Inclusion of maintenance energy improves the intracellular flux predictions of CHO

Abstract

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 pFBA using the CHO GSMM and a wide range of 13C 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.

How reliable are Chinese hamster ovary (CHO) cell genome‐scale metabolic models?

“…Firstly, it may be a result of the use of a generic biomass equation, which did not effectively capture the true biomass composition of cells resulting in an underprediction of fluxes. It may also be due to the lack of an ATP maintenance constraint, which has been shown to improve intracellular flux predictions (Széliová, Štor, et al, 2020 ). Finally, it may be indicative of the “metabolic efficiency” of these models compared to in vivo metabolism, meaning lower flux values through core reactions are able to satisfy the model constraints.…”

“…To compare CHO cell model predictions with experimental flux data, the 13 C‐labeled reactions were mapped to the reactions in our metabolic models. The draft mapping was modified from Széliová, Štor, et al ( 2020 ) and is available in Supporting Information: Table S4 . One‐to‐one mapping was not possible for most reactions as the 13 C‐labeling lumped several reactions into one.…”

“…While some attempts have been made to measure CHO cell GEM predictive performance (Schinn et al, 2021b ; Széliová, 2021a , 2021b ), the effectiveness of CHO cell GEMs at capturing both intracellular flux distributions and extracellular phenotypes has not been comprehensively assessed. In this paper we present an evaluation of extracellular and intracellular predictive capabilities of CHO cell GEMs, using a holistic 13 C‐labeled fluxomic assessment methodology.…”

Genome‐Scale Metabolic Model's multi‐objective solving algorithm based on the inflexion point of Pareto front including maximum energy utilization and its application in Aspergillus niger DS03043

“…Löffler et al (2016) showed that several fluxes exist in the ATP supply inside the cell that is not related to cell growth. The study of Széliová et al (2021) on CHO cells showed that minimizing the absorption of nonessential nutrients is similar to maximizing growth and that the idea of minimizing fluxes and their sum in pFBA is the same as that of cells attempting to maximize resource utilization. Chen et al (2019) found that amino acid consumption estimated by the “minimization of essential nutrients” method is more accurate and demonstrated that minimization of non‐essential nutrient intake is more appropriate for CHO 's modeling objectives.…”

“…When Kadir et al (2010) combined these data, they found that ATP production and dilution rates had a linear relationship, with a slope of approximately 250. Széliová et al (2021) predicted the flux of maintenance in CHO cells, and also provided the total ATP production of CHO at different growth rates. The current work also found that the slope of the specific growth rate and ATP yield is approximately 180.…”