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

Hills, Anna E. and Patel, Ashvin and Boyd, Paul and James, David C. (2001) Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells. Biotechnology and Bioengineering, 75 (2). pp. 239-251. ISSN 0006-3592. (The full text of this publication is not available from this repository)

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Official URL
http://dx.doi.org/10.1002/bit.10022

Abstract

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.

Item Type: Article
Additional information: 0006-3592 (Print) Journal Article
Uncontrolled keywords: Amidohydrolases/metabolism Animals Anthranilic Acids/metabolism Antibodies, Monoclonal/*metabolism Carrier Proteins/chemistry Cell Adhesion Molecules Chromatography, High Pressure Liquid Culture Media, Serum-Free Cytidine Monophosphate N-Acetylneuraminic Acid/analysis Galactose/metabolism Glucosamine/metabolism Glycosylation Humans Immunoglobulin Fc Fragments/chemistry/*drug effects/metabolism Immunoglobulin G/*chemistry/metabolism Mannose/chemistry Membrane Proteins Mice Nucleotides/analysis/*metabolism Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Polysaccharides/analysis/*metabolism Recombinant Proteins/chemistry/*metabolism Tumor Cells, Cultured
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group
Depositing User: Sue Davies
Date Deposited: 30 Oct 2008 18:35
Last Modified: 23 May 2014 13:10
Resource URI: http://kar.kent.ac.uk/id/eprint/6497 (The current URI for this page, for reference purposes)
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