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Current achievements in the production of complex biopharmaceuticals with moss bioreactors

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Abstract

Transgenic plants are promising alternatives for the low-cost and safe pathogen-free production of complex recombinant pharmaceutical proteins (molecular farming). Plants as higher eukaryotes perform posttranslational modifications similar to those of mammalian cells. However, plant-specific protein N-glycosylation was shown to be immunogenic, a fact that represents a drawback for many plant systems in biopharmaceutical production. The moss Physcomitrella patens offers unique properties as a contained system for protein production. It is grown in the predominant haploid gametophytic stage as tissue suspension cultures in photobioreactors. Efficient secretory signals and a transient transfection system allow the secretion of freshly synthesized proteins to the surrounding medium. The key advantage of Physcomitrella compared to other plant systems is the feasibility of targeted gene replacements. By this means, moss strains with non-immunogenic humanized glycan patterns were created. Here we present an overview of the relevant aspects for establishing moss as a production system for recombinant biopharmaceuticals.

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Abbreviations

GMP:

Good manufacturing practice

ADCC:

Antibody-dependent cellular cytotoxicity

HAS:

Human serum albumin

VEGF:

Vascular endothelial growth factor

CHO:

Chinese Hamster Ovary

References

  1. Goeddel DV, Kleid DG, Bolivar F, Heyneker HL, Yansura DG, Crea R, Hirose T, Kraszewski A, Itakura K, Riggs AD (1979) Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci USA 76:106–110

    Article  CAS  Google Scholar 

  2. Martial JA, Hallewell RA, Baxter JD, Goodman HM (1979) Human growth hormone: complementary DNA cloning and expression in bacteria. Science 205:602–607

    Article  CAS  Google Scholar 

  3. Schmidt FR (2004) Recombinant expression systems in the pharmaceutical industry. Appl Microbiol Biotechnol 65:363–372

    Article  CAS  Google Scholar 

  4. Andersen DC, Krummen L (2002) Recombinant protein expression for therapeutic applications. Curr Opin Biotechnol 13:117–123

    Article  CAS  Google Scholar 

  5. Butler M (2005) Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol 68:283–291

    Article  CAS  Google Scholar 

  6. Walsh G, Jefferis R (2006) Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol 24:1241–1252

    Article  CAS  Google Scholar 

  7. Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152–158

    Article  CAS  Google Scholar 

  8. Ma JK, Drake PM, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4:794–805

    Article  CAS  Google Scholar 

  9. Ma JK, Chikwamba R, Sparrow P, Fischer R, Mahoney R, Twyman RM (2005) Plant-derived pharmaceuticals––the road forward. Trends Plant Sci 10:580–585

    Article  CAS  Google Scholar 

  10. Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22:1415–1422

    Article  CAS  Google Scholar 

  11. Schillberg S, Twyman RM, Fischer R (2005) Opportunities for recombinant antigen and antibody expression in transgenic plants-technology assessment. Vaccine 23:1764–1769

    Article  CAS  Google Scholar 

  12. Sijmons PC, Dekker BM, Schrammeijer B, Verwoerd TC, van den Elzen PJ, Hoekema A (1990) Production of correctly processed human serum albumin in transgenic plants. Biotechnology (NY) 8:217–221

    Article  CAS  Google Scholar 

  13. During K, Hippe S, Kreuzaler F, Schell J (1990) Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum. Plant Mol Biol 15:281–293

    Article  CAS  Google Scholar 

  14. Walsh G (2006) Biopharmaceutical benchmarks 2006. Nat Biotechnol 24:769–776

    Article  CAS  Google Scholar 

  15. Schellekens H (2002) Bioequivalence and the immunogenicity of biopharmaceuticals. Nat Rev Drug Discov 1:457–462

    Article  CAS  Google Scholar 

  16. Shields RL, Lai J, Keck R, O’Connell LY, Hong K, Meng YG, Weikert SH, Presta LG (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem 277:26733–26740

    Article  CAS  Google Scholar 

  17. Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M, Yamasaki M, Hanai N, Shitara K (2003) The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 278:3466–3473

    Article  CAS  Google Scholar 

  18. Bardor M, Faveeuw C, Fitchette AC, Gilbert D, Galas L, Trottein F, Faye L, Lerouge P (2003) Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha(1,3)-fucose and core xylose. Glycobiology 13:427–434

    Article  CAS  Google Scholar 

  19. Garcia-Casado G, Sanchez-Monge R, Chrispeels MJ, Armentia A, Salcedo G, Gomez L (1996) Role of complex asparagine-linked glycans in the allergenicity of plant glycoproteins. Glycobiology 6:471–477

