Ammonium ion and glucosamine dependent increases of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells
✍ Scribed by Martin Gawlitzek; Ulrich Valley; Roland Wagner
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 184 KB
- Volume
- 57
- Category
- Article
- ISSN
- 0006-3592
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✦ Synopsis
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 Nglycosylation site (created by a single amino acid exchange of Gln 100 to Asn), was produced under different defined protein-free culture conditions in the presence or absence of either glutamine, NH 4 Cl, 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 ␣1-6-linked fucose; 37% of the antenna were found to be substituted with terminal ␣2-3-linked N-acetylneuraminic acid. In the presence of 15 mM exogenously added NH 4 Cl, 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 NH 3 /NH 4 + and glucosamine concentrations.