ISSN:
0006-3592
Keywords:
γ-IFN
;
sialylation
;
glycosylation
;
sialidase
;
CHO cell culture
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
Since sialic acid content is known to be a critical determinant of the biological properties of glycoproteins, it is essential to characterize and monitor sialylation patterns of recombinant glycoproteins intended for therapeutic use. This study reports site- and branch-specific differences in sialylation of human interferon-γ (IFN-γ) derived from Chinese hamster ovary (CHO) cell culture. Sialylation profiles were quantitated by reversed-phase HPLC separations of the site-specific pools of tryptic glycopeptides representing IFN-γ's two potential N-linked glycosylation sites (i.e., Asn25 and Asn97). Although sialylation at each glycosylation site was found to be incomplete, glycans of Asn25 were more heavily sialylated than those of Asn97. Furthermore, Man(α1-3) arms of the predominant complex biantennary structures were more favorably sialylated than Man(α1-6) branches at each glycosylation site. When the sialylation profile was analyzed throughout a suspension batch culture, sialic acid content at each site and branch was found to be relatively constant until a steady decrease in sialylation was observed coincident with loss of cell viability. The introduction of a competitive inhibitor of sialidase into the culture supernatant prevented the loss of sialic acid after the onset of cell death but did not affect sialylation prior to cell death. This finding indicated that incomplete sialylation prior to loss of cell viability could be attributed to incomplete intracellular sialylation while the reduction in sialylation following loss of cell viability was due to extracellular sialidase activity resulting from cell lysis. Thus, both intracellular and extracellular processes defined the sialic acid content of the final product. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 390-398, 1977.
Additional Material:
4 Ill.
Type of Medium:
Electronic Resource
