Calcium Binding to a Factor IX Fc Fusion Protein and Effects on Higher‐Order Structure

There is significant scope for more meaningful evaluation of higher‐order structure in defining the quality of biopharmaceutical products [Bush L. 2010. Biopharm Int 23(4):14]. We have used isothermal titration calorimetry (ITC) to characterize the Ca2+‐binding isotherm of a recombinant human factor IX Fc fusion protein (rFIXFc) and the parent recombinant human factor IX molecule (rFIX). Circular dichroism, intrinsic fluorescence, and Fourier transform infrared spectroscopies detected characteristic spectral changes that appear qualitatively consistent with the previously characterized behavior of the factor IX molecule. Sedimentation velocity and dynamic light scattering measurements were recorded in the presence and absence of Ca2+ over the protein concentration range 1-10 mg/mL. ITC of Ca2+ binding to rFIXFc reveals a distinctive exothermic‐binding isotherm, which is interpreted as consistent with two high‐affinity and approximately 14 lower‐affinity Ca2+ sites reported in the literature for human factor IX (Schmidt AE, Bajaj SP. 2003. Trends Cardiovasc Med 13(1):39-45). Analysis of accelerated degradation samples showed significant alterations in Ca2+ binding, which correlates with significant loss of biopotency and fragmentation by gel chip capillary electrophoresis. Collectively, these data establish a close correspondence in the Ca2+‐binding characteristics of rFIXFc and its parent rFIX molecule. The utility of ITC to provide a highly pertinent and selective biophysical signature of structure-function for a therapeutic factor protein is discussed. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4597-4606, 2011