Conclusion
We found the glycosylation heterogeneity of hyperglycosylated recombinant human interferon-β (rhIFN-β), R27T. The glycosylation site occupancy showed approx. 94% double-glycosylated form R27T with 6% single-glycosylated R27T. Microheterogeneity of R27T N-glycans had a mixture of bi-, tri-, and tetra-antennary glycans, some with lactosamine extensions, but neither outer arm fucose nor α-galactose was detected. Sialic acid major variants, Neu5Ac and Neu5Gc 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 biantennary structure. These findings could assist the development of second-generation rhIFN-β products, and help us better understand glycosylation CQAs and their biological significance.