Bioassay

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

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.

Cite this article

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 . Quality control of anti-CD79b antibody-vc-MMAE*[J]. Chinese Journal of Pharmaceutical Analysis, 2023 , 43(10) : 1727 -1736 . DOI: 10.16155/j.0254-1793.2023.10.11

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