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A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells

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Abstract

Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-α MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.

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References

  1. Reichert, J. M. (2008). Monoclonal antibodies as innovative therapeutics. Current Pharmaceutical Biotechnology, 9, 423–430.

    CAS  PubMed  Google Scholar 

  2. Reichert, J. M., Rosensweig, C. J., Faden, L. B., & Dewitz, M. C. (2005). Monoclonal antibody successes in the clinic. Nature Biotechnology, 23, 1073–1078.

    CAS  PubMed  Google Scholar 

  3. Rodrigues, M. E., Costa, A. R., Henriques, M., Azeredo, J., & Oliveira, R. (2013). Advanced and drawbacks of adaptation to serum-free culture of CHO-K1 cells for monoclonal antibody production. Applied Biochemistry and Biotechnology, 169, 1279–1291.

    CAS  PubMed  Google Scholar 

  4. Nelson, A. L., Dhimolea, E., & Reichert, J. M. (2010). Development trends for human monoclonal antibody therapeutics. Nature Reviews Drug Discovery, 9, 767–774.

    CAS  PubMed  Google Scholar 

  5. Farid, S. S. (2008). Economic drivers and trade-offs in antibody purification processes. BioPharm International, 21, 37–42.

    Google Scholar 

  6. Farid, S. S. (2007). Process economics of industrial monoclonal antibody manufacture. Journal of Chromatography B, 848, 8–18.

    CAS  Google Scholar 

  7. Bradley, J. (2008). TNF-mediated inflammatory disease. The Journal of Pathology, 214, 149–160.

    CAS  PubMed  Google Scholar 

  8. Parameswaran, N., & Patial, S. (2010). Tumor necrosis factor-α signaling in macrophages. Critical Reviews™ Eukaryotic Gene Expression, 20(2), 87–103.

    CAS  Google Scholar 

  9. Clark, I. A. (2007). How TNF was recognized as a key mechanism of disease. Cytokine & Growth Factor Reviews, 18, 335–343.

    CAS  Google Scholar 

  10. Balabashin, D., Kovalenko, E., Toporova, V., Aliev, T., Panina, A., Svirshchevskaya, E., Dolgikh, D., & Kirpichnikov, M. (2015). Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains. Cytotechnology, 67, 761–772.

    CAS  PubMed  Google Scholar 

  11. Horiuchi, T., Mitoma, H., Harashima, S.-I., Tsukamoto, H., & Shimoda, T. (2010). Transmembrane TNF-α: structure, function and interaction with anti-TNF agents. Rheumatology, 49, 1215–1228.

    CAS  PubMed  Google Scholar 

  12. Jayapal, K. P., Wlaschin, K. F., Hu, W. S., & Yap, M. G. S. (2007). Recombinant protein therapeutics from CHO cells—20 years and counting. Chemical Engineering Progress, 103(10), 40–47.

    CAS  Google Scholar 

  13. Lis, K., Kuzawińska, O., & Bałkowiec-Iskra, E. (2014). Tumor necrosis factor inhibitors—state of knowledge. Archives of Medical Science, 10, 1175–1185.

    PubMed  Google Scholar 

  14. (2012). Biologic drugs set to top 2012 sales. Nat Med, 18 , 636–636.

  15. Willrich, M. A., Murray, D. L., & Snyder, M. R. (2015). Tumor necrosis factor inhibitors: clinical utility in autoimmune diseases. Translational Research, 165, 270–282.

    CAS  PubMed  Google Scholar 

  16. Ghaderi, D., Zhang, M., Hurtado-Ziola, N., & Varki, A. (2012). Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation. Biotechnology and Genetic Engineering Reviews, 28, 147–176.

    CAS  PubMed  Google Scholar 

  17. Swiech, K., Picanço-Castro, V., & Covas, D. T. (2012). Human cells: new platform for recombinant therapeutic protein production. Protein Expression and Purification, 84, 147–153.

    CAS  PubMed  Google Scholar 

  18. Estes, S., & Melville, M. (2013). Advances in biochemical engineering biotechnology, vol 139: mammalian cell cultures for biologics manufacturing. In W. Zhou & A. Kantardjieff (Eds.), Mammalian cell line developments in speed and efficiency (pp. 11–33). Berlin Heidelberg: Springer-Verlag.

    Google Scholar 

  19. Kim, J. Y., Kim, Y.-G., & Lee, G. M. (2012). CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Applied Microbiology and Biotechnology, 93, 917–930.

