Investigation of physiological response of CHO cells to pH shifts during a batch fermentation process

Abstract

The usage of Chinese hamster ovary (CHO) cells to produce monoclonal antibodies is very common in the biopharmaceutical industry. Due to this rapidly increasing market, efficient, reproducible and controlled cultivation processes are needed. For better understanding of these processes, it is important to focus on critical process parameters (CPP) that mainly affect cell performance. A CPP that has strong impact on cell cultivation is pH. During large scale fermentation process changes of pH emerge due to alkali addition to the cell culture, either to control pH or to induce a pH shift. Nonetheless, the addition of alkali can lead to irreversible disruptions of the cells, resulting in loss of product quality and/or quantity. To emphasize the impacts of these changes on cell cycle and glucose uptake, batch cultures with varying pH settings were performed. These fermentations included pH shifts from 7.0 to 6.8, 7.2, 7.8 and 9.0 with regards to the aforementioned subjects. Cell cycle analysis is a common task in many biological approaches, but the effect of altered pH environment on the cell cycle progression is not fully investigated yet. A deeper insight into variations of the amount of cells in the three cell cycle phases, G1, S and G2/M, depending on the extracellular pH is what this thesis tries to point out. An assay was developed for glucose uptake by using 2-NBDG as a fluorescent glucose analogue. The amount of glucose taken up by the cells should be represented by the compound. Further, the distribution of the cell population was under investigation to find alterations between low, mid and high consumer of glucose after the pH was shifted. Concerning the cell cycle, a reduction of pH from 7.0 to 6.8 did not resulted in any accumulation of cells in the G1-phase. In contrast, higher pH caused a strongly increased amount of cells in the G1-phase, indicating an arrest of cells. A pH of 7.8 only caused co-occurring limitation of the nutrients glucose and glutamine. This missing of both sources mainly affected cells passing the S-phase. Additionally, concerning the specific uptake of 2-NBDG (Δq2-NBDG) led to alterations at pH 7.8 only, whereas the other pH shifts hold similar values between control and shifted cells. The increased values at pH 7.8 corresponded to the results of the higher specific glucose uptake rate regarding the shifted cells. ii The results did not reveal variations between respective consumers in any fermentation. But during the death phase it could be assumed that amount of low consumer increased in shifted cells and cells at standard conditions. At pH 6.8 the control cells showed an elevated amount of low consumer compared to shifted cells. The change of extracellular pH resulted in long term effects on the cell metabolism. Hence, decelerated or elevated nutrient consumption was detected, but increased product formation was not exhibited.