Abstract
Metabolic flux analysis with its ability to quantify cellular metabolism is an attractive tool for accelerating cell line selection, medium optimization, and other bioprocess-development activities. In the stoichiometric flux estimation approach, unknown fluxes are determined using intracellular metabolite mass balance expressions and measured extracellular rates. The simplicity of the stoichiometric approach extends its application to most cell culture systems, and the steps involved in metabolic flux estimation by the stoichiometric method are presented in detail in this chapter. Specifically, overdetermined systems are analyzed because the extra measurements can be used to check for gross measurement errors and system consistency. Cell-specific rates comprise the input data for flux estimation, and the logistic modeling approach is described for robust specific rate estimation in batch and fed-batch systems. A simplified network of mammalian cell metabolism is used to illustrate the flux estimation procedure, and the steps leading up to the consistency index determination are presented. If gross measurement errors are detected, a technique for determining the source of gross measurement error is also described. A computer program that performs most of the calculations described in this chapter is presented and references to flux estimation software are provided. The procedure presented in this chapter should enable rapid metabolic flux estimation in any mammalian cell bioreaction network by the stoichiometric approach.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Stephanopoulos, G., Aristodou, A., and Nielsen, J. (1998) Metabolic Engineering. Principles and Methodologies, Academic Press, San Diego.
Stephanopoulos, G. (2002) Metabolic engineering: perspective of a chemical engineer. AIChE J. 48, 920–926.
Stephanopoulos, G. and Stafford, D. E. (2002) Metabolic engineering: a new frontier of chemical reaction engineering. Chem. Eng. Sci. 57, 2595–2602.
Bonarius, H. P., Hatzimanikatis, V., Meesters, K. P. H., de Gooijer, C. D., Schmid, G., and Tramper, J. (1996) Metabolic flux analysis of hybridoma cells in different culture media using mass balances. Biotechnol. Bioeng. 50, 229–318.
Follstad, B. D., Balcarcel, R. R., Stephanopoulos, G., and Wang, D. I. (1999) Metabolic flux analysis of hybridoma continuous culture steady state multiplicity. Biotechnol. Bioeng. 63, 675–683.
Varma, A. and Palsson, B. O. (1994) Metabolic flux balancing: Basic concepts, scientific and practical use. Bio/Technology 12, 994–998.
Bonarius, H. P., Ozemere, A., Timmerarends, T., Skrabal, P., Tramper, J., Schmid, G., and Heinzle, E. (2001) Metabolic-flux analysis of continuously cultured hybridoma cells using 13CO2 mass spectrometry in combination with 13C-Lactate nuclear magnetic resonance spectroscoopy and metabolite balancing. Biotechnol. Bioeng. 74, 528–538.
Forbes, N. S., Clark, D. S., and Blanch, H. W. (2001) Using isotopomer path tracing to quantify metabolic fluxes in pathway models containing reversible reactions. Biotechnol. Bioeng. 74, 196–211.
Wiechert, W. (2001) 13C Metabolic flux analysis. Metab. Eng. 3, 195–206.
Zupke, C. and Stephanopoulos, G. (1995) Intracellular flux analysis in hybridomas using mass balances and In Vitro 13C NMR. Biotechnol. Bioeng. 45, 292–303.
Szyperski, T., Bailey, J. E., and Wüthrich, K. (1996) Detecting and dissecting metabolic fluxes using biosynthetic fractional 13C labeling and two-dimensional NMR spectroscopy. Trends Biotechnol. 14, 453–458.
Cruz, H. J., Moreira, J. L., and Carrondo, M. J. (1999) Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells. Biotechnol. Bioeng. 66, 104–113.
Cruz, H. J., Ferreira, A. S., Freitas, C. M., Moreira, J. L., and Carrondo, M. J. (1999) Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture. Appl. Microbiol. Biotechnol. 51, 579–585.
Altamirano, C., Illanes, A., Casablancas, A., Gámez, X., Cairo, J. J., and GÒdia, C. (2001) Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture. Biotechnol. Prog. 17, 1032–1041.
