Skip to main content

Advertisement

SpringerLink
Log in

Techno-Economic Analysis of Horseradish Peroxidase Production Using a Transient Expression System in Nicotiana benthamiana

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Despite the advantages of plant-based transient expression systems relative to microbial or mammalian cell systems, the commercial production of recombinant proteins using plants has not yet been achieved to any significant extent. One of the challenges has been the lack of published data on the costs of manufacture for products other than biopharmaceuticals. In this study, we report on the techno-economic analysis of the production of a standard commercial enzyme, namely, horseradish peroxidase (HRP), using a transient expression system in Nicotiana benthamiana. Based on the proven plant yield of 240 mg HRP/kg biomass, a biomass productivity of 15-kg biomass/m2/year and a process yield of 54 % (mg HRP product/mg HRP in biomass), it is apparent that HRP can be manufactured economically via transient expression in plants in a large-scale facility (>5 kg HRP/year). At this level, the process is competitive versus the existing technology (extraction of the enzyme from horseradish), and the product is of comparable or improved activity, containing only the preferred isoenzyme C. Production scale, protein yield and biomass productivity are found to be the most important determinants of overall viability.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Lico, C., Santi, L., Twyman, R. M., Pezzotti, M., & Avesani, L. (2012). The use of plants for the production of therapeutic human peptides. Plant Cell Reports, 31, 439–451.

    Article  CAS  Google Scholar 

  2. Viana, A. A., Pelegrini, P. B., & Grossi-de-Sa, M. F. (2012). Plant biofarming: novel insights for peptide expression in heterologous systems. Biopolymers, 98, 416–427.

    Article  Google Scholar 

  3. Fischer, R., Drossard, J., Commandeur, U., Schillberg, S., & Emans, N. (1999). Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants. Biotechnology and Applied Biochemistry, 30(Pt 2), 101–108.

    CAS  Google Scholar 

  4. Fischer, R., & Emans, N. (2000). Molecular farming of pharmaceutical proteins. Transgenic Research, 9, 279–299.

    Article  CAS  Google Scholar 

  5. Daniell, H., Singh, N. D., Mason, H., & Streatfield, S. J. (2009). Plant-made vaccine antigens and biopharmaceuticals. Trends in Plant Science, 14, 669–679.

    Article  CAS  Google Scholar 

  6. Chen, Q., Lai, H., Hurtado, J., Stahnke, J., Leuzinger, K., & Dent, M. (2013). Agroinfiltration as an effective and scalable strategy of gene delivery for production of pharmaceutical proteins. Advanced Techniques in Biology & Medicine, 1, 103. doi:10.4172/atbm.1000103.

    Article  CAS  Google Scholar 

  7. Gleba, Y. Y., Tusé, D. & Giritch, A. (2014). Plant viral vectors. In K Palmer & Y Gleba, Current topics in microbiology and immunology (pp. 155–192). New York: Springer.

  8. Rybicki, E. P. (2010). Plant-made vaccines for humans and animals. Plant Biotechnology Journal, 8, 620–637.

    Article  CAS  Google Scholar 

  9. Fischer, R., Stoger, E., Schillberg, S., Christou, P., & Twyman, R. M. (2004). Plant-based production of biopharmaceuticals. Current Opinion in Plant Biology, 7, 152–158.

    Article  CAS  Google Scholar 

  10. Kathuria, S., Sriraman, R., Nath, R., Sack, M., Pal, R., Artsaenko, O., Talwar, G., Fischer, R., & Finnern, R. (2002). Efficacy of plant-produced recombinant antibodies against HCG. Human Reproduction, 17, 2054–2061.

    Article  CAS  Google Scholar 

  11. Vaquero, C., Sack, M., Schuster, F., Finnern, R., Drossard, J., Schumann, D., Reimann, A., & Fischer, R. (2002). A carcinoembryonic antigen-specific diabody produced in tobacco. The FASEB Journal, 16, 408–410.

    CAS  Google Scholar 

  12. Meyers, A., Chakauya, E., Shephard, E., Tanzer, F. L., Maclean, J., Lynch, A., Williamson, A.-L., & Rybicki, E. P. (2008). Expression of HIV-1 antigens in plants as potential subunit vaccines. BMC Biotechnology, 8, 53.

    Article  Google Scholar 

  13. Maclean, J., Koekemoer, M., Olivier, A., Stewart, D., Hitzeroth, I., Rademacher, T., Fischer, R., Williamson, A.-L., & Rybicki, E. (2007). Optimization of human papilloma virus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16L1 gene variants and different cell-compartment localization. Journal of General Virology, 88, 1460–1469.

    Article  CAS  Google Scholar 

  14. Ma, J. K., Chikwamba, R., Sparrow, P., Fischer, R., Mahoney, R., & Twyman, R. M. (2005). Plant-derived pharmaceuticals–the road forward. Trends in Plant Science, 10, 580–585.

    Article  CAS  Google Scholar 

  15. Schillberg, S., Twyman, R. M., & Fischer, R. (2005). Opportunities for recombinant antigen and antibody expression in transgenic plants—technology assessment. Vaccine, 23, 1764–1769.

    Article  CAS  Google Scholar 

  16. Thomas, D. R., Penney, C. A., Majumder, A., & Walmsley, A. M. (2011). Evolution of plant-made pharmaceuticals. International Journal of Molecular Sciences, 12, 3220–3236.

