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  Tools for Metabolic and Enzymatic Characterization of Mammalian Cell

Wahl, A., Ritter, J., Janke, R., & Reichl, U. (2008). Tools for Metabolic and Enzymatic Characterization of Mammalian Cell. Poster presented at ForSys Meeting, Berlin, Germany.

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 Creators:
Wahl, A.1, Author              
Ritter, J.1, Author              
Janke, R.1, Author              
Reichl, U.1, 2, Author              
Affiliations:
1Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society, ou_1738140              
2Otto-von-Guericke-Universität Magdeburg, ou_1738156              

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 Abstract: The metabolic activity of mammalian cells used in production processes for recombinant proteins, antibodies or viral vaccines is not well understood. During growth mammalian cells produce considerable amounts of toxic by-products that inhibit cell growth and prevent high cell densities. An understanding of the metabolic interactions could help to reduce by-product formation and increase cell densities and productivity. Cellular systems regulate metabolic activity through various mechanisms. In our approach we focus on the intracellular concentrations, flux estimations and enzyme activity measurements as these units drive the biochemical reactions. Flux Analysis under different cultivation conditions is applied to characterize the flux phenotypes. Extracellular carbohydrates, organic acids as well as amino acids are measured. A comprehensive reaction network has been developed to estimate the intracellular flux distribution that takes into account compartments and active transport. Of special interest on the metabolome level is the intracellular energy state, the nucleotides ATP, ADP and AMP as well as GXP and UXP. For the anchorage-dependent MDCK cell line a reproducible extraction and measurement protocol has been developed (Ritter et al., 2006 & 2008). For enzyme activity measurements a high throughput platform developed at the MPI for Molecular Plant Physiology (Gibon et al. 2004) is adapted to mammalian cells. The final aim is to model the metabolic network dynamics including allosteric regulatory mechanisms (Wahl et al., 2006), enzyme modifications and protein levels to better understand the intracellular metabolic interactions and to apply the model for process optimization (e.g. medium development, feeding strategies). Gibon, Y.; Blaesing, O.E.; Hannemann, J.; Carillo, P.; Höhne, M.; Hendriks, J.H.M.; Palacios, N.; Cross, J.; Selbig, J. & Stitt, M. A Robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. Plant Cell, 2004, 16, 3304-3325. Ritter, J.B.; Genzel, Y. & Reichl, U. High-performance anion-exchange chromatography using on-line electrolytic eluent generation for the determination of more than 25 intermediates from energy metabolism of mammalian cells in culture. J Chromatogr B, 2006, 843, 216-226. Ritter, J.B.; Genzel, Y. & Reichl, U. Simultaneous extraction of several metabolites of energy metabolism and related substances in mammalian cells: Optimization using Experimental Design. J Anal Biochem, 2008, 373, 349-369 Wahl, S.A.; Haunschild, M.D.; Oldiges, M. & Wiechert, W. Unravelling the regulatory structure of biochemical networks using stimulus response experiments and large-scale model selection. Syst Biol, 2006, 153, 275-285 Wahl, A.; Sidorenko, Y.; Dauner, M.; Genzel, Y. & Reichl, U. Metabolic model for an anchorage-dependent MDCK cell line. Characteristic Growth Phases and Minimum Substrate Consumption Flux Distribution, Biotechnol Bioeng, in press.

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Language(s): eng - English
 Dates: 2008
 Publication Status: Not specified
 Pages: -
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 Table of Contents: -
 Rev. Type: -
 Identifiers: eDoc: 367115
Other: WahlRitterJankeReichl2008a
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Title: ForSys Meeting
Place of Event: Berlin, Germany
Start-/End Date: 2008-06-19 - 2008-06-20

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