检定研究院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 [2]中国药科大学,南京211198

出　　处：《药物分析杂志》2023年第10期1727-1736,共10页Chinese Journal of Pharmaceutical Analysis

基　　金：国家药典标准提高课题:人用抗体偶联药物制品总论(2022S04)。

摘　　要：目的:建立抗体偶联药物(ADC)抗CD79b单抗-vc-MMAE(维泊妥珠单抗)的质控方法。方法:采用细胞杀伤法测定抗CD79b单抗-vc-MMAE的生物学活性;采用表面等离子共振法(SPR)测定其亲和力参数(K_(D));采用酶联免疫吸附测定法(ELISA)测定其与CD79b的相对效价;对抗CD79b单抗及抗CD79b单抗-vc-MMAE进行肽图鉴别;采用分子排阻色谱法(SEC-HPLC)和十二烷基硫酸钠毛细管电泳法(CE-SDS)分析其纯度;采用毛细管等点聚焦电泳法(iCIEF)分析其电荷异质性;采用液质联用法(UPLC-MS)测定其相对分子质量和药物抗体偶联比(DAR);采用疏水色谱法(HIC-HPLC)进一步确认其DAR;采用反相色谱法(RP-HPLC)测定游离小分子药物。结果:抗CD79b单抗-vc-MMAE的生物学活性相对效价为(99.32±3.99)%,K_(D)为(4.27±0.27)×10^(-9)mol·L^(-1),结合活性相对效价为(106.01±2.88)%,抗CD79b单抗和抗CD79b单抗-vc-MMAE的参比品与其样品的肽图图谱一致,SEC-HPLC主峰纯度为(99.32±0.05)%,非还原CE-SDS的重链-重链-轻链-轻链(HHLLC)纯度为(6.89±0.19)%,重链-重链-轻链(HHLC)纯度为(27.21±0.15)%,重链-重链(HHC)纯度为(18.33±0.06)%等,还原CE-SDS轻链和重链纯度为(95.47±0.16)%,iCIEF主峰峰面积百分比为(73.57±0.55)%,主峰等电点7.53±0.00,液质联用法测定抗CD79b单抗-vc-MMAE分别偶联0、2、4、6个小分子药物的相对分子质量为147891、150527、153161和155796,DAR为3.60,HIC法测得的DAR为3.53±0.01,RP-HPLC测定游离小分子药物浓度为(0.039±0.003)μg·mL^(-1)。结论:建立了抗CD79b单抗-vc-MMAE的质控方法,对该产品的安全性、有效性进行控制,为该类ADC药物的质量检测提供参考。Objective:To establish the quality control method of an antibody-drug conjugate(ADC),anti-CD79b antibody-vc-MMAE(polatuzumab vedotin).Methods:The biological activity of anti-CD79b antibody-vc-MMAE was determined by cell killing method.The affinity constant(K_(D))was determined by surface plasmon resonance(SPR).The binding activity was determined by enzyme linked immunosorbent assay(ELISA).Anti-CD79b antibody and anti-CD79b antibody-vc-MMAE were identified by peptide mapping.Size heterogeneity was measured by size exclusion-high performance liquid chromatography(SEC-HPLC)and capillary electrophoresis-sodium dodecyl sulfonate(CE-SDS).The charge heterogeneity of the ADC was analyzed by imaged capillary isoelectric focusing(iCIEF).Relative molecular mass and drug-to-antibody ratio(DAR)were determined by ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS).DAR was further confirmed by hydrophobic interaction chromatography-high performance liquid chromatography(HIC-HPLC).The contents of free small molecule drugs were determined by reversed phase-high performance liquid chromatography(RP-HPLC).Results:The biological activity relative potency of anti-CD79b antibody-vc-MMAE was(99.32±3.99)%,and K_(D) was(4.27±0.27)×10^(-9)mol·L^(-1).The binding activity relative potency was(106.01±2.88)%.The peptide mapping of reference samples of anti-CD79b antibody and anti-CD79b antibody-vc-MMAE was consistent with those of their samples.The purity of main peak of SEC-HPLC was(99.32±0.05)%.The purity of heavy/heavy/light/light chains(HHLLC),heavy/heavy/light chains(HHLC)and heavy/heavy chains(HHC)of non-reduced CE-SDS were(6.89±0.19)%,(27.21±0.15)% and(18.33±0.06)% respectively.The purity of HLC peaks of reduced-CE-SDS was(95.47±0.16)%.The peak area percent of main peak of iCIEF was(73.57±0.55)%,and the isoelectric point of main peak was 7.53±0.00.The relative molecular masses of conjugated 0,2,4 and 6 free small drugs of anti-CD79b-vc-MMAE were 147891,150527,153161 and 155796 respectively,and the DAR

关 键 词：抗体偶联药物 质量控制 药物抗体偶联比 生物学活性 游离小分子药物 分化群79b 连接子 大小异质性 电荷异质性 

分 类 号：R917[医药卫生—药物分析学]