    Article  CAS  Google Scholar 

  17. GEN. (2012). Protalix, Pfizer report FDA approval of plant-derived Gaucher disease ERT Elelyso. Genetic Engineering & Biotechnology News. GEN. http://www.genengnews.com/gen-news-highlights/protalix-pfizer-report-fda-approval-of-plant-derived-gaucher-disease-ert-elelyso/81246710/. Accessed 3 April 2014.

  18. InVitria. (2010). Lactoferrin is a multi-functional protein that maintains cell health and is found in milk and other bodily fluids. InVitria. http://www.invitria.com/cell-culture-products-services/lactoferrin.html. Accessed 3 April 2014.

  19. Spök, A., & Karner, S. (2008). In A. J. Stein & E. Rodríguez-Cerezo (Eds.), Plant molecular farming: opportunities and challenges. Brussels: Institute for Prospective Technological Studies.

    Google Scholar 

  20. GEN. (2013). Plant-based protein biomanufacturing. Genetic Engineering & Biotechnology News. GEN. http://www.genengnews.com/gen-articles/plant-based-protein-biomanufacturing/4734/. Accessed 1 August 2014.

  21. Buyel, J., & Fischer, R. (2012). Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs. Biotechnology and Bioengineering, 109, 2575–2588.

    Article  CAS  Google Scholar 

  22. Hendricks, F., & Thiel, L. (2011). CSIR biopharming platform: pre-feasibility study report. Pretoria: CSIR.

    Google Scholar 

  23. Kushad, M. M., Guidera, M., & Bratsch, A. D. (1999). Distribution of horseradish peroxidase activity in horseradish plants. HortScience, 34, 127–129.

    CAS  Google Scholar 

  24. Veitch, N. C. (2004). Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry, 65, 249–259.

    Article  CAS  Google Scholar 

  25. Krel, H. W. (1991). In J. Lobarzewski, H. Greppin, C. Pelel, & T. Gaspar (Eds.), Biochemical, molecular, and physiological aspects of plant peroxidases (pp. 470–478). Geneva: Imprimerie Nationale.

    Google Scholar 

  26. Dewan, S. S. (2011). Market research report on medical enzymes: technologies and global markets. Wellesley: BCC Research.

    Google Scholar 

  27. Barnard, A. (2012). The optimization of the extraction and purification of horseradish peroxidase from horseradish roots. MSc Thesis, Stellenbosch University, Stellenbosch, South Africa.

  28. Backhurst, J. R., & Harker, J. H. (1973). Process plant design (1st ed.). New York: American Elsevier.

    Google Scholar 

  29. Choi, D., Chipman, D. C., Bents, S. C., & Brown, R. C. (2010). A techno-economic analysis of polyhydroxyalkanoate and hydrogen production from syngas fermentation of gasified biomass. Applied Biochemistry and Biotechnology, 160, 1032–1046.

    Article  CAS  Google Scholar 

  30. Peters, M., Timmerhaus, K. & West, R. (2003). Plant design and economics for chemical engineers. 5th ed. McGraw-Hill Education, New York.

  31. Giacomelli, G. (2011). Designing the greenhouse to meet your expectations: what’s your technology level? CEAC, the University of Arizona.

  32. Pannell, D. J. (1997). Sensitivity analysis of normative economic models: theoretical framework and practical strategies. Agricultural Economics, 16, 139–152.

    Article  Google Scholar 

  33. Estrada, J. M., Kraakman, N., Lebrero, R., & Muñoz, R. (2012). A sensitivity analysis of process design parameters, commodity prices and robustness on the economics of odour abatement technologies. Biotechnology Advances, 30, 1354–1363.

    Article  CAS  Google Scholar 

  34. de Araujo, A. C., Gallani, S., Mulas, M., & Skogestad, S. (2013). Sensitivity analysis of optimal operation of an activated sludge process model for economic controlled variable selection. Industrial & Engineering Chemistry Research, 52, 9908–9921.

    Article  Google Scholar 

  35. Wilken, L. R., & Nikolov, Z. L. (2012). Recovery and purification of plant-made recombinant proteins. Biotechnology Advances, 30, 419–433.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

DW acknowledges the National Intellectual Property Management Organisation and the Research Contracts and Intellectual Property Services of the University of Cape Town for funding this study. EPR acknowledges the Technology Innovation Agency and the Department of Science and Technology for funding the research.

Conflict of Interest

The authors declare that they have no competing interests deriving from the subject matter of this work.

Authors’ Contributions

DW undertook the analyses and wrote the draft manuscript. EPR had overall responsibility for the work that provided the basis for the study, participated in its design and helped to draft the manuscript. SMH performed the laboratory work and edited the manuscript. All authors read and approved the final manuscript.

Author information

Authors and Affiliations

  1. Department of Engineering and Technology Management, University of Pretoria, Pretoria, 0002, Gauteng, South Africa

    David Richard Walwyn

  2. Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Private Bag X3, Rondebosch, 7701, Cape Town, South Africa

    Suzanne M. Huddy & Edward P. Rybicki

  3. Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa

    Edward P. Rybicki

Authors
  1. David Richard Walwyn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Suzanne M. Huddy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Edward P. Rybicki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David Richard Walwyn.

Electronic supplementary material

The data sets and methodology supporting the results of this article are included within the article, the supplementary material, and in the cited work [23].

ESM 1

(DOCX 18 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Walwyn, D.R., Huddy, S.M. & Rybicki, E.P. Techno-Economic Analysis of Horseradish Peroxidase Production Using a Transient Expression System in Nicotiana benthamiana . Appl Biochem Biotechnol 175, 841–854 (2015). https://doi.org/10.1007/s12010-014-1320-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-014-1320-5

Keywords

Search

Navigation