    CAS  PubMed  Google Scholar 

  20. Barnes, L. M., Bentley, C. M., & Dickson, A. J. (2001). Characterization of the stability of recombinant protein production in the GS-NS0 expression system. Biotechnology and Bioengineering, 73, 261–270.

    CAS  PubMed  Google Scholar 

  21. Page, M. J., & Sydenham, M. A. (1991). High level expression of the humanized monoclonal antibody Campath-1H in Chinese hamster ovary cells. Nature Biotechnology, 9, 64–68.

    CAS  Google Scholar 

  22. Al-Rubeai, M. (1998). Advances in biochemical engineering biotechnology, vol 59: bioprocess and algae reactor technology, apoptosis. In T. Scheper (Ed.), Apoptosis and cell culture technology (pp. 225–249). Berlin Heidelberg: Springer-Verlag.

    Google Scholar 

  23. Arden, N., & Betenbaugh, M. J. (2004). Life and death in mammalian cell culture: strategies for apoptosis inhibition. Trends in Biotechnology, 22, 174–180.

    CAS  PubMed  Google Scholar 

  24. Al-Rubeai, M., & Singh, R. P. (1998). Apoptosis in cell culture. Current Opinion in Biotechnology, 9, 152–156.

    CAS  PubMed  Google Scholar 

  25. Goswami, J., Sinskey, A., Steller, H., Stephanopoulos, G., & Wang, D. (1999). Apoptosis in batch cultures of Chinese hamster ovary cells. Biotechnology and Bioengineering, 62, 632–640.

    CAS  PubMed  Google Scholar 

  26. Morrow, K. J. (2007). Advances in antibody manufacturing using mammalian cells. Biotechnology Annual Review, 13, 95–113.

    CAS  PubMed  Google Scholar 

  27. Chiang, G. G., & Sisk, W. P. (2005). Bcl-xL mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells. Biotechnology and Bioengineering, 91, 779–792.

    CAS  PubMed  Google Scholar 

  28. Charbonneau, J., & Gauthier, E. (2001). Protection of hybridoma cells against apoptosis by a loop domain-deficient Bcl-xL protein. Cytotechnology, 37, 41–47.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Jung, D., Côté, S., Drouin, M., Simard, C., & Lemieux, R. (2002). Inducible expression of Bcl-XL restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures. Biotechnology and Bioengineering, 79, 180–187.

    CAS  PubMed  Google Scholar 

  30. Figueroa Jr., B., Sauerwald, T., Oyler, G., Hardwick, J. M., & Betenbaugh, M. J. (2003). A comparison of the properties of a Bcl-x L variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures. Metabolic Engineering, 5, 230–245.

    CAS  PubMed  Google Scholar 

  31. Tey, B., Singh, R., Piredda, L., Piacentini, M., & Al-Rubeai, M. (2000). Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures. Journal of Biotechnology, 79, 147–159.

    CAS  PubMed  Google Scholar 

  32. Fussenegger, M., Fassnacht, D., Schwartz, R., Zanghi, J. A., Graf, M., Bailey, J. E., & Pörtner, R. (2000). Regulated overexpression of the survival factor bcl-2 in CHO cells increases viable cell density in batch culture and decreases DNA release in extended fixed-bed cultivation. Cytotechnology, 32, 45–61.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Mastrangelo, A. J., Hardwick, J. M., Zou, S., & Betenbaugh, M. J. (2000). Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults. Biotechnology and Bioengineering, 67, 555–564.

    CAS  PubMed  Google Scholar 

  34. Dorai, H., Kyung, Y. S., Ellis, D., Kinney, C., Lin, C., Jan, D., Moore, G., & Betenbaugh, M. J. (2009). Expression of anti-apoptosis genes alters lactate metabolism of Chinese hamster ovary cells in culture. Biotechnology and Bioengineering, 103, 592–608.

    CAS  PubMed  Google Scholar 

  35. Zustiak, M. P., Jose, L., Xie, Y., Zhu, J., & Betenbaugh, M. J. (2014). Enhanced transient recombinant protein production in CHO cells through the co-transfection of the product gene with Bcl-xL. Biotechnology Journal, 9, 1164–1174.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Hauser, H. and Wagner, R. (2014). Animal cell biotechnology: In Biologics Production, 1st ed., Walter de Gruyter GmbH & Co KG.

