Biochemical and functional characterization of a recombinant monomeric factor VIII-Fc fusion protein
Open accessAbstract
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) .
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Expressing rFVIIIFc and rFIXFc in HEK293 resulted in greater tyrosine sulfation and glutamic acid γ-carboxylation compared to CHO cells and excluded α-gal and Neu5Gc from the manufactured products.
“Expressing these glycoproteins in HEK293 resulted in greater tyrosine sulfation and glutamic acid γ-carboxylation compared to CHO cells and excluded any α-gal and Neu5Gc from the manufactured products (Berkner, 1993;Kannicht et al., 2013;Peters et al., 2013;McCue et al., 2014McCue et al., , 2015)).”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Peters et al., 2013
The Materials and methods section reports that rFVIIIFc was produced in HEK293 cells, and the Results section reports nearly complete tyrosine sulfation. Neither section establishes greater sulfation than in CHO cells. The paper does not substantiate the sentence’s claims about rFIXFc, γ-carboxylation, or exclusion of α-gal and Neu5Gc. (LLM, conf=0.88)
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Cited by
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Therapeutic glycoprotein production in mammalian cells
Lalonde, M.-E., & Durocher, Y. (2017) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2017.04.028 · Cited by 296 · see citing works
Nguyen, G. N., George, L. A., Siner, J. I., Davidson, R. J., Zander, C. B., Zheng, X. L., Arruda, V. R., Camire, R. M., & Sabatino, D. E. (2017) · Journal of Thrombosis and Haemostasis · doi:10.1111/jth.13543 · Cited by 50 · see citing works
- Authors
- PETERS, R. T., TOBY, G., LU, Q., LIU, T., KULMAN, J. D., LOW, S. C., BITONTI, A. J., & PIERCE, G. F.
- Affiliations
- Biogen (United States) (US)
- Corresponding author
- PIERCE, G.F.
- Venue
- Journal of Thrombosis and Haemostasis
- Publisher
- Elsevier BV
- Published
- 2013-01
- Volume
- 11
- Issue
- 1
- Pages
- 132-141
- ISSN
- 1538-7836 (ISSN-L)
- PMC
- PMC3588154
- OpenAlex
- W1595085358
- Cited by
- 126
- References
- 0
- Citations by year
- 2026: 3 · 2025: 3 · 2024: 3 · 2023: 4 · 2022: 7 · 2021: 7 · 2020: 12 · 2019: 8 · 2018: 13 · 2017: 10 · 2016: 20 · 2015: 18 · 2014: 11 · 2013: 7
- FWCI
- 7.68
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Journal of Thrombosis and Haemostasis (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- Biogen
- Topics
- Health Sciences › Medicine › Hematology › Hemophilia Treatment and Research
- Health Sciences › Medicine › Hematology › Platelet Disorders and Treatments
- Health Sciences › Medicine › Critical Care and Intensive Care Medicine › Trauma, Hemostasis, Coagulopathy, Resuscitation
- Keywords
- recombinant factor VIII, hemophilia A, Fc fusion, protein half-life extension, von Willebrand factor binding, factor VIII activity, B-domain-deleted factor VIII, chromogenic assay, rotational thromboelastometry, post-translational modification
- MeSH terms
- Amino Acid Sequence, Animals, Binding Sites, Blood Coagulation, Cloning, Molecular, Coagulants, Cysteine Endopeptidases, Disease Models, Animal, Factor VIII, Half-Life, Hemophilia A, Humans, Immunoglobulin Fc Fragments, Male, Neoplasm Proteins, Partial Thromboplastin Time, Peptide Mapping, Protein C, Protein Conformation, Recombinant Fusion Proteins, Mass Spectrometry, Structure-Activity Relationship, Thrombelastography, von Willebrand Factor, Protein Engineering, Mice, Protein Stability
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