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.