Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therap eutic immunoglobulin G (IgG) molecules, specifically terminal galactosylati on and sialylation, may affect both pharmacokinetic behavior and effector f unctions of recombinant therapeutic antibodies. We investigated the hypothe sis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism, In control cultures, N-glycans associated wit h the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta -galactosylated (ave rage 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N -acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylatio n evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, w e included either 10 mM glucosamine or 10 mM galactose in the culture mediu m. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhe xosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Asso ciated with these alterations in cellular UDP-sugar content was a significa nt reduction (57%) in the galactosylation of Fc-derived oligosaccharides. T he proportion of high-mannose-type N-glycans (specifically Man5, the substr ate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In c ontrast, inclusion of 10 mM galactose in culture specifically stimulated UD P-Gal content almost five-fold. However, this resulted in only a minimal, i nsignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sial ylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold incr ease in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylat ion during batch culture showed that beta1,4-linked galactosylation decline d slightly during culture, although, in the latter stages of culture, the r elease of proteases and glycosidases by lysed cells were likely to have con tributed to the more dramatic drop in galactosylation. These data demonstra te: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic contr ol of Fc N-glycosylation. (C) 2001 John Wiley & Sons, Inc.