Show simple item record

dc.contributor.authorBurleigh, Susan C
dc.contributor.authorvan de Laar, Teun
dc.contributor.authorStroop, Corne JM
dc.contributor.authorvan Grunsven, Wout MJ
dc.contributor.authorO'Donoghue, Niaobh
dc.contributor.authorRudd, Pauline M
dc.contributor.authorDavey, Gavin P
dc.date.accessioned2011-11-17T12:32:28Z
dc.date.available2011-11-17T12:32:28Z
dc.date.issued2011-10-18
dc.identifierhttp://dx.doi.org/10.1186/1472-6750-11-95
dc.identifier.citationBMC Biotechnology. 2011 Oct 18;11(1):95
dc.identifier.urihttp://hdl.handle.net/10147/189826
dc.description.abstractAbstract Background The 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. Results Chinese 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. Conclusions The 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.
dc.titleSynergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells
dc.typeJournal Article
dc.language.rfc3066en
dc.rights.holderBurleigh et al.; licensee BioMed Central Ltd.
dc.description.statusPeer Reviewed
dc.date.updated2011-11-17T12:05:42Z
refterms.dateFOA2018-08-22T15:00:40Z
html.description.abstractAbstract Background The 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. Results Chinese 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. Conclusions The 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.


Files in this item

Thumbnail
Name:
1472-6750-11-95.xml
Size:
98.51Kb
Format:
XML
Thumbnail
Name:
1472-6750-11-95.pdf
Size:
3.965Mb
Format:
PDF

This item appears in the following Collection(s)

Show simple item record