Recombinant pharmaceutical protein production in plants: unraveling the therapeutic potential of molecular pharming

Abstract

There is an increasing demand for the generation of recombinant pharmaceutical proteins for a wide array of therapeutic applications. In comparison to bacterial, yeast and animal cells, the production of recombinant proteins in plants with economic and therapeutic importance has only started recently. The most important prerequisite of any expression systems is that it should be simple and inexpensive. In this regard, plant-based expression has emerged an as accepted alternative to conventional expression platforms due to economic feasibility, rapid scalability, higher stability of recombinant proteins, safety due to lack of harmful substances (human, animal pathogens and pyrogens) and capability of producing proteins with desired secondary modifications. Heterologous expression using plants has played a pivotal role in the development of a myriad of recombinant proteins, including neutraceuticals and monoclonal antibodies being utilized in various therapeutic approaches. This paper presents an overview about the current status, various strategies and advantages of pharmaceutical protein production in plant expression systems. We also present a summary of expression of therapeutic monoclonal antibodies, vaccines, clinical trials and the regulatory aspects of plant-based expression. Furthermore, the challenges encountered in plant expression such as costs associated with existing purification strategies are discussed.

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Abbreviations

AAD:

Antibiotic-associated diarrhea

Mab:

Monoclonal antibody

ScFv:

Single chain variable fragment

PMP:

Plant-made pharmaceuticals

References

  1. Altman A, Hasegawa PM (2011) Plant biotechnology and agriculture: prospects for the 21st century. Academic Press, London

    Google Scholar 

  2. Aviezer D, Brill-Almon E, Shaaltiel Y, Hashmueli S, Bartfeld D, Mizrachi S, Liberman Y, Freeman A, Zimran A, Galun E (2009) A plant-derived recombinant human glucocerebrosidase enzyme—a preclinical and phase I investigation. PLoS One 4:e4792

    Article  PubMed  PubMed Central  Google Scholar 

  3. Bartlett JG, Snape JW, Harwood WA (2009) Intron-mediated enhancement as a method for increasing transgene expression levels in barley. Plant Biotechnol J 7:856–866

    CAS  Article  PubMed  Google Scholar 

  4. Bock R (2015) Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology. Ann Rev Plant Biol 66:211–241

    CAS  Article  Google Scholar 

  5. Bosch D, Castilho A, Loos A, Schots A, Steinkellner H (2013) N-Glycosylation of plant-produced recombinant proteins. Curr Pharm Des 19:5503–5512

    CAS  Article  PubMed  Google Scholar 

  6. Broz A, Huang N, Unruh G (2013) Plant-based protein biomanufacturing. Genet Eng Biotechnol News 33:4

    Article  Google Scholar 

  7. Chen R (2012) Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol Adv 30:1102–1107

    CAS  Article  PubMed  Google Scholar 

  8. Chen Q, Lai H (2014) Gene delivery into plant cells for recombinant protein production. Biomed Res Int 2014:932161

    Google Scholar 

  9. Chen Q, Santi L, Zhang C (2014) Plant made biologics. Biomed Res Int. doi:10.1155/2014/418064

    Google Scholar 

  10. Chiaiese P, Palomba F, Tatino F, Lanzillo C, Pinto G, Pollio A, Filippone E (2011) Engineered tobacco and microalgae secreting the fungal laccase POXA1b reduce phenol content in olive oil mill wastewater. Enzyme Microb Technol 49:540–546

    CAS  Article  PubMed  Google Scholar 

  11. Chung ND, Kim NS, Giap doV, Jang SH, Oh SM, Jang SH, Kim TG, Jang YS, Yang MS (2014) Production of functional human vascular endothelial growth factor (165) in transgenic rice cell suspension cultures. Enzyme Microb Technol 63:58–63

  12. Cummings JF, Guerrero ML, Moon JE, Waterman P, Nielsen RK, Jefferson S, Gross FL, Hancock K, Katz JM, Yusibov V (2014) Safety and immunogenicity of a plant produced recombinant monomer hemagglutinin-based influenza vaccine derived from influenza A (H1N1)pdm09 virus: a phase 1 dose-escalation study in healthy adults. Vaccine 32:2251–2259

    CAS  Article  PubMed  Google Scholar 

  13. Decker EL, Parsons J, Reski R (2014) Glyco-engineering for biopharmaceutical production in moss bioreactors. Front Plant Sci 5:346

