Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

Abstract

BackgroundThe glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins.ResultsChinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG.ConclusionsThe use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms

“…7). Burleigh et al reported that lack of glutamine in the medium coupled with reduced glycolysis causes a reduction in the sialylation and branching of N-glycans in recombinant glycoproteins (38). A significant difference between our finding and this finding is that the Min6 insulinoma cell line contained a high level of nucleotide sugars.…”

Tailoring recombinant protein quality by rational media design

“…The same authors report no significant glycan differences in continuous culture between two steady states by decreasing the Gln concentration from 8 to 0 mM. Nonetheless, between additional steady states (steady state 3: 8 mM Gln, steady state 4: 0 mM Gln) a significant glycan species distribution occurred thus showing the impact of the metabolic flux on glycosylation 117 . Following the addition of sucrose, which led to higher levels of high mannose glycans (cf.…”

“…In contrast to human IgG, sialic acids are attached to the terminal galactose residues of antibodies expressed in CHO cells via an α-2,3 linkage exclusively 68 . It was reported that by reducing glutamine concentrations to 0 mM in CHO-K1 batch and perfusion cultures, sialylation was inhibited, thus leading to more abundant neutral N-linked glycans 117 . Dimethyl sulfoxide (DMSO) from 1 to 8% (v/v) reduced sialylation but at the same time negatively impacted cell proliferation 139 .…”

Mannose metabolism in recombinant CHO cells and its effect on IgG glycosylation

“…For this reason, it is generally desirable to select a production cell line that produces IgG with low HM glycosylation. However, since glycosylation is sensitive to small, potentially unforeseen changes in cell culture and manufacturing (Burleigh et al, 2011;Chee Furng Wong et al, 2005;Crowell et al, 2007;Hossler et al, 2009;Li et al, 2010;Muthing et al, 2003;Walsh and Jefferis, 2006), it would be advantageous to predict the potential amount of IgG HM glycosylation either before or during the development of the manufacturing process. Therefore, understanding the causes for HM glycosylation in CHO cells is needed.…”