PURIFICATION OF FACTOR-VIII AND VON-WILLEBRAND-FACTOR FROM HUMAN PLASMA BY ANION-EXCHANGE CHROMATOGRAPHY

Factor VIII (anti-hemophilia A factor) is isolated from human plasma. Purification is carried out by a combination of precipitation and chro matographic procedures. After precipitation, the first step in virus i nactivation is achieved through the effect of a non-ionic detergent su ch as Tween 80, and a solvent, e.g. tri-n-butylphosphate (TnBP). By su bsequent anion-exchange chromatography, a highly enriched product is i solated, consisting of a complex formed by factor VIII and von Willebr and factor (FVIII-vWF). This treatment also removes the virus-inactiva ting reagents to quantities in the low ppm range. The second step in v irus inactivation is aimed specifically at the non-enveloped viruses a nd consists of pasteurization at temperatures higher than 60 degrees C for 10 h. Through the addition of stabilizers, between 80% and 90% of the initial activity of FVIII is preserved during the modified pasteu risation. Along with the possibly denatured proteins the stabilizers, such as sugars, amino acids and bivalent cations, are subsequently rem oved by ion-exchange chromatography. The two-fold virus inactivation, by solvent/detergent treatment and subsequent pasteurisation, allows t he destruction of both lipid-enveloped and non-enveloped viruses. Duri ng the procedure FVIII is stabilized through the high content of vWF. The complex consisting of FVIII and vWF can be dissociated by adding c alcium ions. Subsequently both glycoproteins from this complex are sep arated from one another by further anion-exchange chromatography.