Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity

Kyriakopoulos, Sarantos; Polizzi, Karen M.; Kontoravdi, Cleo

doi:10.1016/j.jbiotec.2013.09.007

Cited by 14 publications

(17 citation statements)

References 27 publications

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“…Cys, Gly, and Glu are precursors for synthesis of the cellular redox buffer glutathione (GSH; γ‐ l ‐glutamyl‐ l ‐cysteinylglycine; Griffith, ). Therefore, although our data do not support the observation that SLC1A2, SLC43A2, and SLC6A6 are specifically increased in MAb‐producing cells (Kyriakopoulos et al, ), our data do confirm increased abundance of AAT transcripts associated with glutathione synthesis in MAb‐producing CHO cells during stationary phase growth. However, other mRNAs encoding enzymes involved in glutathione synthesis (e.g., glutamate‐cysteine ligase, glutamate‐cysteine ligase catalytic subunit (GCLC), and glutamate‐cysteine ligase regulatory subunit (GCLM) genes; GSH synthetase; glutathione synthetase [GSS gene]) were not significantly increased in IgG pool C cells (data not shown), in contrast to Orellana et al () who reported increased abundance of glutathione synthetic mRNAs and proteins in MAb‐producing cells.…”

Section: Resultscontrasting

confidence: 99%

“…Exponential phase CHO cell cultures were seeded at 2 × 10 5 cells/ml in 10 ml of CD‐CHO medium in 50‐ml vented TubeSpin ® Bioreactors (TPP, Trasadingen, Switzerland) maintained at 170 rpm, 37°C, and 5% (vol/vol) CO 2 . After 24 hr, cells were recovered by centrifugation at 200 g for 5 min and the medium was replaced with fresh CD‐CHO medium containing well‐characterized inhibitors targeting highly expressed AATs in CHO and cancer cells (Bhutia et al, ; Kyriakopoulos et al, ; Table ), specifically (a) 2‐(methylamino)isobutyric acid (MeAIB; Sigma‐Aldrich, Poole, UK), which inhibits members of the SLC38 family— SLC38A1, SLC38A2, SLC38A4, and SLC38A10 (Hellsten, Hägglund, Eriksson, & Fredriksson, ; Sugawara et al, ; Varoqui, Zhu, Yao, Ming, & Erickson, ; Yao et al, ); (b) l ‐γ‐glutamyl‐p‐nitroanilide (GPNA; Sigma‐Aldrich) which inhibits SLC1A5 (Esslinger, Cybulski, & Rhoderick, ; Nicklin et al, ); (c) 2‐amino‐2‐norbornanecarboxylic acid (BCH; Sigma‐Aldrich), which inhibits SLC7A5, SLC7A8, SLC43A1, and SLC43A2 (Babu et al, ; Bodoy et al, ; Kanai et al, ; Pineda et al, ); (d) sulfasalazine (SAS; Sigma‐Aldrich) which inhibits SLC7A11 (Chung et al, ; Gout, Buckley, Simms, & Bruchovsky, ; Timmerman et al, ); and (e) N ‐[(3 R )‐3‐([1,1′‐Biphenyl]‐4‐yloxy)‐3‐(4‐fluorophenyl)propyl]‐ N ‐methylglycine hydrochloride (ALX‐5407; Tocris, Bristol, UK) which inhibits SLC6A5 and SLC6A9 (Atkinson et al, ). GPNA, SAS, and ALX‐5407 HCl were all solubilized in 0.2% (vol/vol) dimethyl sulfoxide (Sigma‐Aldrich) before addition to CD‐CHO medium at their final concentrations.…”

Section: Methodsmentioning

confidence: 99%

“…Genome sequencing projects have now predicted the existence of almost all AAT genes in CHO cells (Lewis et al, , Xu et al, ), but variation in relative expression levels can derive from both CHO cell‐type specific differences in constitutive expression and physiological regulation of expression during production processes. A recent study using quantitative reverse‐transcription polymerase chain reaction to profile AAT expression in CHO cell lines transfected with the glutamine synthetase (GS) expression vector revealed high expression of SLC7A5 (Kyriakopoulos, Polizzi, & Kontoravdi, ) but (unlike many cancer cells) relatively low expression of SLC1A5. However, both SLC38A2 (Asn and Ser transport) and SLC1A3 (Asp and Glu transport) expressions were comparatively high, suggesting a GS‐CHO cell‐specific switch in transport mechanism for nonessential amino acids.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Control of amino acid transport into Chinese hamster ovary cells

