Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells.
Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation 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, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (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 control of Fc N-glycosylation.
Hills
A E
AE
Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
Patel
A
A
Boyd
P
P
James
D C
DC
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry; D015815:Cell Adhesion Molecules; D002851:Chromatography, High Pressure Liquid; D016895:Culture Media, Serum-Free; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000032:analysis; D005690:Galactose / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D008358:Mannose / Q000737:chemistry; D008565:Membrane Proteins; D051379:Mice; D009711:Nucleotides / Q000032:analysis / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000032:analysis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D062367:ortho-Aminobenzoates / Q000378:metabolism; D006023:Glycoproteins
D000581:Amidohydrolases; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D016895:Culture Media, Serum-Free; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D005690:Galactose; D005944:Glucosamine; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008358:Mannose; D008565:Membrane Proteins; D009711:Nucleotides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; C106607:FCGBP protein, human; C046179:glycosylated IgG; D006023:Glycoproteins; C000219:anthranilamide