PURIFICATION OF FACTOR-VIII VON-WILLEBRAND-FACTOR FROM HUMAN PLASMA ON IMMOBILIZED LENTIL LECTIN

Human factor VIII/von Willebrand factor (FVIII/vWF) was shown to bind to immobilized lectins from Arachis, Ulex, Concanavalia, and Lens spec ies. The protein/lectin interaction displayed higher affinities for th e lectins from the last two species. However, the Lens culinaris lecti n immobilized on Sepharose 4B (LCA-Sepharose) provided a more selectiv e and flexible affinity system for the purification of FVIII/vWF than Concanavalia lectin. Chromatography on LCA-Sepharose of a purified FVI II containing a small proportion of vWF required a weak acidic medium (pH 6.3) and relatively slow kinetics (about 20 cm/h flow rate). The b ound FVIII was specifically dissociated from LCA-Sepharose by methyl-a lpha-D-mannopyranoside, and to a lesser extent by other monosaccharide s such as D-glucose, methyl-alpha-D-glucopyranoside, D-mannose, and D- galactose. Application to whole plasma resulted in a capacity for FVII I/vWF of about 28 U/ml gel. Specific activities for eluted FVIII and V WF were 3 and 2.2 IU/mg protein, respectively, with respective FVIII:c and vWF:RCo recoveries of 57 and 40% from starting plasma. Coagulatio n factors II, X, VII, IX, V, and XI and fibrinogen were eliminated in the LCA matrix breakthrough fraction, improving the stability of the p urified FVIII molecule. Purity of the LCA eluate was further enhanced by ion-exchange chromatography on DEAE-Fractogel TSR 650 M which reduc ed the amount of protein contaminants and provided a FVIII/vWF fractio n with higher specific activity (45-80 IU/mg protein depending on the chromatographic conditions). The overall process yield was 45 and 25% for FVIII:c and VWF:RCo, respectively. (C) 1994 Academic Press, Inc.