title: Top-down mass spectrometry and hydrogen/deuterium exchange for comprehensive structural characterization of interferons: implications for biosimilars.
abstract: Rapid development in biopharmaceuticals has put high demands on analytical tools that can provide accurate and comprehensive characterization of protein drugs, including biosimilars. Although the enzyme digestion based "bottom-up" approach is usually the method of choice for this purpose, it only gives peptide-level information and sequence coverage is often incomplete. In this work, we used top-down MS with electron capture dissociation (ECD) to characterize both the primary and higher order structures of a therapeutic protein interferon and its variants. Accurate mass measurement at the intact protein level combined with top-down ECD fragmentation enabled unambiguous protein sequence confirmation and identification of all PTMs. Combining hydrogen/deuterium exchange and rapid disulfide reduction with top-down ECD on the LC time scale, we have investigated the differences in higher order structure between the protein variants, as well as the impact of PTMs on protein conformation.
authors.affiliation: University of Victoria - Genome BC Proteomics Centre, University of Victoria, Victoria, BC, Canada.
authors.forename: Jingxi
authors.initials: J
authors.lastname: Pan
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D000595:Amino Acid Sequence; D059451:Biosimilar Pharmaceuticals / Q000032:analysis / Q000737:chemistry*; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004220:Disulfides; D006801:Humans; D007372:Interferons / Q000032:analysis / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry
chemical_list: D059451:Biosimilar Pharmaceuticals; D004220:Disulfides; D011994:Recombinant Proteins; D007372:Interferons
keywords: Biomedicine; Biopharmaceutical; Electron capture dissociation; Hydrogen/deuterium exchange; Post-translational modification; Top-down