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

Abstract

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.

Figure 1

N-glycosylation site occupancy heterogeneity reflects the R27T size and pI differences. (A) SDS-PAGE and IEF analysis of glycoproteins. Lanes 2–4 show the main target peaks from R27T purification, and lanes 5–6 show intermediate peaks comprising mainly non- or partially glycosylated protein. Lane 7 is the singly glycosylated Rebif reference. (B) Microchip analysis (C) cIEF analysis.

Figure 2

Exoglycosidase digestion sequencing of the R27T N-glycan by HILIC–HPLC arrows indicate digestion. The following exoglycosidases were used: sialidase (a368S; specific for α2-3, -6, -8, and -9 sialic acids); beta-galactosidase (b4G; specific for β1-4 galactose); α-galactosidase (a36G; specific for α1-3/6 galactose); fucosidase (a34F; specific for α1-3 and -4 fucose); fucosidase (a6F; specific for α1-6 > 2 fucose); N-acetylglucosaminidase (sph; specific for β-GlcNAc). Glycan structures were allocated by a combination of elution position (expressed as GU value) and subsequent GU values of peaks following digestion with specific exoglycosidases.

Figure 3

HILIC–HPLC profiles of R27T WAX fractions before and after sialidase digestion.

Figure 4

MALDI-MS spectra from permethylated R27T. m/z [M + Na]⁺.