重组人源化抗DR5单克隆抗体质控方法的建立

Development of a method for quality control of recombinant humanized anti-DR5 monoclonal antibody

目的建立重组人源化抗DR5单克隆抗体的质控方法。方法利用Colo205细胞杀伤试验测定重组人源化抗DR5单克隆抗体的生物学活性;SDS毛细管凝胶电泳(capillary electrophoresis sodium dodecyl sulfate,CE-SDS)和分子排阻高效液相色谱(size exclusion high performance liquid chromatography,SE-HPLC)分析纯度;成像毛细管等点聚焦电泳(imaged capillary isoElectric focusing,iCIEF)法测定等电点及电荷异质性;毛细管区带电泳(capillary zone dlectrophoresis,CZE)和肽图法进行鉴别试验;切糖后对N-糖进行2-氨基苯甲酰胺(2-amino benzamide,2-AB)标记,采用亲水相互作用液相色谱(hydrophilic interaction liquid chromatography,HILIC)柱分离并结合质谱对其糖型进行分析。结果重组人源化抗DR5单克隆抗体生物学活性的EC50值为(9.09±1.03)ng/ml,RSD为11.33%。非还原CE-SDS主峰面积为(90.35±0.19)%,RSD为0.21%;还原CE-SDS重链和轻链峰面积共为(96.37±0.20)%,RSD为0.21%;SE-HPLC主峰面积为(99.14±0.13)%,RSD为0.13%。iCIEF分析主峰等电点为(8.95±0.00),RSD为0.00%;CZE和肽图法可对制品做出鉴别。糖谱分析方法相对灵敏。结论建立了重组人源化抗DR5单克隆抗体的质控方法,该方法具有保证产品安全、有效、质量可控的特点,为我国单克隆抗体的质量检测提供了参考依据。

Objective To develop a method for quality control of recombinant humanized anti-DR5 monoclonal antibody（mAb）.Methods The bioactivity of recombinant humanized anti-DR5 mAb was evaluated by determining its cytotoxic effect on Colo205 cells,while purity was analyzed by capillary electrophoresis sodium dodecyl sulfate（CE-SDS）and size exclusion high performance liquid chromatography（SE-HPLC）,and the PI value and charge heterogeneity by imaged capillary isoelectric focusing（iCIEF）.Peptide map and capillary zone electrophoresis（CZE）were used for identity test.The released glycan was labeled with 2-amino benzamide（2-AB） and analyzed by hydrophilic interaction liquid chromatography（HILIC）and mass spectrometry.Results The EC50 of recombinant humanized anti-DR5 mAb in bioassay was（9.09 ± 1.03）ng / ml,with a RSD of 11.33%.The main peak area percentage showed by non-reduced CE-SDS was（90.35 ± 0.19）%,with a RSD of 0.21%.The total peak area percentage of heavy and light chains showed by reduced CE-SDS was（96.37 ± 0.20）%,with a RSD of 0.21%.The main peak area percentage showed by SE-HPLC was（99.14 ± 0.13）%,with a RSD of 0.13%.The isoelectric point of the main peak showed by iCIEF was（8.95 ±0.00）,with a RSD of 0.00%.CZE and peptide mapping could be applied to the identification of the mAb,while the glycan mapping analysis was relatively sensitive.Conclusion A method for quality control of recombinant humanized anti-DR5 mAb was developed,which ensured the safety,effectiveness and quality controllability of the product and provided a reference for quality control of other domestic monoclonal antibody products.