    Article  CAS  Google Scholar 

  20. van Ree R, Cabanes-Macheteau M, Akkerdaas J, Milazzo JP, Loutelier-Bourhis C, Rayon C, Villalba M, Koppelman S, Aalberse R, Rodriguez R, Faye L, Lerouge P (2000) Beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem 275:11451–11458

    Article  Google Scholar 

  21. Westphal S, Kolarich D, Foetisch K, Lauer I, Altmann F, Conti A, Crespo JF, Rodriguez J, Enrique E, Vieths S, Scheurer S (2003) Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato. Eur J Biochem 270:1327–1337

    Article  CAS  Google Scholar 

  22. Faye L, Boulaflous A, Benchabane M, Gomord V, Michaud D (2005) Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming. Vaccine 23:1770–1778

    Article  CAS  Google Scholar 

  23. Decker EL, Reski R (2004) The moss bioreactor. Curr Opin Plant Biol 7:166–170

    Article  CAS  Google Scholar 

  24. Frank W, Decker EL, Reski R (2005) Molecular tools to study Physcomitrella patens. Plant Biol 7:220–227

    Article  CAS  Google Scholar 

  25. Schulte J, Reski R (2004) High throughput cryopreservation of 140,000 Physcomitrella patens mutants. Plant Biol 6:119–127

    Article  CAS  Google Scholar 

  26. Egener T, Granado J, Guitton MC, Hohe A, Holtorf H, Lucht JM, Rensing SA, Schlink K, Schulte J, Schween G, Zimmermann S, Duwenig E, Rak B, Reski R (2002) High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library. BMC Plant Biol 2:6

    Article  Google Scholar 

  27. Schween G, Hohe A, Koprivova A, Reski R (2003) Effects of nutrients, cell density and culture techniques on protoplast regeneration and early protonema development in a moss, Physcomitrella patens. J Plant Physiol 160:209–212

    Article  CAS  Google Scholar 

  28. Hohe A, Decker EL, Gorr G, Schween G, Reski R (2002) Tight control of growth and cell differentiation in photoautotrophically growing moss (Physcomitrella patens) bioreactor cultures. Plant Cell Rep 20:1135–1140

    Article  CAS  Google Scholar 

  29. Hohe A, Reski R (2002) Optimisation of a bioreactor culture of the moss Physcomitrella patens for mass production of protoplasts. Plant Sci 163:69–74

    Article  CAS  Google Scholar 

  30. Lucumi A, Posten C (2006) Establishment of long-term perfusion cultures of recombinant moss in a pilot tubular photobioreactor. Proc Biochem 41:2180–2187

    Article  CAS  Google Scholar 

  31. Baur A, Reski R, Gorr G (2005) Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens. Plant Biotechnol J 3:331–340

    Article  CAS  Google Scholar 

  32. Huether CM, Lienhart O, Baur A, Stemmer C, Gorr G, Reski R, Decker EL (2005) Glyco-engineering of moss lacking plant-specific sugar residues. Plant Biol 7:292–299

    Article  CAS  Google Scholar 

  33. Koprivova A, Stemmer C, Altmann F, Hoffmann A, Kopriva S, Gorr G, Reski R, Decker EL (2004) Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans. Plant Biotech J 2:517–523

    Article  CAS  Google Scholar 

  34. Schaaf A, Reski R, Decker EL (2004) A novel aspartic proteinase is targeted to the secretory pathway and to the vacuole in the moss Physcomitrella patens. Eur J Cell Biol 83:145–152

    Article  CAS  Google Scholar 

  35. Schaaf A, Tintelnot S, Baur A, Reski R, Gorr G, Decker EL (2005) Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells. BMC Biotechnol 5:30

    Article  Google Scholar 

  36. Jost W, Link S, Horstmann V, Decker EL, Reski R, Gorr G (2005) Isolation and characterisation of three moss-derived beta-tubulin promoters suitable for recombinant expression. Curr Genet 47:111–120

    Article  CAS  Google Scholar 

  37. Weise A, Rodriguez-Franco M, Timm B, Hermann M, Link S, Jost W, Gorr G (2006) Use of Physcomitrella patens actin 5′ regions for high transgene expression: importance of 5′ introns. Appl Microbiol Biotechnol 70:337–345

    Article  CAS  Google Scholar 

  38. Baur A, Kaufmann F, Rolli H, Weise A, Luethje R, Berg B, Braun M, Baeumer W, Kietzmann M, Reski R, Gorr G (2005) A fast and flexible PEG-mediated transient expression system in plants for high level expression of secreted recombinant proteins. J Biotechnol 119:332–342