    Article  PubMed  PubMed Central  Google Scholar 

  14. Desai PN, Shrivastava N, Padh H (2010) Production of heterologous proteins in plants. Strategies for optimal expression. Biotechnol Adv 28:427–435

    CAS  Article  PubMed  Google Scholar 

  15. Dirisala VR, Jeevan A, Bix G, Yoshimura T, McMurray DN (2012) Molecular cloning and expression of the IL-10 gene from guinea pigs. Gene 498:120–127

    CAS  Article  PubMed  Google Scholar 

  16. Dirisala VR, Jeevan A, Ramasamy SK, McMurray DN (2013) Molecular cloning, expression and in silico structural analysis of guinea pig IL-17. Mol Biotechnol 55:277–287

    CAS  Article  PubMed  Google Scholar 

  17. Dirisala VR, Jeevan A, Ly LH, McMurray DN (2015) Molecular and biochemical characterization of recombinant guinea pig tumor necrosis factor-alpha (TNF-α). Mediat Inflamm (article ID 619480)

  18. Dolleweerd CJV, Teh AYH, Banyard AC et al (2014) Engineering, expression in transgenic plants and characterization of E559, a rabies virus-neutralizing monoclonal antibody. J Infect Dis 210:200–208

    Article  PubMed  PubMed Central  Google Scholar 

  19. Dugdale B, Mortimer CL, Kato M, James TA, Harding RM, Dale JL (2013) In plant activation: an inducible, hyperexpression platform for recombinant protein production in plants. Plant Cell 25:2429–2443

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Egan AN, Schlueter J, Spooner DM (2012) Applications of next-generation sequencing in plant biology. Am J Bot 99:175–185

    CAS  Article  PubMed  Google Scholar 

  21. Egelkrout E, Rajan V, Howard JA (2012) Overproduction of recombinant proteins in plants. Plant Sci 184:83–101

    CAS  Article  PubMed  Google Scholar 

  22. Feller T, Pascal T, Koch N, Spiegel H, Addai-Mensah Fischer R et al (2013) Plant based production of recombinant plasmodium surface protein Pf38 and evaluation of its potential as a vaccine candidate. PLoS One 8:e79920

    Article  PubMed  PubMed Central  Google Scholar 

  23. Ferreira AM, Gentile P, Chiono V, Ciardelli G (2012) Collagen for bone tissue regeneration. Acta Biomater 8:3191–3200

    CAS  Article  PubMed  Google Scholar 

  24. Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J (2012) GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnol Adv 30:434–439

    CAS  Article  PubMed  Google Scholar 

  25. Fischer R, Schillberg S, Buyel JF, Twyman RM (2013) Commercial aspects of pharmaceutical protein production in plants. Curr Pharm Des 19:5471–5477

    CAS  Article  PubMed  Google Scholar 

  26. Fox JL (2003) Puzzling industry response to ProdiGene fiasco. Nat Biotechnol 21:3–4

    CAS  Article  PubMed  Google Scholar 

  27. Fujiwara Y, Aiki Y, Yang L, Takaiwa F, Kosaka A, Tsuji NM, Shiraki K, Sekikawa K (2010) Extraction and purification of human interleukin-10 from transgenic rice seeds. Protein Expr Purif 72:125–130

    CAS  Article  PubMed  Google Scholar 

  28. Gecchele E, Merlin M, Brozzetti A, Falorni A, Pezzotti M, Avesani L (2015) A comparative analysis of recombinant protein expression in different biofactories: bacteria, insect cells and plant systems. J Vis Exp 97:e52459–e52459

    Google Scholar 

  29. Gelse K, Pöschl E, Aigner T (2003) Collagens—structure, function, and biosynthesis. Adv Drug Deliv Rev 55:1531–1546

    CAS  Article  PubMed  Google Scholar 

  30. Gleba A, Giritch YY (2011) Plant viral vectors for protein expression. Horizon Scientific Press, London

  31. Gleba YY, Tuse D, Giritch A (2014) Plant viral vectors for delivery by Agrobacterium. Plant viral vectors book. Springer, Berlin, pp 155–192

  32. Hassouneh W, MacEwan SR, Chilkoti A (2012) Fusions of elastin-like polypeptides to pharmaceutical proteins. Methods Enzymol 502:215–237

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Hefferon K (2013) Plant-derived pharmaceuticals for the developing world. Biotechnol J 8:1193–1202

