One of the most important limitations of mammalian cell-based processes is the secretion and accumulation of lactate as a by-product of their metabolism. Among the cell lines commonly used in industrial bioprocesses, HEK293 has been gaining importance over the last years. Up recently, HEK293 cells were known to consume lactate in late stages of cell culture usually when glucose and/or glutamine were depleted from media. Remarkably, in both scenarios, no significant cell growth was reported. However, we have observed a different metabolic behavior regarding lactate production and consumption in HEK293 cultures. HEK293 cells were able to co-metabolize glucose and lactate simultaneously, even in exponentially growing cell cultures. Our deep study of the effects of environmental conditions on lactate metabolism revealed that pH was the key to trigger the metabolic shift from lactate production to lactate and glucose concomitant consumption. Remarkably, this shift could be triggered at will when pH was set at 6.8. Even more interesting was the fact that lowering pH to 6.6 and supplementing media with exogenous lactate resulted in co-consumption of glucose and lactate from the beginning of cell culture, without affecting cell growth or protein productivity. On the contrary, cell growth was clearly hampered at this low pH if extracellular lactate was lacking. From our results, we hypothesize that HEK293 cells metabolize extracellular lactate as a strategy for pH detoxification, by means of co-transporting extracellular protons together with lactate into the cytosol. This novel hypothesis for unraveling lactate metabolism in HEK293 cells could open a door to re-direct genetic engineering strategies in order to obtain more efficient cell lines and also to further develop animal cell technology applications.

BACKGROUND Compared with other methods, the stationary liquid mass balance method for oxygen uptake rate (OUR) determination offers advantages in terms of estimation accuracy and reduction of stress. However, the need for sophisticated instrumentation, like mass flow controllers and gas analysers, has historically limited wider implementation of such a method. In this paper, a new simplified method based on inexpensive valves for the continuous estimation of OUR in animal cell cultures is evaluated. The determination of OUR values is based on accurate operation of the dissolved oxygen (DO) control loop and monitoring of its internal variables. RESULTS The method developed was tested empirically in 2 L bioreactor HEK293 batch cultures. OUR profiles obtained by a dynamic method, global mass balance method and the developed simplified method were monitored and compared. The results show how OUR profile obtained with the proposed method better follows the off‐line cell density determination. The OUR estimation frequency was also increased, improving the method capabilities and applications. The theoretical rationale of the method was extended to the sensitivity analysis which was analytically and numerically approached. CONCLUSIONS The results showed the proposed method to be not only cheap, but also a reliable alternative to monitor the metabolic activity in bioreactors in many biotechnological processes, being a useful tool for high cell density culture strategies implementation based on OUR monitoring. © 2017 Society of Chemical Industry

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