title: 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 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.
authors.affiliation: CSL Behring GmbH, Preclinical Research and Development, Marburg, Germany. Electronic address: Stefan.Schmidbauer@cslbehring.com.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schmidbauer
authors.affiliation: CSL Behring GmbH, Preclinical Research and Development, Marburg, Germany.
authors.forename: Reinhild
authors.initials: R
authors.lastname: Witzel
authors.affiliation: CSL Behring GmbH, Preclinical Research and Development, Marburg, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Robbel
authors.affiliation: CSL Behring GmbH, Preclinical Research and Development, Marburg, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Sebastian
authors.affiliation: GlycoThera GmbH, Protein Chemistry and Mass Spectrometry, Hannover, Germany.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Grammel
authors.affiliation: CSL Behring GmbH, Preclinical Research and Development, Marburg, Germany.
authors.forename: Hubert J
authors.initials: HJ
authors.lastname: Metzner
authors.affiliation: CSL Behring GmbH, Preclinical Research and Development, Marburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schulte
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D007141:Immunoglobulin Fc Fragments; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C587014:factor VIII-Fc fusion protein; C005662:tyrosine O-sulfate; D014443:Tyrosine; D005169:Factor VIII
keywords: Factor VIII; Physicochemical; Recombinant; rVIII-SingleChain