重组人促红素的连续聚乙二醇化反应

Consecutive PEGylation Reaction Method of Recombinant Human Erythropoietin

为了达到提高重组人促红素(EPO)的聚乙二醇(PEG)化反应单取代率的目的,建立了一种EPO的连续PEG化反应方法。首先,对保存于4℃冰箱24个月的PEG-EPO反应液进行分析,结果表明其具有很好的长期稳定性。同时,圆二色谱(CD)测试结果显示,EPO在不同的缓冲液体系中,其蛋白质二级结构特征略有不同。接着,在不改变缓冲液体系的情况下,进行了EPO的直接二次PEG化反应;另外,采用双缓冲液体系,进行EPO的二次PEG化反应。结果表明,一次PEG化反应得到的PEG-EPO反应液体系具有很高的稳定性,不能直接进行二次PEG化反应;应用双缓冲液体系,则可实现EPO的连续PEG化反应,所得PEG-EPO反应液的单取代率高于54%。最后,采用双缓冲液体系,成功地进行了EPO的连续3次PEG化反应。对纯化得到的单取代产物(monoPEG-EPO),进行了高效液相色谱、荧光光谱、电泳、唾液酸含量、体内活性以及PEG化修饰位点等测试。测试结果表明,EPO在经过连续3次PEG化反应后,仍然可以很好地保留其生物活性和蛋白质结构,同时其PEG化修饰位点也没有发生显著改变。这些研究结果为开发一种新型的高单取代率的EPO的PEG化反应方法提供了基础,有望降低PEG-EPO长效药物的制备成本。

To establish a useful PEGylation reaction method of recombinant human Erythropoietin（EPO） with a high yield of mono substitution product（mono-PEG-EPO）,a consecutive PEGylation reaction method has been developed.At first,the reaction mixture of PEG-EPO after stored under 4 ℃ for 24 months has been analyzed and it showed a significant long-term stability.Meanwhile,the circular dichroism test results of EPO in various buffer solutions suggested that the character of secondary structure of EPO is slightly different.And then the direct second-time PEGylation of EPO under the same buffer solution,and the consecutive second-times PEGylation of EPO in different dual buffer solution systems were conducted.After that,consecutive three-times PEGylation of EPO was carried out successfully in a dual buffer solution system.The resulting purified mono-PEG-EPO products have been analyzed,by different methods,including HPLC test,FL spectrum test,electrophoresis test,content of sialic acid test,in vivo activity test and PEGylation position test.From these research results,it could be concluded that the reaction mixture of first PEGylation of EPO can not conduct the second-set PEGylation directly.However,applying with dual buffer solution system,consecutive PEGylation of EPO can be carried out with a yield of mono-PEG-EPO above 54% without obvious change of PEGylation position.And the purified monoPEG-EPO products maintain their biological activity and protein structure integrity.These findings provide a basis for the development of a new type of PEG-EPO manufacturing process with a high yield of mono-PEG-EPO product to reduce the drug production cost.