抗PD-L1单抗的质量控制研究

目的：研究建立抗程序性死亡受体配体1（programmed cell death protein ligand 1，PD-L1）单抗的质量控制方法。方法：利用成像毛细管等电聚焦电泳（imaging capillary isoelectric focusing electrophoresis，iCIEF）、肽图对抗PD-L1单抗进行鉴别；利用还原/非还原十二烷基磺酸钠毛细管电泳（capillary electrophoresis-sodium dodecyl sulfonate，CE-SDS）、分子排阻高效液相色谱（size exclusion-high performance liquid chromatography，SEC-HPLC）、离子交换高效液相色谱（ion exchange-high performance liquid chromatography，IEC-HPLC）对抗PD-L1单抗的纯度进行控制；利用细胞结合抑制法和转基因细胞法测定抗PD-L1单抗的活性；利用光阻法和液流成像分析法对不溶性微粒进行检测和分析。结果：抗PD-L1单抗的iCIEF检测结果具有特征性主峰，肽图检测具有相应特征峰，起到很好的鉴别作用。还原CE-SDS的轻链与重链峰面积之和的百分比为（98.37±0.21）%，非还原CE-SDS主峰峰面积百分比为（96.33±0.15）%；SEC-HPLC单体的峰面积百分比为（99.43±0.02）%；IEC-HPLC酸性区峰面积百分比、主峰峰面积百分比、碱性区峰面积百分比分别为（13.63±0.40）%、（80.00±0.36）%、（6.37±0.15）%。细胞结合抑制法与转基因细胞法的活性检测均呈现剂量反应曲线，反映抗PD-L1单抗剂量依赖性地阻断程序性死亡受体1（programmed cell death protein 1，PD-1）与PD-L1的结合及后续的生物学效应。光阻法检测≥10 μm及≥25 μm的不溶性微粒数分别为（1.33±0.58）粒·mL^-1和（0.00±0.00）粒·mL^-1；液流成像分析法检测2~10 μm、≥10 μm及≥25 μm的微粒数分别为（1 243.67±242）粒·mL^-1、（45.67±16.07）粒·mL^-1和（10.00±5.00）粒·mL^-1，其中，蛋白聚体占87.45%，非蛋白聚体占12.55%。结论：研究建立了抗PD-L1单抗的多种质控方法，为国内抗PD-L1单抗的质量检测提供了参考依据。  

Objective:To investigate and establish quality control methods for anti-programmed cell death protein ligand 1(PD-L1) monoclonal antibody(mAb). Methods:Imaging capillary isoelectric focusing electrophoresis(iCIEF) and peptide mapping were used for identification of the anti-PD-L1 mAb. The purity was measured using mothods including reduced/non-reduced capillary electrophoresis-sodium dodecyl sulfonate(CE-SDS),size exclusion-high performance liquid chromatography (SEC-HPLC) and ion exchange-high performance liquid chromatography(IEC-HPLC). The activity of anti-PD-L1 mAb was determined by cell binding inhibition method and transgenic cell method,and the sub-visible particles were detected and analyzed by light obscuration and flow imaging method. Results:The iCIEF and peptide mapping data showed the characteristic peaks of anti-PD-L1 mAb,which played a promising role in product identification. The area percentage of the sum of both light chain and heavy chain was (98.37±0.21)%,as detected by reduced CE-SDS assay;the area percentage of the main peak detected by non-reduced CE-SDS was (96.33±0.15)%. The area percentage of monomer peak detected by SEC-HPLC was (99.43±0.02)%. The area percentage of the acid peak,the main peak and the basic peak were (13.63±0.40)%,(80.00±0.36)% and (6.37±0.15)%,respectively. In bio-specific activity assays,both cell binding inhibition method and transgenic cell method assays showed dose-response curves,reflecting dose-dependent blockade of the binding between PD-L1 and programmed cell death protein 1(PD-1),as well as the subsequent anti-PD-L1 mAb-induced biological effect. The detected amount of sub-visible particles with diameters of ≥ 10 μm, ≥ 25 μm by light obscuration were (1.33±0.58) particle·mL^-1 and (0.00±0.00) particle·mL^-1,respectively;the detected amount of sub-visible particles with diameters of 2-10 μm, ≥ 10 μm and ≥ 25 μm by flow imaging method were (1 243.67±242) particle·mL^-1,(45.67±16.07) particle·mL^-1 and (10.00±5.00) particle·mL^-1,respectively,in which 87.45% were protein aggregates while 12.55% were non-protein particles. Conclusion:A series of representive quality control methods for anti-PD-L1 mAb have been established,which can provide references for the quality control of anti-PD-L1 mAb in China. 

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