Structural Comparability Between Recombinant FVIII-Fc and Its Isolated FVIII and Fc Constituents

Abstract 1135

rFVIIIFc is a recombinant fusion protein comprising human B domain-deleted (BDD) coagulation factor VIII covalently attached to the Fc domain of human immunoglobulin G1 (IgG1). rFVIIIFc is currently in a phase 3 clinical study as a novel long-lasting FVIII molecule designed to potentially reduce dosing frequency and provide prolonged protection from bleeds for patients with hemophilia A receiving prophylaxis or on-demand treatment, or undergoing surgery. In this study, we evaluated the structural comparability of BDD rFVIII and rFVIIIFc using small angle X-ray scattering (SAXS) and hydrogen/deuterium exchange (HDX) mass spectrometry. SAXS analysis of BDD rFVIII and rFVIIIFc, performed at the Advanced Light Source Synchrotron, provided the electron pair radius distribution of each molecule and accurate but inherently low resolution structural information. Both BDD rFVIII and rFVIIIFc were monomeric in solution. BDD rFVIII had a radius of gyration (Rg) of 39.7 Å, compared with a Rg of 38 Å calculated from its published atomic coordinates (2R7E.pdb). rFVIIIFc had a larger Rg of 51 Å, which was consistent with a simple computational model of BDD rFVIII tethered to Fc that predicted an Rg of 50 Å. HDX mass spectrometric analysis yielded 94% coverage of the BDD rFVIII component and 87% coverage of the Fc component. Rates of deuterium exchange within peptides derived from each of these components in the context of the rFVIIIFc fusion protein were very similar to those of the isolated BDD rFVIII and Fc constituents. Taken together, the results of SAXS and HDX analyses support the conclusion that fusion of the Fc moiety to BDD rFVIII does not alter the higher order structure of either component.