Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [13C, 1H] COSY NMR spectroscopy.

The physiological state of CHO cells in perfusion culture was quantified by determining fluxes through the bioreaction network using (13)C glucose and 2D-NMR spectroscopy. CHO cells were cultivated in a 2.5L perfusion bioreactor with glucose and glutamine as the primary carbon and energy sources. The reactor was inoculated at a cell density of 8 x 10(6)cells/mL and operated at approximately 10 x 10(6)cells/mL using unlabeled glucose for the first 13 days. The second phase lasted 12 days and the medium consisted of 10% [U-(13)C]glucose, 40% labeled [1-(13)C]glucose with the balance unlabeled. After the culture attained isotopic steady state, biomass samples from the last 3 days of cultivation were considered representative and used for flux estimation. They were hydrolyzed and analyzed by 2D [(13)C, (1)H] COSY measurements using the heteronuclear single quantum correlation sequence with gradients for artifacts suppression. Metabolic fluxes were determined using the 13C-Flux software package by minimizing the residuals between the experimental and the simulated NMR data. Normalized residuals exhibited a Gaussian distribution indicating good model fit to experimental data. The glucose consumption rate was 5-fold higher than that of glutamine with 41% of glucose channeled through the pentose phosphate pathway. The fluxes at the pyruvate branch point were almost equally distributed between lactate and the TCA cycle (55% and 45%, respectively). The anaplerotic conversion of pyruvate to oxaloacetate by pyruvate carboxylase accounted for 10% of the pyruvate flux with the remaining 90% entering the TCA cycle through acetyl-CoA. The conversion of malate to pyruvate catalyzed by the malic enzyme was 70% higher than that for the anaplerotic reaction catalyzed by pyruvate carboxylase. Most amino acid catabolic and biosynthetic fluxes were significantly lower than the glycolytic and TCA cycle fluxes. Metabolic flux data from NMR analysis validated a simplified model where metabolite balancing was used for flux estimation. In this reduced flux space, estimates from these two methods were in good agreement. This simplified model can routinely be used in bioprocess development experiments to estimate metabolic fluxes with much reduced analytical investment. The high resolution flux information from 2D-NMR spectroscopy coupled with the capability to validate a simplified metabolite balancing based model for routine use make (13)C-isotopomer analysis an attractive bioprocess development tool for mammalian cell cultures.