A positive correlation between the flux of TCA cycle and productivity of Chinese Hamster Ovary (CHO) cells has been reported. Earlier work in this laboratory revealed that supplementation with nutrients that enter the TCA cycle (combination of glucose (Glc), pyruvate (Py), aspartate (Asp), asparagine (Asn) and glutamate (Glu)) significantly increased maximum viable cell density and antibody production of recombinant CHO cells. Increased amounts of extracellular citrate was associated with feeding conditions. It was hypothesized that increased flux through the TCA cycle and related metabolism was linked to enhanced growth and/or productivity of CHO cells. Therefore, the aim of this thesis is to clarify these relationships to provide routes to improve the efficiency of CHO cells by nutrient supplementation and metabolic engineering. The relationship between growth, antibody production and metabolite profiles of CHO-LB01 cells was examined in response to individual supplementation with Asn, Asp, Glu, Py and β-hydroxybutyrate (HB). Feeding HB significantly increased antibody titre while Asn feeding increased maximum cell density but led to earlier cell death. Both nutrients increased the amounts of TCA cycle intermediates and decreased the amounts of lactate, glycerol, sorbitol and amino acids. Moreover, oxygen consumption rate was increased in the presence of Asn or HB. This finding inferred that increased production of the TCA cycle intermediates in cells fed Asn or HB correlated with enhanced flux of the TCA cycle leading to enhanced oxidative metabolism. Combination of Asn or HB with Glc further improved cell growth, increased antibody titre and enhanced metabolic responses to feeds (TCA cycle intermediates). Based on these results, inhibition of sorbitol production using chemical reagent (EPBC) and siRNA designed against Akr1b1 and overexpression of malate dehydrogenase II (MDH II) were undertaken in order to increased flow of carbon atoms to TCA cycle and/or increased flux in the TCA cycle, respectively. Inhibition of sorbitol production was achieved in the presence of EBPC but there was no improvement of cell culture performance and accumulation of TCA cycle intermediates remained the same. CHO cells transfected with exogenous Mdh2 did not show improved cell culture performance. Whilst stable clones exhibited variable MDH II expression at protein level (and antibody titre), overexpression of exogenous MDH II could not be confirmed by Western blot. One CHO-MDH II clone showed greater antibody titre and exhibited similar metabolite profiling with cells fed Asn or HB. This contrasted to the majority of clones that were low producers. Comparison by RNA-Seq transcriptomic profiling of high- and low-producing CHO-MDH II clones showed that the majority of differentially expressed genes were genes related to cytoskeleton-related element and cell signaling pathways. Overall, these results confirmed the relationship between increased the amount of TCA cycle intermediates and increased antibody production. Increased amount of TCA cycle intermediates could result in increased the flow of TCA cycle lead to enhance energy and antibody production. In addition, this work represents the first study on addition of HB offers a simple effective strategy to increase antibody production.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:728149 |
| Date | January 2017 |
| Creators | Porncharoennop, Chompoonuth |
| Contributors | Dickson, Alan |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/metabolite-profiling-associated-with-productive-recombinant-cho-cell-culture(3bb6bdaf-d8dc-4249-b77b-159e9e77307a).html |
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