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-lineand off-line data to estimate metabolic fluxes in an established bioreaction network. The utility of thisapproach towards optimizing bioreactor operation was demonstrated for CHO cells cultivated in 15 Lperfusion reactors at 20 × 106 cells/mL. Medium compositionand 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 tolower glucose and glutamine concentrations, the pyruvate flux into the TCA cycle was increased 30% whilethe pyruvate flux through lactate was decreased 30%, suggesting steady-state multiplicity. By appropriatelyaltering cellular metabolism, perfusion bioreactors can operate at lower perfusion rates without significantaccumulation of inhibitory metabolites such as lactate. Changes in glucose, lactate and glutamine uptake/productionrates had significant effects on the calculation of other fluxes in the network. Sensitivity analysis ofthese key metabolic fluxes highlighted the need for accurate and reliable real-time sensors. Overall, rapidobservation 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 metabolicfluxes and represents a step towards realizing the potential of metabolic flux analysis for acceleratedbioprocess optimization.