Metabolic control at the cytosol–mitochondria interface in different growth phases of CHO cells |
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| Institution: | 1. Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstraße 1, D-39106 Magdeburg, Germany;2. Insilico Biotechnology AG, Meitnerstraße 8, D-70563 Stuttgart, Germany;3. European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SD, Cambridge, United Kingdom;1. Product Attribute Sciences, Amgen, Inc. , One Amgen Center Drive, Thousand Oaks, CA 91320, USA;2. Drug Substance Development, Amgen, Inc. , One Amgen Center Drive, Thousand Oaks, CA 91320, USA;3. Drug Substance Development, Amgen, Inc. , 1201 Amgen Court West, Seattle, WA 98119, USA;1. Waterloo Institute for Nanotechnology, Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada;2. Department of Biology, University of Waterloo, Waterloo, Ontario, Canada;3. Institute of Applied Biotechnology, University of Applied Sciences Biberach, Biberach, Germany;4. Biotechnology Research Institute, National Research Council of Canada, Montreal, QC, Canada;5. Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada;6. Chenomx Inc., Edmonton, Alberta, Canada;1. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, Queensland, Australia;2. ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria;3. Institute for Applied Microbiology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria;1. University of Applied Sciences Biberach, Institute of Applied Biotechnology, Hubertus-Liebrecht-Straße 35, 88400 Biberach, Germany;2. Boehringer Ingelheim Pharma GmbH & Co KG, Cell Culture Development CMB, Birkendorfer Straße 65, 88397 Biberach, Germany;3. Boehringer Ingelheim Pharma GmbH & Co KG, Early Stage Bioprocess Development, Birkendorfer Straße 65, 88397 Biberach, Germany |
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| Abstract: | Metabolism at the cytosol–mitochondria interface and its regulation is of major importance particularly for efficient production of biopharmaceuticals in Chinese hamster ovary (CHO) cells but also in many diseases. We used a novel systems-oriented approach combining dynamic metabolic flux analysis and determination of compartmental enzyme activities to obtain systems level information with functional, spatial and temporal resolution. Integrating these multiple levels of information, we were able to investigate the interaction of glycolysis and TCA cycle and its metabolic control. We characterized metabolic phases in CHO batch cultivation and assessed metabolic efficiency extending the concept of metabolic ratios. Comparing in situ enzyme activities including their compartmental localization with in vivo metabolic fluxes, we were able to identify limiting steps in glycolysis and TCA cycle. Our data point to a significant contribution of substrate channeling to glycolytic regulation. We show how glycolytic channeling heavily affects the availability of pyruvate for the mitochondria. Finally, we show that the activities of transaminases and anaplerotic enzymes are tailored to permit a balanced supply of pyruvate and oxaloacetate to the TCA cycle in the respective metabolic states. We demonstrate that knowledge about metabolic control can be gained by correlating in vivo metabolic flux dynamics with time and space resolved in situ enzyme activities. |
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| Keywords: | Mammalian cell culture CHO Compartmentation Enzyme activities Metabolic flux analysis Mitochondria Metabolic switches |
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