液相色谱-高分辨质谱法评价人干扰素α2b层析工艺对杂质蛋白的去除效果  

Evaluation of removal efficiency of human interferonα2b chromatography process for impurity proteins using liquid chromatography-high resolution mass spectrometry

作　　者：郭莹[1] 龙珍 黄敏 于雷[1] 李响[1] 裴德宁[1] 周勇[1] GUO Ying;LONG Zhen;HUANG Min;YU Lei;LI Xiang;PEI Dening;ZHOU Yong(National Institutes for Food and Drug Control,Beijing 100050,China;不详)

机构地区：[1]中国食品药品检定研究院,北京100050 [2]赛默飞世尔科技(中国)有限公司,北京100080

出　　处：《中国生物制品学杂志》2025年第2期179-184,共6页Chinese Journal of Biologicals

基　　金：国家质量基础设施体系(2021YFF0600804)。

摘　　要：目的采用液相色谱-高分辨质谱(liquid chromatography-high resolution mass spectrometry,LC-HRMS)法评价人干扰素α2b(human interferonα2b,hIFNα2b)纯化过程中层析工艺对杂质蛋白[IFN相关蛋白及宿主细胞蛋白(host cell protein,HCP)]的去除效果,以期为hIFNα2b生产工艺的改进提供实验依据。方法采用LC-HRMS法对hIFNα2b层析工艺过程中的IFN相关蛋白(包括脱酰胺IFN、氧化IFN、乙酰化IFN)及HCP进行完整蛋白水平分析,对hIFNα2b翻译后修饰位点及HCP多肽水平进行检测。结果疏水层析(hydrophobic interaction chromatography,HIC,M柱)可去除HCP;阴离子交换层析(anion exchange chromatography,AEC)可去除HCP、脱酰胺IFN和乙酰化IFN;HIC(B柱)可去除氧化IFN。hIFNα2b的主要乙酰化位点(K34、K121、K131和K134)、主要氧化位点(M16、M21、M59和M148)及主要脱酰胺位点(N45、N156、Q5、Q21、Q46、Q124)中的Q5和Q21均位于蛋白表面,易受环境条件的影响,发生乙酰化、氧化等反应。结论在hIFNα2b纯化工艺中,层析纯化可有效去除IFN相关蛋白和HCP,表明LC-HRMS法可用于IFN纯化工艺的研究。Objective To evaluate the removal efficiency of chromatography process for impurity proteins[IFN-related proteins and host cell proteins(HCPs)]in the purification process of human interferon α2b(h IFNα2b)by liquid chromatogra-phyhigh resolution mass spectrometry(LC-HRMS),in order to provide experimental basis for improving the production process of h IFNα2b.Methods The whole protein levels of IFN-related proteins(including deamidated IFN,oxidized IFN and acetylated IFN)and HCPs during the chromatography process of h IFNα2b were analyzed by LC-HRMS,and the h IFNα2b post-translational modification sites and HCPs were detected on the polypeptide levels.Results HCPs could be removed by hydrophobic interaction chromatography(HIC)M column,HCPs,deamidated IFN and acetylated IFN could be removed by anion exchange chromatography(AEC),and oxidized IFN could be removed by HIC(B column).The major acetylation sites(K34,K121,K131 and K134),major oxidation sites(M16,M21,M59 and M148),as well as Q5 and Q21 of major deamidation sites(N45,N156,Q5,Q21,Q46 and Q124)of h IFNα2b were located on the surface of the protein,which were susceptible to environmental conditions and subjected to acetylation and oxidation.Conclusion In the purification process of h IFNα2b,chromatography purification can effectively remove IFN-related proteins and HCPs,which also indicated that LC-HRMS technology can be used for the research of IFN purification process.

关 键 词：人干扰素α2b 相关蛋白 宿主细胞蛋白质 层析工艺 液相色谱-高分辨质谱 

分 类 号：R917[医药卫生—药物分析学]