Metabolic Engineering

Metabolic Engineering: Application 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 studied the growth and metabolism of human breast cancer cells. We quantified 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. 

© Harvey Blanch 2013