(462a) Applications of ‘Omics Technologies for Enhanced Process Robustness in a Chemically Defined Fed-Batch Industrially Relevant CHO Cell Culture Process

Authors: 
McElearney, K., Biogen
Gilbert, A., Biogen
Krishnan, S., Tufts University
Alden, N., Tufts University
Lee, K., Tufts University
Kshirsagar, R., Biogen
We have previously reported on an undesirable high lactate phenotype, caused by overfeeding a mAb-producing CHO cell line, in a chemically-defined process. To better understand this phenomenon, we utilized metabolic flux analysis (MFA) using isotopic labeling with multiple tracers (glucose and glutamate) and untargeted metabolomics. The results from these analyses provided new insights into our cell culture process. We first identified two supplements capable of delaying the onset of the high lactate phenotype by ~3 days. The addition of the first supplement increased the peak viable cell density by 15% and harvest titer by 25%. The second supplement increased both peak viable cell density and harvest titer by 25%. Following this observation, untargeted metabolomics was performed to investigate the mechanism of action for these supplements by comprehensively characterizing the impact of the supplements on the levels of intracellular and extracellular metabolites. Focusing on central carbon metabolism, we conducted isotopic labeling experiments with glucose and glutamate tracers, and analyzed the results using MFA to characterize the fluxes in and around the TCA cycle. We identified overflow of malate and citrate from the mitochondria as a potential mechanism underlying growth inefficiency, which was attenuated by the two medium supplements. In addition, we assessed the redox potential in our cell culture, identified supplements capable of preventing a redox imbalance, and confirmed their ability to delay the high lactate phenotype in our production culture. Utilizing the insight gained through our systems biology approach, we evaluated the proposed mechanism of action by reformulating our nutrient feed to implement the inhibition of mitochondrial metabolite overflow independent of the two supplements.