Metabolic flux analysis of mammalian cells in high cell density perfusion cultures targetting real-time application

The impact of changes in dilution rate and medium composition on the metabolism of CHO cells was studied in high cell density perfusion reactors. Medium composition and dilution rates were varied during the course of the experiment and nutrient and metabolite concentrations were monitored over time. This information was subsequently used in a comprehensive stoichiometric model and metabolic fluxes in the model were computed using the linear least squares method. Our results indicated that exposure to lower nutrient concentrations at reduced dilution rates caused a shift in metabolism towards a more efficient state. Specifically, a 30% increase in the pyruvate flux through the TCA cycle was observed when the dilution rate was reduced to half while a comparable reduction in the pyruvate flux to lactate was also observed with no loss in productivity. Efficient metabolism coupled with lower production of waste metabolites suggest that perfusion reactors can be operated at lower specific perfusion rates resulting in lower medium usage, higher product titer, and increased process simplicity. Current efforts in our laboratory are focussed on real-time implementation of metabolic flux analysis so that information on changes in cellular physiology and metabolism can be obtained in real-time. Copyright (C) 2001 IFAC.