IN-VITRO CHARACTERIZATION OF HIGH-PURITY FACTOR-IX CONCENTRATES FOR THE TREATMENT OF HEMOPHILIA-B

This study employed sodium dodecyl sulfate-polyacrylamide gel electrop horesis (SDS-PAGE) analysis and immunoblotting to assess the purity of seven high purify factor IX concentrates: Aimafix (Aima), AlphaNine-S D (Alpha Therapeutic), Factor IX VHP (Biotransfusion), Immunine (Immun o), Mononine (Armour Pharmaceutical), Nanotiv (Kabi Pharmacia), and 9M C (Blood Products Laboratory). The mean specific activity of these pro ducts ranged from 68 U factor IX/mg (Aimafix) to 246 U factor IX/mg (M ononine). SDS-PAGE analysis showed that the highest purity product, Mo nonine, had a single contaminating band under non-reducing conditions. Two additional bands were detected when this product was analyzed und er reducing conditions. All other products had multiple contaminating bands that were more apparent under reducing than non-reducing conditi ons. The immunoblot for factor IX showed a dominant factor IX band for all products. In addition, visible light chain of factor IX was detec ted for AlphaNine-SD, Factor IX VHP, Immunine, Mononine, Nanotiv, and 9MC, suggesting that the factor IX in these products had undergone par tial activation to factor IXa. Another contaminating band was visible at 49,500 for all of the products except 9MC. In addition to this band , high molecular weight contaminants were apparent for some products, most notably AlphaNine-SD. The identity of these bands is unknown. Imm unoblotting failed to demonstrate factor VII as a contaminant of any o f the high purity products, although factor VIIa could be detected in some lots of Immunine, Nanotiv, and 9MC by a clot-based assay. Factor X contaminated Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Nanotiv , and 9MC, but activation products of factor X were not detected. Prot hrombin contaminated all of the products except Mononine. Activation p roducts of prothrombin were identified for three of four lots of Immun ine and for one lot of Factor IX VHP. These results thus demonstrate t hat high purity factor IX concentrates differ substantially in the deg ree to which they are contaminated by potentially thrombogenic materia ls.