Intracellular pH monitoring as a tool for the study of hybridoma cell behavior in batch and continuous bioreactor cultures

We show in this paper the results obtained when studying the behavior of hybridoma cells by monitoring of the pH(i) during batch and continuous bioreactor cultures. A first set of experiments, consisting of a batch culture and a continuous culture at variable dilution rate, was set up under normal physiological operating conditions. Significant pH(i) variations were measured during these cultures. For the batch culture, maximal pH(i) values around 7.60 were associated with the middle of the growth phase, while lower values were found in the culture beginning and during the decay phase, respectively 7.47 and 7.40. For the continuous culture, pH(i) increased with increasing dilution rate, ranging between 7.40 and 7.60 for dilution rates between 0.010 and 0.040 h(-1). These pH(i) variations were found for both cultures to be linked to variations of the specific growth rate of the cells. The observed link between pH(i) and cell growth provided us a general framework for the study of the effect of suboptimal operating conditions on cell behavior, all having a particular interest in animal cell culture technology, First, our results indicate that a decrease of the medium pH of its normal value of 7.00 to 6.70 did not necessarily result in cytoplasmic acidification, at least not after prolonged exposition times in continuous culture, and this despite a pronounced growth inhibitory effect. This effect can therefore rather be explained by the combination of the increased demand for maintenance energy associated with the higher Delta pH gradient maintained across the cell membrane and the decreased supply of energy, in particular via the glucose metabolism. Second, our results indicate that also increased ammonium ion concentrations do not lower pH(i). This observation, together with the results of batch cultures carried out at a more alkaline pH(i) indicates that the form NH3, and not the form NH4+, has a negative effect on cell growth of our hybridoma cell line. Third, in the case of an osmolality increase, a significant pH(i) increase was observed, with mean values of 7.35 at the lower osmolalities 335 and 370 mOsm/kg and 7.45 at the higher osmolalities of 400, 425, and 450 mOsm/kg. This higher pH(i) might account at least partially for the increased monoclonal antibody production observed at hyperosmolality and coincided furthermore with a faster glutamine consumption rate.