Preparation of antihemophilic factor and fibronectin from human plasma cryoprecipitate

Abstract

Starting with human plasma cryoprecipitate, fibronectin was separated from antihemophilic factor (AHF) by fractional precipitation under mild conditions, resulting in excellent recovery of AHF in the supernatant solution of the cryoprecipitate. Separation of fibronectin enabled accelerated sterile filtration of the supernatant solution containing AHF even after 3- to 4-fold concentration (by ultrafiltration) to desired potency. The sterile AHF concentrate, dispensed at 1000 U/vial and lyophilized, was completely dissolved within 3 min upon addition of 30 ml of pure water. The expected increment in circulating factor VIII and its hemostatic effects were found following i.v. infusion into factor VIII-deficient patients. Yield of AHF of 5 successive batches, each starting with the cryoprecipitate from some 12,000 U of fresh-frozen plasma, averaged 51%. The fibronectin precipitate was purified by affinity on insolubilized gelatin with chaotropic elution at pH 5.5 followed by removal of the chaotrope by diafiltration. Thermal denaturation of adventitious fibrinogen resulted in electrophoretically pure fibronectin which, following lyophilization and reconstitution with pure water, retained biological properties in an in vitro assay designed to reflect opsonic activity. The yield of fibronectin for 7 successive batches, each starting with the cryoprecipitate from some 900 U of fresh-frozen plasma, averaged 10%.