Geoghegan

Arnall

Hatton³

et al. 2018

Biotech & Bioengineering

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Amino acid transporters (AATs) represent a key interface between the cell and its environment, critical for all cellular processes: Energy generation, redox control, and synthesis of cell and product biomass. However, very little is known about the activity of different functional classes of AATs in Chinese hamster ovary (CHO) cells, how they support cell growth and productivity, and the potential for engineering their activity and/or the composition of amino acids in growth media to improve CHO cell performance in vitro. In this study, we have comparatively characterized AAT expression in untransfected and monoclonal antibody (MAb)‐producing CHO cells using transcriptome analysis by RNA‐seq, and mechanistically dissected AAT function using a variety of transporter‐specific chemical inhibitors, comparing their effect on cell proliferation, recombinant protein production, and amino acid transport. Of a possible 56 mammalian plasma membrane AATs, 16 AAT messenger RNAs (mRNAs) were relatively abundant across all CHO cell populations. Of these, a subset of nine AAT mRNAs were more abundant in CHO cells engineered to produce a recombinant MAb. Together, upregulated AATs provide additional supply of specific amino acids overrepresented in MAb biomass compared to CHO host cell biomass, enable transport of synthetic substrates for glutathione synthesis, facilitate transport of essential amino acids to maintain active protein synthesis, and provide amino acid substrates for coordinated antiport systems to maintain supplies of proteinogenic and essential amino acids.

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Section: Resultscontrasting

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Control of amino acid transport into Chinese hamster ovary cells

Geoghegan

Arnall

Hatton³

et al. 2018

Biotech & Bioengineering

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“…A direct correlation is not compulsory; for example, for glucose transporters, it has been shown that increased gene expression does not necessarily lead to higher glucose uptake rates, which better correlate with mitochondrial phosphorylation activity [38]. For amino acid consumption, a recent study reported substantial gene expression variation for amino acid transporters among different CHO cell lines, whereas the rates of consumption and production remained unchanged [39].…”

Section: Discussionmentioning

confidence: 99%

Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization

Silva

Rodrigues

Correia

et al. 2015

Stem Cells Translational Medicine

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Human embryonic stem cells (hESCs) have an enormous potential as a source for cell replacement therapies, tissue engineering, and in vitro toxicology applications. The lack of standardized and robust bioprocesses for hESC expansion has hindered the application of hESCs and their derivatives in clinical settings. We developed a robust and well-characterized bioprocess for hESC expansion under fully defined conditions and explored the potential of transcriptomic and metabolomic tools for a more comprehensive assessment of culture system impact on cell proliferation, metabolism, and phenotype. Two different hESC lines (feeder-dependent and feeder-free lines) were efficiently expanded on xeno-free microcarriers in stirred culture systems. Both hESC lines maintained the expression of stemness markers such as Oct-4, Nanog, SSEA-4, and TRA1-60 and the ability to spontaneously differentiate into the three germ layers. Whole-genome transcriptome profiling revealed a phenotypic convergence between both hESC lines along the expansion process in stirred-tank bioreactor cultures, providing strong evidence of the robustness of the cultivation process to homogenize cellular phenotype. Under low-oxygen tension, results showed metabolic rearrangement with upregulation of the glycolytic machinery favoring an anaerobic glycolysis Warburg-effect-like phenotype, with no evidence of hypoxic stress response, in contrast to two-dimensional culture. Overall, we report a standardized expansion bioprocess that can guarantee maximal product quality. Furthermore, the "omics" tools used provided relevant findings on the physiological and metabolic changes during hESC expansion in environmentally controlled stirred-tank bioreactors, which can contribute to improved scale-up production systems. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:731-742 SIGNIFICANCEThe clinical application of human pluripotent stem cells (hPSCs) has been hindered by the lack of robust protocols able to sustain production of high cell numbers, as required for regenerative medicine. In this study, a strategy was developed for the expansion of human embryonic stem cells in welldefined culture conditions using microcarrier technology and stirred-tank bioreactors. The use of transcriptomic and metabolic tools allowed detailed characterization of the cell-based product and showed a phenotypic convergence between both hESC lines along the expansion process. This study provided valuable insights into the metabolic hallmarks of hPSC expansion and new information to guide bioprocess design and media optimization for the production of cells with higher quantity and improved quality, which are requisite for translation to the clinic.

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“…This study highlighted an upregulation of genes related to the GSH pathway (Orellana et al, ). Similarly, GSH‐related amino acid transporters have been reported to have higher expression during stationary phase, when the specific productivity is higher (Kyriakopoulos, Polizzi, & Kontoravdi, ). Moreover, it has been shown that GSH plays a role in the maintenance of the redox status within CHO cell ER by preventing the formation of nonnative disulfide bonds (Chakravarthi & Bulleid, ).…”

Section: Use Of Cell Engineering To Reduce Oxidative Stressmentioning

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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Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity

Cited by 14 publications

References 27 publications

Control of amino acid transport into Chinese hamster ovary cells

Control of amino acid transport into Chinese hamster ovary cells

Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization

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

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