Concomitant consumption of glucose and lactate: A novel batch production process for CHO cells

Martínez-Monge, I.; Comas, P.; Triquell, J.; Casablancas, Antoni; Lecina, Martí; Paredes, C.; Cairó, Jordi J.

doi:10.1016/j.bej.2019.107358

Cited by 16 publications

(29 citation statements)

References 51 publications

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“…Depending on the cell lines and media, required amount of NAD+ for mitochondrial LDH to oxidize lactate might be produced by relevant fluxes through other NADH-producing enzymes such as MDH and ICDH. As all these NAD+-requiring mitochondrial enzymes are located in outermembrane space in mitochondria (Passarella et al, 2014) (Martínez-Monge et al, 2019), and highlighted that route 2 displays higher metabolic similarity with early growth phase in terms of cytosolic and mitochondrial NADH regeneration. In fact, fed-batch cultivation frequently shows such distinctive metabolic feature in the growth phase such that active glucose uptake and lactate consumption or marginal production are accompanied by active cell growth (Freund and Croughan, 2018;Konakovsky et al, 2016).…”

Section: Discussionmentioning

confidence: 97%

Enzyme capacity-based genome scale modelling of CHO cells

Yeo

Hong

Lee

2019

Preprint

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Chinese hamster ovary (CHO) cells are most prevalently used for producing recombinant therapeutics in biomanufacturing. Recently, more rational and systems approaches have been increasingly exploited to identify key metabolic bottlenecks and engineering targets for cell line engineering and process development based on the CHO genome-scale metabolic model which mechanistically characterizes cell culture behaviours. However, it is still challenging to quantify plausible intracellular fluxes and discern metabolic pathway usages considering various clonal traits and bioprocessing conditions. Thus, we newly incorporated enzyme kinetic information into the updated CHO genome-scale model (iCHO2291) and added enzyme capacity constraints within the flux balance analysis framework (ecFBA) to significantly reduce the flux variability in biologically meaningful manner, as such improving the accuracy of intracellular flux prediction. Interestingly, ecFBA could capture the overflow metabolism under the glucose excess condition where the usage of oxidative phosphorylation is limited by the enzyme capacity. In addition, its applicability was successfully demonstrated via a case study where the clone-and media-specific lactate metabolism was deciphered, suggesting that the lactatepyruvate cycling could be beneficial for CHO cells to efficiently utilize the mitochondrial redox capacity. In summary, iCHO2296 with ecFBA can be used to confidently elucidate cell cultures and effectively identify key engineering targets, thus guiding bioprocess optimization and cell engineering efforts as a part of digital twin model for advanced biomanufacturing in future.

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Section: Discussionmentioning

confidence: 97%

Enzyme capacity-based genome scale modelling of CHO cells

Yeo

Hong

Lee

2019

Preprint

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“…The methodology followed to calculate the specific consumption and production rates for the metabolites analyzes is detailed in a report by Martínez-Monge et al (2019) [33].…”

Section: Estimation Of Specific Consumption and Production Ratesmentioning

confidence: 99%

“…The model was derived from the complete Hybridoma genome-scale metabolic model constructed from a generic genome-scale metabolic model of Mus musculus [35,36]. The adaptation process to obtain a reduced model which is able to perform flux analysis for the experiments performed in hybridoma was detailed in Martínez-Monge et al (2019) [33]. The base metabolic model did not contain the biomass formation equation, since the defined objective function depended on maximizing or minimizing of the ATP yield.…”

Section: Reduced Genome-scale Metabolic Modelmentioning

confidence: 99%

“…The base metabolic model did not contain the biomass formation equation, since the defined objective function depended on maximizing or minimizing of the ATP yield. So, the base model was modified by adding 14 reactions involved in the synthesis of all of the metabolite precursors needed for biomass formation, as detailed in Martínez-Monge et al (2019) [33]. The final model is composed of 362 internal and 36 external reactions that collectively involve 395 metabolites.…”

Section: Reduced Genome-scale Metabolic Modelmentioning

confidence: 99%

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The Effect of the Expression of the Antiapoptotic BHRF1 Gene on the Metabolic Behavior of a Hybridoma Cell Line

et al. 2021

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One of the most important limitations of mammalian cells-based bioprocesses, and particularly hybridoma cell lines, is the accelerated metabolism related to glucose and glutamine consumption. The high uptake rates of glucose and glutamine (i.e., the main sources of carbon, nitrogen and energy) lead to the production and accumulation of large amounts of lactate and ammonia in culture broth. Lactate and/or ammonia accumulation, together with the depletion of the main nutrients, are the major causes of apoptosis in hybridoma cell cultures. The KB26.5 hybridoma cell line, producing an IgG3, was engineered with BHRF1 (KB26.5-BHRF1), an Epstein–Barr virus-encoded early protein homologous to the antiapoptotic protein Bcl-2, with the aim of protecting the hybridoma cell line from apoptosis. Surprisingly, besides achieving effective protection from apoptosis, the expression of BHRF1 modified the metabolism of the hybridoma cell line. Cell physiology and metabolism analyses of the original KB26.5 and KB26.5-BHRF1 revealed an increase of cell growth rate, a reduction of glucose and glutamine consumption, as well as a decrease in lactate secretion in KB26.5-BHRF1 cells. A flux balance analysis allowed us to quantify the intracellular fluxes of both cell lines. The main metabolic differences were identified in glucose consumption and, consequently, the production of lactate. The lactate production flux was reduced by 60%, since the need for NADH regeneration in the cytoplasm decreased due to a more than 50% reduction in glucose uptake. In general terms, the BHRF1 engineered cell line showed a more efficient metabolism, with an increase in biomass volumetric productivity under identical culture conditions.

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“…A reduced version of a CHO GeM was used by Xing et al [49] to adjust the concentrations of certain amino acids in the culture media, by Junghans et al [50] to propose changes to media and feed that could improve bioprocess efficiency and by Templeton et al [51] to predict high producers based on intracellular flux distribution. The CHO GeM [6] has been used to study glucose and lactate metabolism using FBA coupled with dynamic equations to simulate the consumption and secretion rates of essential metabolites [52] , and aid the design of new feeding strategies by analysing intracellular fluxes [53] . This approach has also been employed to model batch CHO cell culture conditions [54] and to study metabolic shifts as a result of switching from physiological temperature to mild hypothermic conditions [55] .…”

Section: Advances In Gem Development and Applicationmentioning

confidence: 99%