Large-scale preparation of thrombin from human plasma
Large-scale preparation of thrombin from human plasma
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DOI：
10.1016/j.thromres.2007.12.027
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发表时间：
2008-01-01
影响因子：
7.5
通讯作者：
Karlsson, Goran
中科院分区：
文献类型：
--
作者：
Aizawa, Peter;Winge, Stefan;Karlsson, Goran
凝血酶由人血浆制备（批量1200 L）。首先，凝血酶原通过以下分离技术分离：冷沉淀、离子交换色谱（二乙基氨基乙基，DEAE-IEX）、肝素亲和色谱、第二DEAE-IEX步骤和固定化金属亲和色谱（IMAC）。凝血酶原活化为凝血酶后，经疏水层析（HIC）纯化，超滤浓缩。该方法具有成本效益，因为在血浆衍生因子IX（FIX）的商业生产中，可以使用来自其中一个步骤（肝素亲和层析）的废物级分。最终凝血酶制剂的纯度约为75%（比活性约为2400 IU/mg蛋白质），足以满足其在纤维蛋白胶中的预期用途。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），因子X（FX）活性分析和分析HIC也被用来表征凝血酶。使用三种基本上不同的技术来降低任何病毒活性，即：溶剂/洗涤剂（S/D）处理、巴氏灭菌和病毒过滤（纳滤）。本文提出的生产工艺适用于大规模生产具有高度病毒安全性的凝血酶。(C)2008爱思唯尔有限公司保留所有权利。
Thrombin was prepared from human blood plasma (batch size 1200 L). First, prothrombin was isolated by the following separation techniques: cryoprecipitation, ion-exchange chromatography (diethyl aminoethyl, DEAE-IEX), heparin affinity chromatography, a second DEAE-IEX step, and immobilized metal-affinity chromatography (IMAC). Prothrombin was then activated to thrombin, which was purified by hydrophobic interaction chromatography (HIC) and concentrated by ultrafiltration. This process is cost-effective because a waste fraction can be used from one of the steps (heparin affinity chromatography) in the commercial production of plasma-derived Factor IX (FIX). The final thrombin preparation had a purity of approximately 75% (specific activity approximately 2400 IU/mg protein), which is sufficient for its intended purpose in a fibrin glue. Sodium dodecyl sulfate polyacrylamide get electrophoresis (SDS-PAGE), Factor X (FX) activity analysis, and analytical HIC were also used to characterize the thrombin. Three substantially different techniques were used to reduce any viral activity, namely: solvent/detergent (S/D) treatment, pasteurization, and virus filtration (nanofiltration). The manufacturing process presented here would be suitable for large-scale production of thrombin with a high degree of virus safety. (C) 2008 Elsevier Ltd. All rights reserved.