Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells

Abstract

The present work aims at characterizing the regulatory mechanisms of metabolism and product formation of BHK cells producing a recombinant antibody/cytokine fusion protein. This work was carried out through the achievement of several steady‐states in chemostat cultures, corresponding to different glucose and glutamine levels in the feed culture medium. Results obtained indicate 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 GlcGlc and K GlnGln were estimated to be 0.4 and 0.15 mM, respectively, while q maxGlc and q maxGln were estimated to be 1.8 and 0.55 nmol 10−6cells min−1, respectively. At very low glucose concentrations, the glucose‐to‐lactate yield decreased markedly showing a metabolic shift towards lower lactate production; also, the glucose‐to‐cells yield was increased. At very low‐glutamine concentrations, the glutamine‐to‐ammonia and glutamine‐to‐cells yields increased, showing a more efficient glutamine metabolism. Overall, amino acid consumption was increased under low glucose or glutamine concentrations. Metabolic‐flux analysis confirmed the metabolic shifts by showing increases in the fluxes of the more energetically efficient pathways, at low‐nutrient concentrations. No effect of glucose or glutamine concentrations on the cell‐specific productivity was observed, even under metabolically shifted metabolism; therefore, it is possible to confine the cells to a more efficient metabolic state maintaining the productivity of the recombinant product of interest, and consequently, increasing final product titers by increasing cell concentration and culture length. This work is intended to be a model approach to characterize cell metabolism in an integrated way; it is highly valuable for the establishment of operating strategies in mammalian cell fermentations in which cell metabolism is to be confined to a desired state. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 104–113, 1999.

Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production

“…YLac/Glc varies between 1.8 y 2.1 mol mol-1, while YAmm/Gln varied between 1.05 a 1.09 mol mol-1 revealing the highly inefficient use of these nutrients. YLac/Glc between 1.6 a 1.8 mol mol-1 and YAmm/Gln around 0.9 mol mol-1 have been reported for several cell lines (Xie and Wang, 1996;Cruz et al 1999;Lee et al 2003) grown on serum-based media, which can explain the difference with the values here reported. However, YLac/Glc for hybridoma cells grown on serum-free media are higher than those obtained in serum-based medium (Hiller et al 1994), which is the same tendency observed in this case with CHO cells.…”

Cell Culture Processes in Monoclonal Antibody Production

“…[4][5][6][7][8] The selection of expression system is determined by its ability to deliver high productivity with acceptable product quality attributes and the preferences of individual companies, which is often influenced by their historical experiences.…”

Modelling of Mammalian Cells and Cell Culture Processes

“…One key advantage of stoichiometric models is that they can account for competing reactions, which is useful for studying the relative activity of certain pathways under various culture conditions and this has been their major use. For example, the existence of more than one physiological steady state, known as steady-state multiplicity, has been observed by a number of investigators using stoichiometric models and metabolic flux analysis (Follstad et al 1999;Europa et al 2000;Cruz et al 1999;Zhou et al 1997;Linz et al 1997;Paredes et al 1999).…”