The impact of the timing of induction on the metabolism and productivity of CHO cells in culture

• Effects of low and high cell density at induction on the productivity of CHO cells.
• Induced cultures characterized using 13C-substrates and isotopomer analysis.
• Culture productivity correlated with changes in metabolic flux distribution.

Efficient inducible mammalian expression systems are becoming increasingly available and are particularly attractive from a process optimization point of view in that they allow decoupling the growth and production phases. In biphasic processes, the timing of induction is one of the most critical factors to consider for maximizing the productivity, since it will affect both the cumulative biomass concentration and the cell specific productivity. In an effort to assess how these two factors must be balanced for optimal productivity, we have performed a metabolic characterization of CHO cells expressing a recombinant antibody and harboring an efficient inducible expression system. Cells taken at different stages of growth were transferred and induced in fresh medium at their corresponding cell densities, and the kinetics of growth, nutrient consumption and product formation were compared during the production phase. Low cell density inductions achieved lower maximum cell concentrations, but exhibited higher cell specific productivity and greater culture longevity, and ultimately led to increased final product titers. To gain more physiological insights into the observed differences, 13C metabolic flux analysis was performed to characterize and compare the metabolism of cells induced at respectively low and high cell concentrations. A number of key intracellular fluxes were found to be affected by the cell density and the corresponding availability of nutrients during the induction phase. While glucose utilization efficiency is increased in high cell density induction, thereby reducing the specific lactate production rate, it appears to be compensating for the decreased catabolic rates of amino acids. The comprehensive metabolic characterization performed in this work can help guide the development of an efficient feeding strategy pre- and post-induction.