Glycosylation Heterogeneity of Hyperglycosylated Recombinant Human Interferon-β (rhIFN-β)

[Image: see text] We previously developed a biobetter version of rhIFN-β (R27T) that possesses an additional glycosylation site compared with rhIFN-β 1a. Herein, we characterized N-glycosylation heterogeneity of R27T, which includes both N-glycan site occupancy heterogeneity (macro-heterogeneity) and complexity of carbohydrate moieties (micro-heterogeneity). N-glycan site occupancy manifested as distinct differences in size and isoelectric point. The analysis of complex carbohydrate moieties of R27T involved the common biopharmaceutical glycosylation critical quality attributes such as core fucosylation, antennary composition, sialylation, N-acetyllactosamine extensions, linkages, and overall glycan profiles using weak anion-exchange and hydrophilic interaction high-performance liquid chromatography with 2-aminobenzoic acid-labeled N-glycans. The double-glycosylated form accounted for approx. 94% R27T, while the single-glycosylated form accounted for 6% R27T. N-glycans consisted of a mixture of bi-, tri-, and tetra-antennary glycans, some with N-acetyllactosamine extensions, but neither outer arm fucose nor α-galactose was detected. Sialic acid major variants, N-acetyl- and N-glycolyl-neuraminic acid, were more abundant in R27T than in Rebif. The major N-glycan, accounting for ∼42% of total N-glycans, had a di-sialylated, core-fucosylated bi-antennary structure.