Introduction
Several immunomodulatory treatments have been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) for relapsing-remitting forms of multiple sclerosis (RRMS) (Chen, Wu, & Watson, 2018; Diebold & Derfuss, 2016; Goodin et al., 2002; Madsen, 2017). Among them, recombinant human interferon-β (rhIFN-β) has long been used as an effective first therapeutic intervention and disease-modifying therapy (DMT) for RRMS (Borden et al., 2007; Kappos et al., 2007; Kappos et al., 2006). Though almost three decades have passed since the introduction of rhIFN-β therapies, they remain important for the management of MS due to their good long-term safety profile and cost-efficacy (Castro-Borrero et al., 2012; Dumitrescu, Constantinescu, & Tanasescu, 2018; Gasperini & Ruggieri, 2011). However, there are direct and indirect limitations for clinical use including the need for frequent injections, and high immunogenicity and aggregation propensity, the latter of which is responsible for the therapeutic effect of the protein (Grossberg, Oger, Grossberg, Gehchan, & Klein, 2011; Hartung, Munschauer, & Schellekens, 2005; van Beers, Jiskoot, & Schellekens, 2010). To address these issues, in our previous study, an additional single-glycosylation site was introduced at amino acid 25 in rhIFN-β 1a, resulting in R27T in which Arg at position 27 is mutated to Thr (Song et al., 2014). The additional glycosylation site increases the half-life and in vitro biological activity, as well as thermostability, allowing less frequent dosing (Lee et al., 2018; Song et al., 2014).
As observed for R27T, glyco-engineering of therapeutic proteins can enhance in vivo activities by improving pharmacokinetic properties, solubility, thermal stability, and protease resistance, and reducing the immunogenicity, all of which may improve clinical outcomes (Ghaderi, Zhang, Hurtado-Ziola, & Varki, 2012; Hossler, Khattak, & Li, 2009; Sareneva, Pirhonen, Cantell, & Julkunen, 1995; Sola & Griebenow, 2010; Walsh & Jefferis, 2006; Wright & Morrison, 1997). However, despite its importance, it is very difficult to accurately determine the influence of glycosylation on glycoproteins due to its inherent complexity. Potential glycosylation sites such as Asn residues within Asn-X-Ser/Thr consensus sequences are not always occupied by oligosaccharides in mammalian cells because a consensus sequence alone is essential but not sufficient for N-linked protein glycosylation, resulting in site occupancy heterogeneity (macroheterogeneity) (Jenkins, Parekh, & James, 1996; Zhang, Li, Lu, & Liu, 2017). In addition, the composition of attached oligosaccharides can also vary considerably, although a core pentasaccharide unit (Man3GlcNAc2) is typically linked to an asparagine (Asn) residue via a chitobiose (GlcNAc2) (Barry et al., 2013). The glycan structure can be highly variable in terms of antennary structure, monosaccharide composition, and sialylation depending on host cell type, cell culture, and manufacturing conditions (Becerra-Arteaga & Shuler, 2007; Joosten & Shuler, 2003; Nam, Zhang, Ermonval, Linhardt, & Sharfstein, 2008; Wurm, 2004). This results in inherent structural complexity and variability (microheterogeneity) (Zhang et al., 2017). Since the complexity of glycosylation heterogeneity makes it difficult to understand structure-function relationships, it is imperative to characterize glycosylation variability. Herein, we performed a comprehensive characterization of the main R27T glycoforms. Glycosylation analysis can identify glycosylation parameters that may influence drug safety and efficacy profiles via critical quality attributes (CQAs). In the present study, we investigated common biopharmaceutical glycosylation CQAs including site occupancy heterogeneity, core fucosylation, antennary composition, sialylation, lactosamine extensions, linkages, and overall glycan profiles of R27T glycan.