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

Chevallier, Valentine; Schoof, Erwin M.; Malphettes, Laetitia; Andersen, Mikael Rørdam; Workman, Christopher T.

doi:10.1002/bit.27495

Cited by 17 publications

(10 citation statements)

References 43 publications

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“…Our data compares favorably with Turan et al 57 MSX inhibits the activity of the enzyme GCL, which catalyzes the synthesis of reduced glutathione (GSH) from its precursor amino acids: glutamic acid, cysteine, and glycine. 59 Alteration in the amount of glutathione (either GSH or GSGG forms) has been shown to have dramatic consequences on productivity 39,60,61 and oxidative status 62 in RI derived cell lines.…”

Section: Product Quality For Mabs Produced From the Hd7876 Hostmentioning

confidence: 99%

CHOK1SV GS‐KO SSIexpression system: A combination of theFer1L4locus and glutamine synthetase selection

Feary

Moffat

Casperson

et al. 2021

Biotechnol Progress

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There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional "random integration" (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates.Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene.The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0-4.5 g/L) and robust expression stability (100% of clones stable through the 50 generation "manufacturing window" which supports commercial manufacturing at 12,000 L bioreactor scale).

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Section: Product Quality For Mabs Produced From the Hd7876 Hostmentioning

confidence: 99%

CHOK1SV GS‐KO SSIexpression system: A combination of theFer1L4locus and glutamine synthetase selection

Feary

Moffat

Casperson

et al. 2021

Biotechnol Progress

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show abstract

“…However, overexpression of the catalytic subunit of γ-glutamyl cysteine synthetase (GCL, the bottleneck enzyme in GSH biosynthesis) in CHO cells, which led to an increased intracellular concentration of GSH, did not improve the production of a monoclonal antibody in transient expression assays [ 67 ]. A subsequent study of a monoclonal antibody-producing CHO cell line where its GSH content was down-modulated by limiting cysteine supply or by chemical inhibition of GCL has shown a severe impairment in the productivity of the heterologous protein [ 68 ]. The parallel proteomic analysis conducted in this study further revealed additional metabolic adaptations of the cells in response to GSH depletion that involved the downregulation of a subset of glutathione S-transferase enzymes, TCA-cycle enzymes, and lipid biosynthesis (cholesterol in particular), and the upregulation of several antioxidant enzymes.…”

Section: Discussionmentioning

confidence: 99%

Generation and Characterization of Stable Redox-Reporter Mammalian Cell Lines of Biotechnological Relevance

Perelmuter

Tiscornia

Comini

et al. 2022

Sensors

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Cellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeostasis of cells. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathione, the major low molecular mass thiol in mammals. In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. The hGM-CSF production was not affected by the expression of the biosensor. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxidant reagents). Under batch culture conditions, a significant and progressive oxidation of the biosensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity.

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“…The protein samples were prepared according to Chevallier et al [ 47 ]. P. rubra S4059 wild type and GH19 mutant were grown in 20 mL MMM with crystalline chitin for 2 days at 25 °C, 200 rpm.…”

Section: Methodsmentioning

confidence: 99%

Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059

et al. 2021

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Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

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

Cited by 17 publications

References 43 publications

CHOK1SV GS‐KO SSIexpression system: A combination of theFer1L4locus and glutamine synthetase selection

CHOK1SV GS‐KO SSIexpression system: A combination of theFer1L4locus and glutamine synthetase selection

Generation and Characterization of Stable Redox-Reporter Mammalian Cell Lines of Biotechnological Relevance

Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059

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