171. Quantification of Human FVIII and Estimation of Its Molecular Weight: Potential Applications in Gene Therapy for Hemophilia A

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Current protocols for measurement of FVIII rely on chromogenic or coagulation assays, and are not designed for quantification of FVIII antigen. Development of assays for measuring FVIII antigen may be necessary and beneficial for determining the efficiency of newer therapeutic approaches for Hemophilia A such as gene therapy. Herein, we describe the development of proprietary assays for quantification of human FVIII in plasma matrices from heterologous species, and estimation of the human FVIII molecular weight.A variety of monoclonal antibodies against the FVIII heavy and light chain were screened for their ability to recognize human FVIII from normal human plasma pool, or CHO cell derived recombinant human FVIII-B domain deleted (FVIII-BDD) spiked in mouse and non-human primate (NHP) plasma. Based on the initial screen, a monoclonal each against the human FVIII heavy or the light chain was selected for enrichment, followed by detection using a sheep anti-FVIII polyclonal antibody. Using this sandwich ELISA procedure, a standard curve was generated for human FVIII-BDD antigen in the range of 100 ng/ml - 0.1 ng/ml, and minimal cross reactivity was observed when tested with mouse and NHP plasma pools containing endogenous FVIII antigen. Furthermore, individual mouse strain plasma pools, and individual NHP samples representing different genetic origins were also screened for cross-reactivity to endogenous FVIII antigen and found to be negative. The sandwich ELISA was used to quantify FVIII antigen in defined plasma samples from normal or Hemophilia A human subjects, and antigen levels correlated with the reported FVIII activity.The ELISA procedure was modified and adapted for estimation of the human FVIII antigen molecular weight. FVIII heavy and light chains, from normal human plasma pool or recombinant human FVIII-B domain deleted (FVIII-BDD), were enriched followed by resolution using a denaturing reducing PAGE and western blotting. The apparent migration of heavy and light chain was at the appropriate molecular weights; 90-185 kDa for heavy chain and 70 kDa for light chain. In case of heavy chain from normal human plasma pool, a ladder of differing molecular weights was seen and may be indicative of the differential furin and thrombin dependent processing of the B-domain.Thus, these newly developed assays for quantification of human FVIII and estimation of its molecular weights will be useful for further characterization of gene therapy for Hemophilia A.