Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production

Henry, Matthew; MacDonald, Michael A.; Orellana, Camila A.; Gray, Peter P.; Gillard, Marianne; Baker, Kym; Nielsen, Lars K.; Marcellin, Esteban; Mahler, Stephen M.; Martínez, Verónica

doi:10.1002/bit.27269

Cited by 34 publications

(44 citation statements)

References 225 publications

(302 reference statements)

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“…The metabolite 2‐HB was strongly elevated in these cultures, indicating that it might be correlated to the improved productivity similar to its chemical analogons such as 3‐HB and NaB. Further reduction of ROS species via media optimization of antioxidant compounds such as vitamins, thiols, or α‐ketoacids as well as genetic engineering approaches could further improve culture longevity in HSD processes (Chevallier et al, 2020; Henry et al, 2020). At the same time strategies to induce cell cycle arrest and thereby increase specific productivities via temperature shifts or media components such as sodium butyrate, pentanoic acid, or resveratrol (Kumar et al, 2007; McHugh et al, 2020; Toronjo‐Urquiza et al, 2019) could enhance the productivity of HSD cultures.…”

Section: Discussionmentioning

confidence: 98%

Application of metabolic modeling for targeted optimization of high seeding density processes

Brunner

Kolb

Keitel

et al. 2021

Biotech & Bioengineering

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Process intensification by application of perfusion mode in pre‐stage bioreactors and subsequent inoculation of cell cultures at high seeding densities (HSD) has the potential to meet the increasing requirements of future manufacturing demands. However, process development is currently restrained by a limited understanding of the cell's requirements under these process conditions. The goal of this study was to use extended metabolite analysis and metabolic modeling for targeted optimization of HSD cultivations. The metabolite analysis of HSD N‐stage cultures revealed accumulation of inhibiting metabolites early in the process and flux balance analysis led to the assumption that reactive oxygen species (ROS) were contributing to the fast decrease in cell viability. Based on the metabolic analysis an optimized feeding strategy with lactate and cysteine supplementation was applied, resulting in an increase in antibody titer of up to 47%. Flux balance analysis was further used to elucidate the surprisingly strong synergistic effect of lactate and cysteine, indicating that increased lactate uptake led to reduced ROS formation under these conditions whilst additional cysteine actively reduced ROS via the glutathione pathway. The presented results finally demonstrate the benefit of modeling approaches for process intensification as well as the potential of HSD cultivations for biopharmaceutical manufacturing.

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Section: Discussionmentioning

confidence: 98%

Application of metabolic modeling for targeted optimization of high seeding density processes

Brunner

Kolb

Keitel

et al. 2021

Biotech & Bioengineering

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“…through MOMP) before the activation of caspases. Moreover, caspases are not involved in caspaseindependent apoptosis [50].…”

Section: Discussionmentioning

confidence: 99%

Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability

et al. 2020

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Background Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell in the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. Results Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. Conclusion These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.

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“…Chinese hamster ovary (CHO) cells are the preferred expression system for recombinant therapeutic protein (RTP) production, and nearly 70% of RTPs are currently generated by CHO cells (Pereira et al, 2018;Stolfa et al, 2018). In particular, CHO cells are used to produce recombinant therapeutic proteins and monoclonal antibodies (mAbs), such as adalimumab, bezlotoxumab, avelumab, dupilumab, durvalumab, ocrelizumab, and brodalumab (Kaneyoshi et al, 2019a;Henry et al, 2020). CHO cells have the following advantages over other systems: (1) they are not susceptible to human virus infection and have high safety (Lalonde and Durocher, 2017);…”

Section: Introductionmentioning

confidence: 99%

Serum-Free Medium for Recombinant Protein Expression in Chinese Hamster Ovary Cells

Liu

Fan

Lin

et al. 2021

Front. Bioeng. Biotechnol.

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At present, nearly 70% of recombinant therapeutic proteins (RTPs) are produced by Chinese hamster ovary (CHO) cells, and serum-free medium (SFM) is necessary for their culture to produce RTPs. In this review, the history and key components of SFM are first summarized, and its preparation and experimental design are described. Some small molecule compound additives can improve the yield and quality of RTP. The function and possible mechanisms of these additives are also reviewed here. Finally, the future perspectives of SFM use with CHO cells for RTP production are discussed.

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Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production

Cited by 34 publications

References 225 publications

Application of metabolic modeling for targeted optimization of high seeding density processes

Application of metabolic modeling for targeted optimization of high seeding density processes

Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability

Serum-Free Medium for Recombinant Protein Expression in Chinese Hamster Ovary Cells

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