干扰素α-2a的复性DOE研究

Refolding Reasearch of Interferon α-2a via Design of Experiment(DOE) Method

通过DOE(Design of experiment)的方法研究干扰素α-2a(IFNα-2a)蛋白的复性条件,以获得适宜的工业生产条件。以包涵体(Inclusion body,IB)形式在大肠杆菌体内表达的干扰素α-2a蛋白,经过变性、复性后折叠形成天然结构的活性蛋白。通过稀释复性的方法,并对复性液的pH、精氨酸浓度及复性时间进行DOE设计和考察,获得了上述复性参数的工艺范围:复性时间50~60 h,pH(9.9±0.1),精氨酸浓度0.3~0.5 mol/L。复性率为35%~42%,19 min杂质(峰后)小于15%。最终结果表明,精氨酸能有效抑制复性过程中蛋白质的聚集,提高干扰素α-2a折叠效率;从而提高产品收率及该产品的经济效益;pH值的合理选择和复性时间的有效延长可降低19 min杂质含量,进而降低纯化成本。

The renaturation conditions of Interferonα-2 a（IFN α-2 a） was researched via design of experiment（DOE） method to obtain suitable process parameters for industry-scale production. IFN α-2 a was expressed in the form of inclusion bodies in Escherichia coli,which could transform into the folded protein with native structure after denaturation and renaturation. Protein refolding by dilution was mainly described in this article,and the process parameters such as pH value of renaturation solution,concentration of arginine and refolding time were investigated via design of experiment（DOE） method in the form of CCF model. The results were obtained as follows： refolding time varied between 50 and 60 h,pH value varied between 9. 8 and 10. 0,concentration of arginine varied between0. 3 and 0. 5 mol/L,renaturation yield varied between 35 and 42 percent,and impurity content of 19 min was less than 15 percent.The final results indicated that arginine could effectively inhibit the aggregation of proteins during refolding process to improve the refolding efficiency of IFN α-2 a,which would increase the product yield and the economic value of the product. Simultaneously the reasonable selection of pH value and the prolonged refolding time could reduce the impurity content of 19 min,which would lower the cost of the subsequent purification.