Dataset Information

Over-expression of a Codon Optimized Yeast Cytosolic Pyruvate Carboxylase (PYC2) in CHO Cells for an Augmented Lactate Metabolism.

ABSTRACT: Monoclonal antibodies are the most demanding biotherapeutic drugs now a days used for the cure of various critical illnesses. Chinese hamster ovary (CHO) cells are one of the main hosts used for the large scale production of these antibodies. However, the cell line and production processes are the key factors to determine the cost and affordability of these antibodies. The metabolic waste lactic acid and ammonium are accumulated during a cell culture process and adversely affects productivity as well as product quality. To control the lactate metabolism of mAb (IgG1-kappa) producing CHO clones, we super-transfected the cells with a mammalian construct bearing codon optimized yeast cytosolic pyruvate carboxylase (PYC2) and a strong fusion promoter for optimal expression of PYC2 enzyme. A pool study was also performed for the assessment of cell's performance, post-translational modification of a mAb and its expression in a CHO clone. The current study resulted an improved mAb titer up to 5%, galactosylation up to 2.5-folds, mannosylation up to twofold and marginal improved main and basic peaks in the charge variant profile at the cell pool stage. Such, approach may be suitable for the implementation in CHO cells producing recombinant protein for a better process control for the production of biotherapeutics.

SUBMITTER: Gupta SK

PROVIDER: S-EPMC5511841 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Over-expression of a Codon Optimized Yeast Cytosolic Pyruvate Carboxylase (PYC2) in CHO Cells for an Augmented Lactate Metabolism.

Gupta Sanjeev K SK Sharma Ankit A Kushwaha Hiralal H Shukla Pratyoosh P

Frontiers in pharmacology 20170717

Monoclonal antibodies are the most demanding biotherapeutic drugs now a days used for the cure of various critical illnesses. Chinese hamster ovary (CHO) cells are one of the main hosts used for the large scale production of these antibodies. However, the cell line and production processes are the key factors to determine the cost and affordability of these antibodies. The metabolic waste lactic acid and ammonium are accumulated during a cell culture process and adversely affects productivity as ...[more]

PMID: 28769797

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Project description:In order to obtain a global view of energy metabolism pathways of the sulfate-reducer Desulfovibrio vulgaris Hildenborough and the proteins involved therein whole-genome microarrays were used to compare the transcriptional response of cells grown with hydrogen/sulfate, pyruvate/sulfate, lactate/thiosulfate, and pyruvate with limiting sulfate, relative to growth in standard lactate/sulfate condition. Growth with hydrogen/sulfate showed the largest number of differently expressed genes and the largest changes in expression levels. The most up-regulated energy metabolism genes were those coding for the periplasmic [NiFeSe] hydrogenase, followed by the Ech hydrogenase, and the most down-regulated were genes coding for the Coo hydrogenase. The results point to the involvement of formate cycling and the ethanol pathway during growth on hydrogen, whereas there is evidence for CO cycling during growth on lactate and pyruvate, but not on H2. Growth with thiosulfate showed the hallmarks of a reduced energy status of the cells with down regulation of the ATP synthase and the Qmo and Dsr complexes., whereas growth with pyruvate showed the smallest differences but an increased role for the Ech hydrogenase.that in this case functions in reverse from the case of growth with H2. The multiple periplasmic hydrogenases and formate dehydrogenases, do not display the same regulation pattern showing that their metabolic roles are not totally interchangeable. This result together with the observation that several genes coding for proteins that have not been biochemically characterised were considerably affected in this study, reveals a more complex energy metabolism than previously considered and provides guidance for further studies. Keywords: Growth protocol Desulfovibrio vulgaris from our laboratory culture collection was cultured at 37°C with pyruvate as the only substrate (without sulfate as the electron acceptor) to mid-log phase. Cultures were also cultivated similarly in Lactate-Sulfate medium to mid-log phase. Gene expression profiles of cultures grown under pyruvate fermentation were compared with those of the cultures grown via sulfate reduction. Total RNA was harvested from four replicate cultures for microarray analysis. RNA extraction, purification, and labeling were performed independently on each cell sample.Two samples of each total RNA preparation were labeled, one with Cy3-dUTP and another with Cy5-dUTP for microarray hybridization.

Dataset Information

Over-expression of a Codon Optimized Yeast Cytosolic Pyruvate Carboxylase (PYC2) in CHO Cells for an Augmented Lactate Metabolism.

Publications

Over-expression of a Codon Optimized Yeast Cytosolic Pyruvate Carboxylase (PYC2) in CHO Cells for an Augmented Lactate Metabolism.

Similar Datasets

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