Preparation of antihemophilic factor and fibronectin from human plasma cryoprecipitate

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 three- to fourfold concentration (by ultrafiltration) to desired potency. The sterile AHF concentrate, dispensed at 1000 u per vial and lyophilized, was completely dissolved within 3 minutes upon addition of 30 ml of pure water. The expected increment in circulating factor VIII and its hemostatic effects were found following intravenous infusion into factor VIII-deficient patients. Yield of AHF of five successive batches, each starting with the cryoprecipitate from some 12,000 units of fresh-frozen plasma, averaged 51 percent. 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 seven successive batches, each starting with the cryoprecipitate from some 900 units of fresh-frozen plasma, averaged 10 percent.