Application of 13C flux analysis to identify high-productivity CHO metabolic phenotypes

Templeton, Neil; Smith, Kevin D.; Pereira, Allison G. McAtee; Dorai, Haimanti; Betenbaugh, Michael J.; Lang, Stanley; Young, Jamey D.

doi:10.1016/j.ymben.2017.01.008

Cited by 47 publications

(50 citation statements)

References 33 publications

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“…Limiting cytosolic aspartate availability either by depletion of AGC1 (aspartate–glutamate transporter across the mitochondrial membrane) or SLC1A3 (glutamate–aspartate transporter across the plasma membrane) is detrimental for growth under glutamine limitation. Metabolic flux analysis in CHO cells also showed higher flux from aspartate to oxaloacetate in glutamine‐deplete conditions when GS was overexpressed . Our results indicate that growth of QL is limited by aspartate in glutamine‐replete conditions.…”

Section: Discussionsupporting

confidence: 51%

“…Metabolic flux analysis in CHO cells also showed higher flux from aspartate to oxaloacetate in glutamine-deplete conditions when GS was overexpressed. 47 Our results indicate that growth of QL is limited by aspartate in glutamine-replete conditions. Recent reports have also identified aspartate as a limiting metabolite for tumor growth both in vitro and in vivo.…”

Section: Ql Cells Were Able To Grow Better Under Serine Deprivationmentioning

confidence: 61%

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Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine‐replete conditions and can be inhibited by serine

Maralingannavar

Parmar

Panchagnula

et al. 2019

Biotechnology Progress

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Passaging and expansion of animal cells in lean maintenance medium could result in periods of limitation of some nutrients. Over time, such stresses could possibly result in selection of cells with metabolic changes and contribute to heterogeneity. Here, we investigate whether selection of Chinese Hamster Ovary (CHO) cells under glutamine limitation results in changes in growth under glutamine‐replete conditions. In glutamine‐limiting medium, compared to control cells passaged in glutamine‐rich medium, the selected cells showed higher glutamine synthetase (GS) activity and attained a higher peak viable cell density (PVCD). Surprisingly, in glutamine‐replete conditions, selected cells still showed a higher GS activity but a lower PVCD. We show that in glutamine‐replete medium, PVCD of selected cells was restored on (a) inhibition of GS activity with methionine sulfoximine, (b) supplementation of aspartate—without affecting GS activity, and (c) supplementation of serine, which is reported to inhibit GS in vitro. Consistent with the reported effect of serine, inhibition of GS activity was observed upon serine supplementation along with reduced growth of cells under glutamine‐limiting conditions. The latter observation is important for the design of glutamine‐free culture medium and feed used for GS‐CHO and GS‐NS0. In summary, we show that CHO cells selected under glutamine limitation have superfluous GS activity in glutamine‐replete medium, which negatively affects their PVCD. This may be due to its effect on availability of aspartate which was the limiting nutrient for the growth of selected cells in glutamine‐replete conditions.

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Section: Discussionsupporting

confidence: 51%

Section: Ql Cells Were Able To Grow Better Under Serine Deprivationmentioning

confidence: 61%

Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine‐replete conditions and can be inhibited by serine

Maralingannavar

Parmar

Panchagnula

et al. 2019

Biotechnology Progress

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“…While these metrics generate practical data about cellular responses to media alterations, they do not provide quantitative readouts of the intracellular metabolic response to media manipulations. 13 C metabolic flux analysis (MFA) bridges the gap between extracellular manipulations and intracellular metabolic pathway perturbations, providing a rigorous analytical approach to quantify changing metabolic phenotypes across different experimental conditions …”

Section: Introductionmentioning

confidence: 99%

¹³C Flux Analysis Reveals that Rebalancing Medium Amino Acid Composition can Reduce Ammonia Production while Preserving Central Carbon Metabolism of CHO Cell Cultures

