Metabolic-flux analysis of continuously cultured hybridoma cells using (CO2)-C-13 mass spectrometry in combination with C-13-lactate nuclear magneticresonance spectroscopy and metabolite balancing

Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO2, and ammonia. In this study, the in tracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste pr oducts derived from glucose. Continuously cultured hybridoma cells are grow n in medium containing either 1-C-13-, 2-C-13-, or 6-C-13-glucose. The upta ke and production rates of amino acids, glucose, ammonia, O-2, and CO2 as w ell as the cellular composition are measured. In addition, the C-13 distrib ution of the lactate produced and alanine produced by the hybridomas is det ermined by H-1-NMR spectroscopy, and the (CO2)-C-13/(CO2)-C-12 ratio is mea sured by on-line mass spectrometry. These data are used to calculate the in tracellular fluxes of the glycolysis, the pentose phosphate pathway, the TC A cycle, and fluxes involved in amino acid metabolism. It is shown that: (i ) approximately 20% of the glucose consumed is channeled through the pentos e shunt; (ii) the glycolysis pathway contributes the most to lactate produc tion, 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 est imated to be 10% of the glucose uptake rate. Based on these flux data sugge stions are made to engineer a more efficient glucose metabolism in mammalia n cells. (C) 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 528-538, 20 01.