    CAS  PubMed  Google Scholar 

  34. Hiatt A, Cafferkey R, Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342:76–78

    CAS  Article  PubMed  Google Scholar 

  35. Howard JA, Hood EE (2014) Commercial plant-produced recombinant protein products: case studies. Springer, New York

  36. Hull AK, Criscuolo CJ, Mett V, Groen H, Steeman W, Westra H, Chapman G, Legutki B, Baillie L, Yusibov V (2005) Human-derived, plant-produced monoclonal antibody for the treatment of anthrax. Vaccine 23:2082–2086

    CAS  Article  PubMed  Google Scholar 

  37. Jain A, Saini V, Kohli DV (2013) Edible transgenic plant vaccines for different diseases. Curr Pharm Biotechnol 14:594–614

    CAS  Article  PubMed  Google Scholar 

  38. Jansen PLM, Bruijne JD (2012) Controlled-release interferon alpha 2b, a new member of the interferon family for the treatment of chronic hepatitis C. Expert Opin Investig Drugs 21:111–118

    CAS  Article  PubMed  Google Scholar 

  39. Jez J, Castilho A, Grass J, Vorauer-Uhl K, Sterovsky T, Altmann F, Steinkellner H (2013) Expression of functionally active sialylated human erythropoietin in plants. Biotechnol J 8:371–382

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Kaldis A, Ahmad A, Reid A, McGarvey B, Brandle J, Ma S, Jevnikar A, Kohalmi SE, Menassa R (2013) High-level production of human interleukin-10 fusions in tobacco cell suspension cultures. Plant Biotechnol J 11:535–545

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Kim H, Jeon J, Lee KJ, Ko K (2014) N-Glycosylation modification of plant-derived virus-like particles: an application in vaccine. Biomed Res Int (article ID 249519)

  42. Kittur FS, Hung CY, Darlington DE, Sane DC, Xie J (2012) N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin. Plant Cell Rep 31:1233–1243

    CAS  Article  PubMed  Google Scholar 

  43. Klimyuk V, Pogue G, Herz S, Butler J, Haydon H (2014) Production of recombinant antigens and antibodies in Nicotiana benthamiana using magnifection technology: GMP-compliant facilities for small- and large-scale manufacturing. Curr Top Microbiol Immunol 375:127–154

    CAS  PubMed  Google Scholar 

  44. Komarnytsky S, Borisjuk NV, Borisjuk LG, Alam MZ, Raskin I (2000) Production of recombinant proteins in tobacco guttation fluid. Plant Physiol 124:927–934

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. Kwon KC, Verma D, Singh ND, Herzog R, Daniell H (2013) Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells. Adv Drug Deliv Rev 65:782–799

    CAS  Article  PubMed  Google Scholar 

  46. Lahrtz F (2015) How to successfully patent therapeutic antibodies. J Biomol Screen 20:484–491

    CAS  Article  PubMed  Google Scholar 

  47. Lai H, Engle M, Fuchs A et al (2010) Monoclonal antibodies produced in plants efficiently treats west Nile virus infection in mice. Proc Natl Acad Sci USA 9:2419–2424

    Article  Google Scholar 

  48. Lee JH, Park DY, Lee KJ et al (2013) Intracellular Reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody. PLoS One 8:e68772

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Leuzinger K, Dent M, Hurtado J et al (2013) Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J Vis Exp 77:e5052

    Google Scholar 

  50. Li WL, Li K, Li J, Lin X, Sun X, Tang K (2011) Expression of biologically active human insulin-like growth factor 1 in Arabidopsis thaliana seeds via oleosin fusion technology. Biotechnol Appl Biochem 58:139–146

    CAS  Article  PubMed  Google Scholar 

  51. Luchakivskaya Y, Kishchenko O, Gerasymenko I, Olevinskaya Z, Simonenko Y, Spivak M, Kuchuk M (2011) High-level expression of human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants. Plant Cell Rep 30:407–415

    CAS  Article  PubMed  Google Scholar 

  52. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J (2013) Recent advances in host plants and expression cassettes structure and function in plant molecular pharming. Biodrugs 28:145–159

    Article  Google Scholar 

  53. Makvandi-Nejad S, McLean MD, Hirama T, Almquist KC, MacKenzie CR, Hall JC (2005) Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scFv) antibody against Salmonella enterica serotype Paratyphi B. Transgenic Res 14:785–792

    CAS  Article  PubMed  Google Scholar 

  54. Maxmen A (2012) Drug-making plant blooms. Nature 485:160

    CAS  Article  PubMed  Google Scholar 

  55. Merlin M, Gecchele E, Capaldi S, Pezzotti M, Avesani L (2014) Comparative evaluation of recombinant protein production in different biofactories: the green perspective. Biomed Res Int (article ID 136419)

