Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph
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...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2016-01-01
|
| Series: | Advances in Hematology |
| Online Access: | http://dx.doi.org/10.1155/2016/7678901 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832556661702656000 |
|---|---|
| author | Dougald M. Monroe Richard J. Jenny Kevin E. Van Cott Shelly Buhay Laura L. Saward |
| author_facet | Dougald M. Monroe Richard J. Jenny Kevin E. Van Cott Shelly Buhay Laura L. Saward |
| author_sort | Dougald M. Monroe |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-2d65397166a646c596e5cbe7ffed7bce |
| institution | Kabale University |
| issn | 1687-9104 1687-9112 |
| language | English |
| publishDate | 2016-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advances in Hematology |
| spelling | doaj-art-2d65397166a646c596e5cbe7ffed7bce2025-02-03T05:44:37ZengWileyAdvances in Hematology1687-91041687-91122016-01-01201610.1155/2016/76789017678901Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 PolymorphDougald M. Monroe0Richard J. Jenny1Kevin E. Van Cott2Shelly Buhay3Laura L. Saward4School of Medicine, Department of Hematology/Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USAHaematologic Technologies, Incorporated, Essex Junction, VT 05452, USADepartment of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USABiosciences Division, Emergent BioSolutions Incorporated, Winnipeg, MB, R3T 5Y3, CanadaBiosciences Division, Emergent BioSolutions Incorporated, Winnipeg, MB, R3T 5Y3, CanadaThe 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.http://dx.doi.org/10.1155/2016/7678901 |
| spellingShingle | Dougald M. Monroe Richard J. Jenny Kevin E. Van Cott Shelly Buhay Laura L. Saward Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph Advances in Hematology |
| title | Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph |
| title_full | Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph |
| title_fullStr | Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph |
| title_full_unstemmed | Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph |
| title_short | Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph |
| title_sort | characterization of ixinity r trenonacog alfa a recombinant factor ix with primary sequence corresponding to the threonine 148 polymorph |
| url | http://dx.doi.org/10.1155/2016/7678901 |
| work_keys_str_mv | AT dougaldmmonroe characterizationofixinitytrenonacogalfaarecombinantfactorixwithprimarysequencecorrespondingtothethreonine148polymorph AT richardjjenny characterizationofixinitytrenonacogalfaarecombinantfactorixwithprimarysequencecorrespondingtothethreonine148polymorph AT kevinevancott characterizationofixinitytrenonacogalfaarecombinantfactorixwithprimarysequencecorrespondingtothethreonine148polymorph AT shellybuhay characterizationofixinitytrenonacogalfaarecombinantfactorixwithprimarysequencecorrespondingtothethreonine148polymorph AT lauralsaward characterizationofixinitytrenonacogalfaarecombinantfactorixwithprimarysequencecorrespondingtothethreonine148polymorph |