Physicochemical characterisation of rVIII-SingleChain, a novel recombinant single-chain factor VIII

Abstract rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational mo...

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Bibliographic Details
Published in:Thrombosis research 2015-08, Vol.136 (2), p.388-395
Main Authors: Schmidbauer, Stefan, Witzel, Reinhild, Robbel, Lars, Sebastian, Petra, Grammel, Nicolas, Metzner, Hubert J, Schulte, Stefan
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Binding Sites
CHO Cells
Cricetinae
Cricetulus
Factor VIII
Factor VIII - chemistry
Factor VIII - genetics
Hematology, Oncology and Palliative Medicine
Humans
Immunoglobulin Fc Fragments - chemistry
Immunoglobulin Fc Fragments - genetics
Molecular Sequence Data
Molecular Weight
Peptide Fragments - chemistry
Physicochemical
Protein Binding
Recombinant
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
rVIII-SingleChain
Tyrosine - analogs & derivatives
Tyrosine - chemistry
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Summary:Abstract rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational modifications of FVIII products are N -glycosylation of asparagine residues and tyrosine sulphations. Here, the physicochemical properties, thrombin cleavage products and post-translational modifications of rVIII-SingleChain were investigated and compared against commercially available recombinant FVIII (rFVIII) products with a predominant two-chain structure (B-domain deleted rFVIII and full-length rFVIII). rVIII-SingleChain was expressed in Chinese hamster ovary (CHO) cells and purified by chromatographic methods. Physicochemical properties of rVIII-SingleChain or thrombin-derived cleavage products were assessed using size-exclusion chromatography, reversed-phase chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Analysis of the respective carbohydrate structures was performed after release of N -glycans by PNGase F followed by fluorescence labelling and high-performance liquid chromatography. Proteolysis by trypsin generated the corresponding peptides, which were analysed for sulphated tyrosines by liquid chromatography-electrospray ionisation time of flight-mass spectrometry. rVIII-SingleChain was shown to be of high purity and homogeneity, and presented a well-defined single-chain molecule with predominant β-sheet conformation. The coagulation-relevant thrombin-activation products of rVIII-SingleChain were comparable with those obtained by activation of commercially available rFVIII products. rVIII-SingleChain post-translational modifications were similar to other CHO cell-derived rFVIII products for N -glycopattern and tyrosine sulphation. In conclusion, rVIII-SingleChain is of high homogeneity and purity, and provides an expected cleavage pattern on activation, setting the basis for optimal efficacy in the patient.
ISSN:0049-3848
1879-2472
DOI:10.1016/j.thromres.2015.05.005