Investigation of mammalian cell metabolism by quantification of key metabolic enzyme activities

Beschreibung

During growth of mammalian cells in glutamine-containing media, large amounts of lactate and ammonia are secreted into the culture supernatant. These toxic by-products not only affect cell viability and productivity but often also prevent groth to high cell densities. However, different production cell lines typically used for manufacturing of biopharmaceuticals and viral vaccines can also grow on pyruvate (Pyr) instead of glutamine (Gln) in the culture medium. As a consequence, these cells not only release no ammonia but glucose (Gluc) consumption and lactate production are also reduced significantly. One possibility to better understand cell metabolism and the cellular regulatory mechanisms that govern cell growth is to perform enzyme activity studies. Although the analysis of enzymes is more complex and high-throughput technologies for enzyme activities are not commonly available, enzyme activities provide information on pathway flux rates, which is important for the understanding of metabolic networks. Furthermore, the integration of enzyme activity data into mathematical models of cell metabolism together with proteome and metabolome data by systems biology approaches will significantly contribute towards a better understanding of metabolic pathways relevant for cell line and media optimization. The objective of this thesis was to characterize the effect of different culture conditions on key enzyme activities of central metabolic pathways in the Madin-Darby canine kidney (MDCK) production cell line. On the basis of a high-throughput platform for measuring enzyme activities in plant cell extracts, the work first involved the development of a platform to determine enzyme activities in mammalian cells including grouping of various enzymes that share a common detection method.

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Robert Janke

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MDCK enzymatic cycling enzyme activities metabolism

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