重组人胰岛素制备过程中转肽工艺的研究

Transpeptidation Sudy on the Recombinant Human Insulin Preparation

目的:酵母表达体系制备重组人胰岛素的工艺中,转肽反应属于酶促半合成反应,过程复杂,成本高昂,通过实验研究提高反应效率,降低反应成本。方法:通过优化转肽反应中胰蛋白酶和双保护苏氨酸(Thr(But)OBut)的比例,寻找有利的反应条件,为进一步的条件确定奠定基础;从提高转肽反应物的反应效率出发,调整工艺顺序,将一步转肽反应调整为两步转肽反应;以胰蛋白酶和双保护苏氨酸两种反应物,进行综合成本分析比较;同时采用药典方法测定两种不同工艺制备的重组人胰岛素,进行生物活性的比较分析。结果:通过实验研究,一步转肽反应的收率最高可以达到74%,而两步转肽收率的收率最高可以达到90%;两步转肽反应相比一步转肽反应,酶量减少70%,双保护苏氨酸的使用量降低62.5%,同时提高了转肽反应中胰岛素原的反应浓度,达到12.5 mM,有着进一步开发的潜质;生物活性测定两种工艺生产的重组人胰岛素均符合中国药典的要求。结论:两步转肽反应对比一步转肽反应,提高了反应效率,产物的纯度较高,降低了反应的成本,有利于提高国产胰岛素的市场竞争力。

Objective： The study was conducted to improve the efficiency of transpeptidation and reduce costs in producing recombinant human insulin with pichia pastoris expression system,that transpeptidation is one kind of complex trypsin-catalyzed semisynthesis reaction.Methods： Experiment design was optimized to find the trypsin and dual protection threonine（Thr（But）OBut） content in the reaction system,which was the base for decided reaction condition.For improvement the use ratio of the reactants,procedure of transpeptidation was changed from one step to two steps,and cost analysis was done on the basis of trypsin and dual protection threonine price.Comparative analysis of biological activity was carried on at end of experiment with the test method according to the Chinese pharmacopeia.Results： The yield ratio of one-step transpeptidation was up to 74 %,and the ratio of two-step transpepdation was up to 94 %.The consumption of trypsin and dual protection threonine was cut by 70 %,62.5 % respectively.The reaction concentration of insulin also increased to 12.5 mm.The biological activity results of refined products were in accordance with the requirement of Chinese pharmacopoeia.Conclusion： Compared with one-step transpeptidation,the efficiency of transpeptidation was improved,purity of product higher,and the costs cut into two-step transpeptidation,which would be helpful in improving the market competitiveness of domestically produced insulin.