Elucidating the effects of postinduction glutamine feeding on the growth and productivity of CHO cells

Abstract

Inducible mammalian expression systems are increasingly being used for the production of valuable therapeutics. In such system, maximizing the product yield is achieved by carefully balancing the biomass concentration during the production phase and the specific productivity of the cells. These two factors are largely determined by the availability of nutrients and/or the presence of toxic waste metabolites in the culture environment. Glutamine is one of the most important components of cell culture medium, since this substrate is an important building block and source of energy for biomass and recombinant protein production. Its metabolism, however, ultimately leads to the formation of ammonia, a well known inhibitor of cellular growth and productivity. In this work, we show that nutrient feeding post-induction can greatly enhance the product yield by alleviating early limitations encountered in batch. Moreover, varying the amount of glutamine in the feed yielded two distinct culture behaviors post-induction; whereas excess glutamine allowed to reach greater cell concentrations, glutamine-limited fed-batch led to increased cell specific productivity. These two conditions also showed distinctive lactate metabolism. To further assess the physiological impact of glutamine levels on the cells, a comparative (13) C-metabolic flux analysis was conducted and a number of key intracellular fluxes were found to be affected by the amount of glutamine present in the feed during the production phase. Such information may provide useful clues for the identification of physiological markers of cell growth and productivity that could further guide the optimization of inducible expression systems.

Mechanisms driving the lactate switch in Chinese hamster ovary cells

“…(d) The H + concentration inside the cell is reduced. This is supported by metabolic profiling data which show accumulation of alanine(Sheikholeslami, Jolicoeur, & Henry, 2014;Zhang, Shen, & Zhang, 2004) which sometimes undergoes its own metabolic shift similar to that of lactate(Ma et al, 2009). Lactate is transported alongside protons into the cell, despite a lactate gradient not being present.…”

Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes

“…D the osmolality values of the control cultures and cultures at elevated pCO 2 started to diverge after the metabolic shift and increased osmolality is known to potentially affect cell metabolism. Since glutamine metabolism has been shown before to correlate with the lactate metabolic shift in the batch cultivations and other studies and furthermore ammonia metabolite profiles were strongly different between cultures at low and high pCO₂, the role of glutamine was investigated in more detail. Differences in the ammonia profiles could be directly linked to variations in the specific glutamine consumption rate.…”

Integrated 13 C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli

“…Studies have shown that COMPLETE-MFA can significantly improve not only flux precision but also flux observability in complex network models (Crown and Antoniewicz, 2013a). Parallel labeling experiments are also increasingly used for validation of metabolic network models, including in microbial (Au et al, 2014; Leighty and Antoniewicz, 2012), mammalian (Ahn and Antoniewicz, 2013; Sheikholeslami et al, 2014), and plant systems (Allen and Young, 2013; Alonso et al, 2007; Schwender et al, 2006). …”