Abstract
Cellular physiology and metabolism were monitored using a quasi real-time combination of on-lineand off-line data to estimate metabolic fluxes in an established bioreaction network. The utility of thisapproach towards optimizing bioreactor operation was demonstrated for CHO cells cultivated in 15 Lperfusion reactors at 20 × 106 cells/mL. Medium compositionand dilution rates were changed to obtain several steady states with varying glucose and glutamine concentrations.When cells were restored to initial culture medium and perfusion rate conditions after being exposed tolower glucose and glutamine concentrations, the pyruvate flux into the TCA cycle was increased 30% whilethe pyruvate flux through lactate was decreased 30%, suggesting steady-state multiplicity. By appropriatelyaltering cellular metabolism, perfusion bioreactors can operate at lower perfusion rates without significantaccumulation of inhibitory metabolites such as lactate. Changes in glucose, lactate and glutamine uptake/productionrates had significant effects on the calculation of other fluxes in the network. Sensitivity analysis ofthese key metabolic fluxes highlighted the need for accurate and reliable real-time sensors. Overall, rapidobservation of metabolic fluxes can be a valuable tool for bioprocess development, monitoring and control.The framework presented in this study offers a convenient means for quasi real-time estimation of metabolicfluxes and represents a step towards realizing the potential of metabolic flux analysis for acceleratedbioprocess optimization.
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References
Konstantinov K, Chuppa S, Sajan E, Tsai Y, Yoon S, Golini F (1994) Real-time biomass-concentration monitoring in animal-cell cultures. Trends Biotechnol 12:324
Trampler F, Sonderhoff SA, Pui PW, Kilburn DG, Piret JM (1994) Acoustic cell filter for high density perfusion culture of hybridoma cells. Bio/Technol 12:281
Glacken MW, Huang C, Sinskey AJ (1989) Mathematical description of hybridoma culture kinetics III. Simulation of fed-batch reactors. J Biotechnol 10:39
Zhou WC, Rehm J, Europa AF, Hu WS (1997) Alteration of mammalian cell metabolism by dynamic nutrient feeding. Cytotechnol 24:99
Bonarius HP, de Gooijer CD, Tramper J, Schmid G (1995) Determination of the respiration quotient in mammalian cell culture in bicarbonate buffered media. Biotechnol Bioeng 45:524
Nyberg GB, Balcarcel RR, Follstad BD, Stephanopoulos G, Wang DI (1999) Metabolism of peptide amino acids by Chinese hamster ovary cells grown in a complex medium. Biotechnol Bioeng 62:324
Zupke C, Sinskey AJ, Stephanopoulos G (1995) Intracellular flux analysis applied to the effect of dissolved oxygen on hybridomas. Appl Microbiol Biotechnol 44:27
Stephanopoulos G, Vallino JJ (1991) Network rigidity and metabolic engineering in metabolite overproduction. Science 252:1675
Vallino JJ, Stephanopoulos G (1993) Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction. Biotechnol Bioeng 41:633
Varma A, Palsson BO (1994) Metabolic flux balancing: Basic concepts, scientific and practical use. Bio/Technol 12:994
Wiechert W (2001) 13C Metabolic flux analysis. Metabol Engin 3:195
Cooney CR, Wang HY, Wang DIC (1977) Computer-aided material balancing for prediction of fermentation parameters. Biotechnol Bioeng 19:55
Wang HY, Cooney C, Wang DIC (1977) Computer-aided baker's yeast fermentations. Biotechnol Bioeng 19:69
Wang HY, Cooney C, Wang DIC (1979) Computer control of baker's yeast production. Biotechnol Bioeng 21:975
Erickson LE (1979) Application of mass-energy balance in on-line data analysis. Biotechnol Bioeng Symp 9:48
Spruytenburg R, Dunn IJ, Bourne JR (1979) Computer control of glucose feed to a continuous culture of Sacchromyces cerevisiae using the respiratory quotient. Biotechnol Bioeng Symp 9:359
