Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation.
Chinese Hamster Ovary cells
bioprocessing
cell cycle arrest
monoclonal antibodies
perfusion
Journal
Biotechnology and bioengineering
ISSN: 1097-0290
Titre abrégé: Biotechnol Bioeng
Pays: United States
ID NLM: 7502021
Informations de publication
Date de publication:
11 Dec 2023
11 Dec 2023
Historique:
revised:
25
10
2023
received:
27
08
2023
accepted:
19
11
2023
medline:
11
12
2023
pubmed:
11
12
2023
entrez:
11
12
2023
Statut:
aheadofprint
Résumé
Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Advance Queensland Women's Research Assistance Program
ID : WRAP213-2019RD1
Organisme : Australian Research Council Training Centre for Biopharmaceutical Innovation
ID : IC160100027
Organisme : Novo Nordisk Fonden
ID : NNF10CC1016517
Organisme : Novo Nordisk Fonden
ID : NNF14OC0009473
Informations de copyright
© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.
Références
Bruderer, R., Bernhardt, O. M., Gandhi, T., Miladinović, S. M., Cheng, L.-Y., Messner, S., Ehrenberger, T., Zanotelli, V., Butscheid, Y., Escher, C., Vitek, O., Rinner, O., & Reiter, L. (2015). Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues. Molecular & cellular proteomics: MCP, 14(5), 1400-1410. https://doi.org/10.1074/mcp.M114.044305
Carrillo-Cocom, L. M., Genel-Rey, T., Araíz-Hernández, D., López-Pacheco, F., López-Meza, J., Rocha-Pizaña, M. R., Ramírez-Medrano, A., & Alvarez, M. M. (2015). Amino acid consumption in naïve and recombinant CHO cell cultures: Producers of a monoclonal antibody. Cytotechnology, 67(5), 809-820. https://doi.org/10.1007/s10616-014-9720-5
Chen, J. (2016). The Cell-Cycle arrest and apoptotic functions of p53 in tumor initiation and progression. Cold Spring Harbor Perspectives in Medicine, 6(3), a026104. https://doi.org/10.1101/cshperspect.a026104
Chen, Z.-L., Wu, B.-C., Liu, H., Liu, X.-M., & Huang, P.-T. (2004). Temperature shift as a process optimization step for the production of pro-urokinase by a recombinant Chinese hamster ovary cell line in high-density perfusion culture. Journal of Bioscience and Bioengineering, 97(4), 239-243. https://doi.org/10.1016/S1389-1723(04)70198-X
Chevallier, V., Andersen, M. R., & Malphettes, L. (2020). Oxidative stress-alleviating strategies to improve recombinant protein production in CHO cells. Biotechnology and Bioengineering, 117(4), 1172-1186. https://doi.org/10.1002/bit.27247
Clincke, M. F., Mölleryd, C., Zhang, Y., Lindskog, E., Walsh, K., & Chotteau, V. (2013). Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor. Part I. Effect of the cell density on the process. Biotechnology Progress, 29(3), 754-767. https://doi.org/10.1002/btpr.1704
Coronel, J., Heinrich, C., Klausing, S., Noll, T., Figueredo-Cardero, A., & Castilho, L. R. (2020). Perfusion process combining low temperature and valeric acid for enhanced recombinant factor VIII production. Biotechnology Progress, 36(1), e2915. https://doi.org/10.1002/btpr.2915
Dickinson, B. C., & Chang, C. J. (2011). Chemistry and biology of reactive oxygen species in signaling or stress responses. Nature Chemical Biology, 7, 504-511. https://doi.org/10.1038/nchembio.607
Espinosa-Diez, C., Miguel, V., Mennerich, D., Kietzmann, T., Sánchez-Pérez, P., Cadenas, S., & Lamas, S. (2015). Antioxidant responses and cellular adjustments to oxidative stress. Redox Biology, 6, 183-197. https://doi.org/10.1016/j.redox.2015.07.008
Golabgir, A., Gutierrez, J. M., Hefzi, H., Li, S., Palsson, B. O., Herwig, C., & Lewis, N. E. (2016). Quantitative feature extraction from the Chinese hamster ovary bioprocess bibliome using a novel meta-analysis workflow. Biotechnology Advances, 34(5), 621-633. https://doi.org/10.1016/j.biotechadv.2016.02.011
