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Compartment-specific 13C metabolic flux analysis reveals boosted NADPH availability coinciding with increased cell-specific productivity for IgG1 producing CHO cells after MTA treatment.

ABSTRACT: Increasing cell-specific productivities (CSPs) for the production of heterologous proteins in Chinese hamster ovary (CHO) cells is an omnipresent need in the biopharmaceutical industry. The novel additive 5'-deoxy-5'-(methylthio)adenosine (MTA), a chemical degradation product of S-(5'-adenosyl)-ʟ-methionine (SAM) and intermediate of polyamine biosynthesis, boosts the CSP of IgG1-producing CHO cells by 50%. Compartment-specific 13C flux analysis revealed a fundamental reprogramming of the central metabolism after MTA addition accompanied by cell-cycle arrest and increased cell volumes. Carbon fluxes into the pentose-phosphate pathway increased 22 fold in MTA-treated cells compared to that in non-MTA-treated reference cells. Most likely, cytosolic ATP inhibition of phosphofructokinase mediated the carbon detour. Mitochondrial shuttle activity of the α-ketoglurarate/malate antiporter (OGC) reversed, reducing cytosolic malate transport. In summary, NADPH supply in MTA-treated cells improved three fold compared to that in non-MTA-treated cells, which can be regarded as a major factor for explaining the boosted CSPs.

SUBMITTER: Wijaya AW

PROVIDER: S-EPMC8638276 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Abstract We previously demonstrated that increased monoclonal antibody productivity in dihydrofolate reductase (DHFR)-amplified CHO cells correlates with phosphorylated transcription factor-cytomegalovirus (CMV) promoter interactions. In this article, we extend the characterization to include CMV promoter methylation and its influence on NFκB and CREB1 transcription factor binding to the CMV promoter in two families of DHFR-amplified CHO cell lines. CMV promoter methylation was determined using bisulfite sequencing. To overcome Sanger-sequencing limitations due to high CG bias and multiple transgenes copies, pyrosequencing was used to determine the frequency of methylated cytosines in regions proximal to and containing the NFκB and CREB1 transcription-factor consensus binding sites. Chromatin immunoprecipitation was performed to interrogate transcription factor–DNA interactions. Antibodies to CREB1 and NFκB were used to immunoprecipitate formaldehyde-crosslinked protein-DNA fractions, followed by reverse transcription quantitative real-time polymerase chain reaction to quantitate the number of copies of CMV-promoter DNA bound to the various transcription factors. The relative unmethylated fraction at the CREB1 and NFκB consensus binding sites determined by pyrosequencing was correlated with transcription factor binding as determined by chromatin immunoprecipitation. Azacytidine treatment reduced methylation in all treated samples, though not at all methylation sites, while increasing transcription. Distinct promoter methylation patterns arise upon clonal selection in different families of cell lines. In both cell line families, increased methylation was observed upon amplification. In one family, the NFκB binding-site methylation was accompanied by increased CREB1 interaction with the promoter. In the other cell line family, lower methylation frequency at the NFκB consensus binding site was accompanied by more NFκB recruitment to the promoter region. Statement of Significance: In this work, we have characterized the methylation pattern in various positions along the cytomegalovirus (CMV) promoter driving the expression of the mAb heavy chain and light chain in different clones. The interplay between the methylated regions of transcription-factor consensus binding sites and the nuclear proteins influences the transcript levels, leading to higher productivity phenotypes. Our workflow demonstrates a way to identify influencers of high transcriptional rates and identifies opportunities for transcription factor screening and rational synthetic promoter design, toward enhancing specific productivity in commercially applicable manufacturing processes.

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Compartment-specific 13C metabolic flux analysis reveals boosted NADPH availability coinciding with increased cell-specific productivity for IgG1 producing CHO cells after MTA treatment.

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