  37. Bos, A. B., Duque, J. N., Bhakta, S., Farahi, F., Chirdon, L. A., Junutula, J. R., Harms, P. D., & Wong, A. W. (2014). Development of a semi-automated high throughput transient transfection system. Journal of Biotechnology, 180, 10–16.

    CAS  PubMed  Google Scholar 

  38. Codamo, J., Munro, T. P., Hughes, B. S., Song, M., & Gray, P. P. (2011). Enhanced CHO cell-based transient gene expression with the epi-CHO expression system. Molecular Biotechnology, 48, 109–115.

    CAS  PubMed  Google Scholar 

  39. Hacker, D. L., Kiseljak, D., Rajendra, Y., Thurnheer, S., Baldi, L., & Wurm, F. M. (2013). Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells. Protein Expression and Purification, 92, 67–76.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Lee, S., & Lee, G. M. (2013). Bcl-2 overexpression in CHO cells improves polyethylenimine-mediated gene transfection. Process Biochemistry, 48, 1436–1440.

    CAS  Google Scholar 

  41. Sou, S. N., Polizzi, K. M., & Kontoravdi, C. (2013). Evaluation of transfection methods for transient gene expression in Chinese hamster ovary cells. Advances in Bioscience and Biotechnology, 4, 1013–1019.

    Google Scholar 

  42. Ehrhardt, C., Schmolke, M., Matzke, A., Knoblauch, A., Will, C., Wixler, V., & Ludwig, S. (2006). Polyethylenimine, a cost-effective transfection reagent. Signal Transduction, 6, 179–184.

    CAS  Google Scholar 

  43. Longo, P. A., Kavran, J. M., Kim, M.-S., & Leahy, D. J. (2013). Transient mammalian cell transfection with polyethylenimine (PEI). Methods in Enzymology, 529, 227.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Zaric, V., Weltin, D., Erbacher, P., Remy, J. S., Behr, J. P., & Stephan, D. (2004). Effective polyethylenimine-mediated gene transfer into human endothelial cells. The Journal of Gene Medicine, 6, 176–184.

    CAS  PubMed  Google Scholar 

  45. Obata, Y., Ciofani, G., Raffa, V., Cuschieri, A., Menciassi, A., Dario, P., & Takeoka, S. (2010). Evaluation of cationic liposomes composed of an amino acid-based lipid for neuronal transfection. Nanomedicine: Nanotechnology, Biology and Medicine, 6, 70–77.

    CAS  Google Scholar 

  46. Gulce Iz, S., Calimlioglu, B., & Deliloglu, S. I. D. (2012). Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis. Electronic Journal of Biotechnology, 15(5), 2–2.

    Google Scholar 

  47. Bertrand, R., Solary, E., O’Connor, P., Kohn, K. W., & Pommier, Y. (1994). Induction of a common pathway of apoptosis by staurosporine. Experimental Cell Research, 211, 314–321.

    CAS  PubMed  Google Scholar 

  48. Nalbantsoy, A., Karaboz, I., Ivanova, R., & Deliloglu Gurhan, I. (2010). Isolation and purification of O and H antigens from Salmonella Enteritidis as diagnostic tools. Annals of Microbiology, 60, 565–571.

    CAS  Google Scholar 

  49. Ayyildiz Tamis, D., Deliloglu Gurhan S.I. (2016). Optimization of humanized monoclonal antibody against human tumor necrosis factor-α production. PhD Thesis, Ege University, Institute of Science, Thesis reference number: 10117485.

  50. Toledo, J. R., Prieto, Y., Oramas, N., & Sánchez, O. (2009). Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors. Applied Biochemistry and Biotechnology, 157, 538–544.

    CAS  PubMed  Google Scholar 

  51. Haldankar, R., Li, D., Saremi, Z., Baikalov, C., & Deshpande, R. (2006). Serum-free suspensin large-scale transient transfection of CHO cells in WAVE bioreactors. Molecular Biotechnology, 34, 191–199.

    CAS  PubMed  Google Scholar 

  52. Rosser, M. P., Xia, W., Hartsell, S., McCAMAN, M., Zhu, Y., Wang, S., Harvey, S., Bringmann, P., & Cobb, R. R. (2005). Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expression and Purification, 40, 237–243.

    CAS  PubMed  Google Scholar 

  53. Ye, J., Kober, V., Tellers, M., Naji, Z., Salmon, P., & Markusen, J. F. (2009). High-level protein expression in scalable CHO transient transfection. Biotechnology and Bioengineering, 103, 542–551.

