目的 应用超高效液相色谱-行波离子淌度串联四极杆飞行时间质谱仪(UPLC-VION)高分辨质谱联用技术对不同表达体系重组人血白蛋白和不同生产工艺下人血白蛋白在完整蛋白水平进行分析,探讨质谱分析技术在白蛋白产品鉴别实验上的实际应用。方法 采用ACQUITY UPLC超高效液相色谱系统,ACQUITY UPLC BEH C4色谱柱脱盐处理,电喷雾离子化 (ESI)VION IMS QTof高分辨率质谱和UNIFI软件,对重组和血浆来源人血白蛋白进行分析。质谱信号经MaxEnt1去卷积化软件处理后,获得样品平均相对分子质量以及相对强度图谱。样品包括血浆来源人血白蛋白(2个厂家)、酵母表达系统(毕赤及酿酒酵母)生产的重组人血白蛋白(3个厂家)、转基因水稻表达的重组人血白蛋白(1个厂家)共6批,分别进行了测定和数据分析。结果 UPLC-VION高分辨质谱联用分析结果显示,不同厂家和不同表达载体纯化得到的重组人血白蛋白均能检测到66 437的信号,误差低于1,与人血白蛋白理论序列平均相对分子质量一致;不同厂家和不同工艺的样品具有特征性的质谱图,即不同来源人血白蛋白具有不均一性。结论 UPLC/VION超高效液相色谱-质谱联用方法专属性好,灵敏度高,能够区分出不同厂家及不同工艺得到的重组或血浆来源人血白蛋白,可作为白蛋白类药品质量控制的鉴别实验方法。
Abstract
OBJECTIVE To evaluate the applicability of UPLC/MS method for the identification test of human serum albumin (HSA) products including plasma derived and recombinant HSA samples.METHODS ACQUITY UPLC with Vion IMS QTof LC/MS system was used combined with on-line HSA sample desalting with ACQUITY UPLC BEH C18 column. The acquired multiply-charged mass spectrum was processed with MaxEnt1 automatic protein deconvolution software in UNIFI, which can transfer the raw mass spectrometry data to zero charge molecular mass or mass distribution of the intact protein.RESULTS Intact protein mass analysis not only provided the accurate mass of HSA, but also provided an overall view of the heterogeneity of HSA and the relative amounts of various forms. From this study, a very specific mass signal 【(66 437±1), which is the theoretical average MW of human serum albumin】 was obtained from all the six HSA samples. And the characteristic spectra of different samples were also got.CONCLUSION UPLC/MS method has very good specificity and high sensitivity and can distinguish HSA products made by different manufacturers and processes. The total analytical time is 10 min, which is ideal for the QC identification test of HSA products.
关键词
重组人血白蛋白 /
人血白蛋白 /
质量控制 /
鉴别实验 /
超高效液相色谱 /
串联四极杆飞行时间质谱 /
平均相对分子质量 /
完整蛋白分析
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Key words
recombinant human albumin /
plasma derived human serum albumin /
quality control /
identification test /
ultra-performance liquid chromatography /
QTof high resolution mass spectrometry /
average mass molecular weight /
intact mass analysis
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中图分类号：
R917
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参考文献
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脚注
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基金
重大新药创制项目资助(2018ZX09101001,2018ZX09301004)
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