Nyberg, G. B., Balcarcel, R. R., Follstad, B. D., Stephanopoulos, G., and Wang, D. I. (1999) Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium, Biotechnol. Bioeng. 62, 324–335.
Bonarius, H. P., Houtman, J. H., Schmid, G., de Gooijer, C. D., and Tramper, J. (2000) Metabolic-flux analysis of hybridoma cells under oxidative and reductive stress using mass balance. Cytotechnology 32, 97–107.
Europa, A. F., Gambhir, A., Fu, P. C., and Hu, W. S. (2000) Multiple steady states with distinct cellular metabolism in continuous culture of mammalian cells. Biotechnol. Bioeng. 67, 25–34.
Zupke, C., Sinskey, A. J., and Stephanopoulos, G. (1995) Intracellular flux analysis applied to the effect of dissolved oxygen on hybridomas. Appl. Microbiol. Biotechnol. 44, 27–36.
Bonarius, H. P., Timmerarends, B., de Gooijer, C. D., and Tramper, J. (1998) Metabolite-balancing techniques vs. 13C tracer experiments to determine metabolic fluxes in hybridoma cells. Biotechnol. Bioeng. 58, 258–262.
Nadeau, I., Sabatié, J., Koehl, M., Perrier, M., and Kamen, A. (2000) Human 293 cell metabolism in low glutamine-supplied culture: Interpretation of metabolic changes through metabolic flux analysis. Metab. Eng. 2, 277–292.
de Graaf, A.A., Mahle, M., Möllney, M., Wiechert, W., Stahmann, P., and Sahm, H. (2000) Determination of full 13C isotopomer distributions for metabolic flux analysis using heteronuclear spin echo difference NMR spectroscopy. J. Biotechnol. 77, 25–35.
Schmidt, K., Carlsen, M., Nielsen, J., and Villadsen, J. (1997) Modeling isotopomer distributions in biochemical networks using isotopomer mapping matrices. Biotechnol. Bioeng. 56, 831–840.
van Winden, W. A., Heijnen, J. J., and Verheijen, P. J. T. (2002) Cumulative bandomers: A new concept in fluxanalysis from 2D[13C, 1H] COSY NMR data. Biotechnol. Bioeng. 80, 731–745.
Wiechert, W., Möllney, M., Petersen, S., and de Graaf, A. A. (2001) A universal framework for 13C metabolic flux analysis. Metab. Eng. 3, 265–283.
Xie, L. and Wang, D. I. C. (1996) Material balance studies on animal cell metabolism using a stoichiometrically based reaction network. Biotechnol. Bioeng. 52, 579–590.
Burgard, A. P. and Maranas, C. (2001) Review of the enzymes and metabolic pathways (EMP) database. Metab. Eng. 3, 193–194.
Bree, M. A., Dhurjati, P., Geoghegan, R. F., and Robnett, B. (1988) Kinetic modeling of hybridoma cell growth and immunoglobulin production in a large-scale suspension culture. Biotechnol. Bioeng. 32, 1067–1072.
Dalili, M., Sayles, G. D., and Ollis, D. F (1990) Glutamine-limited batch hybridoma growth and antibody production: experiment and model. Biotechnol. Bioeng. 36, 74–82.
Linz, M., Zeng, A. P., Wagner, R., and Deckwer, W. D. (1997) Stoichiometry, kinetics and regulation of glucose and amino acid metabolism of a recombinant BHK cell line in batch and continuous culture. Biotechnol. Prog. 13, 453–463.
Altamirano, C., Paredes, C., Illanes, A., Cairo, J. J., and GÒdia, C. (2004) Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium. J. Biotechnol. 110, 171–179.
Zhou, W.C., Chen, C.-C., Buckland, B., and Aunins, J. G. (1997) Fed-batch culture of recombinant NSO myeloma cells with high monoclonal antibody production. Biotechnol. Bioeng. 55, 783–792.
