hide
Free keywords:
-
Abstract:
Animal cell culture processes are needed for the production of monoclonal antibodies, recombinant proteins and viral vaccines. To increase the production yields genetic modifications of the cell lines or improvement of the cultivation conditions are needed. A systematic knowledge of control and capacity of cellular metabolism can be applied for directed modifications. In addition to the measurement of extra- and intracellular metabolites (Ritter et al., 2006), metabolic flux analysis (Sidorenko et al., 2008) and monitoring of key enzyme activities provide functional insights into cellular metabolism and product formation.
In order to measure enzyme activities a high-throughput platform (Gibon et al., 2004) was adapted to adherent cells. Enzymatic cycling systems allow to determine low amount of product formed or substrate consumed. NAD(H) that is a product/substrate in many reactions can be amplified by a cycling reaction, that couples alcohol dehydrogenase to phenazine ethosulfate. Due to the continuous reoxidation of NAD, multiple electrons can be transferred to the tetrazolium salt MTT by theoretically one molecule to form colored insoluble formazan. With a set of cycling assays enzyme activities of glycolysis, gluconeogenesis, tricarboxylic acid cycle, glutaminolysis and the pentose phosphate pathway could be measured in cell extracts of an anchorage-dependent MDCK cell line.
The platform will now be applied to study enzyme activities under different cultivation conditions (e.g. glutamine- and pyruvate-containing growth medium). Differences have already been observed by flux analysis (Genzel et al., 2005; Sidorenko et al., 2008). Furthermore, the data obtained by enzyme activity measurements will be used to generate mathematical models of cellular metabolism.
Genzel, Y., Ritter, J.B., König, S., Alt, R. and Reichl, U. 2005. Substitution of Glutamine by Pyruvate To Reduce Ammonia Formation and Growth Inhibition of Mammalian Cells. Biotechnol. Prog., 21, 58-69.
Gibon, Y., Blaesing, O. E., Hannemann, J., Carillo, P., Hohne, M., Hendriks, J. H. M., Palacios, N., Cross, J., Selbig, J. and Stitt, M. 2004. 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. The Plant Cell, 16, 3304-3325.
Ritter, J.B., Genzel, Y. and Reichl, U. 2006. 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, 216-226.
Sidorenko, Y., Wahl, A., Dauner, M., Genzel, Y. and Reichl, U. 2008. Comparison of Metabolic Flux Distributions for MDCK Cell Growth in Glutamine- and Pyruvate-Containing Media. Biotechnol. Prog., 24 (2), 311-320.
