Transgenic Research

, Volume 19, Issue 2, pp 241–256 | Cite as

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals

  • Richard Colgan
  • Christopher J. Atkinson
  • Matthew Paul
  • Sally Hassan
  • Pascal M. W. Drake
  • Amy L. Sexton
  • Simon Santa-Cruz
  • David James
  • Keith Hamp
  • Colin Gutteridge
  • Julian K-C. Ma
Original Paper

Abstract

Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth temperature. Overall productivity is therefore correlated to biomass production, although other factors such as purification burden, extractability protein stability and quality also need to be considered in the optimal design of cultivation conditions.

Keywords

Contained cultivation Nicotiana tabacum Molecular farming Recombinant antibody 

References

  1. Boyd MR, Gustafson KR, McMahon JB, Shoemaker RH, O’Keefe BR, Mori T, Gulakowski RJ, Wu L, Rivera MI, Laurencot CM, Currens MJ, Cardellina JH, Buckheit RW Jr, Nara PL, Pannell LK, Sowder RC, Henderson LE (1997) Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development. Antimicrob Agents Chemother 41:1521–1530PubMedGoogle Scholar
  2. Colleluori DM, Tien D, Kang F, Pagliei T, Kuss R, McCormick T, Watson K, McFadden K, Chaiken I, Buckheit RW Jr, Romano JW (2005) Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development. Protein Expr Purif 39:229–236CrossRefPubMedGoogle Scholar
  3. Elbers IJ, Stoopen GM, Bakker H, Stevens LH, Bardor M, Molthoff JW, Jordi WJ, Bosch D, Lommen A (2001) Influence of growth conditions and developmental stage on N-glycan heterogeneity of transgenic immunoglobulin G and endogenous proteins in tobacco leaves. Plant Physiol 126:1314–1322CrossRefPubMedGoogle Scholar
  4. Geada D, Valdes R, Escobar A, Ares DM, Torres E, Blanco R, Ferro W, Dorta D, Gonzalez M, Aleman MR, Padilla S, Gomez L, Del CN, Mendoza O, Urquiza D, Soria Y, Brito J, Leyva A, Borroto C, Gavilondo JV (2007) Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco. Biologicals 35:309–315CrossRefPubMedGoogle Scholar
  5. Hassan S, van Dolleweerd CJ, Ioakeimidis F, Keshavarz-Moore E, Ma JK (2008) Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants. Plant Biotechnol J 6:733–748CrossRefPubMedGoogle Scholar
  6. Henkes S, Sonnewald U, Badur R, Flachmann R, Stitt M (2001) A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism. Plant Cell 13:535–551CrossRefPubMedGoogle Scholar
  7. Ma JK, Lehner T, Stabila P, Fux CI, Hiatt A (1994) Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants. Eur J Immunol 24:131–138CrossRefPubMedGoogle Scholar
  8. Ma JK, Hiatt A, Hein M, Vine ND, Wang F, Stabila P, van DC, Mostov K, Lehner T (1995) Generation and assembly of secretory antibodies in plants. Science 268:716–719CrossRefPubMedGoogle Scholar
  9. Ma JK, Barros E, Bock R, Christou P, Dale PJ, Dix PJ, Fischer R, Irwin J, Mahoney R, Pezzotti M, Schillberg S, Sparrow P, Stoger E, Twyman RM (2005) Molecular farming for new drugs and vaccines. Current perspectives on the production of pharmaceuticals in transgenic plants. EMBO Rep 6:593–599CrossRefPubMedGoogle Scholar
  10. Sexton A, Drake PM, Mahmood N, Harman SJ, Shattock RJ, Ma JK (2006) Transgenic plant production of Cyanovirin-N, an HIV microbicide. FASEB J 20:356–358PubMedGoogle Scholar
  11. Smith R, Lehner T, Beverley PC (1984) Characterization of monoclonal antibodies to Streptococcus mutans antigenic determinants I/II, I, II, and III and their serotype specificities. Infect Immun 46:168–175PubMedGoogle Scholar
  12. Sparrow PA, Irwin JA, Dale PJ, Twyman RM, Ma JK (2007) Pharma-planta: road testing the developing regulatory guidelines for plant-made pharmaceuticals. Transgenic Res 16:147–161CrossRefPubMedGoogle Scholar
  13. Spok A, Twyman RM, Fischer R, Ma JK, Sparrow PA (2008) Evolution of a regulatory framework for pharmaceuticals derived from genetically modified plants. Trends Biotechnol 26:506–517CrossRefPubMedGoogle Scholar
  14. Stevens LH, Stoopen GM, Elbers IJ, Molthoff JW, Bakker HA, Lommen A, Bosch D, Jordi W (2000) Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco. Plant Physiol 124:173–182CrossRefPubMedGoogle Scholar
  15. Valdes R, Gomez L, Padilla S, Brito J, Reyes B, Alvarez T, Mendoza O, Herrera O, Ferro W, Pujol M, Leal V, Linares M, Hevia Y, Garcia C, Mila L, Garcia O, Sanchez R, Acosta A, Geada D, Paez R, Luis VJ, Borroto C (2003) Large-scale purification of an antibody directed against hepatitis B surface antigen from transgenic tobacco plants. Biochem Biophys Res Commun 308:94–100CrossRefPubMedGoogle Scholar
  16. van Dolleweerd CJ, Chargelegue D, Ma JK (2003) Characterization of the conformational epitope of Guy’s 13, a monoclonal antibody that prevents Streptococcus mutans colonization in humans. Infect Immun 71:754–765CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Richard Colgan
    • 1
  • Christopher J. Atkinson
    • 1
  • Matthew Paul
    • 2
  • Sally Hassan
    • 2
  • Pascal M. W. Drake
    • 2
  • Amy L. Sexton
    • 2
  • Simon Santa-Cruz
    • 3
  • David James
    • 3
  • Keith Hamp
    • 4
  • Colin Gutteridge
    • 1
  • Julian K-C. Ma
    • 2
  1. 1.East Malling ResearchEast MallingUK
  2. 2.CMM, 2nd Floor Jenner WingSt. George’s Hospital Medical SchoolLondonUK
  3. 3.Empharm Ltd.East MallingUK
  4. 4.Unigro Ltd.Gay Dawn OfficesFawkhamUK

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