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Title: Metabolomics study of CHO cell cultures
Keywords: Metabolomics, LC-MS, CHO, recombinant protein, metabolic engineering, apoptosis
Issue Date: 23-Apr-2010
Citation: CHONG POOI KAT, WILLIAM (2010-04-23). Metabolomics study of CHO cell cultures. ScholarBank@NUS Repository.
Abstract: Chinese hamster ovary (CHO) cells are predominantly used for the production of recombinant proteins. Currently, with the exception of ammonia and lactate, there is little knowledge of other metabolites that are released by CHO cells during culture. This thesis describes the development of a liquid chromatography-mass spectrometry metabolomics strategy to identify extracellular metabolites in recombinant CHO fed-batch cultures. 11 out of 12 identified metabolites were reported for the first time in CHO culture medium. Amongst the metabolites, malate accumulation was the highest at 329 ?M. Malate efflux was found to be due to the supply of aspartate and possibly an enzymatic bottleneck at malate dehydrogenase II (MDH II). Subsequent metabolic engineering to overexpress MDH II in CHO cells resulted in 3 fold increases in intracellular ATP and NADH. In addition, integral cell number and product titer increased by up to 1.9 and 1.2 fold respectively. This is the first report of a metabolic engineering target that was successfully identified using a metabolomics approach. In addition, a panel of extracellular metabolites exhibited good correlation with intracellular caspase activity. Some of these metabolites, such as oxidized glutathione and AMP, were shown to induce apoptosis. This result could offer the first insight into why CHO cells in fed-batch cultures eventually senesce and die, even though nutrients were not limiting and ammonia and lactate were below inhibitory levels. These findings demonstrate how a metabolomics-based strategy enabled the identification of key cell growth and death-related metabolites in recombinant CHO cultures, thereby guiding metabolic engineering efforts to improve culture performance and product titer.
Appears in Collections:Ph.D Theses (Open)

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