Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO2, and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste products derived from glucose. Continuously cultured hybridoma cells are grown in medium containing either 1-13C-, 2-13C-, or 6-13C-glucose. The uptake and production rates of amino acids, glucose, ammonia, O2, and CO2 as well as the cellular composition are measured. In addition, the 13C distribution of the lactate produced and alanine produced by the hybridomas is determined by 1H-NMR spectroscopy, and the 13CO2/12CO2 ratio is measured by on-line mass spectrometry. These data are used to calculate the intracellular fluxes of the glycolysis, the pentose phosphate pathway, the TCA cycle, and fluxes involved in amino acid metabolism. It is shown that: (i) approximately 20␘f the glucose consumed is channeled through the pentose shunt; (ii) the glycolysis pathway contributes the most to lactate production, and most of the CO2 is produced by the TCA cycle; (iii) the pyruvate-carboxylase flux is negligibly small; and (iv) the malic-enzyme flux is estimated to be 10␘f the glucose uptake rate. Based on these flux data suggestions are made to engineer a more efficient glucose metabolism in mammalian cells
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