In vitro biological characterization of IFN-β-1a major glycoforms

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 physico-chemical and biological characterization by means of mass spectrometry (ESI-MS), sialic acid content, thermal denaturation and various in vitro bioassays (anti-proliferative, 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 biand higherantennarity 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 are having 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 sheds new light on the role of the glycosylation heterogeneity, in particular with regard to antennarity, on biological properties of glycoproteins.