摘要
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-1 Ra)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-1 Ra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.
Abstract
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.
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