COMPARISON OF N-LINKED OLIGOSACCHARIDES OF RECOMBINANT HUMAN TISSUE KALLIKREIN PRODUCED BY CHINESE-HAMSTER OVARY CELLS ON MICROCARRIER BEADS AND IN SERUM-FREE SUSPENSION-CULTURE

Glycosylation heterogeneity in recombinant human tissue kallikrein (r- HuTK) produced by Chinese hamster ovary (CHO) cells from microcarrier culture and from a serum-free suspension cell recycle process has been compared. Significant differences in the degree of sialylation were o bserved in glycoform distribution and oligosaccharide heterogeneity. H igh-performance liquid chromatography with a pellicular anion exchange column under low pH eluant conditions was used to characterize the nu mber and types of N-linked complex type oligosaccharides present. The oligosaccharides were released by N-glycanase and, after reduction, we re resolved into a number of peaks containing one, two, three, and fou r sialic acids with an additional subfractionation based on the nature of the antennary structure. The microcarrier process resulted in a re duced amount of sialylated oligosaccharide species as compared to the suspension cell process. Removal of sialic acid followed by chromatogr aphy of the asialooligosaccharides under high pH anion-exchange condit ions indicated that the same antennary structures were present but in slightly different relative amounts. The oligosaccharide profiles are indicative of a highly complex array of microheterogeneity present, en compassing mono-, di-, tri-, and tetrasialylated complex type oligosac charides.