人血白蛋白中杂蛋白的生物质谱分析

Analysis of Impurity Proteins by Biomass Spectrometry in Human Serum Albumin

目的建立基于电喷雾质谱(ESI-Q-TOF MS)技术检测人血白蛋白中杂蛋白的方法,考察国内上市的27家企业的28批白蛋白产品中的未知蛋白组分。方法按2015年版《中国药典》四部通则3121方法,采用凝胶过滤色谱柱(Hiprep16/60Sepharyl S-200HR),对人血白蛋白中的各组分进行分离,并收集其中的二聚体、多聚体组分;经除盐、浓缩和胰蛋白酶酶解后,采用BEH C18(2.1 mm×100 mm,130 )色谱柱,以水(含0.1%甲酸)-乙腈流动相梯度洗脱,采用色谱-质谱(MS)技术对多肽进行二级质谱序列测定,并利用搜库软件(PLGS3.0)分析鉴定人血白蛋白中的杂蛋白。结果利用所建立的方法对27家企业的制品进行测定,共检测出23种杂蛋白,其中载脂蛋白A-Ⅱ(apolipoprotein A-Ⅱ)、人结合珠蛋白(haptoglobin)、人血色素结合蛋白(hempoexin)、α-1B-糖蛋白(alpha-1B-glycoprotein)与α-2-HS-糖蛋白(alpha-2HS-glycoprotein)等5种蛋白为所有企业产品共有的杂蛋白;α白蛋白(VE结合蛋白)、N-乙酰-L-丙氨酸酰胺酶、触珠蛋白相关蛋白,血红蛋白(β亚基,α亚基)、转甲状腺素蛋白等蛋白大部分企业产品均存在。各企业杂蛋白数量分布在8~17种之间,27家企业产品杂蛋白种类数量差异较大。结论本方法可用于人血白蛋白中未知组分的研究,为人血白蛋白的生产工艺优化提供了指导。

OBJECTIVE To establish a method for detecting the impurity components in human serum albumin by electrospray ionization mass spectrometry(ESI-Q-TOF MS) and investigate the impurity components in 28 batches of post-marketing albumin products from 27 companies.METHODS According to Chinese Pharmacopoeia’s General Principles 4121,gel filtration column(Hiprep16/60 Sepharyl S-200 HR) was used to separate the protein components.The dimer and multimer components were collected,then desalted,concentrated and digested by trypsin.The peptide was eluted by BEH C18(2.1 mm × 100 mm,130■) column with mobile phase consisting of water(0.1% formic acid)-acetonitrile in gradient elution mode.Chromatography-mass spectrometry was used to determine the secondary sequences of the peptides and the searching software(PLGS3.0) was used to identify the impurity protein components in albumin.RESULTS A total of 23 impurity proteins were detected,among which apolipoprotein A-Ⅱ,haptoglobin,and hempoexin,α-1 B-glycoprotein and α-2-HS-glycoprotein were the impurity proteins common to all enterprise products;α-albumin(VE binding protein),N-acetyl-L-alanine amidase,haptoglobin-related proteins,hemoglobin(β subunit,α subunit),transthyretin and other proteins were found in the products of most companies.The number of heterologous proteins in each enterprise was between 8-17,and the numbers of impurity proteins in 27 companies were quite different.CONCLUSION This method can be used for the investigation of unknown components in human serum albumin,which provides a guidance for the optimization of human albumin production process.