Large-scale pH heterogeneities and accumulated CO2 : investigating their impact on CHO cells with small-scale systems

Abstract

The highly sensitive mammalian cell cultures set limits in terms of tolerable fluid dynamic stress conditions, mostly caused by agitation and aeration in large-scale reactors. The resulting prolonged mixing times create pH gradients due to addition of base from top of the bioreactor and the bad CO2 removal capacity in large-scale can lead to CO2 accumulation in the fermentation broth. These heterogeneities of important fermentation parameters might seriously affect the processes of recombinant protein production by mammalians, regarding cell physiology and most importantly product quantity and its critical quality attributes. Accordingly, the goal of this master thesis was to investigate the impact of these large-scale pH heterogeneities and CO2 accumulation on Chinese hamster ovary cells` physiology and overall process performance. These two scale up phenomena were simulated in separate fed-batch experiments. For applying (i) pH perturbations, a two-compartment system was established and characterized. The (ii) CO2 simulations were realized by elevated pCO2 set-points in a single bioreactor system. (i) The control strategy including the desired mixing respectively residence times in the two-compartment system fulfilled the prior declared requirements, thus, making the results reliable. The pH shots in the two-compartment system with an amplitude of around 9.06 occurring every 3 to 9 h severely affected cell growth and viability, especially in the first phase of exponential cell growth. Further, the metabolism of the waste products lactate and ammonium were impaired, meaning, they were not consumed during fermentation. (ii) For the investigation of the impact of accumulated CO2 two pair of fermentations were performed. Both runs with elevated pCO2 set-points, were lacking the usual metabolic shift from lactate production to consumption compared to control runs. Additionally, the growth performance and specific productivity were affected, hence, leading to a lower monoclonal antibody product titer in the fermentation broth. It could be shown for the first time, that pH heterogeneities in large-scale processes could have a huge impact on the specific growth and productivity of Chinese hamster ovary cells. Accumulated CO2 is additionally leading to changes in metabolism. These important findings have to be considered during the development of industrial processes.