Baker, K.N. and Rendall, M.H. and Hills, A.E. and Hoare, M. and Freedman, R.B. and James, D.C. (2001) Metabolic Control of Recombinant Protein N-glycan Processing in NS0 and CHO Cells. Biotechnology and Bioengineering, 73 (3). pp. 188-202. ISSN 0006-3592.
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Chinese hamster ovary and murine myeloma NS0 cells are currently favored host cell types for the production of therapeutic recombinant proteins. In this study, we compared N-glycan processing in GS-NS0 and GS-CHO cells producing the same model recombinant glycoprotein, tissue inhibitor of metalloproteinases 1. By manipulation of intracellular nucleotide-sugar content, we examined the feasibility of implementing metabolic control strategies aimed at reducing the occurrence of murine-specific glycan motifs on NS0-derived recombinant proteins, such as Galalpha1,3Galbeta1,4GlcNAc. Although both CHO and NS0-derived oligosaccharides were predominantly of the standard complex type with variable sialylation, 30% of N-glycan antennae associated with NS0-derived TIMP-1 terminated in alpha1,3-linked galactose residues. Furthermore, NS0 cells conferred a greater proportion of terminal N-glycolylneuraminic (sialic) acid residues as compared with the N-acetylneuraminic acid variant. Inclusion of the nucleotide-sugar precursors, glucosamine (10 mM, plus 2 mM uridine) and N-acetylmannosamine (20 mM), in culture media were shown to significantly increase the intracellular pools of UDP-N-acetylhexosamine and CMP-sialic acid, respectively, in both NS0 and CHO cells. The elevated UDP-N-acetylhexosamine content induced by the glucosamine/uridine treatment was associated with an increase in the antennarity of N-glycans associated with TIMP-1 produced in CHO cells but not N-glycans associated with TIMP-1 from NS0 cells. In addition, elevated UDP-N-acetylhexosamine content was associated with a slight decrease in sialylation in both cell lines. The elevated CMP-sialic acid content induced by N-acetylmannosamine had no effect on the overall level of sialylation of TIMP-1 produced by both CHO and NS0 cells, although the ratio of N-glycolylneuraminic acid:N-acetylneuraminic acid associated with NS0-derived TIMP-1 changed from 1:1 to 1:2. These data suggest that manipulation of nucleotide-sugar metabolism can promote changes in N-glycan processing that are either conserved between NS0 and CHO cells or specific to either NS0 cells or CHO cells.
|Additional information:||0006-3592 (Print) Comparative Study Journal Article|
|Uncontrolled keywords:||Animals Bioreactors CHO Cells Cell Division/drug effects Chromatography, High Pressure Liquid Cricetinae Galactose/metabolism Glucosamine/pharmacology Glycosylation/drug effects Hexosamines/pharmacology Mice N-Acetylneuraminic Acid/metabolism Nucleotides/metabolism Polysaccharides/chemistry/*metabolism Recombinant Proteins/metabolism Sialyltransferases/metabolism Tissue Inhibitor of Metalloproteinase-1/chemistry/*metabolism Tumor Cells, Cultured|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group|
|Depositing User:||Sue Davies|
|Date Deposited:||30 Oct 2008 18:41|
|Last Modified:||14 Jan 2010 14:24|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/6498 (The current URI for this page, for reference purposes)|
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