(133d) Proteomic and Metabolic Flux Analysis of Recombinant CHO Cell Cultures

Chinese Hamster Ovary (CHO) cells cultures are the major source for producing therapeutic proteins that require post-translational modification. The cell culture process can dramatically influence the cell culture performance, including cell growth, protein yield and quality. In this presentation, we report fluxes under two process conditions that yield different lactic acid profiles.  In one process, the lactic acid production was highest during the growth phase of the cell culture, and decreased as the cell culture progressed. Later, it changed to lactate consumption around mid stationary phase, but then changed to lactate excretion during the death phase. In contrast, the second process had lower lactic acid production during growth phase and complete reutilization during the stationary phase. Fluxes were evaluated for the two processes using ¹³C-MFA using isotopically labeled glucose. The lower lactic acid producing process was found to have lower flux towards the pentose phosphate pathway as compared to higher lactic acid producing process. Further, oxygen uptake profiles and TCA cycle fluxes for the two processes indicated that for the higher lactic acid producing process, the mitochondrial capacity was limited as compared to the glucose uptake.  An untargeted proteomic analysis was also performed to understand the causes of the underlying shifts in these fluxes and lactic acid production profiles. Partial least squares analysis was used to correlate the changes in metabolic proteins to key fluxes and split ratios. Some key proteins were identified that were correlated to the changing flux profiles. For the high lactic acid production process, the changes in the protein profiles related to mitochondrial function (Pyruvate dehydrogenase, alpha- keto dehydrogenase) and ATP production were correlated to the TCA cycle flux and lactic acid production. The lactate dehydrogenase expression pattern was not correlated or had a minor effect on lactic acid production flux, in both processes. This indicated the control of the lactic acid production, was allosteric and dependent on the substrate availability.  Further, for both the processes, proteins that play a role in mitochondrial oxidative stress and oxidative stress in general were found to be correlated to the flux profiles.  This indicated that the proteins that affect redox potential and energy charge of the cells play a role in changing flux profiles over the cell culture.