Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein

Autor: Qi Lu, John Kulman, Glenn F. Pierce, Robert T. Peters, Low Susan, Alan J. Bitonti, Garabet G. Toby, Tongyao Liu
Rok vydání: 2013
Předmět:
Male
Protein Conformation
Protein Engineering
Mass Spectrometry
law.invention
Mice
law
hemic and lymphatic diseases
Cloning
Molecular
medicine.diagnostic_test
biology
Protein Stability
Chemistry
long-acting
Hematology
Neoplasm Proteins
Thrombelastography
Cysteine Endopeptidases
Biochemistry
Recombinant DNA
Partial Thromboplastin Time
Half-Life
Partial thromboplastin time
medicine.drug
congenital
hereditary
and neonatal diseases and abnormalities
Fc fusion
Recombinant Fusion Proteins
Context (language use)
Hemophilia A
Peptide Mapping
Structure-Activity Relationship
Thrombin
Von Willebrand factor
In vivo
von Willebrand Factor
medicine
Animals
Humans
Amino Acid Sequence
Blood Coagulation
rFVIIIFc
Binding Sites
Factor VIII
Coagulation
Coagulants
Fusion protein
Immunoglobulin Fc Fragments
Disease Models
Animal
biology.protein
Ex vivo
Protein C
Zdroj: Journal of Thrombosis and Haemostasis
ISSN: 1538-7836
DOI: 10.1111/jth.12076
Popis: 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 IgG1.
Databáze: OpenAIRE
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