Metabolic engineering of Chinese hamster ovary cells towards reduced biosynthesis and accumulation of novel growth inhibitors in fed-batch cultures

Mulukutla, Bhanu Chandra; Mitchell, J. R.; Geoffroy, Pauline; Harrington, Cameron; Krishnan, Manisha; Kalomeris, Taylor; Morris, Caitlin; Zhang, Lin; Pegman, Pamela; Hiller, Gregory

doi:10.1016/j.ymben.2019.03.001

Cited by 52 publications

(49 citation statements)

References 28 publications

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“…Using a chemical standard, we confirmed the presence of 4-hydroxyphenyllactate in the CHO cell samples analyzed in this study, even though there is no documented gene associated with CHO cell metabolism that can catalyze the reaction with 4-hydroxyphenyllactate as product. Our result is supported by other recent papers that report on the presence of this metabolite in CHO cell cultures [54,55].…”

Section: Discussionsupporting

confidence: 92%

Biological Filtering and Substrate Promiscuity Prediction for Annotating Untargeted Metabolomics

et al. 2020

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Mass spectrometry coupled with chromatography separation techniques provides a powerful platform for untargeted metabolomics. Determining the chemical identities of detected compounds however remains a major challenge. Here, we present a novel computational workflow, termed extended metabolic model filtering (EMMF), that aims to engineer a candidate set, a listing of putative chemical identities to be used during annotation, through an extended metabolic model (EMM). An EMM includes not only canonical substrates and products of enzymes already cataloged in a database through a reference metabolic model, but also metabolites that can form due to substrate promiscuity. EMMF aims to strike a balance between discovering previously uncharacterized metabolites and the computational burden of annotation. EMMF was applied to untargeted LC–MS data collected from cultures of Chinese hamster ovary (CHO) cells and murine cecal microbiota. EMM metabolites matched, on average, to 23.92% of measured masses, providing a > 7-fold increase in the candidate set size when compared to a reference metabolic model. Many metabolites suggested by EMMF are not catalogued in PubChem. For the CHO cell, we experimentally confirmed the presence of 4-hydroxyphenyllactate, a metabolite predicted by EMMF that has not been previously documented as part of the CHO cell metabolic model.

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Section: Discussionsupporting

confidence: 92%

Biological Filtering and Substrate Promiscuity Prediction for Annotating Untargeted Metabolomics

et al. 2020

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“…The cell culture fluid was centrifuged for 30 min at 17,000 g in a 3K Amicon® 0.5‐ml Filter (Merck). The sample preparation and analysis were performed by reverse‐phase ultraperformance liquid chromatography as described previously (Mulukutla et al, 2019) using a TUV detector (Waters) set at 254 nm.…”

Section: Methodsmentioning

confidence: 99%

Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis

Chevallier

Schoof

Malphettes

et al. 2020

Biotech & Bioengineering

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Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have demonstrated a relationship between GSH metabolism and clone productivity. However, a thorough investigation is required to understand this relationship and potentially highlight new targets for cell engineering. In this study, we have modu-This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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“…The use of cell engineering to reduce byproduct generation is another option. For example, it has recently been shown that phenyl lactate production could be reduced by cell engineering of the phenylalanine‐tyrosine catabolic pathway (Mulukutla et al, ). Finally, continuous processes are another potential solution to issues arising from the accumulation of waste in the medium.…”

Section: Discussionmentioning

confidence: 99%

Oxidative stress‐alleviating strategies to improve recombinant protein production in CHO cells

Chevallier

Andersen

Malphettes

2019

Biotech & Bioengineering

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Large scale biopharmaceutical production of biologics relies on the overexpression of foreign proteins by cells cultivated in stirred tank bioreactors. It is well recognized and documented fact that protein overexpression may impact host cell metabolism and that factors associated with large scale culture, such as the hydrodynamic forces and inhomogeneities within the bioreactors, may promote cellular stress. The metabolic adaptations required to support the high‐level expression of recombinant proteins include increased energy production and improved secretory capacity, which, in turn, can lead to a rise of reactive oxygen species (ROS) generated through the respiration metabolism and the interaction with media components. Oxidative stress is defined as the imbalance between the production of free radicals and the antioxidant response within the cells. Accumulation of intracellular ROS can interfere with the cellular activities and exert cytotoxic effects via the alternation of cellular components. In this context, strategies aiming to alleviate oxidative stress generated during the culture have been developed to improve cell growth, productivity, and reduce product microheterogeneity. In this review, we present a summary of the different approaches used to decrease the oxidative stress in Chinese hamster ovary cells and highlight media development and cell engineering as the main pathways through which ROS levels may be kept under control.

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Metabolic engineering of Chinese hamster ovary cells towards reduced biosynthesis and accumulation of novel growth inhibitors in fed-batch cultures

Cited by 52 publications

References 28 publications

Biological Filtering and Substrate Promiscuity Prediction for Annotating Untargeted Metabolomics

Biological Filtering and Substrate Promiscuity Prediction for Annotating Untargeted Metabolomics

Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis

Oxidative stress‐alleviating strategies to improve recombinant protein production in CHO cells

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