Probing the metabolism of an inducible mammalian expression system using extracellular isotopomer analysis

In an effort to quantitatively assess the impact of recombinant protein expression on the primary metabolism of mammalian cells in culture, we have employed an efficient inducible expression system and conducted a comparative study of the intracellular flux map distribution with and without the induction of recombinant protein synthesis. Cells were grown in parallel semi-continuous cultures with various singly and uniformly labeled substrates and the resulting mass isotopomer distributions of lactate and extracellular amino acids were measured by mass spectrometry. These distributions were used to quantify the main intracellular fluxes. The analysis revealed that, under mild hypothermic conditions, the onset of protein expression is correlated with small but significant changes in several key pathways related to ATP and NADPH formation. More specifically, we observed that induced cells exhibit a more efficient utilization of glucose, characterized by an increased flux of pyruvate into the TCA cycle. In contrast, the catabolic rates of most amino acids remained relatively unaffected. Such analysis is instrumental to guide the identification of robust biomarkers of productivity, as well as the development of medium formulations optimized for recombinant protein production.