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Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein
Background: Hemophilia A results from a deficiency in factor VIII activity. Current treatment regimens require frequent dosing, owing to the short half-life of FVIII. A recombinant FVIII–Fc fusion protein (rFVIIIFc) was molecularly engineered to increase the half-life of FVIII, by 1.5–2-fold, in sev...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Blackwell Publishing Ltd
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3588154/ https://www.ncbi.nlm.nih.gov/pubmed/23205847 http://dx.doi.org/10.1111/jth.12076 |
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author | PETERS, R T TOBY, G LU, Q LIU, T KULMAN, J D LOW, S C BITONTI, A J PIERCE, G F |
author_facet | PETERS, R T TOBY, G LU, Q LIU, T KULMAN, J D LOW, S C BITONTI, A J PIERCE, G F |
author_sort | PETERS, R T |
collection | PubMed |
description | Background: Hemophilia A results from a deficiency in factor VIII activity. Current treatment regimens require frequent dosing, owing to the short half-life of FVIII. A recombinant FVIII–Fc fusion protein (rFVIIIFc) was molecularly engineered to increase the half-life of FVIII, by 1.5–2-fold, in several preclinical animal models and humans. Objective: To perform a biochemical and functional in vitro characterization of rFVIIIFc, with existing FVIII products as comparators.Methods: rFVIIIFc was examined by utilizing a series of structural and analytic assays, including mass spectrometry following lysyl endopeptidase or thrombin digestion. rFVIIIFc activity was determined in both one-stage clotting (activated partial thromboplastin time) and chromogenic activity assays, in the context of the FXase complex with purified components, and in both in vitro and ex vivo rotational thromboelastometry (ROTEM) assays performed in whole blood. Results: rFVIIIFc contained the predicted primary structure and post-translational modifications, with an FVIII moiety that was similar to other recombinant FVIII products. The von Willebrand factor-binding and specific activity of rFVIIIFc were also found to be similar to those of other recombinant FVIII molecules. Both chromogenic and one-stage assays of rFVIIIFc gave similar results. Ex vivo ROTEM studies demonstrated that circulating rFVIIIFc activity was prolonged in mice with hemophilia A in comparison with B-domain-deleted or full-length FVIII. Clot parameters at early time points were similar to those for FVIII, whereas rFVIIIFc showed prolonged improvement of clot formation. Conclusions: rFVIIIFc maintains normal FVIII interactions with other proteins necessary for its activity, with prolonged in vivo activity, owing to fusion with the Fc region of IgG(1). |
format | Online Article Text |
id | pubmed-3588154 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Blackwell Publishing Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-35881542013-03-11 Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein PETERS, R T TOBY, G LU, Q LIU, T KULMAN, J D LOW, S C BITONTI, A J PIERCE, G F J Thromb Haemost Coagulation Background: Hemophilia A results from a deficiency in factor VIII activity. Current treatment regimens require frequent dosing, owing to the short half-life of FVIII. A recombinant FVIII–Fc fusion protein (rFVIIIFc) was molecularly engineered to increase the half-life of FVIII, by 1.5–2-fold, in several preclinical animal models and humans. Objective: To perform a biochemical and functional in vitro characterization of rFVIIIFc, with existing FVIII products as comparators.Methods: rFVIIIFc was examined by utilizing a series of structural and analytic assays, including mass spectrometry following lysyl endopeptidase or thrombin digestion. rFVIIIFc activity was determined in both one-stage clotting (activated partial thromboplastin time) and chromogenic activity assays, in the context of the FXase complex with purified components, and in both in vitro and ex vivo rotational thromboelastometry (ROTEM) assays performed in whole blood. Results: rFVIIIFc contained the predicted primary structure and post-translational modifications, with an FVIII moiety that was similar to other recombinant FVIII products. The von Willebrand factor-binding and specific activity of rFVIIIFc were also found to be similar to those of other recombinant FVIII molecules. Both chromogenic and one-stage assays of rFVIIIFc gave similar results. Ex vivo ROTEM studies demonstrated that circulating rFVIIIFc activity was prolonged in mice with hemophilia A in comparison with B-domain-deleted or full-length FVIII. Clot parameters at early time points were similar to those for FVIII, whereas rFVIIIFc showed prolonged improvement of clot formation. Conclusions: rFVIIIFc maintains normal FVIII interactions with other proteins necessary for its activity, with prolonged in vivo activity, owing to fusion with the Fc region of IgG(1). Blackwell Publishing Ltd 2013-01 /pmc/articles/PMC3588154/ /pubmed/23205847 http://dx.doi.org/10.1111/jth.12076 Text en © 2012 International Society on Thrombosis and Haemostasis http://creativecommons.org/licenses/by/2.5/ Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. |
spellingShingle | Coagulation PETERS, R T TOBY, G LU, Q LIU, T KULMAN, J D LOW, S C BITONTI, A J PIERCE, G F Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein |
title | Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein |
title_full | Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein |
title_fullStr | Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein |
title_full_unstemmed | Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein |
title_short | Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein |
title_sort | biochemical and functional characterization of a recombinant monomeric factor viii–fc fusion protein |
topic | Coagulation |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3588154/ https://www.ncbi.nlm.nih.gov/pubmed/23205847 http://dx.doi.org/10.1111/jth.12076 |
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