Large-scale preparation of thrombin from human plasma.

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 gel 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.