地特胰岛素原料药的生产工艺优化  

Optimization of Production Process for Diethylene Insulin Active Pharmaceutical Ingredient

作　　者：陆乐 徐荣发 翁文锃 周展[1] LU Le;XU Rongfa;WENG Wenzeng;ZHOU Zhan(College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310058,China;Hangzhou Jiuyuan Gene Engineering Co.,Ltd.,Hangzhou 310018,China)

机构地区：[1]浙江大学药学院,杭州310058 [2]杭州九源基因工程股份有限公司,杭州310018

出　　处：《中国现代应用药学》2024年第18期2502-2509,共8页Chinese Journal of Modern Applied Pharmacy

摘　　要：目的 构建并表达地特胰岛素原,开发原料药生产工艺,优化柱层析、酶切反应、修饰反应等关键工艺,提升和稳定回收率,并制备得到和原研质量一致的地特胰岛素原料药。方法 通过建立DesB30工程菌来发酵表达地特胰岛素原,通过离子交换层析粗提捕获胰岛素原,再通过酶切反应切除链接肽,反相层析提纯后定点修饰十四烷基脂肪酸链,得到地特胰岛素,最后通过两步高压反相层析精纯,结晶冻干得到地特胰岛素原料药粉。结果 优化离子交换层析,提高胰岛素原的洗脱浓度,操作便于生产;优化酶切反应和修饰反应,提升并稳定回收率和目的蛋白纯度,保障最终产品质量。优化后工艺总收率达25%,目的蛋白纯度>99%。结论 优化后工艺稳定性良好,多批次制备样品的分子量、等电点、氨基酸序列、二硫键、胰岛受体亲和力等多项指标和原研质量一致,可供产业化生产参考。OBJECTIVE To construct and express Detemir proinsulin,develop raw material production processes,optimize key processes to improve and stabilize recovery rates,and manafacture the raw materials with the same quality as the original drug.METHODS By establishing DesB30 engineering bacteria for fermentation and expression of Detemir proinsulin.Capturing Detemir proinsulin through ion exchange chromatography.Cut off the linked short peptides through enzyme reaction.Reverse phase chromatography after targeted modification of fatty acid chains to obtain Levemir.Finally,through two steps of reverse phase chromatography and freeze-drying,the raw material powder was obtained.RESULTS Optimization exchange chromatography to increase the elution concentration of proinsulin,and convenient for production operations;Optimize enzyme reaction and modification reaction,improve and stabilize recovery rates and target protein purity,to ensure the quality of the final product.The overall yield of this process reaches 25%,and the purity of the target protein>99%.CONCLUSION The molecular weight,isoelectric point,amino acid sequence,disulfide bond,and insulin receptor affinity of the samples prepared by this process are consistent with the quality of the original drug.The process is robustness and can be used as a reference for large-scale production.

关 键 词：地特胰岛素原 地特胰岛素 原料药 酶切反应 修饰反应 DesB30 

分 类 号：R945[医药卫生—微生物与生化药学]