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Poster

Monitoring Energy Metabolism : The Challenge of Intracellular Metabolite Analysis of Mammalian Cells in Culture

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons86451

Ritter,  J. B.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86303

Genzel,  Y.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons86448

Reichl,  U.
Otto-von-Guericke-Universität Magdeburg;
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Zitation

Ritter, J. B., Genzel, Y., & Reichl, U. (2006). Monitoring Energy Metabolism: The Challenge of Intracellular Metabolite Analysis of Mammalian Cells in Culture. Poster presented at Cell Culture Engineering X, Whistler, Canada.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0013-9A4A-3
Zusammenfassung
Many strategies for optimization in animal cell culture focus on an improvement of growth conditions by either adapting media or fermentation strategies or genetically modifying cell lines. Cellular energy metabolism can be assumed to be one of the key issues for processes with living organisms, however, intracellular fluxes and control of this complex metabolic network is still far from being completely understood. Standard monitoring methods only include the analysis of extracellular metabolites like the waste products lactate and ammonia as well as the substrates glucose and glutamine. Furthermore, monitoring of amino acids in culture broth has become a common analytical technique. However, to gain additional knowledge about the physiological conditions of cells in culture, the analysis of intracellular metabolite pools and fluxes and together with modelling approaches to understand the data seems to be essential. In this work, we are investigating intracellular concentrations of more than 20 intracellular metabolites from central metabolism, including organic acids, sugar phosphates and nucleotides in mammalian cells during growth and after viral infection in a vaccine production process. In particular, we are interested in differences between adherent and suspension cells as well as different media. The analysis is performed using an anion-exchange chromatography system (DX-320, Dionex, Idstein, Germany) with conductivity and UV detection. This method allows the simultaneous detection of intermediates from glycolysis, like glucose-6-phosphate, fructose-1,6-bisphosphate, 3-phosphoglycerate and PEP, from citric acid cycle, e.g. citrate, succinate and malate, but also nucleotides including tri-, di-, monophosphates. Moreover, as important precursors for glycosylation of cellular and viral proteins, some sugar-phosphate levels can be measured. Sample preparation turned out to need thorough investigation to confirm quality of results. The analysis of intracellular metabolites of especially adherent cells poses several problems. Various sample preparation procedures have been tested. To verify measured concentrations, ATP, as an important intracellular metabolite, was chosen to be assayed with an alternative method in parallel. ATP concentrations determined by chromatography are cross-checked by using a luciferin/luciferase luminescence assay (Perkin Elmer, Boston, US). By using chromatograms as a fingerprint for anionic, extracted metabolic intermediates, further information can be gained, since several unknown peaks additionally show a dynamic behaviour during cultivation. The increase or decrease of a certain peak or peaks, even though unknown, might indicate limitations or metabolic changes and thus be used for controlling fermentations.