English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Changes in specific enzyme activities of MDCK cells during growth under different cultivation conditions

Janke, R., Genzel, Y., Wahl, A., & Reichl, U. (2010). Changes in specific enzyme activities of MDCK cells during growth under different cultivation conditions. Poster presented at 8th European Symposium on Biochemical Engineering Science (ESBES), Bologna, Italy.

Item is

Basic

show hide
Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0013-8F1B-F Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0025-1B54-D
Genre: Poster

Files

show Files

Locators

show

Creators

show
hide
 Creators:
Janke, R.1, Author              
Genzel, Y.1, Author              
Wahl, A.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              

Content

show
hide
Free keywords: -
 Abstract: Various producer cell lines used for large scale production of biopharmaceuticals like monoclonal antibodies, hormones or viral vaccines often show inefficient use of nutrients for growth. They have a very high demand in substrates, mainly glucose and glutamine, which results in release of large amounts of lactate and ammonium. This metabolic imbalance often not only decreases cell productivity and viability but also inhibits growth to high cell densities. Thus, new strategies including optimization of cultivation conditions, design of media and modification of specific properties of cells by systems biology approaches should be developed to increase cell densities and process yields. For the growth of adherent MDCK cells distinct differences in glycolysis and tricarboxylic acid cycle were found for extra- and intracellular metabolite concentrations and metabolic fluxes when comparing the influence of glutamine or pyruvate addition to cell culture media [1-4]. The objective of this study was to further elucidate these differences by establishing a high-throughput platform for enzyme activity measurements of mammalian cells [5]. This method, which uses four cycling assays, allowed the determination of 28 key enzyme activities of central carbon metabolism in extracts of MDCK cells. Whereas, a comparison of Pyr and Gln-containing medium showed an up-regulation of glutamine synthetase activity by a factor of 4, the activity of glutaminase was down-regulated by a factor of 6 in GMEM medium. Decreased activities were also found for ATP-citrate lyase, phosphoenolpyruvate carboxykinase and the glutaminolytic enzymes aspartate- and alanine transaminase with Pyr as carbon source. Under all conditions, very low activities of pyruvate dehydrogenase and pyruvate carboxylase were measured and confirmed by comparing normal mouse kidney tissue with MDCK cells. Other enzymes like lactate dehydrogenase (LDH) and malate dehydrogenase showed less distinct changes in activity. In all media the activities of the glycolytic enzymes hexokinase and phosphofructokinase were relatively low, whereas maximum activities of LDH and pyruvate kinase were comparatively high. In contrast, most enzyme activities measured in serum-free Episerf medium were increased. Based on the established assay metabolic states of production cell lines can now be further characterized. This can then be used to validate mathematical models of cellular metabolism and to improve our understanding of intracellular metabolic interactions relevant for process characterization and optimization. [1] Genzel, Y., Ritter, J.B., König, S., Alt, R. and Reichl, U. 2005. Biotechnol. Prog., 21, 58-69. [2] Ritter, J.B., Genzel, Y. and Reichl, U. 2006. Journal of Chromatography B, 843, 216-226. [3] Sidorenko, Y., Wahl, A., Dauner, M., Genzel, Y. and Reichl, U. 2008. Biotechnol Prog, 24(2), 311-320. [4] Wahl, A., Sidorenko, Y., Dauner, M., Genzel, Y. & Reichl, U. 2008. Biotechnology and Bioengineering, 101(1), 135-152. [5] Janke, R., Genzel, Y., Wahl, A & Reichl, U. 2010. Metabolic Engineering, submitted.

Details

show
hide
Language(s): eng - English
 Dates: 2010
 Publication Status: Not specified
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: -
 Identifiers: eDoc: 494457
 Degree: -

Event

show
hide
Title: 8th European Symposium on Biochemical Engineering Science (ESBES)
Place of Event: Bologna, Italy
Start-/End Date: 2010-09-06 - 2010-09-08

Legal Case

show

Project information

show

Source

show