Data for engineering lipid metabolism of Chinese hamster ovary (CHO) cells for enhanced recombinant protein production

Budge, James D.; Knight, Tanya J.; Povey, Jane F.; Roobol, Joanne; Brown, Ian R.; Singh, Gurdeep; Dean, Andrew P.; Turner, Sarah J.; Jaques, Colin; Young, Robert J.; Racher, Andrew J.; Smales, C. Mark

doi:10.1016/j.dib.2020.105217

Cited by 4 publications

(4 citation statements)

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“…We have previously shown, using an alternative antibiotic based selection marker, that genetic engineering to modify lipid metabolism by over-expression of SCD1 or SREBF1 can be employed in CHO cells to increase recombinant product yields ( Budge et al, 2019 , 2020 ). In the present study, the P5CS/proline selection system was used to genetically engineer the CHOK1SV GSKO™ host cell line through overexpression of the SCD1 gene as an exemplar of the system as our previous study showed SCD1 over-expression tended to give greater enhancement of recombinant protein production than SREBF1.…”

Section: Resultsmentioning

confidence: 99%

“…We then successfully employed the selection system to facilitate genetic over-expression engineering of either sterol regulatory element binding protein 1 (SREBF1) or stearoyl CoA desaturase 1 (SCD1) lipid modifying genes in the CHOK1SV GSKO™ host cell line. We have previously shown how manipulation of these lipid-altering genes can improve cellular processes involved in recombinant therapeutic expression ( Budge et al, 2019 , 2020 ). Importantly, we show that the utilisation of the P5CS based system does not affect the subsequent use of the GS based selection system to generate recombinant biotherapeutic expressing CHO cell lines producing industrially relevant amounts of recombinant protein.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

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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“…Lipids were extracted from (a) total cell pellets (1 × 10 6 viable cells containing 298.9 g of protein when harvested at day 3 and 477.2 g of protein when harvested at day 5) that had been stored at − 80 °C, and (b) from the pooled exosomes isolated from 7 × 1 ml of culture (i.e. we took 7 × 75 μl exosome aliquots each isolated from 1 ml of culture for each time point and then pooled these together) essentially as previously described (Budge et al 2020a , 2020b ). The lipids were then extracted from these samples by adding a chloroform:methanol (2:1) solution (3 ml) to the cells/exosome preparations, which was then left for 20 min before adding Milli-Q grade H 2 O (500 l) and then centrifuging the mixture for 5 min at 1500 g. The chloroform (lower) lipid-containing phase was then removed, dried under N 2 and stored at − 80 °C (Llorente et al 2013 ).…”

Section: Methodsmentioning

confidence: 99%

“…Here we report on the isolation of exosomes from CHO cells, an industrially relevant and widely used host for biopharmaceutical protein production (Budge et al 2020a ; Vito et al 2020 ). We have utilized a commercially available polymer based precipitation (PBP) technique, Total Exosome Isolation (TEI), to isolate and enrich extracellular exosome vesicles from batch culture.…”

Section: Introductionmentioning

confidence: 99%

Isolation and characterisation of exosomes from Chinese hamster ovary (CHO) cells

Skrika-Alexopoulos

Smales

2023

Biotechnol Lett

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Exosomes have previously been isolated from Chinese hamster ovary (CHO) cells and their anti-apoptotic properties reported. However, to further facilitate the study of CHO cell derived exosomes and allow their comparison across studies, it is necessary to characterise and define such exosomes using at least three criteria that can act as a reference for the generation of CHO cell produced exosomes. Here we report on the isolation of exosomes from CHO cells, an industrially relevant and widely used cell host for biopharmaceutical protein production, during the exponential and stationary phase of growth during batch culture using a Total Exosome Isolation (TEI) method. The resulting vesicles were characterized and visualized using a diverse range of techniques including Dynamic Light Scattering (DLS), Zeta potential, Electron Microscopy and immunoblotting, and their protein and RNA content determined. We also generated the lipid fingerprint of isolated exosomes using MALDI-ToF mass spectroscopy. We confirmed the presence of nano sized extracellular vesicles from CHO cells and their subsequent characterization revealed details of their size, homogeneity, surface charge, protein and RNA content. The lipid content of exosomes was also found to differ between exosomes isolated on different days of batch culture. This analysis provides a profile and characterisation of CHO cell exosomes to aid future studies on exosomes from CHO cells and improving the manufacturing of exosomes for biotherapeutic application.

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Expression of mammalian proteins for diagnostics and therapeutics: a review

et al. 2022

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Background Antibodies have proven to be remarkably successful for biomedical applications. They play important roles in epidemiology and medicine from diagnostics of diseases to therapeutics, treating diseases from incessant chronic diseases such as rheumatology to pandemic outbreaks. With no end in sight for the demand for antibody products, optimizations and new techniques must be expanded to accommodate this. Methods and Results This review discusses optimizations and techniques for antibody production through choice of discovery platforms, expression systems, cell culture mediums, and other strategies to increase expression yield. Each system has its own merits and demerits, and the strategy chosen is critical in addressing various biological aspects. Conclusions There is still insufficient evidence to validate the efficacy of some of these techniques, and further research is needed to consolidate these industrial production systems. There is no doubt that more strategies, systems, and pipelines will contribute to enhance biopharmaceutical production.

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Data for engineering lipid metabolism of Chinese hamster ovary (CHO) cells for enhanced recombinant protein production

Cited by 4 publications

References 2 publications

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

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

Isolation and characterisation of exosomes from Chinese hamster ovary (CHO) cells

Expression of mammalian proteins for diagnostics and therapeutics: a review

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