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

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.

Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab

“…In early years the MABs were deglycosylated and analyzed with, e.g., 1 H NMR [10], sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [11], hydrophobic interaction chromatography [11], and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) [12]. Radio labeling of the released glycans, and analyzing them by a combination of paper electrophoresis and gel-filtration chromatography [13], with normal phase high-performance liquid chromatography (HPLC) [14,15] or anion-exchange HPLC [16] coupled with a fluorescence detector have also been described. Additionally, capillary electrophoresis coupled with different detectors has been described [17][18][19].…”

Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs

“…Furthermore, though the quantitation of glycoforms using peak intensities from deconvoluted ESI-TOF MS spectra has been reported by our group [14] and others [15], concerns remain regarding the accuracy and reproducibility of this method. An alternative approach involves chromatographic analysis of glycans released from the protein through enzymatic [16] (e.g., peptide Nglycosidases) or chemical (e.g., ␤-elimination) procedures [11]. This "N-glycan release assay" is relatively straightforward and well-established, but information on the site of the protein-carbohydrate bond and potential asymmetry in glycosylation of the two heavy chains is lost with this approach.…”

Tailoring recombinant protein quality by rational media design

“…The lower degree of galactosylation at that time of the culture increases the substrate demand in order to reach a comparable percentage of galactosylated mAb to B, C and D at the end. It has been reported that despite 5-fold higher intracellular UDP-galactose levels, galactosylation was not significantly improved 123 , which highlights the importance to take enzyme activity and gene expression into account too. The cofactor for galactosyltransferase, manganese, increases the GalT activity and thus considerably promotes galactosylation 23,52 .…”

“…In these conditions only minor modifications of other glycoforms or quality attributes were induced 52 . In the absence of synergy, the impact of the galactose concentration on increased galactosylation is limited 123 . More recent results however displayed an increase of galactosylation from 14 to 25% of monoclonal antibodies expressed in CHO DG44-derived fed-batch suspension cell cultures, supplementing medium and feeds to reach a final media concentration of 20 mM of galactose 124 .…”

“…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 .…”