PROPERTIES OF FACTOR VIII IN IMMUNO-AFFINITY PURIFIED FACTOR VIII CONCENTRATE

PROPERTIES OF FACTOR VIII IN IMMUNO-AFFINITY PURIFIED FACTOR VIII CONCENTRATE Kimihito Koshihara, Kazuhiko Kagawa, Hisashi Yorifuji, Katsuyuki Fukutake, Masao Hada, Shojiro Ikematsu and Michio Fujimaki Department of Clinical Pathology, Tokyo Medical College For the treatment of hemophilia A patients, a highly purified factor VIII concentrate has been developed. The concentrate utilized oragnic solvent/detergent mixture as a means to inactivte virus, immuno-affinity chromatography with an anti-factor VIII monoclonal antibody and ion-exchange chromatography to purify the final products. The objective of this study was to determine whether the sophisticated manufacturing processes might modify the protein to introduce potentially harmful substance. The properties of factor VIII protein in six lots of the preparation were evaluated using clotting assay for factor VIII, two different ELISA techniques for VIII:Ag with two tag monoclonal antibodies that bound different epitopes on the light chain, ELISA for vWF:Ag with polyclonal antibody and vWF multimeric analyses. In addition, the polypeptides of heavy and light chains were analyzed using SDS-PAGE and western blotting methods with two monoclonal antibodies to each chain comparing with a conventional wet-heated preparation. The VIII: C levels of purified factor VIII concentrates measured in our laboratory were close enough to their labeled VIII:C. However, there was little difference between VIII:C and VIII: Ag in each lot, there was no apparent rule for the discrepancy. A small amount of vWF:Ag was detected in the preparations and the multimeric analyses for vWF showed increased satellite bands and decreased large size multimers in comparison with normal pooled plasma. The polypeptides of heavy and light chains were well maintained in every lot as well as those in a conventional material. After digestion by thrombin, heavy and light chains in both products were cleaved identically. Based on these observations, the immuno-affinity purified factor VIII concentrate was proved to have highly specific factor VIII activity without any unexpected protein modification.