Summary. An intermediate‐ and a high‐purity factor‐VIII concentrate for clinical use have been prepared on a large scale by cryoethanol precipitation, extraction of the precipitate with tris buffer, and fractionation with polyethylene glycol. With bench‐scale fractionation, the intermediate material is 22‐fold purified on the average and the mean yield is 63%, while the high‐purity factor VIII is 274‐fold purified with a mean yield of 62%. With fractionation of 100 1. or more of fresh frozen plasma, the intermediate material shows a 30% yield and 14‐30‐fold purification; the high‐purity factor VIII shows an 18% yield and 125–350‐fold purification using 5.8 g/100 ml polyethylene glycol (PEG). A yield of nearly 30% should be possible with PEG, 4–5 g/100 ml. Both factor‐VIII preparations are stable for over a year in the lyophilized state at 4°C. Other plasma proteins can be fractionated from the residual plasma by routine Cohn procedures.
Summary. Intermediate‐ and high‐purity antibaemophilic factor (factor‐VIII) concentrates developed by the American National Red Cross (ANRC) have undergone extensive clinical investigation. When administered to severely affected haemophiliacs, the recovery in vivo and the metabolic half‐disappearance time were similar to those achieved with plasma.
The bleeding disorder in hemophilia A results from a deficiency or abnormality of Factor VIII (FVIII), a member of the coagulation cascade. FVIII is a large glycoprotein (approximately 350,000 daltons) that is activated by a series of proteolytic cleavages. During activation, a large internal domain (B domain) is removed, resulting in an active complex comprised of the amino and carboxyl subunits of the parental molecule. Using a bovine papillomavirus expression vector system, we have established stable, genetically engineered cell lines harboring either full-length FVIII cDNA or variant FVIII cDNA (delta FVIII), the latter containing an extensive deletion in the region encoding the B domain. We demonstrate that the two recombinant FVIII molecules manifest the biological attributes of native FVIII. Relative to full-length FVIII transformants, cells harboring delta FVIII cDNA are five to eight times more efficient in expressing coagulant activity. This difference is due to a post-transcriptional event.
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