Towards industrial application of quasi real-time metabolic flux analysis for mammalian cell culture.

Cellular physiology and metabolism were monitored using a quasi real-time combination of on-line and off-line data to estimate metabolic fluxes in an established bioreaction network. The utility of this approach towards optimizing bioreactor operation was demonstrated for CHO cells cultivated in 15 L perfusion reactors at 20 x 10(6) cells/mL. Medium composition and dilution rates were changed to obtain several steady states with varying glucose and glutamine concentrations. When cells were restored to initial culture medium and perfusion rate conditions after being exposed to lower glucose and glutamine concentrations, the pyruvate flux into the TCA cycle was increased 30% while the pyruvate flux through lactate was decreased 30%, suggesting steady-state multiplicity. By appropriately altering cellular metabolism, perfusion bioreactors can operate at lower perfusion rates without significant accumulation of inhibitory metabolites such as lactate. Changes in glucose, lactate and glutamine uptake/production rates had significant effects on the calculation of other fluxes in the network. Sensitivity analysis of these key metabolic fluxes highlighted the need for accurate and reliable real-time sensors. Overall, rapid observation of metabolic fluxes can be a valuable tool for bioprocess development, monitoring and control. The framework presented in this study offers a convenient means for quasi real-time estimation of metabolic fluxes and represents a step towards realizing the potential of metabolic flux analysis for accelerated bioprocess optimization.

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