COMPETITIVE ELISA TO EVALUATE THE IMMUNOGENICITY OF DOUBLE-VIRUS-INACTIVATED FACTOR-VIII IN RABBITS

Viral inactivation is a critical step during the manufacture of factor VIII formulations for use in humans. Indeed, viral inactivation proce dures may alter the three-dimensional structure of factor VIII resulti ng in the formation of new epitopes which, in turn, may lead to the sy nthesis of inhibiting antibodies, and hence to a decreased therapeutic activity. A rabbit model was used to compare the immunogenicity of a solvent/detergent(SD)-inactivated formulation with a double-inactivate d (SD plus heating in solution, SDP) formulation of factor VIII. Two g roups of five rabbits were immunized by six s.c. injections of each fo rmulation. The serum of each animal was incubated with various amounts of the competing antigens, e.g. factor VIII SD or factor VIII SDP. Th e remaining free polyclonal antibodies were assayed by ELISA. Curves o btained with both antigens were compared for each serum. Both factor V III SD and factor WI SDP decreased the amount of antibodies raised to either formulation in a dose-dependent manner without observable diffe rences. These results suggest that no new epitopes were present on fac tor VIII SDP as compared with factor VIII SD and that no epitope delet ions occurred, supporting the view that the double-inactivation proced ure does not change the immunogenicity of factor VIII SD. This model i s proposed as a tool to detect changes in the immunogenicity of protei ns which may be modified.