(724d) Investigation of pH-Induced Metabolic Switch By Flux Balance Analysis and Gene Expression Analysis

Mammalian cells, used for the production of large, complex and post-translationally modified proteins, generally rely on glucose and glutamine as main carbon and nitrogen sources and consequently produce lactate and ammonia as waste products. The accumulation of lactate and ammonia results in reduced cell growth in batch and fed-batch culture. Additionally the high production of lactate also demonstrates the inefficient metabolism of mammalian cells marked by the strong dependence on the glycolytic pathway as energy source, thus resulting in lower net energy production [1]. Different bioprocess strategies to reduce lactate production by keeping low glucose concentrations [2], [3] or by feeding alternative carbon sources such as galactose [4], have been described.

Our goal is to systematically study the effect of process parameters (pH, osmolarity, dissolved gases and hydrodynamic forces) on cell metabolism, in particular the metabolic switch from lactate production to lactate consumption. Under pH-shifted culture conditions we observed a switch to lactate consumption at pH 6.8 and an increased lactate production above pH 7.5. For comparison, we also performed glucose-limited cultivations, which also resulted in a switch to lactate consumption. By applying flux balance analysis, we investigated changes in the metabolic pathways due to shifted process conditions and compared them to glucose-limited conditions. Furthermore we also aim to find intracellular triggers of metabolic switches and therefore apply transcriptome analysis to evaluate the over- or underexpression of certain metabolic genes as well as cell and mitochondrial transporter genes. With this we aim to validate our flux balance analysis as well as to identify possible flux constraints.

Finally, the knowledge gained will be used to construct a kinetic metabolic model that is able to describe the effect of operating parameters, in particular pH, as well as glucose limitation on cell metabolism.

[1]      O. Warburg, “On the origin of cancer cells.,” Science (New York, N.Y.), vol. 123, no. 3191, pp. 309–14, Feb. 1956.

[2]      H. J. Cruz, a S. Ferreira, C. M. Freitas, J. L. Moreira, and M. J. Carrondo, “Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture.,” Applied microbiology and biotechnology, vol. 51, no. 5, pp. 579–85, May 1999.

[3]      W. Zhou, J. Rehm, and W. S. Hu, “High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding.,” Biotechnology and bioengineering, vol. 46, no. 6, pp. 579–87, Jun. 1995.

[4]      C. Altamirano, C. Paredes, J. J. Cairó, and F. Gòdia, “Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine.,” Biotechnology progress, vol. 16, no. 1, pp. 69–75.