Identification of chemical modification types of associated proteins in recombinant human interleukin-1 receptor antagonist preparations and precipitation
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.
Recombinant human interleukin-1 receptor antagonist(rhIL-1Ra)Chemical modification typeOxidation modificationDehydration cyclization