MOLECULAR INTEGRITY OF FVIII IN COMMERCIAL FVIII CONCENTRATES

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Since FVIII is highly susceptible to proteolysis, purification processes used in the manufacture of FVIII may result in fragmentation, activation and/or degradation of the FVIII molecule. The molecular integrity of a variety of currently available FVIII concentrates as well as several preparations which are in development is being determined. An immunoprecipitation procedure has been developed for isolating FVIII from commercial concentrates. Polyclonal rabbit IgG against recombinant FVIII has been purified by Protein A chromatography and absorbed with immobilized PPF to remove antibodies to other proteins. The resulting IgG preparation has been coupled to CNBr activated agarose. This solid phase immunoabsorbent is being used to immunoprecipitate FVIII from commercial concentrates. Immunoprecipitates are being electrophoresed on SDS PAGE. FVIII protein is being visualized using the Western blot technique with enzyme linked antibodies. Western blots of FVIII from the various concentrates are being compared with regard to the size and relative distribution of FVIII polypeptides.