IMMOBILIZED HIRUDIN AND HIRUDIN-BASED PEPTIDES USED FOR THE PURIFICATION OF RECOMBINANT HUMAN THROMBIN PREPARED FROM RECOMBINANT HUMAN PROTHROMBIN

A simple and efficient activation-affinity purification system was dev eloped to obtain thrombin from recombinant CHO cells expressing human prothrombin. In this method, a controllable process for the activation of recombinant prothrombin is directly coupled with a purification st rategy for the recombinant thrombin generated. At a constant how rate and with a contact time limited to few seconds, recombinant prothrombi n was filtered through immobilized trypsin. In a closed flow system, t he recombinant thrombin generated was filtered through newly designed thrombin-specific affinity gels. Hirudin, the most specific thrombin i nhibitor, and hirudin-based peptides were covalently immobilized to Se pharose, thus creating thrombin-specific affinity gels that immediatel y absorb the thrombin generated from the activation mixture. Prothromb in and incompletely activated molecules did not bind to the affinity g el and were recirculated for a further activation cycle. Due to the sp ecificity of the affinity gels for thrombin and the elimination of thr ombin from the activation mixture, proteolytic degradation and autocat alytic inactivation of the recombinant thrombin was prevented. Recombi nant thrombin was isolated from the hirudin-based affinity gels by cha otrope salt elution, resulting in high yields of highly pure, active t hrombin. Affinity purification of thrombin was not deleteriously affec ted by contamination of the starting material with other proteins. Act ivation and affinity purification were equally effective for recombina nt and human plasma-derived prothrombin as well as for human and recom binant thrombin. (C) 1996 Academic Press, Inc.