RP-HPLC法测定可溶性CD95-Fc融合蛋白中异天冬氨酸的含量  

Determination of isoaspartate in soluble CD95-Fc fusion protein by RPHPLC

作　　者：于雷[1] 陶磊[1] 毕华[1] 李响[1] 饶春明[1] YU Lei;TAO Lei;BI Hua;LI Xiang;RAO Chunming(Division of Recombinant Biological Products, National Institute of Food and Drug Control, Beijing 100050, China)

机构地区：[1]中国食品药品检定研究院重组药物室,北京100050

出　　处：《中国医药导报》2019年第6期109-112,共4页China Medical Herald

基　　金：国家科技重大专项项目(2015ZX09501008-001);中国食品药品检定研究院中青年发展研究基金课题(2017B3)

摘　　要：目的测定可溶性CD95-Fc融合蛋白中异天冬氨酸(IsoAsp)含量。方法采用ISOQUANT誖异天冬氨酸检测试剂盒结合反相高效液相色谱法测定样品中IsoAsp含量,采用Phenomenex Synergi 4μm Hydro RP-80 C18色谱柱(150 mm×3 mm,4μm),以7.9 mmol/L磷酸二氢钾-1.1 mmol/L磷酸氢二钾-10%甲醇为流动相A,以甲醇为流动相B,梯度洗脱,流速为0.43 mL/min。结果对照品和3批原液中IsoAsp含量相近(0.30～0.32 mol/mol sCD95-Fc),3批成品中IsoAsp含量明显增高(0.51～0.53 mol/mol sCD95-Fc);25℃和37℃处理24 h后成品中IsoAsp含量无明显变化,60℃处理后明显增加。结论该法适用于可溶性CD95-Fc融合蛋白中IsoAsp含量检测,高温处理可加快样品中IsoAsp生成。Objective To determine the content of isoaspartate (IsoAsp) in soluble CD95-Fc fusion protein. Methods IsoAsp content in samples was determined by ISOQUANT^RIsoaspartic Acid Detection Kit combined with reversed-phase high performance liquid chromatography. Phenomenex Synergi 4 μm Hydro RP-80 C18 column (150 mm × 3 mm, 4 μm) was used. Potassium dihydrogen phosphate-1.1 mmol/L potassium dihydrogen phosphate-10% methanol was used as mobile phase A, methanol as mobile phase B, gradient elution was performed at a flow rate of 0.43 mL/min. Results The content of IsoAsp in reference material was similar to that in three batches of raw liquor (0.30-0.32 mol/mol sCD95-Fc). The content of IsoAsp in three batches of finished products increased significantly (0.51-0.53 mol/mol sCD95-Fc). The content of IsoAsp in finished products did not change significantly after 24 hours of treatment at 25℃ and 37℃, but increased significantly after treatment at 60℃. Conclusion This method is suitable for the determination of IsoAsp in soluble CD95-Fc fusion protein. High temperature treatment can accelerate the formation of IsoAsp in samples.

关 键 词：可溶性CD95-Fc 异天冬氨酸 脱酰胺修饰 反相高效液相色谱 

分 类 号：R914[医药卫生—药物化学]