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.
Aizawa
Peter
P
Octapharma AB, SE-11275 Stockholm, Sweden.
Winge
Stefan
S
Karlsson
Göran
G
D002624:Chemistry, Clinical / Q000379:methods*; D002845:Chromatography / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005164:Factor IX / Q000737:chemistry; D005170:Factor X / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D006801:Humans; D008024:Ligands; D011516:Prothrombin / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013917:Thrombin / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
D003902:Detergents; D008024:Ligands; D012997:Solvents; D011516:Prothrombin; D005164:Factor IX; D005170:Factor X; D006493:Heparin; D012685:Sepharose; D013917:Thrombin