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

Abstract

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 observed in glycoform distribution and oligosaccharide heterogeneity. High‐performance liquid chromatography with a pellicular anion‐exchange column under low pH eluant conditions was used to characterize the number and types of N‐linked complex type oligosaccharides present. The oligosaccharides were released by N‐gly‐canase and, after reduction, were resolved into a number of peaks containing one, two, three, and four sialic acids with an additional subfractionation based on the nature of the antennary structure. The microcarrier process resulted in a reduced amount of sialylated oligosaccharide species as compared to the suspension cell process. Removal of sialic acid followed by chromatography of the asialooligosaccharides under high pH anion‐exchange conditions 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, encompassing mono‐, di‐, tri‐, and tetrasialylated complex type oligosaccharides.