Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells

The present work aims at characterizing the regulatory mechanisms of metabo lism and product formation of BHK cells producing a recombinant antibody/cy tokine fusion protein. This work was carried out through the achievement of several steady-states in chemostat cultures, corresponding to different gl ucose and glutamine levels in the feed culture medium. Results obtained ind icate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on residual glucose and glutamine concentrations, respectively. Similar dependence was also observed for lactate and ammonia productions. K -Glc(Glc) and K-Gln(Gln) were estimated to be 0.4 and 0.15 mM, respectively , while q(Glc)(max) and q(Gln)(max) were estimated to be 1.8 and 0.55 nmol 10(-6) cells min(-1), respectively. At very low glucose concentrations, the glucose-to-lactate yield decreased markedly showing a metabolic shift towa rds lower lactate production; also, the glucose-to-cells yield was increase d. At very low-glutamine concentrations, the glutamine-to-ammonia and gluta mine-to-cells yields increased, showing a more efficient glutamine metaboli sm. Overall, amino acid consumption was increased under low glucose or glut amine concentrations. Metabolic-flux analysis confirmed the metabolic shift s by showing increases in the fluxes of the more energetically efficient pa thways, at low-nutrient concentrations. No effect of glucose or glutamine c oncentrations on the cell-specific: productivity was observed, even under m etabolically shifted metabolism; therefore, it is possible to confine the c ells to a more efficient metabolic state maintaining the productivity of th e recombinant product of interest, and consequently, increasing final produ ct titers by increasing cell concentration and culture length. This work is intended to be a model approach to characterize cell metabolism in an inte grated way; it is highly valuable for the establishment of operating strate gies in mammalian cell Fermentations in which cell metabolism is to be conf ined to a desired state. (C) 1999 John Wiley & Sons, Inc.