抗CD19单抗的质量控制研究

俞小娟; 武刚; 张峰; 于传飞; 李萌; 王文波; 崔永霏; 郭璐韵; 王兰

文章摘要

俞小娟,武刚,张峰,于传飞,李萌,王文波,崔永霏,郭璐韵,王兰.抗CD19单抗的质量控制研究[J].中国药事,2021,35(9):1007-1015

抗CD19单抗的质量控制研究

Quality Control Study of Recombinant Anti-CD19 Monoclonal Antibody

DOI：10.16153/j.1002-7777.2021.09.007

中文关键词: 抗CD19单抗鉴别纯度检测生物学活性

英文关键词: anti-CD19 mAb identification purity determination biological activity

基金项目:中国食品药品检定研究院中青年发展研究基金(编号2019B1)

作者	单位
俞小娟	中国食品药品检定研究院卫生部生物技术产品检定及标准化重点实验室,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
武刚	中国食品药品检定研究院卫生部生物技术产品检定及标准化重点实验室,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
张峰	中国食品药品检定研究院卫生部生物技术产品检定及标准化重点实验室,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
于传飞	中国食品药品检定研究院卫生部生物技术产品检定及标准化重点实验室,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
李萌	中国食品药品检定研究院卫生部生物技术产品检定及标准化重点实验室,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
王文波	中国食品药品检定研究院卫生部生物技术产品检定及标准化重点实验室,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
崔永霏	中国食品药品检定研究院卫生部生物技术产品检定及标准化重点实验室,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
郭璐韵	中国食品药品检定研究院卫生部生物技术产品检定及标准化重点实验室,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
王兰	中国食品药品检定研究院卫生部生物技术产品检定及标准化重点实验室,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

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中文摘要:

目的：研究并建立针对抗白细胞分化抗原19(Cluster of Differentiation 19，CD19)单抗的关键质量属性质控方法。方法：运用肽图对抗CD19的单抗进行鉴别；采用还原/非还原十二烷基磺酸钠毛细管电泳(Capillary Electrophoresis-sodium Dodecyl sulfonate，CE-SDS)和分子排阻色谱(Size ExclusionHigh Performance Liquidchromatography，SEC-HPLC)进行单抗纯度的控制；运用成像毛细管等电聚焦电泳(Imaging Capillary Isoelectricfocusing Electrophoresis，iCIEF)测定电荷异质性；运用高效液相色谱法(High Performance Liquid Chromatography, HPLC)分析抗CD19单抗的糖基化，采用基于报告基因的抗体依赖的细胞介导的细胞毒效应(Antibody-Dependent Cell-mediated Cytotoxicity，ADCC) 测定生物学活性。其他各项指标均应符合现行版《中华人民共和国药典》以及其他相关要求。结果：肽图检测抗CD19单抗具有相应的特征图谱，能够用于鉴别；还原CE-SDS的轻链与重链峰面积之和百分比为(98.77±0.05)%；非还原CE-SDS主峰峰面积百分比为(96.19±0.05)%，片段百分比为 (3.81±0.05)%；SEC-HPLC单体的峰面积百分比为(99.42±0.001)%，聚合物峰面积百分比为 (0.54±0.001)%；iCIEF分析的主要亚型的峰面积百分比为(66.41±0.38)%，酸性亚型的峰面积百分比为(13.69±0.16)%，碱性亚型的峰面积百分比为(19.90±0.46)%；在糖型分析中，占比最高的三个糖型分别为G0、G0-GN和M5，G0的含量为(64.64±0.61)%，G0-GN的含量为(9.75±0.42)%， M5含量为(6.22±0.35)%，生物学活性的半数最大效应浓度(Concentration for 50% of Maximal Effect， EC₅₀)值为(10.09±1.40)ng·mL^-1。结论：针对抗CD19单抗的关键质量属性，研究并建立了相应的质量控制方法，以确保其安全、有效和质量可控，为该类单抗产品的质量控制方法和策略提供参考依据。

英文摘要:

Objective: To study and establish quality control methods for recombinant anti-cluster of differentiation 19 (CD19) monoclonal antibody (mAb). Methods: Peptide mapping was used for identification of the anti-CD19 mAb. The purity of the mab was controlled by the reduced/non-reduced capillary electrophoresis-sodium dodecyl sulfonate (CE-SDS) and size exclusion-high performance liquidchromatography (SEC-HPLC)methods. Imaging capillary isoelectric focusing electrophoresis (iCIEF) was used to determine the charge heterogeneity. The glycation of anti-CD19 mAb was analyzed by high performance liquid chromatography (HPLC) and the biological activity was measured by antibody-dependent cell-mediated cytotoxicity (ADCC) based on reporter assay. Other indicators should comply with corresponding requirements of Chinese Pharmacopeia 2020 edition and related guidelines. Results: Peptide mapping detection of anti-CD19 mAb had the corresponding characteristic map, which played a good role in the identification. The sum percentage of peak area of light chain and heavy chain of reduced CE-SDS was (98.77 ± 0.05) %, the main peak area percentage of non-reduced CESDS was (96.19 ± 0.05) %, and the fragment percentage was (3.81 ± 0.05) %. The peak area percentage of SECHPLC monomer was (99.42 ± 0.001) % and the percentage of polymer peak area was (0.54 ± 0.001) %. The peak area percentage of the main subtypes analyzed by iCIEF was (66.41 ± 0.38) %, the peak area percentage of acid subtype was (13. 69 ± 0.16)%, and that of alkaline subtype was (19.90 ± 0.46) %. In the analysis of sugar type, the three sugar types with the highest proportion were G0, G0-Gn and M5, respectively. The content of G0 was (64.64 ± 0.61) % , G0-Gn was (9.75 ± 0.42) %, M5 was (6.22 ± 0.35) %. The EC50 value of ADCC activity was (10.09 ± 1.40) ng·mL^-1. Conclusions: According to the key quality attributes of anti-CD19 monoclonal antibody, the corresponding quality control methods are studied and established to ensure its safety, effectiveness and quality control , which provides references for the quality control methods and strategies of anti-CD19 mAb products.

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