Gu, Z., Eils, R., & Schlesner, M. (2016). Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics, 32(18), 2847-2849. https://doi.org/10.1093/bioinformatics/btw313
Hefzi, H., Ang, K. S., Hanscho, M., Bordbar, A., Ruckerbauer, D., Lakshmanan, M., Orellana, C. A., Baycin-Hizal, D., Huang, Y., Ley, D., Martinez, V. S., Kyriakopoulos, S., Jiménez, N. E., Zielinski, D. C., Quek, L. E., Wulff, T., Arnsdorf, J., Li, S., Lee, J. S., … Lewis, N. E. (2016). A consensus genome-scale reconstruction of Chinese hamster ovary cell metabolism. Cell Systems, 3(5), 434-443. https://doi.org/10.1016/j.cels.2016.10.020
Henry, M. N., MacDonald, M. A., Orellana, C. A., Gray, P. P., Gillard, M., Baker, K., Nielsen, L. K., Marcellin, E., Mahler, S., & Martínez, V. S. (2020). Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production. Biotechnology and Bioengineering, 117(4), 1187-1203. https://doi.org/10.1002/bit.27269
Hiller, G. W., Ovalle, A. M., Gagnon, M. P., Curran, M. L., & Wang, W. (2017). Cell-controlled hybrid perfusion fed-batch CHO cell process provides significant productivity improvement over conventional fed-batch cultures. Biotechnology and Bioengineering, 114(7), 1438-1447. https://doi.org/10.1002/bit.26259
Janumyan, Y., Cui, Q., Yan, L., Sansam, C. G., Valentin, M., & Yang, E. (2008). G0 function of BCL2 and BCL-xLRequires BAX, BAK, and p27 phosphorylation by mirk, revealing a novel role of BAX and BAK in quiescence regulation. Journal of Biological Chemistry, 283(49), 34108-34120. https://doi.org/10.1074/jbc.M806294200
Jiang, W., Johnson, C., Simecek, N., López-Álvarez, M. R., Di, D., Trowsdale, J., & Traherne, J. A. (2016). qKAT: A high-throughput qPCR method for KIR gene copy number and haplotype determination. Genome Medicine, 8(1), 99. https://doi.org/10.1186/s13073-016-0358-0
Karnati, S., Lüers, G., Pfreimer, S., & Baumgart-Vogt, E. (2013). Mammalian SOD2 is exclusively located in mitochondria and not present in peroxisomes. Histochemistry and Cell Biology, 140(2), 105-117. https://doi.org/10.1007/s00418-013-1099-4
Kaufman, R. J., Wasley, L. C., Spiliotes, A. J., Gossels, S. D., Latt, S. A., Larsen, G. R., & Kay, R. M. (1985). Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells. Molecular and Cellular Biology, 5(7), 1750-1759. https://doi.org/10.1128/mcb.5.7.1750
Kaufmann, H., Mazur, X., Fussenegger, M., & Bailey, J. E. (1999). Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. Biotechnology and Bioengineering, 63(5), 573-582. https://doi.org/10.1002/(SICI)1097-0290(19990605)63:5%3C573::AID-BIT7%3E3.0.CO;2-Y
Kito, Y., Matsumoto, M., Hatano, A., Takami, T., Oshikawa, K., Matsumoto, A., & Nakayama, K. I. (2020). Cell cycle-dependent localization of the proteasome to chromatin. Scientific Reports, 10(1), 5801. https://doi.org/10.1038/s41598-020-62697-2
Knoops, B., Clippe, A., Bogard, C., Arsalane, K., Wattiez, R., Hermans, C., Duconseille, E., Falmagne, P., & Bernard, A. (1999). Cloning and characterization of AOEB166, a novel mammalian antioxidant enzyme of the peroxiredoxin family*. Journal of Biological Chemistry, 274(43), 30451-30458. https://doi.org/10.1074/jbc.274.43.30451
Ko, P., Misaghi, S., Hu, Z., Zhan, D., Tsukuda, J., Yim, M., Sanford, M., Shaw, D., Shiratori, M., Snedecor, B., Laird, M., & Shen, A. (2018). Probing the importance of clonality: Single cell subcloning of clonally derived CHO cell lines yields widely diverse clones differing in growth, productivity, and product quality. Biotechnology Progress, 34(3), 624-634. https://doi.org/10.1002/btpr.2594
Kruse, J.-P., & Gu, W. (2009). Modes of p53 regulation. Cell, 137(4), 609-622. https://doi.org/10.1016/j.cell.2009.04.050
Kumari, R., & Jat, P. (2021). Mechanisms of cellular senescence: Cell cycle arrest and senescence associated secretory phenotype. Frontiers in Cell and Developmental Biology, 9, 485. https://doi.org/10.3389/fcell.2021.645593