    Article  CAS  Google Scholar 

  39. Strepp R, Scholz S, Kruse S, Speth V, Reski R (1998) Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin. Proc Natl Acad Sci USA 95:4368–4373

    Article  CAS  Google Scholar 

  40. Horstmann V, Huether CM, Jost W, Reski R, Decker EL (2004) Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude. BMC Biotechnol 4:13

    Article  Google Scholar 

  41. Schaefer DG (2002) A new moss genetics: targeted mutagenesis in Physcomitrella patens. Annu Rev Plant Biol 53:477–501

    Article  CAS  Google Scholar 

  42. Zeidler M, Gatz C, Hartmann E, Hughes J (1996) Tetracycline-regulated reporter gene expression in the moss Physcomitrella patens. Plant Mol Biol 30:199–205

    Article  CAS  Google Scholar 

  43. Knight CD, Sehgal A, Atwal K, Wallace JC, Cove DJ, Coates D, Quatrano RS, Bahadur S, Stockley PG, Cuming AC (1995) Molecular responses to abscisic acid and stress are conserved between moss and cereals. Plant Cell 7:499–506

    Article  CAS  Google Scholar 

  44. Bierfreund NM, Reski R, Decker EL (2003) Use of an inducible reporter gene system for the analysis of auxin distribution in the moss Physcomitrella patens. Plant Cell Rep 21:1143–1152

    Article  CAS  Google Scholar 

  45. Saidi Y, Finka A, Chakhporanian M, Zryd JP, Schaefer DG, Goloubinoff P (2005) Controlled expression of recombinant proteins in Physcomitrella patens by a conditional heat-shock promoter: a tool for plant research and biotechnology. Plant Mol Biol 59:697–711

    Article  CAS  Google Scholar 

  46. Kiessling J, Kruse S, Rensing SA, Harter K, Decker EL, Reski R (2000) Visualization of a cytoskeleton-like FtsZ network in chloroplasts. J Cell Biol 151:945–950

    Article  CAS  Google Scholar 

  47. Kiessling J, Martin A, Gremillon L, Rensing SA, Nick P, Sarnighausen E, Decker EL, Reski R (2004) Dual targeting of plastid division protein FtsZ to chloroplasts and the cytoplasm. EMBO Rep 5:889–894

    Article  CAS  Google Scholar 

  48. Richter U, Kiessling J, Hedtke B, Decker E, Reski R, Borner T, Weihe A (2002) Two RpoT genes of Physcomitrella patens encode phage-type RNA polymerases with dual targeting to mitochondria and plastids. Gene 290:95–105

    Article  CAS  Google Scholar 

  49. Nishiyama T, Fujita T, Shin IT, Seki M, Nishide H, Uchiyama I, Kamiya A, Carninci P, Hayashizaki Y, Shinozaki K, Kohara Y, Hasebe M (2003) Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: implication for land plant evolution. Proc Natl Acad Sci USA 100:8007–8012

    Article  CAS  Google Scholar 

  50. Rensing SA, Rombauts S, Van de Peer Y, Reski R (2002) Moss transcriptome and beyond. Trends Plant Sci 7:535–538

    Article  CAS  Google Scholar 

  51. Lang D, Eisinger J, Reski R, Rensing SA (2005) Representation and high-quality annotation of the Physcomitrella patens transcriptome demonstrates a high proportion of proteins involved in metabolism in mosses. Plant Biol 7:238–250

    Article  CAS  Google Scholar 

  52. Rensing SA, Fritzowsky D, Lang D, Reski R (2005) Protein encoding genes in an ancient plant: analysis of codon usage, retained genes and splice sites in a moss, Physcomitrella patens. BMC Genomics 6:43

    Article  Google Scholar 

  53. Franklin SE, Mayfield SP (2004) Prospects for molecular farming in the green alga Chlamydomonas. Curr Opin Plant Biol 7:159–165

    Article  CAS  Google Scholar 

  54. Schaefer DG (2001) Gene targeting in Physcomitrella patens. Curr Opin Plant Biol 4:143–150

    Article  CAS  Google Scholar 

  55. Koprivova A, Altmann F, Gorr G, Kopriva S, Reski R, Decker EL (2003) N-Glycosylation in the moss Physcomitrella patens is organized similarly to higher plants. Plant Biol 5:582–591

    Article  CAS  Google Scholar 

  56. Vietor R, Loutelier-Bourhis C, Fitchette AC, Margerie P, Gonneau M, Faye L, Lerouge P (2003) Protein N-glycosylation is similar in the moss Physcomitrella patens and in higher plants. Planta 218:269–275

