Metabolic control of recombinant protein N‐glycan processing in NS0 and CHO cells

Abstract

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.

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…However, this phenomenon is not universal for all cell lines, such as NS0 cells, which have been reported to have no positive feedback for glucosamine addition (Baker et al, 2001). It is noteworthy that since UDP-GlcNAc competes with CMPNeuAc for transport into the Golgi, elevated UDP-GlcNAc appears to cause a decline in sialylation (Rijcken et al, 1995).…”

“…UDP-GlcNAc is an important intracellular nucleotide sugar in late process of N-glycosylation pathway. Study found that adding glucosamine into the culture medium could elevate the intracellular UDPGlcNAc pool, especially added in conjunction with uridine (Baker et al, 2001). …”

Tailoring recombinant protein quality by rational media design

“…While improved sialylation of IFN-γ was the result of medium supplementation with the specific sialic acid precursor N-acetylmannosamine (ManNAc) at 20 mM in a CHO cell line (cotransfected with genes for dihydrofolate reductase) 134 , it remained unchanged for human tissue inhibitor of metalloproteinases 1 (TIMP-1) at the same ManNAc concentration in GS-NS0 and GS-CHO cells 135 . Likewise, feeding CHO-K1 cells producing EPO with 10 mM ManNAc increased sialylation 136 .…”

“…The biochemical mechanisms of N-glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus are highly complex and involve many different processes, enzymes, substrates and cofactors 24 . Furthermore, the metabolism of the host cell is linked with the glycosylation pattern of the recombinant protein, and hence, metabolic control may come into play to influence N-glycan processing 106,123,135 . Metabolomic analysis of fed-batch cultures revealed increasing ornithine levels coincided with higher high mannose glycan levels 285 .…”

Using cell engineering and omic tools for the improvement of cell culture processes

“…In fact, most species predominantly have Nglycolyl-neuraminic acid (NGNA) rather than NANA. CHO cells have the advantage of the presence of a high percentage of NANA sialylation, but still include some NGNA sialylation (Baker et al 2001). …”