  56. Mortimer E, Maclean JM, Mbewana S, Buys A, Williamson AL, Hitzeroth II, Rybicki EP (2012) Setting up a platform for plant-based influenza virus vaccine production in South Africa. BMC Biotechnol 12:14

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. Nausch H, Mikschofsky H, Koslowski R, Meyer U, Broer I, Huckauf J (2012) High level transient expression of ER-targeted human interleukin-6 in Nicotiana benthamiana. PLoS One 7:e48938

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. Nykiforuk CL, Shen Y, Murray EW, Boothe JG, Busseuil D, Rheaume E, Tardif JC, Reid A, Moloney MM (2011) Expression and recovery of biologically active recombinant apolipoprotein AI (Milano) from transgenic safflower (Carthamus tinctorius) seeds. Plant Biotechnol J 9:250–263

    CAS  Article  PubMed  Google Scholar 

  59. Obembe OO, Popoola JO, Leelavathi S, Reddy SV (2011) Advances in plant molecular farming. Biotechnol Adv 29:210–222

    Article  PubMed  Google Scholar 

  60. Park S, Yi N, Kim YS, Jeong M, Bang S, Choi YD, Kim J (2010) Analysis of five novel putative constitutive gene promoters in transgenic rice plants. J Exp Bot 61:2459–2467

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  61. Paul M (2015) International society for plant molecular farming. Transgenic Res 24:375–380

    CAS  Article  PubMed  Google Scholar 

  62. Paul M, Ma JKC (2011) Plant-made pharmaceuticals: leading products and production platforms. Biotechnol Appl Biochem 58:58–67

    CAS  Article  PubMed  Google Scholar 

  63. Penney CA, Thomas DR, Deen SS, Amanda M, Walmsley M (2011) Plant-made vaccines in support of the millennium development goals. Plant Cell Rep 30:789–798

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  64. Peyret H, Lomonosoff GP (2013) The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants. Plant Mol Biol 83:51–58

    CAS  Article  PubMed  Google Scholar 

  65. Piron R, Koker SD, De Paepe A, Goossens J, Grooten J, Nauwynck H, Depicker A (2014) Boosting in Planta production of antigens derived from the porcine reproductive and respiratory syndrome virus (PRRSV) and subsequent evaluation of their immunogenicity. PLoS One 9:e91386

    Article  PubMed  PubMed Central  Google Scholar 

  66. Platis D, Labrou NE (2009) Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract. Biotechnol J 4:1320–1327

    CAS  Article  PubMed  Google Scholar 

  67. Rigano MM, Scotti N, Cardi T (2012) Unsolved problems in plastid transformation. Bioengineered 3:329–333

    Article  PubMed  PubMed Central  Google Scholar 

  68. Sabalza M, Christou P, Capell T (2014) Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnol Lett 36:2367–2379

    CAS  Article  PubMed  Google Scholar 

  69. Sack M, Hofbauer A, Fischer R, Stoger E (2015) The increasing value of plant made proteins. Curr Opin Biotechnol 32:163–170

    CAS  Article  PubMed  Google Scholar 

  70. Scotti N, Cardi T (2014) Transgene-induced pleiotropic effects in transplastomic plants. Biotechnol Lett 36:229–239

    CAS  Article  PubMed  Google Scholar 

  71. Shanmugaraj BM, Ramalingam S (2014) Plant expression platform for the production of recombinant pharmaceutical proteins. Austin J Biotechnol Bioeng 1:4

    Google Scholar 

  72. Shchelkunov SN, Salyaev RK, Pozdnyakov SG, Rekoslavskaya NI, Nesterov AE, Ryzhova TS, Sumtsova VM, Pakova NV, Mishutina UO, Kopytina TV, Hammond RW (2006) Immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis B and human immunodeficiency viruses. Biotechnol Lett 28:959–967

    CAS  Article  PubMed  Google Scholar 

  73. Shoseyov O, Posen Y, Grynspan F (2014) Human collagen produced in plants: more than just another molecule. Bioengineered 5:49–52

    Article  PubMed  Google Scholar 

  74. Sindarovska YR, Gerasymenko IM, Sheludko YV, Olevinskaya ZM, Spivak NY, Kuchuk NV (2010) Production of human interferon alpha 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression. Cytol Genet 44:313–316

    Article  Google Scholar 

  75. Sparrow P, Broer I, Hood EE, Eversole K, Hartung F, Schiemann J (2013) Risk Assessment and regulation of molecular farming—a comparison between Europe and US. Curr Pharm Des 2013:19

