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.