Team Leader (USP)

PD Dr. Yvonne Genzel
PD Dr. Yvonne Genzel
Phone: +49 391 6110 257
Room: N0.18

Researchers

Dipl.-Biol. (t.o.) Thomas Bissinger
Dipl.-Biol. (t.o.) Thomas Bissinger
Phone: +49 391 6110 131
Room: N 0.07

Additional Information

External collaboration

Dr. Markus Rehberg & Dr. Paulus Wohlfart, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany

Prof. Dr. Leda Castillo, Federal University of Rio de Janeiro, Cell Culture Engineering Laboratory, Rio de Janeiro, Brazil

Prof. Alan Dickson (PhD), University of Manchester, Institute of Biotechnology, Manchester, United Kingdom

Prof. Dr. Jan Korvink, Karlsruhe Institute of Technology, Institute of Microstructure Technology, Karlsruhe, Germany

East China University (Dr. Liu)

Valneva

Metabolic Profiling of Mammalian Cell Culture Systems

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Metabolic Profiling of Mammalian Cell Culture Systems

Motivation

Mammalian cell culture has grown to one of the major production systems for biopharmaceuticals over the last decades. Despite this huge interest of both industry and academia, relatively little is known about the overall regulation linking cell metabolism, cell growth, and productivity. In many cases, modeling approaches like MFA or DOE relying on steady state assumptions are used to describe and predict specific limitations that could decrease cell growth or productivity. A much deeper understanding of cell metabolism can be achieved by dynamic measurements of intracellular metabolite pools, which describe changes in the physiological state of cells over the time course of cultivations. This knowledge is a valuable research tool to understand the function of metabolic pathways within the metabolic network, to investigate changes in energy and precursor pools, and to identify possible bottlenecks for growth and product formation. Using this information, a rational optimization strategy for the improvement of product yields (metabolic engineering) can be performed.

Aim of the project

Highly developed sample preparation methods are crucial for an accurate measurement of intracellular metabolites. In particular, the shutdown of cell metabolism (quenching) and the subsequent isolation of metabolites from the cell extract (extraction) need to be optimized to obtain a detailed snapshot of metabolite concentrations. In a next step, for simultaneous quantification of specific metabolic targets, the extracted metabolites are separated due to their charge and size employing anion exchange chromatography. Compounds are detected via conductivity, UV and MS and matched with a calibration standard to finally calculate compound concentrations.

Fig. 1 Scheme of the experimental process for the quantification of intracellular metabolites. Zoom Image
Fig. 1 Scheme of the experimental process for the quantification of intracellular metabolites.

Metabolic profiling of suspension cells

Since high-cell-density suspension cell cultures are of high interest both for protein and virus production, monitoring of metabolic changes is crucial for process design and optimization. To investigate intracellular dynamics of metabolites in high-cell-density, quenching and extraction procedures established for adherent cells or described elsewhere in literature were adapted or modified.

Metabolic response to viral infection

Viral infection of mammalian cells can have a significant effect on various metabolic pathways leading to an alteration in cell growth and morphology. Changes in metabolic pathways of infected cells can lead to increased substrate consumption and by-product formation. Metabolic profiling can help to locate intracellular changes in the metabolic network and identify possible metabolic bottlenecks. Appropriate media supplementation or feeding strategies could then be applied to avoid nutrition limitation during virus production.

Fig. 2 Reaction network of the central carbon metabolism of adherent MDCK cells. Measured metabolites are highlighted in grey (Modified from Wahl et. al., 2008). Zoom Image
Fig. 2 Reaction network of the central carbon metabolism of adherent MDCK cells. Measured metabolites are highlighted in grey (Modified from Wahl et. al., 2008). [less]

References

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. Journal of Chromatography B 843 (2), pp. 216 - 226 (2006)
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. Analytical Biochemistry 373, pp. 349 - 369 (2008)
Rehberg, M.; Ritter, J. B.; Genzel, Y.; Flockerzi, D.; Reichl, U.: The relation between growth phases, cell volume changes and metabolism of adherent cells during cultivation. Journal of Biotechnology 164 (4), pp. 489 - 499 (2013)
Rehberg, M.; Ritter, J.; Reichl, U.: Glycolysis Is Governed by Growth Regime and Simple Enzyme Regulation in Adherent MDCK Cells. PLoS Computational Biology 10 (10), p. e1003885 (2014)
Kluge, S.; Benndorf, D.; Genzel, Y.; Scharfenberg, K.; Rapp, E.; Reichl, U.: Monitoring changes in proteome during stepwise adaptation of a MDCK cell line from adherence to growth in suspension. Vaccine 33 (35), pp. 4269 - 4280 (2015)
 
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