摘要
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≥10μm及≥25μm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2～10μm、≥10μm及≥25μm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.
作者
郭莎
王开芹
武刚
李萌
崔永霏
俞小娟
张荣建
于传飞
王兰
GUO Sha;WANG Kai-qin;WU Gang;LI Meng;CUI Yong-fei;YU Xiao-juan;ZHANG Rong-jian;YU Chuan-fei;WANG Lan(Division of Monoelonal Antibody,National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 102629,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2019年第1期13-22,共10页
Chinese Journal of Pharmaceutical Analysis
基金
国家科技重大专项重大新药创制项目免疫检查点单抗和其他创新生物技术药的质量评价方法和标准研究(2018ZX09101001-003)
