Inclusion Of Maintenance Energy Improves The Intracellular Flux Predictions Of Cho

Chinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by parsimonious flux balance analysis (pFBA) using the CHO GSMM and a wide range of C-13 metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.Author summary There is an increasing demand for protein pharmaceuticals, especially monoclonal antibodies. Chinese Hamster Ovary (CHO) are currently the leading production host due to their ability to perform human-like post-translational modifications. However, it typically takes several months of trial-and-error approaches to develop a high-producer cell line. Metabolic modelling has the potential to make cell line and process development faster and cheaper by predicting targeted modifications to the cell line genome, cultivation medium or bioprocess. In fact, genome-scale metabolic reconstructions of CHO are already available, and ready for use in cell line development. However, in order to successfully use these models, we need to make sure that they are able to accurately predict metabolic phenotypes. Here we use genome-scale metabolic models of CHO to evaluate the models' ability to correctly predict intracellular flux distributions. We find that a crucial key ingredient for the correct estimation of central carbon fluxes is the non-growth associated maintenance energy (mATP). We estimated mATP computationally and confirmed it experimentally. Adding this single constraint leads to significantly better predictions of intracellular fluxes, especially in glycolysis and the tricarboxylic acid cycle.