Metabolic Control of Recombinant Protein N-glycan Processing in NS0 and CHO Cells

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.