Human coagulation factor IX: assessment of thrombogenicity in animal models and viral safety.

Thromboembolic complications associated with prothrombin complex concentrate treatment may be related to the high levels of factors II and X in these products. We report here results from preclinical safety studies with a human coagulation factor IX product (AlphaNine; Alpha Therapeutic Corp., Los Angeles, Calif.) that contains no detectable factor II or VII and less than 10 units of factor X/100 units of factor IX. This product was manufactured from virally inactivated factor IX complex with a barium citrate adsorption step followed by affinity chromatography yielding factor IX concentrate with a specific activity of about 86 factor IX units/mg protein. Electrophoresis and immunoblot analysis indicated that the factor IX represents about 65% of the protein in this product. The virus inactivation step incorporated into the manufacturing process (incubation with n-heptane at 60 degrees C for 20 hours) was shown to inactivate at least 8.6 logs of type 1 human immunodeficiency virus. The barium citrate adsorption and affinity chromatography steps were found to remove 2.0 logs of the marker virus, vaccinia, and the DEAE ion-exchange chromatography used to produce factor IX complex was found to remove 1.4 logs of the marker virus, Sindbis. Analysis of three separate manufacturing lots with the polymerase chain reaction revealed no evidence of hepatitis C virus. The purified factor IX was nonthrombogenic when tested at doses of 450 units/kilogram in a rabbit stasis (Wessler) model, whereas the prothrombin complex concentrates were found to be thrombogenic at doses of less than 50 units/kg. There was no evidence of DIC in a porcine model after infusion of 200 units/kg of coagulation factor IX, as manifested by negative fibrin monomer tests, the absence of fibrin in blood vessels at autopsy, little or no change in prothrombin times and partial thromboplastin times, and only moderate decreases in platelet levels after infusion.

Separation of human factor X from factor Xa by reversed-phase high-performance liquid chromatography.

Human factor X is the vitamin K-dependent proenzyme of a plasma serine protease that participates in the cascade of events leading to blood coagulation. It is converted to its active form, factor Xa, after specific cleavage by other plasma proteases or the protease from Russel's Viper venom. We have separated Factor X from factor Xa by reversed-phase high-performance liquid chromatography using an increasing gradient of acetonitrile in 0.1% trifluoroacetic acid. The factor X and factor Xa activities were well separated from each other on a wide-pore diphenyl column (Whatman Protesil 300) in less than 30 min. Both factor X and factor Xa activities were found to be essentially unaffected by the solvent system. This system was used to evaluate the purity of several factor X and factor Xa preparations. The kinetics of the Russel's Viper venom catalyzed conversion of factor X to factor Xa was also studied by using this chromatography system. A time-dependent decrease in the protein peak corresponding to factor X and a corresponding increase in the factor Xa protein peak was observed upon incubation with Russel's Viper venom.

Isolation of human factor VIII:C by preparative high-performance size-exclusion chromatography.

Human Factor VIII procoagulant protein (VIII:C) is a plasma protein that participates in the cascade of events leading to blood coagulation. It is absent or defective in patients with hemophilia A. In vivo Factor VIII:C associates with Von Willibrand factor and its multimers to form a high-molecular-weight particle that can be dissociated into a lower-molecular-weight form in the presence of high concentrations of salt. We have been able to purify rapidly Factor VIII:C on a large scale by sequential high-performance size-exclusion chromatography (HPSEC) under conditions of first low salt and then high salt concentration. Reconstituted commercial Factor VIII:C concentrate was purified by chromatography on a preparative HPSEC column (Toyo Soda, 60 X 2.5 cm, 300 ml) in 0.05 M imidazole buffer, (pH 7.0), containing 0.15 M sodium chloride. Factor VIII:C activity was eluted in the void volume in less than 20 min as a high-molecular-weight particle, well separated from low-molecular-weight contaminants. Purification was 20-fold, with a yield of 80%. Up to 4 g of Factor VIII concentrate could be purified at one time in this manner. This material was then concentrated and made 0.35 M in calcium chloride prior to re-chromatography on the same column in a buffer containing 0.30 M calcium chloride. Under these conditions, Factor VIII:C activity was eluted in the inner volume of the column at a position corresponding to a molecular weight of several hundred thousand in less than 1 h. It was well separated from both larger proteins and smaller peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)