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。
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
.
DOI: 10.16153/j.1002-7777.2021.09.007
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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