Development of large-scale fractionation methods. VI. An improved method for preparation of antihemophilic factor.
To increase purity and potency of the antihemophilic factor (AHF) concentrate prepared by the American Red Cross method, the following modifications were introduced: (1) A cold extraction step was incorporated to remove cold-soluble impurities. The cryoprecipitate (cryo) was extracted with 0.02 M Tris buffer, pH 7.0 (4 ml/g cryo) at 0 degrees C. Factor VIII loss in this step was negligible. (2) AHF was then recovered from the cold-insoluble portion of the cryo by extraction at 21 degrees C with the same buffer. To increase the AHF concentration, this second extraction step was carried out with a smaller buffer volume (2 ml instead of 3--4 ml/g cryo). The subsequent steps, deprothrombinization, filtration and lyophilization were essentially unchanged. To further increase factor VIII concentration, the dried AHF concentrate was reconstituted to 40 rather than 50% of the initial volume. AHF concentrate prepared on a large scale by this method was 20- to 30-fold concentrated and 40- to 50-fold purified over plasma at a recovery of about 250 factor VIII units per liter of plasma. The final product was readily soluble, clear and almost colorless upon reconstitution.