title: In vitro biological characterization of IFN-β-1a major glycoforms.
abstract: Recombinant human interferon β-1a (IFN-β-1a) is extensively used as the first-line treatment of relapsing forms of multiple sclerosis. Its glycosylation is recognized as having a complex impact on a wide range of molecule characteristics and functions. The present study reports the enrichment of IFN-β-1a glycoforms and their physicochemical and biological characterization by means of electrospray ionization-mass spectrometry, sialic acid content, thermal denaturation and various in vitro bioassays (antiproliferative, antiviral, immunomodulatory and reporter gene assay). The glycoforms were fractionated by means of cation-exchange chromatography using recombinant IFN-β-1a derived from Chinese Hamster Ovary cell culture as starting material. The obtained fractions contained bi- and higher-antennarity glycans as described in the European Pharmacopoeia monograph (Nr. 1639E, Interferon beta 1a concentrated solution). The in vitro bioassay responses revealed a correlation mainly with the glycan antennarity. It is therefore suggested that all glycoforms have biological activity and play a role in modulating the overall IFN-β biological activity with higher-antennarity glycoforms being able to better sustain IFN-β-1a bioactivity over time. These data indicate the role of IFN-β-1a glycosylation in vivo and shed new light on the role of the glycosylation heterogeneity, in particular with regard to antennarity, on biological properties of glycoproteins.
authors.affiliation: Analytical Development Biotech Products, Merck Serono SpA, an affiliate of Merck KgaA, Darmstadt, Germany, Via Luigi Einaudi, 11, Guidonia Montecelio (Roma) 00012, Italy.
authors.forename: Renato
authors.initials: R
authors.lastname: Mastrangeli
authors.affiliation: Analytical Development Biotech Products, Merck Serono SpA, an affiliate of Merck KgaA, Darmstadt, Germany, Via Luigi Einaudi, 11, Guidonia Montecelio (Roma) 00012, Italy.
authors.forename: Mara
authors.initials: M
authors.lastname: Rossi
authors.affiliation: Analytical Development Biotech Products, Merck Serono SpA, an affiliate of Merck KgaA, Darmstadt, Germany, Via Luigi Einaudi, 11, Guidonia Montecelio (Roma) 00012, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Mascia
authors.affiliation: Global Research and Development, Merck Serono S.A., an affiliate of Merck KgaA, Darmstadt, Germany, Route de la Verrerie 6, Coinsins 1267, Switzerland.
authors.forename: Wolf
authors.initials: W
authors.lastname: Palinsky
authors.affiliation: Analytical Development Biotech Products, Merck Serono SpA, an affiliate of Merck KgaA, Darmstadt, Germany, Via Luigi Einaudi, 11, Guidonia Montecelio (Roma) 00012, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Datola
authors.affiliation: Analytical Development Biotech Products, Merck Serono SpA, an affiliate of Merck KgaA, Darmstadt, Germany, Via Luigi Einaudi, 11, Guidonia Montecelio (Roma) 00012, Italy.
authors.forename: Mariagrazia
authors.initials: M
authors.lastname: Terlizzese
authors.affiliation: Analytical Development Biotech Products, Merck Serono SpA, an affiliate of Merck KgaA, Darmstadt, Germany, Via Luigi Einaudi, 11, Guidonia Montecelio (Roma) 00012, Italy horst.bierau@merckgroup.com.
authors.forename: Horst
authors.initials: H
authors.lastname: Bierau
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001681:Biological Assay; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002461:Cell Line, Transformed; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004847:Epithelial Cells / Q000187:drug effects / Q000821:virology; D017930:Genes, Reporter; D006031:Glycosylation; D006801:Humans; D007155:Immunologic Factors / Q000737:chemistry / Q000494:pharmacology*; D000068556:Interferon beta-1a; D016899:Interferon-beta / Q000737:chemistry / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D018116:Vesiculovirus / Q000187:drug effects / Q000502:physiology
chemical_list: D000970:Antineoplastic Agents; D000998:Antiviral Agents; D007155:Immunologic Factors; D011134:Polysaccharides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D016899:Interferon-beta; D008156:Luciferases; D000068556:Interferon beta-1a
keywords: activity; antennarity; glycosylation; interferon; sialylation