Herwig C, Marison I, Stockar U (2001) On-line stoichiometry and identification of metabolic state under dynamic process conditions. Biotechnol Bioeng 75:345
Konstantinov K (1996) Monitoring and control of the physiological state of cell cultures. Biotechnol Bioeng 52:271
Stephanopoulos G, Aristodou A, Nielsen J (1998) Metabolic Engineering. Principles and Methodologies. Academic, San Diego
Schügerl K (2001) Progress in monitoring, modeling and control of bioprocesses during the last 20 years. J Biotechnol 85:149
Stärk E, Hitzmann B, Schügerl K, Scheper T, Fuchs C, Köster D, Märkl H (2002) In-situ-fluorescence-probes: a useful tool for non-invasive bioprocess monitoring. Adv Biochem Eng/Biotechnol 74:21
Zupke C, Stephanopoulos G (1995) Intracellular flux analysis in hybridomas using mass balances and in vitro 13C NMR. Biotechnol Bioeng 45:292
Klamt S, Schuster S, Gilles ED (2002) Calculability analysis in underdetermined metabolic networks illustrated by a model of the central metabolism in purple non-sulfur bacteria. Biotechnol Bioeng 77:734
Follstad BD, Balcarcel RR, Stephanopoulos G, Wang DI (1999) Metabolic flux analysis of hybridoma continuous culture steady-state multiplicity. Biotechnol Bioeng 63:675
Europa AF, Gambhir A, Fu PC, Hu WS (2000) Multiple steady states with distinct cellular metabolism in continuous culture of mammalian cells. Biotechnol Bioeng 67:25
Cruz HJ, Moreira JL, Carrondo MJ (1999) Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells. Biotechnol Bioeng 66:104
Zielke HR, Zielke CL, Ozand PT (1984) Glutamine: A major energy source for cultured mammalian cells. Federation Proc 43:121
Zielke HR, Ozand PT, Tildon JT, Sevdalian DA, Cornblath M (1978) Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts. J Cell Physiol 95:41
Zhou WC, Rehm J, Hu WS (1995) High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding. Biotechnol Bioeng 46:579
Hassel T, Gleave S, Butler M (1991) Growth inhibition in animall cell culture: the effect of lactate and ammonia. Appl Biochem Biotechnol 30:29
Ozturk S, Thrift J, Blackie J, Naveh D (1997) Real-time monitoring and control of glucose and lactate concentrations in a mammalian cell perfusion reactor. Biotechnol Bioeng 53:372
Rhiel M, Cohen MB, Murhammer DW, Arnold MA (2002) Non-destructive near-infrared spectroscopic measurement of multiple analytes in undiluted samples of serum-based cell culture media. Biotechnol Bioeng 77:73
Rhiel M, Ducommun P, Bolzonella I, Marison I, von Stockar U (2002) Real-time in situ monitoring of freely suspended and immobilized cell cultures based on mid-infrared spectroscopic measurements. Biotechnol Bioeng 77:174
Bonarius HP, Timmerarends B, de Gooijer CD, Tramper J (1998) Metabolite-balancing techniques vs. 13C tracer experiments to determine metabolic fluxes in hybridoma cells. Biotechnol Bioeng 58:258
Schmidt K, Marx A, de Graaf AA, Wiechert W, Sahm H, Nielsen J, Villadsen J (1998) 13C tracer experiments and metabolite balancing for metabolic flux analysis: comparing two approaches. Biotechnol Bioeng 58:254
Bonarius HP, Schmid G, Tramper J (1997) Flux analysis of underdetermined metabolic networks: the quest for the missing constraints. Trends Biotechnol 15:308
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Goudar, C., Biener, R., Zhang, C., Michaels, J., Piret, J., Konstantinov, K. (2006). Towards Industrial Application of Quasi Real-Time Metabolic Flux Analysis for Mammalian Cell Culture. In: Hu, WS. (eds) Cell Culture Engineering. Advances in Biochemical Engineering/Biotechnology, vol 101. Springer, Berlin, Heidelberg. https://doi.org/10.1007/10_020
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DOI: https://doi.org/10.1007/10_020
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