Pereira

Walther

Hollenbach

et al. 2018

Biotechnology Journal

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C metabolic flux analysis (MFA) provides a rigorous approach to quantify intracellular metabolism of industrial cell lines. In this study, C MFA was used to characterize the metabolic response of Chinese hamster ovary (CHO) cells to a novel medium variant designed to reduce ammonia production. Ammonia inhibits growth and viability of CHO cell cultures, alters glycosylation of recombinant proteins, and enhances product degradation. Ammonia production was reduced by manipulating the amino acid composition of the culture medium; specifically, glutamine, glutamate, asparagine, aspartate, and serine levels were adjusted. Parallel C flux analysis experiments determined that, while ammonia production decreased by roughly 40%, CHO cell metabolic phenotype, growth, viability, and monoclonal antibody (mAb) titer were not significantly altered by the changes in media composition. This study illustrates how C flux analysis can be applied to assess the metabolic effects of media manipulations on mammalian cell cultures. The analysis revealed that adjusting the amino acid composition of CHO cell culture media can effectively reduce ammonia production while preserving fluxes throughout central carbon metabolism.

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“…Isotopic-labelling data 13 C-MFA 13 C-MFA at a genome scale [88] 13 C-MFA 13 C-MFA of central carbon metabolism of hepatocellular carcinoma and effect of Hexokinase-2 on the metabolism [82] 13 C-MFA 13 C-MFA at a genome scale [53] 13 C-MFA 13 C-MFA at a genome scale of evolved knockout E. coli strains [89] 13 C-MFA 13 C-MFA of central carbon and amino acid metabolism reveals how changing medium amino acid composition metabolism in CHO cell culture [83] 13 C-MFA Application of 13 C-MFA of central carbon and amino acid metabolism to study CHO cells with high productivity of industrially relevant proteins [84] Metabolomics (single data point)…”

Section: Comment Referencementioning

confidence: 99%

Metabolic Modelling as a Framework for Metabolomics Data Integration and Analysis

et al. 2020

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Metabolic networks are regulated to ensure the dynamic adaptation of biochemical reaction fluxes to maintain cell homeostasis and optimal metabolic fitness in response to endogenous and exogenous perturbations. To this end, metabolism is tightly controlled by dynamic and intricate regulatory mechanisms involving allostery, enzyme abundance and post-translational modifications. The study of the molecular entities involved in these complex mechanisms has been boosted by the advent of high-throughput technologies. The so-called omics enable the quantification of the different molecular entities at different system layers, connecting the genotype with the phenotype. Therefore, the study of the overall behavior of a metabolic network and the omics data integration and analysis must be approached from a holistic perspective. Due to the close relationship between metabolism and cellular phenotype, metabolic modelling has emerged as a valuable tool to decipher the underlying mechanisms governing cell phenotype. Constraint-based modelling and kinetic modelling are among the most widely used methods to study cell metabolism at different scales, ranging from cells to tissues and organisms. These approaches enable integrating metabolomic data, among others, to enhance model predictive capabilities. In this review, we describe the current state of the art in metabolic modelling and discuss future perspectives and current challenges in the field.

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Application of 13C flux analysis to identify high-productivity CHO metabolic phenotypes

Cited by 47 publications

References 33 publications

Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine‐replete conditions and can be inhibited by serine

Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine‐replete conditions and can be inhibited by serine

¹³C Flux Analysis Reveals that Rebalancing Medium Amino Acid Composition can Reduce Ammonia Production while Preserving Central Carbon Metabolism of CHO Cell Cultures

Metabolic Modelling as a Framework for Metabolomics Data Integration and Analysis

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Application of 13C flux analysis to identify high-productivity CHO metabolic phenotypes

Cited by 47 publications

References 33 publications

Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine‐replete conditions and can be inhibited by serine

Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine‐replete conditions and can be inhibited by serine

13C Flux Analysis Reveals that Rebalancing Medium Amino Acid Composition can Reduce Ammonia Production while Preserving Central Carbon Metabolism of CHO Cell Cultures

Metabolic Modelling as a Framework for Metabolomics Data Integration and Analysis

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¹³C Flux Analysis Reveals that Rebalancing Medium Amino Acid Composition can Reduce Ammonia Production while Preserving Central Carbon Metabolism of CHO Cell Cultures