重组人白细胞介素-1受体拮抗剂制剂和沉淀中相关蛋白化学修饰类型的鉴定

Identification of chemical modification types of associated proteins in recombinant human interleukin⁃1 receptor antagonist preparations and precipitation

目的 鉴定重组人白细胞介素-1受体拮抗剂(recombinant human interleukin-1 receptor antagonist,rhIL-1Ra)制剂和室温加速获得的制剂沉淀中相关蛋白的化学修饰类型。方法 利用反相高效液相色谱(reverse phase-high-performance liquid chromatography,RP-HPLC)对rhIL-1Ra制剂和室温加速获得的制剂沉淀进行相关蛋白检测,并按面积归一化法确定相关蛋白的百分比;再用液质联用质谱法对rhIL-1Ra制剂和室温加速获得的制剂沉淀进行相关蛋白的鉴定。结果 RP-HPLC结果显示,rhIL-1Ra制剂中有6个相关蛋白色谱峰,所占百分比依次为0.414%、0.870%、0.871%、0.304%、0.104%、1.182%,rhIL-1Ra室温加速获得的沉淀中有5个相关蛋白色谱峰,所占百分比依次为0.350%、3.603%、1.118%、0.629%、0.866%。液质联用结果显示,rhIL-1Ra制剂6个相关蛋白色谱峰化学修饰类型分别为甲硫氨酸氧化、天冬氨酸脱水环化+甲硫氨酸氧化、甲硫氨酸氧化、甲硫氨酸氧化、天冬氨酸脱水环化、天冬氨酸脱水环化;rhIL-1Ra室温加速获得的沉淀中5个相关蛋白色谱峰化学修饰类型分别为甲硫氨酸氧化、天冬氨酸脱水环化+甲硫氨酸氧化、以甲硫氨酸氧化为主、以天冬氨酸脱水环化+甲硫氨酸氧化为主、以天冬氨酸脱水环化为主。结论 rhIL-1Ra制剂和室温加速获得的制剂沉淀的相关蛋白修饰类型相同,主要存在3种:甲硫氨酸氧化修饰、天冬氨酸脱水环化、天冬氨酸脱水环化+甲硫氨酸氧化,本研究为提高rhIL-1Ra制剂的质量提供了参考。

Objective To identify the types of protein chemical modifications associated with the precipitates of recom⁃binant human interleukin⁃1 receptor antagonist(rhIL⁃1Ra)preparations and preparations obtained at room temperature acceleration.Methods rhIL⁃1Ra products and precipitates in rhIL⁃1Ra products obtained at room temperature acceleration were detected by reverse phase⁃high⁃performance liquid chromatography(RP⁃HPLC),and the percentage of associated proteins was determined by area normalization method.Then the rhIL⁃1Ra product and precipitates in rhIL⁃1Ra product were identified for the associated proteins by liquid mass spectrometry.Results RP⁃HPLC results showed that there were 6 associated protein chromatographic peaks in rhIL⁃1Ra product,and the percentages were 0.414%,0.870%,0.871%,0.304%,0.104%and 1.182%respectively.There were 5 associated protein chromatographic peaks in rhIL⁃1Ra product precipitation,and the percentages were 0.350%,3.603%,1.118%,0.629%and 0.866%respectively.The liquid mass spectrometry results showed that the rhIL⁃1Ra product had 6 protein chromatographic peaks,of which the chemical modi⁃fication types were methionine oxidation,aspartic acid dehydration cyclization+methionine oxidation,methionine oxida⁃tion,methionine oxidation,aspartic acid dehydration cyclization,and aspartic acid dehydration cyclization.There were 5 associated protein chromatographic peaks in the precipitates generated by rhIL⁃1Ra product,and the chemical modification types were methionine oxidation,aspartic acid dehydration cyclization+methionine oxidation,mainly methionine oxida⁃tion,mainly aspartic acid dehydration cyclization+methionine oxidation,and mainly aspartic acid dehydration cyclization.Conclusion The modification types of associated proteins in rhIL⁃1Ra precipitation obtained at room temperature acce⁃leration and rhIL⁃1Ra product were the same,which have three main types,namely methionine oxidation modification,aspartic acid dehydration cyclization,and aspartic acid dehydration cyclization+methionine oxidation.This study provides a reference for improving the quality of rhIL⁃1Ra preparation.