Goudar, C. T., Joeris, K., Konstantinov, K., and Piret, J. M. (2005) Logistic equations effectively model mammalian cell batch and fed-batch kinetics by logically constraining the fit. Biotechnol. Prog. 21, 1109–1118.
Balcarcel, R. R. and Clark, L. M. (2003) Metabolic screening of mammalian cell cultures using well-plates. Biotechnol. Prog. 19, 98–108.
van der Heijden, R. T. J. M., Romein, B., Heijnen, S., Hellinga, C., and Luyben, K. C. A. M. (1994) Linear constraint relations in biochemical reaction systems: II. Diagnosis and estimation gross errors. Biotechnol. Bioeng. 43, 11–20.
Wang, N. S. and Stephanopoulos, G. (1983) Application of macroscopic balances to the identification of gross measurement errors. Biotechnol. Bioeng. 25, 2177–2208.
Klamt, S., Schuster, S., and Gilles, E. D. (2002) Calculability analysis in underdetermined metabolic networks illustrated by a model of the central metabolism in purple nonsulfur bacteria. Biotechnol. Bioeng. 77, 734–751.
Lee, D. Y., Hongsoek, Y., Park, S., and Lee, S. Y (2003) MetaFluxNet: the management of metabolic reaction information and quantitative metabolic flux analysis. Bioinformatics 19, 2144–2146.
Okayasu, T., Ikeda, M., Akimoto, K., and Sorimachi, K. (1997) The amino acid composition of mammalian and bacterial cells. Amino Acids 13, 379–391.
Bonarius, H. P., de Gooijer, C. D., Tramper, J., and Schmid, G. (1995) Determination of the respiration quotient in mammalian cell culture in bicarbonate buffered media. Biotechnol. Bioeng. 45, 524–535.
Frahm, B., Blank, H.-C., Cornand, P., et al. (2002) Determination of dissolved CO2 concentration and CO2 production rate of mammalian cell suspension culture based on off-gas measurement. J. Biotechnol. 99, 133–148.
Fell, D. A. and Small, J. R. (1986) Fat synthesis is adipose tissue. An examination of stoichiometric constraints. J. Biochem. (Tokyo) 238, 781–786.
Majewski, R. A. and Domach, M. M. (1990) Simple constrained optimization view of acetate overflow in E. coli. Biotechnol. Bioeng. 35, 732–738.
Pramanik, J. and Keasling, J. D. (1997) A stoichiometric model of Escherichia coli metabolism. Incorporation of growth-rate-dependent biomass composition and mechanistic energy requirements. Biotechnol. Bioeng. 56, 398–421.
Savinell, J. M. and Palsson, B. O. (1992) Network analysis of intermediary metabolism using linear optimization. I. Development of mathematical formulation. J. Theor. Biol. 154, 421–454.
Savinell, J. M. and Palsson, B. O. (1992) Network analysis of intermediary metabolism using linear optimization. II. Interpretation of hybridoma cell metabolism. J. Theor. Biol. 154, 455–473.
Varma, A., Boesch, B. W., and Palsson, B. O. (1993) Biochemical production capabilities of Escherichia coli. Biotechnol. Bioeng. 42, 59–73.
Madron, F., Veverka, V., and Vanecek, V. (1977) Stastical analysis of material balance of a chemical reactor. AIChE J. 23, 482–486.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Goudar, C.T., Biener, R., Piret, J.M., Konstantinov, K.B. (2007). Metabolic Flux Estimation in Mammalian Cell Cultures. In: Pörtner, R. (eds) Animal Cell Biotechnology. Methods in Biotechnology, vol 24. Humana Press. https://doi.org/10.1007/978-1-59745-399-8_14
Download citation
DOI: https://doi.org/10.1007/978-1-59745-399-8_14
Publisher Name: Humana Press
Print ISBN: 978-1-58829-660-3
Online ISBN: 978-1-59745-399-8
eBook Packages: Springer Protocols