不同表达体系重组人血白蛋白中糖蛋白的多重分析方法测定及质量评价

Determination and Quality Evaluation of Glycoprotein in Recombinant Human Albumin from Different Expression Systems

目的用多种方法测定不同表达体系重组人血白蛋白中糖蛋白的含量,进行初步质量评价。方法选择不同来源样品包括酿酒酵母表达的重组人血白蛋白、毕赤酵母表达的重组人血白蛋白、水稻表达的重组人血白蛋白、血浆来源的人血白蛋白,采用硫酸苯酚法、HPLC峰面积法、ConA结合洗脱HPLC定量法测定样品中糖蛋白含量。结果硫酸苯酚法测定毕赤酵母表达体系的10批次样品,两个企业样品的甘露糖含量均值分别为2.7 mg·g(Pro)^(-1)(A厂,n=4)和1.7 mg·g(Pro)^(-1)(B厂,n=6);HPLC峰面积法测定整体上看毕赤酵母表达的重组人血白蛋白ConA结合蛋白峰面积百分比较高,分别为2.65%(A厂)及0.71%(B厂),而水稻表达的重组人血白蛋白ConA结合蛋白峰面积百分比较低,均值为0.05%(E厂,n=3),血浆来源的人血白蛋白ConA结合蛋白峰面积为0.01%(W厂);ConA结合后洗脱HPLC定量法测定毕赤酵母表达重组人血白蛋白ConA结合蛋白含量分别为27.58(A厂)、21.48(B厂)、32.02 mg·g(Pro)^(-1)(C厂);酿酒酵母来源重组人血白蛋白ConA结合蛋白含量较低,仅为2.29 mg·g(Pro)^(-1)(D厂);水稻来源重组人血白蛋白ConA结合蛋白含量最低,为1.27 mg·g(Pro)^(-1)(E厂);血浆来源人血白蛋白ConA结合蛋白含量为31.16 mg·g(Pro)^(-1)(S厂)。结论就本实验涉及的样品和检测方法所获得的初步结果而言,重组人血白蛋白中均含有糖类蛋白,毕赤酵母表达体系样品中的糖类蛋白含量整体上高于酿酒酵母表达体系,水稻表达的重组人血白蛋白中糖类蛋白含量最低(仅限现有分析条件),血浆来源的人血白蛋白中也含有糖类蛋白。

OBJECTIVE To determine glycoprotein content in recombinant human albumin from different expression systems with different methods. METHODS Recombinant human albumin samples from Saccharomyces cerevisiae expression system, Pichia pastoris expression system, Oryza sativa expression system as well as plasma derived human albumin were investigated by phenol sulfuric acid method, HPLC peak area method and ConA combining elution HPLC method. RESULTS For 10 batches of samples expressed in pichia pastoris expression system, the total contents of mannose were 2.7 mg·g（Pro）-1 （A manufacturer, n=4） and 1.7 mg·g（Pro）-1 （B manufacturer, n=6）, respectively. The HPLC peak area percentages of ConA binding protein in recombinant human albumin from Pichia pastoris expression system were the highest, which showed 2.65% （A manufacturer） and 0.71% （B manufacturer）respectively, the peak area percentage of ConA binding protein in Oryza sativa expression system was 0.05% （E manufacturer, n=3）, and the ConA binding protein peak area of plasma derived human albumin was 0.01% （W manufacturer）. The RESULTS of ConA binding and elution analysis with HPLC method for Quantitative determination of ConA binding protein showed that the ConA binding protein contents in the samples from pichia pastoris expression system were much higher： 27.58 mg·g（Pro）-1 （A manufacturer）, 21.48 mg·g（Pro）-1（B manufacturer）, 32.02 mg·g（Pro）-1（C manufacturer）; the ConA binding protein content in the sample from Saccharomyces cerevisiae expression system was lower, 2.29 mg·g（Pro）-1（D manufacturer）; the ConA binding protein content in the samples from oryza sativa expression system was the lowest, 1.27 mg·g（Pro）-1 （E manufacturer）; the plasma derived human albumin ConA binding protein content was 31.16 mg·g（Pro）-1 （S manufacturer）. CONCLUSION In terms of the RESULTS of the samples and METHODS involved in this study,there were glycosylated or glycol forms of protein in all recombinant human albumin samples from different expression systems; the glycosylated protein content in samples of Pichia pastoris expression system is higher than Saccharomyces cerevisiae expression system, while the glycoformed protein in samples of Oryza sativa expression system is the lowest. Plasma derived human albumin also contains glycoprotein or glycosylated protein.