Kurano, N., Leist, C., Messi, F., Kurano, S., & Fiechter, A. (1990). Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor. 2. Effects of medium components and waste products. Journal of Biotechnology, 15(1), 113-128. https://doi.org/10.1016/0168-1656(90)90055-G
Lin, H., Leighty, R. W., Godfrey, S., & Wang, S. B. (2017). Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed-batch media. Biotechnology Progress, 33(4), 891-901. https://doi.org/10.1002/btpr.2472
Luo, W., & Brouwer, C. (2013). Pathview: An R/Bioconductor package for pathway-based data integration and visualization. Bioinformatics, 29(14), 1830-1831. https://doi.org/10.1093/bioinformatics/btt285
MacDonald, M. A., Barry, C., Groves, T., Martínez, V. S., Gray, P. P., Baker, K., & Nielsen, L. K. (2022). Modeling apoptosis resistance in CHO cells with CRISPR-mediated knockouts of Bak1, Bax, and Bok. Biotechnology and Bioengineering, 119(6), 1380-1391. https://doi.org/10.1002/bit.28062
MacDonald, M. A., Nöbel, M., Martínez, V. S., Baker, K., Shave, E., Gray, P. P., & Marcellin, E. (2022). Engineering death resistance in CHO cells for improved perfusion culture. mAbs, 14(1), 2083465. https://doi.org/10.1080/19420862.2022.2083465
MacDonald, M. A., Nöbel, M., Roche Recinos, D., Martínez, V. S., Schulz, B. L., Howard, C. B., & Munro, T. (2022). Perfusion culture of Chinese Hamster Ovary cells for bioprocessing applications. Critical Reviews in Biotechnology, 42(7), 1099-1115. https://doi.org/10.1080/07388551.2021.1998821
Martínez, V. S., Dietmair, S., Quek, L. E., Hodson, M. P., Gray, P., & Nielsen, L. K. (2013). Flux balance analysis of CHO cells before and after a metabolic switch from lactate production to consumption. Biotechnology and Bioengineering, 110(2), 660-666. https://doi.org/10.1002/bit.24728
Mayrhofer, P., Reinhart, D., Castan, A., & Kunert, R. (2020). Rapid development of clone-specific, high-performing perfusion media from established feed supplements. Biotechnology Progress, 36(2), e2933. https://doi.org/10.1002/btpr.2933
Mulukutla, B. C., Kale, J., Kalomeris, T., Jacobs, M., & Hiller, G. W. (2017). Identification and control of novel growth inhibitors in fed-batch cultures of Chinese hamster ovary cells. Biotechnology and Bioengineering, 114(8), 1779-1790. https://doi.org/10.1002/bit.26313
Orellana, C. A., Marcellin, E., Gray, P. P., & Nielsen, L. K. (2017). Overexpression of the regulatory subunit of glutamate-cysteine ligase enhances monoclonal antibody production in CHO cells. Biotechnology and Bioengineering, 114(8), 1825-1836. https://doi.org/10.1002/bit.26316
Orellana, C. A., Marcellin, E., Schulz, B. L., Nouwens, A. S., Gray, P. P., & Nielsen, L. K. (2015). High-antibody-producing Chinese hamster ovary cells up-regulate intracellular protein transport and glutathione synthesis. Journal of Proteome Research, 14(2), 609-618. https://doi.org/10.1021/pr501027c
Pan, X., Dalm, C., Wijffels, R. H., & Martens, D. E. (2017). Metabolic characterization of a CHO cell size increase phase in fed-batch cultures. Applied Microbiology and Biotechnology, 101(22), 8101-8113. https://doi.org/10.1007/s00253-017-8531-y
Pereira, S., Kildegaard, H. F., & Andersen, M. R. (2018). Impact of CHO metabolism on cell growth and protein production: An overview of toxic and inhibiting metabolites and nutrients. Biotechnology Journal, 13(3), 13. https://doi.org/10.1002/biot.201700499
