Abstract
Although optimization of recombinant protein production is an important part of expression, it is difficult to provide “cookbook” techniques. We will instead outline general approaches to optimization with specific methods described where appropriate. Optimal titers are reached by designing an environment where growth, viability, and specific productivity (protein/cell/time) are balanced so as to give maximum titers (protein/volume of medium). The strategy used will depend on the cell line and the characteristics of the protein being produced. Other considerations are the amount of protein needed, how much time is available for developing the process, and whether production of the protein will need to be repeated. Additionally, purification is much more efficient if the starting material contains a significant proportion (10–80%) of the protein of interest.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Mather, J. and Tsao, M. T. (1990) Expression of cloned proteins in mammalian cells. Regulation of cell-associated parameters, in Large Scale Mammalian Cell Culture Technology (Lubiniecki, A., ed.), Marcel Dekker, New York, pp. 161–177.
Mather, J. P. (1990) Optimizing the cell and culture environment for the production of recombinant proteins, in Gene Expression Technology (Goeddel, D., ed.), Methods in Enzymology, vol. 185, Academic, New York, pp. 157–167.
Arathoon, W. R. and Birch, J. R. (1986) Large-scale cell culture in biotechnology. Science 232, 1396–1395.
Griffiths, J. B. and Rather, A. J. (1994) Cultural and physiological factors affecting expression of recombinant proteins. Cytotechnology 15, 3–9.
Swim, H. E. and Parker, R. F. (1960) Effect of pluronic F68 on growth of fibroblasts in suspension on a rotary shaker. Proc. Sci. Exp. Biol. Med. 103, 252–254.
Ham, R. G. and McKeehan, W. L. (1979) Media and growth requirements, in Methods in Enzymology, vol. 58, Academic, New York, pp. 44–93.
McKeehan, W. L., McKeenan, K. A., and Ham, R. G. (1977) Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein. In Vitro 13, 399–416.
Zetterberg, A. and Engstrom, W. (1981) Effects of alkaline pH and glutamine on cell growth and multiplication, in The Biology of Normal Human Growth (Ritzén, M., ed.), Raven, New York, pp. 47–57.
Bottenstein, J., Hayashi, I., Hutching, S. H., Masui, H., Mather, J., McClure, D. B., Okasa, S., Rizzino, A., Sato, G., Serroro, G., Wolfe, R., and Wu, R. (1979) The growth of cells in serum-free hormone-supplemented media, in Methods in Enzymology, vol. 58, Academic, New York, pp. 94–109.
Barnes, D. and Sato, G. H. (1980) Methods for the growth of cells in serum free medium. Anal. Biochem. 102, 255–270.
Hewlett, G. (1991) Strategies for optimizing serum-free media. Cytotechnology 5, 3–14.
Moore, A., Donahue, C. J., Hooley, J., Stocks, D. L., Bauer, K. D., and Mather, J. P. (1995) Apoptosis in CHO cell batch cultures: examination by flow cytometry. Cytotechnology 17, 1–11.
Franek, F., Vomastek, T., and Dolnikova, J. (1992) Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures. Cytotechnology 9, 117–123.
Kerr, J. F. K., Wyllie, A. H., and Currie, A. H. (1972) Apoptosis, a basic biological phenomenon with wider implications in tissue kinetics. Brit. J. Cancer 26, 239–245.
Arends, M. J. and Wyllie, A. H. (1991) Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Path. 32, 223–254.
Moore, J. V. (1987) Death of cells and necrosis of tumours, in Perspectives in Mammalian Cell Death (Potten, C. S., ed.), Oxford University Press, Oxford, UK, pp. 295–325.
Horan, P. K. and Kappler, J. W. (1977) Automated flourescent analysis for cyrotoxicity assays. J. Immunol. Meth. 18, 309–316.
Goergen, J. L., Marc, A., and Engasser, J. M. (1993) Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release. Cytotechnology 11, 189–195.
Dive, C. and Wyllie, A. H. (1993) Apoptosis and cancer chemotherapy, in Frontiers in Pharmacology: Cancer Chemotherapy (Hickman, J. A. and Tritton, T. R., eds.), Oxford University Press, Oxford, UK, pp. 21–56.
Wyllie, A. H. (1980) Glucocorticoid induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555, 556.
Duke, R. C., Chervenak, R., and Cohen, J. J. (1983) Endogenous endonuclease-induced DNA fragmentation an early event in cell-mediated cytolysis. Proc. Natl. Acad. Sci. USA 80, 6361–6365.
Telford, W. G., King, L. E., and Fraker, P. J. (1992) Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry 13, 137–143.
Gorczyca, W., Bruno, S., Darzynkiewicz, R. J., Gong, J., and Darzynkiewicz, Z. (1992) DNA strand breaks occurring during apoptosis their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int. J. Oncol. 1, 639–648.
Gorczyca, W., Bigman, K., Mittleman, A., Ahmed, T., Gong, J., Melamed, M. R., and Darzynkiewicz, Z. (1993) Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7, 659–670.
Tilly, J. L. and Hsueh, A. J. W. (1993) Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation. J. Cell. Physiol. 154, 519–526.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1997 Humana Press Inc.
About this protocol
Cite this protocol
Mather, J.P., Moore, A., Shawley, R. (1997). Optimization of Growth, Viability, and Specific Productivity for Expression of Recombinant Proteins in Mammalian Cells. In: Tuan, R.S. (eds) Recombinant Gene Expression Protocols. Methods in Molecular Biology, vol 62. Humana Press. https://doi.org/10.1385/0-89603-480-1:369
Download citation
DOI: https://doi.org/10.1385/0-89603-480-1:369
Publisher Name: Humana Press
Print ISBN: 978-0-89603-480-8
Online ISBN: 978-1-59259-548-8
eBook Packages: Springer Protocols