We have characterized a highly purified (HP) factor IX concentrate intended for therapy of hemophilia B. The product has been prepared from pooled human plasma using a large-scale procedure combining three conventional chromatographic steps based on DEAE ion exchange and affinity on immobilized heparin. The specific activity of the product was 119±10 IU factor IX:c/mg protein (n = 15), corresponding to a purification factor of about 9,000. The concentrate was free of the vitamin K-dependent clotting factors II, VII and X and of proteins C and S. Most of the contaminants found in factor IX complex concentrate (PCC) were absent in this new product. High-molecular-weight kininogen, factors VIII, XI, XII or prekallikrein were not detected. There were no activated factors, such as factors IXa, and Xa, no thrombin and no phospholipids. Only two contaminants could be detected: C4 and inter-alpha-trypsin inhibitor (about 0.8 and 1.2mg/1,000IU factor IX:c, respectively). The purity of the product, as compared to PCC, was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar- Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Thrombogenicity tests in rabbits revealed that the HP factor IX tested had a lower thrombogenic power than the PCC tested. The concentrate has been subjected to a 0.3% tri(n-butyl) phosphate-1% Tween 80 treatment for 6h at 25°C during its production to reduce or eliminate the risk of transmission of plasma-borne lipid-enveloped viruses. These conditions inactivated more than 3.8 log(10) of vesicular stomatitis virus and more than 4.3 log(10) of sindbis virus within 1 and 2 h of treatment, respectively. These data demonstrate that a highly purified therapeutic clotting factor IX concentrate can be prepared from human plasma by conventional chromatographic methods.

A human solvent-detergent (SD)-treated factor IX concentrate has been produced from cryoprecipitatepoor plasma using DEAE-Sepharose CL-6B and heparin-Sepharose CL-6B chromatography. The DEAE eluate was incubated with an SD mixture [0.3% tri(n-butyl) phosphate-1% Tween 80, 6-h at 24°C] which was found to inactivate, in less than 1 h, more than 3.8 log(10) of vesicular stomatitis virus and more than 4.8 log(10) of Sindbis virus; the SD was removed by a subsequent heparin adsorption step. The specific activity of the concentrate was 10.9 ± 1.3 IU factor IX: c/mg protein (n= 15). The factor IX coagulant to antigen ratio was 0.7 ± 0.1. The concentrate was essentially free of factors II, VII and X, and protein C. The usual major contaminants of prothrombin complex concentrate (PCC) were absent: the concentrate contained about 94% alpha-1 proteins, and only 4 major proteins were resolved by SDS-PAGE (respective apparent molecular weight: 130, 86, 76 and 69 kilodaltons), and by crossed immunoelectrophoresis against an anti-PCC serum. The nonactivated partial thromboplastin time was equivalent to that of PCC; the product was devoid of factor IXa, of other activated procoagulant factors and of coagulant-active phospholipids (removed with SD in the heparin breakthrough fraction). Animal studies using the Wessler test and acute-toxicity test in rabbits revealed no adverse side effects. SD treatment could thus be used to inactivate viruses in factor IX concentrate and improve the safety of replacement therapy in hemophilia B.