    CAS  PubMed  Google Scholar 

  54. Galbraith, D. J., Tait, A. S., Racher, A. J., Birch, J. R., & James, D. C. (2006). Control of culture environment for improved Polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells. Biotechnology Progress, 22, 753–762.

    CAS  PubMed  Google Scholar 

  55. Thuret, G., Chiquet, C., Herrag, S., Dumollard, J., Boudard, D., Bednarz, J., Campos, L., & Gain, P. (2003). Mechanisms of staurosporine induced apoptosis in a human corneal endothelial cell line. British Journal of Ophthalmology, 87, 346–352.

    CAS  Google Scholar 

  56. Delafosse, L., Xu, P., & Durocher, Y. (2016). Comparative study of polyethylenimines for transient gene expression in mammalian HEK293 and CHO cells. Journal of Biotechnology, 227, 103–111.

    CAS  PubMed  Google Scholar 

  57. Wang, W., Li, W., Ma, N., & Steinhoff, G. (2013). Non-viral gene delivery methods. Current Pharmaceutical Biotechnology, 14, 46–60.

    CAS  PubMed  Google Scholar 

  58. Yin, H., Kanasty, R. L., Eltoukhy, A. A., Vegas, A. J., Dorkin, J. R., & Anderson, D. G. (2014). Non-viral vectors for gene-based therapy. Nature Reviews Genetics, 15, 541–555.

    CAS  PubMed  Google Scholar 

  59. Derouazi, M., Girard, P., Van Tilborgh, F., Iglesias, K., Muller, N., Bertschinger, M., & Wurm, F. M. (2004). Serum-free large-scale transient transfection of CHO cells. Biotechnology and Bioengineering, 87, 537–545.

    CAS  PubMed  Google Scholar 

  60. Abbott, W. M., Middleton, B., Kartberg, F., Claesson, J., Roth, R., & Fisher, D. (2015). Optimisation of a simple method to transiently transfect a CHO cell line in high-throughput and at large scale. Protein Expression and Purification, 116, 113–119.

    CAS  PubMed  Google Scholar 

  61. Singh, R. P., Emery, A. N., & Al-Rubeai, M. (1996). Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor gene bcl-2. Biotechnology and Bioengineering, 52, 166–175.

    CAS  PubMed  Google Scholar 

  62. Fassnacht, D., Rössing, S., Singh, R., Al-Rubeai, M., & Pörtner, R. (1999). Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma. Cytotechnology, 30, 95–106.

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Meents, H., Enenkel, B., Eppenberger, H. M., Werner, R. G., & Fussenegger, M. (2002). Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-xL on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media. Biotechnology and Bioengineering, 80, 706–716.

    CAS  PubMed  Google Scholar 

  64. 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.

    CAS  PubMed  Google Scholar 

  65. Figueroa, B., Chen, S., Oyler, G. A., Hardwick, J. M., & Betenbaugh, M. J. (2004). Aven and Bcl-xL enhance protection against apoptosis for mammalian cells exposed to various culture conditions. Biotechnology and Bioengineering, 85, 589–600.

    CAS  PubMed  Google Scholar 

  66. Majors, B. S., Betenbaugh, M. J., Pederson, N. E., & Chiang, G. G. (2008). Enhancement of transient gene expression and culture viability using Chinese hamster ovary cells overexpressing Bcl-xL. Biotechnology and Bioengineering, 101, 567–578.

    CAS  PubMed  Google Scholar 

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Acknowledgements

This study has been partly funded by TUBITAK through 2209-A project number 1919B011402737 under the supervision of Sultan GULCE IZ, PhD.

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Authors and Affiliations

  1. Department of Bioengineering, Faculty of Engineering, Ege University, 35100, Izmir, Bornova, Turkey

    Sultan Gulce Iz, Muge Anil Inevi, Pelin Saglam Metiner, Duygu Ayyildiz Tamis & Nazli Kisbet

  2. Department of Biotechnology and Bioengineering, Izmir Institute of Technology, 35430, Izmir, Urla, Turkey

    Muge Anil Inevi

  3. Turgut Ilaclari A.S, 34394, Istanbul, Besiktas, Turkey

    Duygu Ayyildiz Tamis

  4. GlaxoSmithKline, 34394, Istanbul, Besiktas, Turkey

    Nazli Kisbet

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  1. Sultan Gulce Iz
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Gulce Iz, S., Inevi, M.A., Metiner, P.S. et al. A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells. Appl Biochem Biotechnol 184, 303–322 (2018). https://doi.org/10.1007/s12010-017-2540-2

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