title: Conformational comparability of factor IX-Fc fusion protein, factor IX, and purified Fc fragment in the absence and presence of calcium.
abstract: A long lasting recombinant factor IX -Fc fusion protein (rFIX-Fc) is being developed for the treatment of hemophilia B and is currently in late stage clinical investigation. By limiting injection frequency and maintaining efficacy, rFIX-Fc shows promise as a new therapeutic option for hemophilia B patients. However, before gaining regulatory approval, rFIX-Fc must undergo rigorous analytical and biological testing, in addition to clinical trials. Included in this testing is the need to understand this protein's higher-order structure and dynamics. In this study, we investigated and compared the biophysical properties of rFIX-Fc, rFIX, and Fc using hydrogen/deuterium exchange mass spectrometry and differential scanning calorimetry. Within the limits of these techniques, our results show that structural comparability exists between rFIX and the FIX region of rFIX-Fc. In addition, changes in the structure and dynamics of both proteins, in response to calcium binding, a requirement for FIX function, are also highly comparable. In the case of Fc and Fc region of rFIX-Fc, conformational comparability is also established. These biophysical results further support the conclusion that fusing an immunoglobulin gamma 1 Fc to rFIX does not significantly alter the higher-order structure of FIX or Fc, Ca binding to FIX, or Fc functionality.
authors.affiliation: Analytical Development, Biogen Idec, Cambridge, Massachusetts 02142, USA. damian.houde@biogenidec.com
authors.forename: Damian
authors.initials: D
authors.lastname: Houde
authors.forename: Steven A
authors.initials: SA
authors.lastname: Berkowitz
mesh_terms: D002118:Calcium / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D005164:Factor IX / Q000737:chemistry*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D013058:Mass Spectrometry; D011487:Protein Conformation
chemical_list: D007141:Immunoglobulin Fc Fragments; D005164:Factor IX; D002118:Calcium