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。
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
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.
[1] BIRRER MJ, MOORE KN, BETELLA I, et al. Antibody-drug conjugate-based therapeutics: state of the science[J]. J Natl Cancer Inst, 2019, 111(6):538
[2] FU Z, LI S, HAN S, et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy[J]. Signal Transduct Target Ther, 2022, 7(1):93
[3] HAFEEZ U, PARAKH S, GAN HK, et al. Antibody-drug conjugates for cancer therapy[J]. Molecules, 2020, 25(20):4764
[4] SEHN LH, HERRERA AF, FLOWERS CR, et al. Polatuzumab vedotin in relapsed or refractory diffuse large B-cell lymphoma[J]. J Clin Oncol, 2020, 38(2):155
[5] LI DW, LEE D, DERE RC, et al. Evaluation and use of an anti-cynomolgus monkey CD79b surrogate antibody-drug conjugate to enable clinical development of polatuzumab vedotin[J]. Br J Pharmacol, 2019, 176(19):3805
[6] THANDRA KC, BARSOUK A, SAGINALA K, et al. Epidemiology of non-Hodgkin’s lymphoma[J]. Med Sci (Basel), 2021, 9(1):5
[7] SAWALHA Y, MADDOCKS K. Profile of polatuzumab vedotin in the treatment of patients with relapsed/refractory non-Hodgkin lymphoma: a brief report on the emerging clinical data[J]. Onco Targets Ther, 2020, 13: 5123
[8] CHEASON BD, NOWAKOWSKI G, SALLES G. Diffuse large B-cell lymphoma: new targets and novel therapies[J]. Blood Cancer J, 2021, 11(4):68
[9] HARRIS LJ, PATEL K, MARTION M. Novel therapies for relapsed or refractory diffuse large B-cell lymphoma[J]. Int J Mol Sci, 2020, 21(22):8553
[10] LIU Y, BARTA SK. Diffuse large B-cell lymphoma: 2019 update on diagnosis, risk stratification, and treatment[J]. Am J Hematol, 2019, 94(5):604
[11] STEGEMANN M, DENKER S, SCHMITT CA. DLBCL 1L—What to expect beyond R-CHOP?[J]. Cancers (Basel), 2022, 14(6):1453
[12] PAPAGEORGIOU SG, THOMOPOULOS TP, LIASKAS A, et al. Monoclonal antibodies in the treatment of diffuse large B-cell lymphoma: moving beyond rituximab[J]. Cancers, 2022, 14(8):1917
[13] CHU PG, ARBER DA. CD79: a review[J]. Appl Immunohistochem Mol Morphol, 2001, 9(2):97
[14] RADAEV S, ZOU Z, TOLAR P, et al. Structural and functional studies of Igalphabeta and its assembly with the B cell antigen receptor[J]. Structure, 2010, 18(8):934
[15] 王兰, 郭莎, 于传飞, 等. 抗体偶联药物质量控制和临床前评价专家共识[J]. 中国药事,2018, 32(7):993
WANG L, GUO S, YU CF, et al. Expert consensus on quality control and pre-clinical evaluation of antibody-drug conjugate[J]. Chin Pharm Aff, 2018, 32(7):993
[16] 李萌, 孙亮, 朱磊, 等. 以Delta样蛋白3为靶点的抗体偶联药物质量研究[J]. 中国药学杂志, 2019, 54(24):2018
LI M, SUN L, ZHU L, et al. Quality control of anti-delta-like protein 3 antibody-MPVP-PBD[J]. Chin Pharm J, 2019, 54(24):2018
[17] 赵雪羽, 李萌, 武刚, 等. 以CD79b为靶点的抗体偶联药物的一级结构表征[J]. 中国药学杂志, 2023, 58(1):45
ZHAO XY, LI M, WU G, et al. Primary structure characterization of anti-CD79b antibody-vc-MMAE[J]. Chin Pharm J, 2023, 58(1):45
[18] 李萌, 赵雪羽, 俞小娟, 等. 以CD79b为靶点抗体偶联药物结合活性的评价研究[J]. 药物分析杂志, 2022, 42(10):1754
LI M, ZHAO XY, YU XJ, et al. Evaluation of drug binding activity of anti-CD79b antibody-vc-MMAE[J]. Chin J Pharm Anal, 2022, 42(10):1754
[19] BECK A, TERRAL G, DEBAENE F, et al. Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates[J]. Expert Rev Proteomics, 2016, 13(2):157