In Vitro Characterization of High Purity Factor IX Concentrates for the Treatment of Hemophilia B

Abstract

SummaryThis study employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting to assess the purity of seven high purity factor IX concentrates: Aimafix (Aima), AlphaNine-SD (Alpha Therapeutic), Factor IX VHP (Biotransfusion), Immunine (Immuno), Mononine (Armour Pharmaceutical), Nanotiv (Kabi Pharmacia), and 9MC (Blood Products Laboratory). The mean specific activity of these products ranged from 68 U factor IX/mg (Aimafix) to 246 U factor IX/mg (Mononine). SDS-PAGE analysis showed that the highest purity product, Mononine, had a single contaminating band under non-reducing conditions. Two additional bands were detected when this product was analyzed under reducing conditions. All other products had multiple contaminating bands that were more apparent under reducing than non-reducing conditions. The immunoblot for factor IX showed a dominant factor IX band for all products. In addition, visible light chain of factor IX was detected for AlphaNine-SD, Factor IX VHP, Immunine, Mononine, Nanotiv, and 9MC, suggesting that the factor IX in these products had undergone partial 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. Immunoblotting failed to demonstrate factor VII as a contaminant of any of the high purity products, although factor Vila 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. Prothrombin contaminated all of the products except Mononine. Activation products of prothrombin were identified for three of four lots of Immunine and for one lot of Factor IX VHP. These results thus demonstrate that high purity factor IX concentrates differ substantially in the degree to which they are contaminated by potentially thrombogenic materials.

Plasma Factor VIla: Measurement and Potential Clinical Significance

“…A recently proposed application of the new sTF-based assay for plasma FVIIa levels is to screen commercial clotting factor concen trates for contamination by potentially thrombogenic materials [30], Another poten tial application is to monitor plasma FVIIa levels during recombinant FVIIa therapy for bleeding disorders such as hemophilia [8,31] , 32-43]. In particular, the Northwick Park Heart Study found a significant correlation between ele vated plasma FVII:C levels and risk of isch emic heart disease [6], Interestingly, long term follow-up of Northwick Park Heart Study participants revealed a strong correla tion between FVII:C and fatal (but not nonfatal) events of ischemic heart disease [42], In another large prospective study, the Prospec tive Cardiovascular Münster study (PRO CAM), a correlation between plasma FV1I:C and coronary events was also reported, but with less significance than the Northwick Park Heart Study [32], Long-term follow-up of PROCAM study participants showed no statistically significant difference in FVILC levels among men who had or had not experi enced coronary events [36].…”

Recombinant Coagulation Factor IX (BeneFIX®)

“…Activated FIX (FIXa) is present in rFIX and pdFIX preparations at low levels as evidenced by immunological and enzymatic analysis (55,56). Analysis of rFIX production batches using a nonactivated partial thromboplastin time assay (as described in the European Pharmacopoeia) shows that FIXa levels are consistently below the European Pharmacopoeia test limits.…”

High-Purity Factor IX Concentrates for Treatment of Hemophilia B: Relative Purity and Thrombogenic Potential

“…Our most recent study [8] extended the analysis by eval uating seven high-purity concentrates tested with a series of sensitive assays for potentially thrombogenic materials, including sodium dodecyl sulfate-polyacrylamide gel elec trophoresis (SD S-P A G E ) under both reducing and nonre- ducing conditions, immunoblotting to detect factor V II, IX , X and prothrombin, chromogenic assay to detect acti vated clotting factors and the highly sensitive clot-based assay o f Morrissey et al [24] to detect factor V ila activity. In addition to AlphaNine-SD and Mononine, the fol lowing high-purity factor IX products were analyzed: Aimafix (Aima) Factor IX V H P (BioTransfusion), Immunine (Immuno), Nanotiv (Kabi Pharmacia) and 9 M C , now known as Replinine (Blood Products Laboratory).…”

“…Evaluation o f these products in in vitro assays [5][6][7][8] and after infusion into animals [9][10][11][12] has suggested that they will be less thrombogenic in vivo. Following infusions in patients with hemophilia B, they have exhibited reduced activation o f the coagulation cas cade [7,[13][14][15].…”

“…Following infusions in patients with hemophilia B, they have exhibited reduced activation o f the coagulation cas cade [7,[13][14][15]. To date, no thrombotic complications associated with use o f these new products have been reported, but the definitive answer regarding their throm bogenic potential awaits further clinical experience, espe cially when one recalls that 8 years o f P C C use had passed before their associated thromboembolic complications were first reported in the literature [1], The uncertainty concerning the thrombogenicity of high-purity factor IX concentrates is underscored by re sults from recent in vitro studies [5,6,8], which suggest that significant differences exist in the degree o f purity among the 'ultra-pure' products. Although the clinical sig nificance is as yet unknown, it is possible that these differ ences in purity may reflect varying thrombogenic poten tials.…”