Playford, E. G., Munro, T., Mahler, S. M., Elliott, S., Gerometta, M., Hoger, K. L., Jones, M. L., Griffin, P., Lynch, K. D., Carroll, H., El Saadi, D., Gilmour, M. E., Hughes, B., Hughes, K., Huang, E., de Bakker, C., Klein, R., Scher, M. G., Smith, I. L., … Broder, C. C. (2020). Safety, tolerability, pharmacokinetics, and immunogenicity of a human monoclonal antibody targeting the G glycoprotein of henipaviruses in healthy adults: A first-in-human, randomised, controlled, phase 1 study. The Lancet Infectious Diseases, 20(4), 445-454. https://doi.org/10.1016/S1473-3099(19)30634-6
Ritacco, F. V., Wu, Y., & Khetan, A. (2018). Cell culture media for recombinant protein expression in Chinese hamster ovary (CHO) cells: History, key components, and optimization strategies. Biotechnology Progress, 34(6), 1407-1426. https://doi.org/10.1002/btpr.2706
Tejwani, V., Andersen, M. R., Nam, J. H., & Sharfstein, S. T. (2018). Glycoengineering in CHO cells: Advances in systems biology. Biotechnology Journal, 13(3), 1700234. https://doi.org/10.1002/biot.201700234
Templeton, N., Dean, J., Reddy, P., & Young, J. D. (2013). Peak antibody production is associated with increased oxidative metabolism in an industrially relevant fed-batch CHO cell culture. Biotechnology and Bioengineering, 110(7), 2013-2024. https://doi.org/10.1002/bit.24858
Templeton, N., Lewis, A., Dorai, H., Qian, E. A., Campbell, M. P., Smith, K. D., Lang, S. E., Betenbaugh, M. J., & Young, J. D. (2014). The impact of anti-apoptotic gene Bcl-2∆ expression on CHO central metabolism. Metabolic Engineering, 25, 92-102. https://doi.org/10.1016/j.ymben.2014.06.010
Templeton, N., Xu, S., Roush, D. J., & Chen, H. (2017). 13C metabolic flux analysis identifies limitations to increasing specific productivity in fed-batch and perfusion. Metabolic Engineering, 44, 126-133. https://doi.org/10.1016/j.ymben.2017.09.010
Tobey, R. A., Oishi, N., & Crissman, H. A. (1990). Cell cycle synchronization: Reversible induction of G2 synchrony in cultured rodent and human diploid fibroblasts. Proceedings of the National Academy of Sciences, 87(13), 5104-5108. https://doi.org/10.1073/pnas.87.13.5104
Villiger-Oberbek, A., Yang, Y., Zhou, W., & Yang, J. (2015). Development and application of a high-throughput platform for perfusion-based cell culture processes. Journal of Biotechnology, 212, 21-29. https://doi.org/10.1016/j.jbiotec.2015.06.428
Walsh, G., & Walsh, E. (2022). Biopharmaceutical benchmarks 2022. Nature Biotechnology, 40(12), 1722-1760. https://doi.org/10.1038/s41587-022-01582-x
Walther, J., Lu, J., Hollenbach, M., Yu, M., Hwang, C., McLarty, J., & Brower, K. (2019). Perfusion cell culture decreases process and product heterogeneity in a Head-to-Head comparison with Fed-Batch. Biotechnology Journal, 14(2), e1700733. https://doi.org/10.1002/biot.201700733
Wolf, M. K. F., Closet, A., Bzowska, M., Bielser, J.-M., Souquet, J., Broly, H., & Morbidelli, M. (2018). Improved performance in mammalian cell perfusion cultures by growth inhibition. Biotechnology Journal, 14(2), 1700722. https://doi.org/10.1002/biot.201700722
Xu, J., Tang, P., Yongky, A., Drew, B., Borys, M. C., Liu, S., & Li, Z. J. (2018). Systematic development of temperature shift strategies for Chinese hamster ovary cells based on short duration cultures and kinetic modeling. mAbs, 11, 191-204. https://doi.org/10.1080/19420862.2018.1525262
Zamani, L., Lundqvist, M., Zhang, Y., Aberg, M., Edfors, F., Bidkhori, G., Lindahl, A., Mie, A., Mardinoglu, A., Field, R., Turner, R., Rockberg, J., & Chotteau, V. (2018). High cell density perfusion culture has a maintained exoproteome and metabolome. Biotechnology Journal, 13(10), 1800033. https://doi.org/10.1002/biot.201800036
Zhu, Z., Bossart, K. N., Bishop, K. A., Crameri, G., Dimitrov, A. S., McEachern, J. A., Feng, Y., Middleton, D., Wang, L. F., Broder, C. C., & Dimitrov, D. S. (2008). Exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody. The Journal of infectious diseases, 197(6), 846-853. https://doi.org/10.1086/528801