Dataset Information

Glycosylation Heterogeneity of Hyperglycosylated Recombinant Human Interferon-? (rhIFN-?).

ABSTRACT: We previously developed a biobetter version of rhIFN-? (R27T) that possesses an additional glycosylation site compared with rhIFN-? 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 ?-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.

SUBMITTER: Song K

PROVIDER: S-EPMC7114697 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Song Kyoung K Moon Dae Bong DB Kim Na Young NY Shin Young Kee YK

ACS omega 20200320 12

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 ...[more]

PMID: 32258897

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Project description:Recombinant human interferon-gamma (Hu-IFN-gamma) produced by Chinese-hamster ovary (CHO) cells was analysed by immunoprecipitation and SDS/PAGE. Up to twelve molecular-mass variants were secreted by this cell line. Three variants were recovered after enzymic removal of all N-linked oligosaccharides or when glycosylation was inhibited by tunicamycin. The presence of three polypeptide forms rather than a single form suggested that proteolytic cleavage had occurred at two sites in both the glycosylated and non-glycosylated forms. Proteolytically cleaved IFN-gamma was more prevalent in cell lysates than in the secreted glycoprotein. In common with naturally produced IFN-gamma, both fully glycosylated IFN-gamma (asparagine residues 28 and 100 occupied) and partially glycosylated product (thought to be substituted at position Asn28) were secreted. This was deduced from the Mr of the glycosylated products and the relative amounts of sialic acid expressed by each variant. In contrast with naturally produced IFN-gamma, non-glycosylated IFN-gamma was also secreted by the transfected CHO cells. When the cells were grown in batch culture in serum-free medium under pH and dissolved-oxygen control, the proportion of non-glycosylated IFN-gamma increased from 3 to 5% after 3 h, to 30% of the total IFN-gamma present after 195 h. This change in the proportion of glycosylated protein produced was not seen when metabolically labelled IFN-gamma was incubated for 96 h with cell-free supernatant from actively growing CHO cells. This implied that an alteration in intracellular glycosylation was occurring rather than a degradation of oligosaccharide side chains after secretion. The decrease in IFN-gamma glycosylation was independent of the glucose concentration in the culture medium, but could be related to specific growth and IFN-gamma production rates, as these declined steadily after 50 h of culture, in line with the increased production of non-glycosylated IFN-gamma.

Dataset Information

Glycosylation Heterogeneity of Hyperglycosylated Recombinant Human Interferon-? (rhIFN-?).

Publications

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

Similar Datasets

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