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.
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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