Ammonium ion and glucosamine dependent increases of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells
Article first published online: 26 MAR 2000
Copyright © 1998 John Wiley & Sons, Inc.
Biotechnology and Bioengineering
Volume 57, Issue 5, pages 518–528, 5 March 1998
How to Cite
Gawlitzek, M., Valley, U. and Wagner, R. (1998), Ammonium ion and glucosamine dependent increases of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells. Biotechnol. Bioeng., 57: 518–528. doi: 10.1002/(SICI)1097-0290(19980305)57:5<518::AID-BIT3>3.0.CO;2-J
- Issue published online: 26 MAR 2000
- Article first published online: 26 MAR 2000
- Manuscript Accepted: 4 AUG 1997
- Manuscript Received: 9 FEB 1996
- EED. Grant Numbers: BIOTEC-92-0304, BIO2-CT943069
- N -glycosylation;
- BHK-21 cells
The effect of different ammonium concentrations and glucosamine on baby hamster kidney (BHK)-21 cell cultures grown in continuously perfused double membrane bioreactors was investigated with respect to the final carbohydrate structures of a secretory recombinant glycoprotein. The human interleukin-2 (IL-2) mutant glycoprotein variant IL-Mu6, which bears a novel N-glycosylation site (created by a single amino acid exchange of Gln100 to Asn), was produced under different defined protein-free culture conditions in the presence or absence of either glutamine, NH4Cl, or glucosamine. Recombinant glycoprotein products were purified and characterized by amino acid sequencing and carbohydrate structural analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry, high-pH anion-exchange chromatography with pulsed amperometric detection, and methylation analysis. In the absence of glutamine, cells secreted glycoprotein forms with preponderantly biantennary, proximal fucosylated carbohydrate chains (85%) with a higher NeuAc content (58%). Under standard conditions in the presence of 7.5 mM glutamine, complex-type N-glycans were found to be mainly biantennary (68%) and triantennary structures (33%) with about 50% containing proximal α1-6-linked fucose; 37% of the antenna were found to be substituted with terminal α2-3-linked N-acetylneuraminic acid. In the presence of 15 mM exogenously added NH4Cl, a significant and reproducible increase in tri- and tetraantennary oligosaccharides (45% of total) was detected in the secretion product. In glutamin-free cultures supplemented with glucosamine, an intermediate amount of high antennary glycans was detected. The increase in complexity of N-linked oligosaccharides is considered to be brought about by the increased levels of intracellular uridine diphosphate-GlcNAc/GalNAc. These nucleotide sugar pools were found to be significantly elevated in the presence of high NH3/NH4+ and glucosamine concentrations. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 518-528, 1998.