膜分离制备右旋糖酐40中细菌内毒素的控制

Control of Bacterial Endoxtoxin for Dextran 40 Prepared by Membrane Separation

目的:将细菌内毒素作为膜分离制备右旋糖酐40工艺过程中的一个标的进行检测,使细菌内毒素含量成为右旋糖酐40原料药的质量指标,与国际标准接轨。方法:右旋糖酐发酵液经过板框过滤、陶瓷膜微滤和超滤等预处理,盐酸水解后通过100,10,50 kDa超滤除杂分离纯化为右旋糖酐40。结果:右旋糖酐发酵液预处理,果糖的去除率79%~89%。6%右旋糖酐发酵液处理液盐酸水解,还原糖的变化为5.72~8.68 mg·ml^(-1),总糖收率90%以上。水解液经过截留相对分子质量(Mr)分别为100,10,50 kDa超滤处理,绝大部分内毒素被去除,100 kDa和50 kDa截留干品(杂质)Mr均在50000以上。截留Mr 50 kDa超滤膜的滤过液经减压浓缩,50%~60%乙醇沉淀制取右旋糖酐40内毒素含量<3.0 EU·g^(-1),优于<10.0 EU·g^(-1)的国际标准,右旋糖酐40 Mr分布达到中国药典标准。结论:右旋糖酐发酵液经过陶瓷膜、超滤预处理之后盐酸水解,超滤(100,10,50 kDa)分离纯化水解液可以得到右旋糖酐40,同时对其细菌内毒素的含量加以控制,右旋糖酐40原料药的细菌内毒素指标优于国际标准。

Objective:To make the content of bacterial endotoxin become a product quality index of dextran 40 API,which is in line with international standards,bacterial endotoxin is detected and controlled as a target in the preparation process of dextran 40 by membrane separation.Methods:Dextran fermentation broth was pretreated by plate frame filtration,ceramic membrane microfiltration and ultrafiltration.After hydrochloric acid hydrolysis,dextran 40 was purified by impurity removal with 100 kDa,10 kDa and 50 kDa ultrafiltration.Results:Dextran fermentation broth was pretreated,and the removal rate of fructose was 79%-89%.The pretreatment solution of dextran fermentation broth was hydrolyzed by hydrochloric acid.The change of reducing sugar was 5.72-8.68 mg·ml^(-1),and the total sugar yield was more than 90%.The hydrolysate was treated with ultrafiltration by molecular weights of 100 kDa,10 kDa and 50 kDa,respectively,and most of the endotoxin was removed.The average molecular weights of dry products(impurities)retained by 100 kDa and 50 kDa were more than 50000.Dextran 40 prepared by concentrating the filtrate by the ultrafiltration membrane at the molecular weight of 50 kDa under reduced pressure and precipitating by 50%-60%ethanol had an endotoxin content<3.0 EU·g^(-1),which was better than the international standard<10.0 EU·g^(-1).The molecular weight distribution of dextran 40 reached the standard of Chinese Pharmacopoeia.Conclusion:Dextran fermentation broth is hydrolyzed by hydrochloric acid after ceramic membrane microfiltration and ultrafiltration.Dextran 40 can be obtained by ultrafiltration(100 kDa,10 kDa and 50 kDa),and the content of bacterial endotoxin can be controlled.The bacterial endotoxin index of dextran 40 API is better than that of the international standard.