生物检定

以CD79b为靶点抗体偶联药物的质量研究*

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  • 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
第一作者 李 萌 Tel:(010)53852176;E-mail:lemon831115@163.com
赵雪羽 Tel:(010)53852199;E-mail:zhaoxueyucpu@163.com
** Tel:(010)53852159;E-mail:iamouran@163.com

收稿日期: 2023-02-07

  网络出版日期: 2024-06-21

基金资助

* 国家药典标准提高课题:人用抗体偶联药物制品总论(2022S04)

Quality control of anti-CD79b antibody-vc-MMAE*

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  • 1. 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;
    2. China Pharmaceutical University, Nanjing 211198, China

Received date: 2023-02-07

  Online published: 2024-06-21

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(KD);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)%,KD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147 891、150 527、153 161和155 796,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。

本文引用格式

李萌, 赵雪羽, 武刚, 杜加亮, 王文波, 郭璐韵, 龙彩凤, 杨雅岚, 付志浩, 俞小娟, 刘春雨, 段茂芹, 徐刚领, 于传飞, 王兰 . 以CD79b为靶点抗体偶联药物的质量研究*[J]. 药物分析杂志, 2023 , 43(10) : 1727 -1736 . DOI: 10.16155/j.0254-1793.2023.10.11

Abstract

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 (KD) 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 KD 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 147 891, 150 527, 153 161 and 155 796 respectively, and the DAR was 3.60. The DAR was 3.53±0.01 determined by HIC-HPLC. The concentration of free small molecule drug was (0.039±0.003) μg·mL-1. Conclusion: The quality control method for anti-CD79b antibody-vc-MMAE was preliminarily established to ensure the safety and effectiveness of the product. It can provide reference for the quality detection of related ADC drugs.

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