An arginase-based system for selection of transfected CHO cells without the use of toxic chemicals

Roca, Berta Capella; Lao, Nga T.; Barron, Niall; Doolan, Padraig; Clynes, Martin

doi:10.1074/jbc.ra119.011162

Cited by 12 publications

(4 citation statements)

References 41 publications

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“…Glycolysis and glutaminolysis provide energy and intermediates for cell proliferation. Polyamines have essential roles in cell proliferation [ 79 ]. TAMs in tumors can secrete putrescine to induce drug resistance of tumor cells.…”

Section: Discussionmentioning

confidence: 99%

Metabolic Reprogramming of Chemoresistant Cancer Cells and the Potential Significance of Metabolic Regulation in the Reversal of Cancer Chemoresistance

Chen

2020

Metabolites

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Metabolic reprogramming is one of the hallmarks of tumors. Alterations of cellular metabolism not only contribute to tumor development, but also mediate the resistance of tumor cells to antitumor drugs. The metabolic response of tumor cells to various chemotherapy drugs can be analyzed by metabolomics. Although cancer cells have experienced metabolic reprogramming, the metabolism of drug resistant cancer cells has been further modified. Metabolic adaptations of drug resistant cells to chemotherapeutics involve redox, lipid metabolism, bioenergetics, glycolysis, polyamine synthesis and so on. The proposed metabolic mechanisms of drug resistance include the increase of glucose and glutamine demand, active pathways of glutaminolysis and glycolysis, promotion of NADPH from the pentose phosphate pathway, adaptive mitochondrial reprogramming, activation of fatty acid oxidation, and up-regulation of ornithine decarboxylase for polyamine production. Several genes are associated with metabolic reprogramming and drug resistance. Intervening regulatory points described above or targeting key genes in several important metabolic pathways may restore cell sensitivity to chemotherapy. This paper reviews the metabolic changes of tumor cells during the development of chemoresistance and discusses the potential of reversing chemoresistance by metabolic regulation.

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Section: Discussionmentioning

confidence: 99%

Metabolic Reprogramming of Chemoresistant Cancer Cells and the Potential Significance of Metabolic Regulation in the Reversal of Cancer Chemoresistance

Chen

2020

Metabolites

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“…It has been shown that utilizing these two systems simultaneously increases the probability that a highly productive cell will be generated—as well as improving the maximum product titer (Li et al, 2010). Recent work has demonstrated the feasibility of additional metabolic selection systems in CHO (Budge et al, 2021; Capella Roca et al, 2019; Pourcel et al, 2020; Sun et al, 2020; Zhang et al, 2020) with one study (Zhang et al, 2022) showing that using 8 selectable markers simultaneously can significantly increase the productivity of the resulting cell lines (although the cells grew very slowly). The orthogonality of these new selection systems (e.g., each requiring the dropout of a different medium component for selective pressure) and/or need for multiple genetic edits led us to explore whether it was possible to increase the selective stringency of glutamine deprivation in a simpler manner, hereby enhancing the selective pressure of one of the most established tools in the clinical cell line generation workflow, without requiring changes from established selection conditions.…”

Section: Introductionmentioning

confidence: 99%

Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium

Òdena

Karottki

et al. 2023

Biotech & Bioengineering

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The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine—the selection medium for Gs‐based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co‐transfection of Gs‐Enbrel and Aspg‐Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5‐fold higher specific productivity and ~3‐fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.

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“…For example, Zhang et al reported the development of a double auxotrophic selection system based on knockout of two enzymes involved in pyrimidine and purine biosynthesis and utilised these for selection and isolation of cell lines expressing a model Fc-fusion and mAb molecule ( Zhang et al, 2020 ). In a different approach, Capella Roca et al have reported the development of an arginase based selection system for CHO cell hosts lacking arginase activity ( Roca et al, 2019 ). Very recently, Sun et al reported a proline based metabolic selection system used to isolate GFP and mAb expressing recombinant CHO cell lines, and although the mAb titres were not high by industrial standards (approximately 3.4 μg/ml) they were comparable to those achieved from antibiotic selection systems including zeocin (2.25 μg/ml) and G418 (1.65 μg/ml) based systems ( Sun et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

A proline metabolism selection system and its application to the engineering of lipid biosynthesis in Chinese hamster ovary cells

Budge

Roobol

Singh

et al. 2021

Metabolic Engineering Communications

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Chinese hamster ovary (CHO) cells are the leading mammalian cell host employed to produce complex secreted recombinant biotherapeutics such as monoclonal antibodies (mAbs). Metabolic selection marker technologies (e.g. glutamine synthetase (GS) or dihydrofolate reductase (DHFR)) are routinely employed to generate such recombinant mammalian cell lines. Here we describe the development of a selection marker system based on the metabolic requirement of CHO cells to produce proline, and that uses pyrroline-5-carboxylase synthetase (P5CS) to complement this auxotrophy. Firstly, we showed the system can be used to generate cells that have growth kinetics in proline-free medium similar to those of the parent CHO cell line, CHOK1SV GS-KO™ grown in proline-containing medium. As we have previously described how engineering lipid metabolism can be harnessed to enhance recombinant protein productivity in CHO cells, we then used the P5CS selection system to re-engineer lipid metabolism by over-expression of either sterol regulatory element binding protein 1 (SREBF1) or stearoyl CoA desaturase 1 (SCD1). The cells with re-engineered proline and lipid metabolism showed consistent growth and P5CS, SCD1 and SREBF1 expression across 100 cell generations. Finally, we show that the P5CS and GS selection systems can be used together. A GS vector containing the light and heavy chains for a mAb was super-transfected into a CHOK1SV GS-KO™ host over-expressing SCD1 from a P5CS vector. The resulting stable transfectant pools achieved a higher concentration at harvest for a model difficult to express mAb than the CHOK1SV GS-KO™ host. This demonstrates that the P5CS and GS selection systems can be used concomitantly to enable CHO cell line genetic engineering and recombinant protein expression.

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An arginase-based system for selection of transfected CHO cells without the use of toxic chemicals

Cited by 12 publications

References 41 publications

Metabolic Reprogramming of Chemoresistant Cancer Cells and the Potential Significance of Metabolic Regulation in the Reversal of Cancer Chemoresistance

Metabolic Reprogramming of Chemoresistant Cancer Cells and the Potential Significance of Metabolic Regulation in the Reversal of Cancer Chemoresistance

Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium

A proline metabolism selection system and its application to the engineering of lipid biosynthesis in Chinese hamster ovary cells

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