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Applicaton of metabolic
engineering to mammalian cells |
Mammalian cells are commonly employed in the production
of proteins and peptides where glycosylation is important to biological
function, or where the product protein is large, multimeric or unsuitable for
production in prokaryotic hosts. Examples include monoclonal antibodies,
clotting factors, growth factors, and EPO. While conditions for growth of
bacterial, yeast and fungal systems are well established, much less is known
about the growth, physiology and regulation of mammalian cells. In earlier
work, we studied the environmental factors important for hybridoma growth and
developed an NMR-based method to quantify intracellular metabolites in
hybridomas grown under steady-state conditions, using a hollow-fiber bioreactor
placed in the bore of the NMR magnet. These studies have been valuable in
guiding industrial bioreactor operation for production of monoclonal antibodies
and other products. The methodology developed now permits the rational design of
balanced media, feed strategies and reactor operation to optimize product yields
in hybridoma, CHO or BHK cell lines.
Expanding this technique, in collaboration with
Professor
Douglas Clark, we are studying the growth and metabolism of human breast cancer
cells. We quantify the physiological response of two breast cancer cell lines,
T47D and MCF7 cells, to estrogen, the commonly-employed anti-cancer drug tamoxifen, and cerulenin, which influences fatty acid metabolism. Cells are
grown on microcarriers under controlled conditions, and growth rates, glucose
uptake, glutamine consumption rates, and other metabolic parameters are observed
in estrogen receptor positive (ER+) and ER- cells. Cells are challenged with
estrogen and tamoxifen.
Intracellular metabolic fluxes are determined using
13C-labelled glucose and glutamine, by measuring intracellular fates and
concentrations of NMR-observable metabolites. The objectives are to
quantitatively determine how metabolism in breast cancer cells differs from that
in normal cells, and the role played by estrogen and the anti-estrogen tamoxifen.
With flux data available, potential enzymes activities can be identified as
targets for drug development. Current Research
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