    Article  CAS  Google Scholar 

  57. Mari A (2002) IgE to cross-reactive carbohydrate determinants: analysis of the distribution and appraisal of the in vivo and in vitro reactivity. Int Arch Allergy Immunol 129:286–295

    Article  CAS  Google Scholar 

  58. Koprivova A, Stemmer C, Altmann F, Hoffmann A, Kopriva S, Gorr G, Reski R, Decker EL (2004) Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans. Plant Biotechnol J 2:517–523

    Article  CAS  Google Scholar 

  59. Nechansky A, Schuster M, Jost W, Siegl P, Wiederkum S, Gorr G, Kircheis R (2007) Compensation of endogenous IgG mediated inhibition of antibody-dependent cellular cytotoxicity by glyco-engineering of therapeutic antibodies. Mol Immunol 44:1826–1828

    Article  Google Scholar 

  60. Gorr G, Jost W (2005) Glycosylation design in transgenic moss for better product efficacy. Bioprocess J 4:26–30

    Google Scholar 

  61. Gorr G, Altmann F (2006) Glycosylation of recombinant proteins in plants. In: Kayser O, Quax W (eds) Medical plant biotechnology. Wiley-VCH, Weinheim, pp 345–374

    Google Scholar 

  62. Bakker H, Bardor M, Molthoff JW, Gomord V, Elbers I, Stevens LH, Jordi W, Lommen A, Faye L, Lerouge P, Bosch D (2001) Galactose-extended glycans of antibodies produced by transgenic plants. Proc Natl Acad Sci USA 98:2899–2904

    Article  CAS  Google Scholar 

  63. Palacpac NQ, Yoshida S, Sakai H, Kimura Y, Fujiyama K, Yoshida T, Seki T (1999) Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns. Proc Natl Acad Sci USA 96:4692–4697

    Article  CAS  Google Scholar 

  64. Lerouge P, Bardor M, Pagny S, Gomord V, Faye L (2000) N-glycosylation of recombinant pharmaceutical glycoproteins produced in transgenic plants: towards an humanisation of plant N-glycans. Curr Pharm Biotechnol 1:347–354

    Article  CAS  Google Scholar 

  65. Shah MM, Fujiyama K, Flynn CR, Joshi L (2003) Sialylated endogenous glycoconjugates in plant cells. Nat Biotechnol 21:1470–1471

    Article  CAS  Google Scholar 

  66. Zeleny R, Kolarich D, Strasser R, Altmann F (2006) Sialic acid concentrations in plants are in the range of inadvertent contamination. Planta 224:222–227

    Article  CAS  Google Scholar 

  67. Delorme E, Lorenzini T, Giffin J, Martin F, Jacobsen F, Boone T, Elliott S (1992) Role of glycosylation on the secretion and biological activity of erythropoietin. Biochemistry 31:9871–9876

    Article  CAS  Google Scholar 

  68. Erbayraktar S, Grasso G, Sfacteria A, Xie QW, Coleman T, Kreilgaard M, Torup L, Sager T, Erbayraktar Z, Gokmen N, Yilmaz O, Ghezzi P, Villa P, Fratelli M, Casagrande S, Leist M, Helboe L, Gerwein J, Christensen S, Geist MA, Pedersen LO, Cerami-Hand C, Wuerth JP, Cerami A, Brines M (2003) Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc Natl Acad Sci USA 100:6741–6746

    Article  CAS  Google Scholar 

  69. 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 6:593–599

    Article  CAS  Google Scholar 

  70. Cox KM, Sterling JD, Regan JT, Gasdaska JR, Frantz KK, Peele CG, Black A, Passmore D, Moldovan-Loomis C, Srinivasan M, Cuison S, Cardarelli PM, Dickey LF (2006) Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 24:1591–1597

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the German Federal Ministry of Education and Research (BMBF grants 0312624 and 0313852), the German Academic Exchange Service (DAAD), and the Wissenschaftliche Gesellschaft of the University of Freiburg.

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  1. Plant Biotechnology, Faculty of Biology, Freiburg University, Schaenzlestr. 1, 79104, Freiburg, Germany

    Eva L. Decker & Ralf Reski

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  1. Eva L. Decker
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Correspondence to Eva L. Decker.

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Decker, E.L., Reski, R. Current achievements in the production of complex biopharmaceuticals with moss bioreactors. Bioprocess Biosyst Eng 31, 3–9 (2008). https://doi.org/10.1007/s00449-007-0151-y

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