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 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-glycanase 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.
Watson
E
E
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
Shah
B
B
Leiderman
L
L
Hsu
Y R
YR
Karkare
S
S
Lu
H S
HS
Lin
F K
FK
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007610:Kallikreins / Q000032:analysis*; D019158:N-Acetylneuraminic Acid; D009844:Oligosaccharides / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis*; D012794:Sialic Acids / Q000032:analysis; D013535:Suspensions
D016895:Culture Media, Serum-Free; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; D013535:Suspensions; D007610:Kallikreins; D019158:N-Acetylneuraminic Acid