干扰素a-2b的聚乙二醇修饰

Interferon a-2b Modified with Polyethylene Glycol

摘要:

采用分子量为20 kD的单甲氧基聚乙二醇丙醛(mPEG-ALD)修饰重组人干扰素a-2b(IFN a-2b), 建立了修饰反应及分离纯化工艺。考察了修饰反应各因素对单修饰转化率以及单修饰产物体外活性的影响, 获得了优化的修饰反应条件, 即在pH 6.5, 20 mmol/L的磷酸氢二钠-柠檬酸缓冲溶液中, 干扰素a-2b的浓度为4 mg/mL, PEG与IFN a-2b的摩尔比为8:1, 4oC时反应20 h; 在优化的反应条件下, 单修饰PEG-IFN a-2b的转化率达到55%。并且, 采用离子交换层析对修饰产物进行分离纯化, 单修饰产品纯度达到97%, 体外活性保留达到未修饰干扰素a-2b的13.4%, 其在SD大鼠体内的循环半衰期得到了较大的延长, 且具有较好的水溶液稳定性。

Abstract:

In order to obtain a more stable PEGylated interferon a-2b, and prolong its half life, interferon a-2b(IFN a-2b) was modified with monomethoxy polyethylene glycol propionaldehyde (mPEG-ALD) 20000. It was found that the optimized reaction condition for the maximum bioactivity and highest PEGylation degree of the mono PEGylated interferon a-2b was as follows: in 20 mmol/L, pH 6.5, citric acid and sodium dihydrogen phosphate buffer, the concentration of IFN a-2b was 4 mg/mL, and the molar ratio of PEG/IFN a-2b was 8:1, and the reaction time was 20 h at 4oC. Under the optimized reaction condition, the mono PEGylation degree reached to 55%. Ion exchange chromatography was used to separate and purify mono PEGylated interferon a-2b from the reaction mixture. The purity of mono PEGylated interferon a-2b was higher than 97% characterized by HPLC. The bioactivity of the mono PEGylated interferon a-2b was 13.4% of the native IFN a-2b, while its half life in SD rat is much longer than the native IFN a-2b. The mono PEGylated interferon a-2b is also stable in aqueous.