Properties of a Highly Punified Human Plasma Factor IX:c Therapeutic Concentrate Prepared by Conventional Chromatography

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 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 1.2 mg/1,000 IU 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 6 h 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 log₁₀ of vesicular stomatitis virus and more than 4.3 log₁₀ 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.