A deamidated interferon-ß variant binds to integrin αvß3.

Human type I interferons are a family of pleiotropic cytokines with antiviral, anti-proliferative and immunomodulatory activities. They signal through the same cell surface receptors IFNAR1 and IFNAR2 yet evoking markedly different physiological effects. One differentiating factor of interferon-beta (IFN-ß) from other type I interferons is the presence of theAsn-Gly-Arg (NGR) sequence motif, which, upon deamidation, converts to Asp-Gly-Arg (DGR) and iso-Asp-Gly-Arg (iso-DGR) motifs. In other proteins, the NGR and iso-DGR motifs are reported as CD13- and αvß3, αvß5, αvß6, αvß8 and α5ß1 integrin-binding motifs, respectively. The scope of this study was to perform exploratory surface plasmon resonance (SPR) experiments to assess the binding properties of a deamidated IFN-ß variant to integrins. For this purpose, integrin αvß3 was selected as a reference model within the iso-DGR- integrin binding members. The obtained results show that deamidated IFN-ß binds integrin αvß3 with nanomolar affinity and that the response was dependent on the deamidation extent. Based on these results, it can be expected that deamidated IFN-ß also binds to other integrin family members that are able to bind to the iso-DGR binding motif. The novel binding properties could help elucidate specific IFN-ß attributes that under physiological conditions may be modulated by the deamidation.

In vitro biological characterization of IFN-ß-1a major glycoforms.

Recombinant human interferon ß-1a (IFN-ß-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-ß-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-ß-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-ß biological activity with higher-antennarity glycoforms being able to better sustain IFN-ß-1a bioactivity over time. These data indicate the role of IFN-ß-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.

Demonstration of physicochemical and functional similarity between the proposed biosimilar adalimumab MSB11022 and Humira®.

Biosimilars are biological products that are highly similar to existing products approved by health authorities. Demonstration of similarity starts with the comprehensive analysis of the reference product and its proposed biosimilar at the physicochemical and functional levels. Here, we report the results of a comparative analysis of a proposed biosimilar adalimumab MSB11022 and its reference product, Humira®. Three batches of MSB11022 and up to 23 batches of Humira® were analyzed by a set of state-of-the-art orthogonal methods. Primary and higher order structure analysis included N/C-terminal modifications, molecular weight of heavy and light chains, C-terminal lysine truncation, disulfide bridges, secondary and tertiary structures, and thermal stability. Purity ranged from 98.4%-98.8% for MSB11022 batches (N = 3) and from 98.4%-99.6% for Humira® batches (N = 19). Isoform analysis showed 5 isoform clusters within the pI range of 7.94-9.14 and 100% glycan site occupancy for both MSB11022 and Humira®. Functional analysis included Fab-dependent inhibition of tumor necrosis factor (TNF)-induced cytotoxicity in L929-A9 cell line and affinity to soluble and transmembrane forms of TNF, as well as Fc-dependent binding to Fcγ and neonatal Fc receptors and C1q complement proteins. All tested physicochemical and functional parameters demonstrated high similarity of MSB11022 and Humira®, with lower variability between MSB11022 and Humira® batches compared with variability within individual batches of Humira®. Based on these results, MSB11022 is anticipated to have safety and efficacy comparable to those of Humira®.