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 13C-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. (c) 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:535-546, 2014