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Advances in Hematology
Volume 2016, Article ID 7678901, 9 pages
Research Article

Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph

1School of Medicine, Department of Hematology/Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
2Haematologic Technologies, Incorporated, Essex Junction, VT 05452, USA
3Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USA
4Biosciences Division, Emergent BioSolutions Incorporated, Winnipeg, MB, Canada R3T 5Y3
5University of Manitoba, Winnipeg, MB, Canada R3T 2N2

Received 29 September 2015; Accepted 14 January 2016

Academic Editor: David Varon

Copyright © 2016 Dougald M. Monroe et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The goal of these studies was to extensively characterize the first recombinant FIX therapeutic corresponding to the threonine-148 (Thr-148) polymorph, IXINITY (trenonacog alfa [coagulation factor IX (recombinant)]). Gel electrophoresis, circular dichroism, and gel filtration were used to determine purity and confirm structure. Chromatographic and mass spectrometry techniques were used to identify and quantify posttranslational modifications. Activity was assessed as the ability to activate factor X (FX) both with and without factor VIIIa (FVIIIa) and in a standard clotting assay. All results were consistent across multiple lots. Trenonacog alfa migrated as a single band on Coomassie-stained gels; activity assays were normal and showed <0.002 IU of activated factor IX (FIXa) per IU of FIX. The molecule has >97%γ-carboxylation and underwent the appropriate structural change upon binding calcium ions. Trenonacog alfa was activated normally with factor XIa (FXIa); once activated it bound to FVIIIa and FXa. When activated to FIXa, it was inhibited efficiently by antithrombin. Glycosylation patterns were similar to plasma-derived FIX with sialic acid content consistent with the literature reports of good pharmacokinetic performance. These studies have shown that trenonacog alfa is a highly pure product with a primary sequence and posttranslational modifications consistent with the common Thr-148 polymorphism of plasma-derived FIX.