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.