    Google Scholar 

  76. Stoger E, Fischer R, Moloney M, Ma JKC (2014) Plant molecular pharming for the treatment of chronic and infectious diseases. Ann Rev Plant Biol 65:743–768

    CAS  Article  Google Scholar 

  77. Sully EK, Whaley KJ, Bohorova N, Bohorov O, Goodman C, Kim Do H, Pauly MH, Velasco J, Hiatt E, Morton J, Swope K, Roy CJ, Zeitlin L, Mantis NJ (2014) A chimeric plantibody passively protects mice against aerosolized ricin challenge. Clin Vaccine Immunol 21:777–782

    Article  PubMed  PubMed Central  Google Scholar 

  78. Thomas DR, Walmsley AM (2014) Improved expression of recombinant plant-made hEGF. Plant Cell Rep 33:1801–1814

    CAS  Article  PubMed  Google Scholar 

  79. Thomas DR, Penney CA, Majumder A, Walmsley AM (2011) Evolution of plant-made pharmaceuticals. Int J Mol Sci 12:3220–3236

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  80. Tusé D, Tu T, McDonald KA (2014) Manufacturing economics of plant-made biologics: case studies in therapeutic and industrial enzymes. Biomed Res Int (article ID 256135)

  81. Vardakoua M, Sainsburyb F, Rigby N, Mulhollanda F, Lomonossoff GP (2012) Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV-HT transient expression system. Protein Expr Purif 81:69–74

    Article  Google Scholar 

  82. Veale MA, Slabbert MM, Van Emmenes L (2012) Agrobacterium-mediated transformation of potato cv. Mnandi for resistance to the potato tuber moth (Phthorimaea operculella). S Afr J Bot 80:67–74

    CAS  Article  Google Scholar 

  83. Webster DE, Thomas MC (2012) Post-translational modification of plant made foreign proteins; glycosylation and beyond. Biotechnol Adv 30:410–418

    CAS  Article  PubMed  Google Scholar 

  84. Werner S, Breus O, Symonenko Y, Marillonnet S, Gleba Y (2011) High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector. Proc Natl Acad Sci USA 108:14061–14066

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  85. Wilken LR, Nikolov ZL (2012) Recovery and purification of plant-made recombinant proteins. Biotechnol Adv 30:419–433

    CAS  Article  PubMed  Google Scholar 

  86. Xu C, Li L, Jin W, Wan Y (2014) Recombinase polymerase amplification (RPA) of CaMV 35S promoter and nos terminator for rapid detection of genetically modified crops. Int J Mol Sci 15:18197–18205

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  87. Yemets AI, Tanasienko IV, Krasylenko YA, Blume YB (2014) Plant-based biopharming of recombinant human lactoferrin. Cell Biol Int 38:989–1002

    CAS  PubMed  Google Scholar 

  88. Yusibov V, Streatfield SJ, Kushnir N (2011) Clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond. Hum Vaccines 7:313–321

    CAS  Article  Google Scholar 

  89. Zhang D, Nandi S, Bryan P, Pettit S, Nguyen D, Santos MA, Huang N (2010) Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.). Protein Expr Purif 74:69–79

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  90. Zimran A, Brill-Almon E, Chertkoff R, Petakov M, Blanco-Favela F et al (2011) Pivotal trial with plant cell-expressed recombinant glucocerebrosidase, taliglucerase alfa, a novel enzyme replacement therapy for Gaucher disease. Blood 118:5767–5773

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

The authors thank Vignan’s University, India for its in-house administrative and financial resources to execute this work. This work was supported in part by a Grant (ECR/2016/000304) from Science and Engineering Research Board, New Delhi, India under the young scientist scheme. We also thank the anonymous reviewers for their comments which helped us in improving the quality of manuscript. We thank Dr. David McMurray, Regents Professor, Texas A & M University System Health Science Center, College Station, USA for providing valuable comments.

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Correspondence to Vijaya R. Dirisala.

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Communicated by A. K. Kononowicz.

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Dirisala, V.R., Nair, R.R., Srirama, K. et al. Recombinant pharmaceutical protein production in plants: unraveling the therapeutic potential of molecular pharming. Acta Physiol Plant 39, 18 (2017). https://doi.org/10.1007/s11738-016-2315-3

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Keywords

  • Therapeutic proteins
  • Heterologous expression
  • Transgenic plants
  • Edible vaccines
  • Clinical trials