Recombinant Factor VIIa (rFVIIa) Mediated Activation of Prothrombin Complex Concentrates (PCCs). Studies on the Comparison of Novoseven with a Biosimilar Product

Introduction

Recombinant factor VIIa (NovoSeven® Copenhagen, Denmark) is used in the management of bleeding in patients with hemophilia. A generic biosimilar version of NovoSeven® is also developed for same indication (AryoSeven™, AryoGen, Tehran-Iran). The two products exhibit comparable compositional and biochemical profile. To compare the activation profile of NovoSeven® and AryoSeven™, two commercially available protein complex concentrates (PCCs) were used. The activation profiles were studied utilizing SDS- gel electrophoresis profile and immunoblotting using anti-rabbit recombinant thrombin antibody.

Materials & Methods

NovoSeven®, AryoSeven™, Profilnine® SD (Grifols Biologicals Inc. USA), and Beriplex® (CSL Behring Canada, Inc.) were obtained commercially. Recombinant human thrombin antibodies were generated in rabbits. Recothrom® was commercially obtained (Zymogenetic, Seatle,Wa- USA and Medicine Company ,Parsippany, NJ –USA). Tissue factor RecombiPlasTin 2G was obtained from Instrumentation Laboratory Company (Bedford- USA).

NovoSeven® and AryoSeven™ were diluted with saline to make working solution of 1mg/ml. Further dilutions were made with saline to obtain 0.5 µg/ml, 1.25µg/ml, and 2.5µg/ml rFVIIa preparations. To make working solution of PCCs, PCCs were diluted to 10 U/ml in saline. The sample mixtures contain 2U/ml PCCs, 0.1, 0.25, 0.5 µg/ml rFVIIa, and with RecombiPlasTin 2G at 1:5 ratio. The samples mixtures incubated in 5, 15, and 30 minutes at 37°^c. SDS-PAGE (Pierce Biotechnology) and immunoblotting carried against Recothrom®.

Results

Profilnine® SD activated by RecombiPlasTin 2G resulted in conversions of prothrombin to prethrombin and thrombin at 5 to 30 minutes incubation time as evident by the generation of distinct bands at 50kDa and 36 kDa. However, addition of rFVIIa at final concentration range of 0.25 and 0.5µg/ml to the same mixture resulted in total conversion of prothrombin to thrombin at 30 minutes with a doublet bands at 36 kDa. Recombinant FVIIa alone failed to generate thrombin in native Profilnine® SD at 5 to 30 minutes as evident by the absence of 36 kDa band in the SDS-PAGE and Immunoblotting studies. Further studies comparing NovoSeven® and AryoSeven™ (FC: 0.1µg/ml) in the activated Profilnine® SD resulted in a comparable and progressive generation of thrombin at 5 and 15 minutes incubation time. At 5 and 15 minutes residual prethrombin bands were noted which was less intense at 15 minute incubation time in both rFVIIa preparations. Although rFVIIa alone unable to generate thrombin in native Beriplex®, the presence of rFVIIa in activated Beriplex did not change the activation profile. Activated Beriplex® by RecombiPlasTin 2G resulted in complete conversion of prothrombin to thrombin at 30 minutes incubation time. A complete generation of thrombin was noted in both activated PCCs in the presence of rFVIIa at final concentrations of 0.25µg/ml and 0.5µg/ml. At 5 minutes residual prothrombin and prethrombin bands were noted in the presence of rFVIIa at final concentration of 0.1µg/ml. However, at 15 minutes prothrombin bands were completely disappeared and prethrombin bands were less intense at same concentration of 0.1µg/ml of rFVIIa. The results obtained by Western blot analysis confirmed the differential activation of Profilnine® SD and Beriplex®. However, both the NovoSeven® and AryoSeven™ exhibited similar activation profiles.

Conclusions

These results indicate that the activation of PCCs by both AryoSeven™ and NovoSeven® result in comparable generation of thrombin. Furthermore, while both rFVIIa produced similar activation profile, PCCs based differences in the activation profile were noted. These studies provide an in vitro profiling of the effects of activated FVIIa on the generation of thrombin and related enzymes to demonstrate biosimilarity of the branded and generic versions of these haemostatic products.