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Title: Characterisation of amino acid transport processes in Chinese Hamster Ovary (CHO) cells
Author: Geoghegan, Darren
ISNI:       0000 0004 5919 0726
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2015
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Amino acid transport is controlled by a system of transporter proteins whose function remains poorly defined in CHO cells. In this thesis, the relationship between amino acid transporter activity and culture performance parameters was investigated and used as a basis to explore new opportunities for improving cell line productivity. Transcriptomic analysis of transporter expression was first performed in two non-producing CHO cell lines and one antibody-producing CHO cell line during fed-batch cultures. Seven transporters were highly expressed across the three cell lines (ASCT1, CAT-1, GLAST, LAT1, SNAT2, xCT and y+LAT2). For all cell lines, the cystine-glutamate xCT transporter was significantly upregulated at stationary phase and formed part of a larger adaptive response to support the cellular availability of glutathione. The antibodyproducing cell line also upregulated ASCT1 and LAT1 transporter expression during stationary phase, which allowed the cells to maintain a high consumption rate of amino acids abundant in the antibody. Cells were next treated with inhibitors that block amino acid transport through individual or groups of transporters. Inhibition results confirmed that xCT transport activity is a key determinant of culture viability in all cell lines, and along with LAT1, also supports specific productivity during late-stage culture in the antibody-producing cell line. A directed evolution strategy was subsequently developed to increase xCT transport capability in the host. Evolved host cells demonstrated an increased capacity for GSH synthesis, increased resistance to a ROS insult, and a heritable improvement in cell growth but were not able to outperform the unevolved host in the transient production of an IgG. Finally, a mechanistic model to describe essential amino acid transport processes in CHO cells was constructed from transcriptomic and inhibitor data. This model can be used to direct future optimisation of amino acid concentrations in culture media to maximise cell growth and antibody production.
Supervisor: James, David C. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available