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This document discusses plant biopharming, which involves producing recombinant proteins in transgenic plants. It provides an overview of the concept, strategies, production systems, applications and case studies of plant biopharming. Specifically: 1. Plant biopharming is more cost effective than traditional systems, with transgenic plants able to produce proteins on a large scale. Common plants used include tobacco, cereals and potatoes. 2. Stable nuclear transformation is the most common method to generate transgenic plants. Applications include producing monoclonal antibodies, industrial enzymes, and edible vaccines in plants. 3. A case study demonstrates the production of highly concentrated and heat-stable hepatitis B surface antigen in transgenic maize, with the
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- 2. Production of antibodies from Costper gram (USD) Mammalian cell 1000.00 Transgenic plants 200.00 According to Monsanto’s Integrated Protein Technologies 2
- 3. Plant biopharming: applicationsand its importance Kalilu S. Donzo 2013-11-199 CPBMB COH, Vellanikkara KAU 3
- 4. Outline Concept Generalstrategy in biopharming Different production systems Downstream processing Applications Case study Biosafety issues Conclusion Future lines 4
- 5. Concept Biopharming Large scaleproduction of recombinant proteins, including therapeutics and industrial proteins, in transgenic plants • Biopharming is also known as molecular pharming 5 (Humphreys et al., 2000)
- 6. …concept Biopharming startedabout 20 years ago with the promise to produce therapeutic molecules like vaccines, antibodies etc. Some therapeutic molecules are very expensive to produce using conventional systems Falls under the category of green biotechnology 6
- 7. Biopharming vs. Biofarming Biopharming PRODUCTIONof active pharmaceutical substances in genetically modified organisms (GMOs) Used exclusively for Pharmaceuticals Biofarming USE of genetically modified organisms (GMOs) as a production platform Used for both pharmaceuticals and others metabolites 7
- 8. 8 1986 1989 1997 The first plant-derivedrecombinant therapeutic HGH protein produced in tobacco and sunflower Full-size IgG produced in tobacco Avidin produced in maize – the first commercialized plant-derived protein Barta et al., 1986 Hiatt et al., 1989 Hood et al., 1997 Milestones 1992 Hepatitis B virus surface antigen produced in tobacco – the first plant-derived vaccine candidate Mason et al., 1992
- 9. 9 2000 2003 2009 Human growth hormoneproduced in tobacco chloroplasts Bovine trypsin – the first marketed plant- derived protein, targeted towards a broad market Highest transient expression of full-sized IgG antibody in plants Staub et al., 2000 Woodard et al., 2003 Vézina et al., 2009 2006 Antibody against Hepatitis B – the first commercialized plant-derived antibody (marketed in Cuba) Vermin and Waltz, 2006 …milestones
- 10. Why plants forbiopharming? 10 Simple, cost-effective and faster High yield Stability – storage Safety - free from animal virus Disadvantages Environmental safety- gene flow and wildlife exposure Health safety concern- some plants produce allergenic compounds like alkaloids
- 11. Expression system Yeast Bacteria Plant viruses Transgenic plants Transgenic animal Animal cell culture Costof maintaining less expensi ve less expensi ve less expensi ve less expensive Expensive Expensi ve Type of storage(Celsiu s) -2.0 -2.0 -2.0 RT Liquid N2 Liquid N2 Gene(protein) size unlimite d unlimite d limited unlimited limited limited Production cost medium medium low low high high Protein yield high medium very high high medium high 11(Ma et al., 2003 ) …why plants for biopharming?
- 12. Plants often used 12 Tobacco Most popular used High biomass yield Rapid scalability lettuce & alfalfa Immediately process Rapid degeneration of proteins in leaves-Less stable (Ma et al., 2003)
- 13. …plants often used 13 Cereal grains- rice and maize To avoid the problem of short shelf-life Easy to transform and manipulate Potatoes First system to be developed for edible vaccine Edible Protein stable in storage tissue (Ma et al., 2003)
- 14. General strategy inbiopharming • Clone a gene of interest • Transform the host species • Grow the host species, recover biomass • Process biomass • Purify product of interest • Deliver product of interest 14
- 15. Different production systems 15 Stable nuclear transformation Plastid transformation Transient transformation Stable transformation for hydroponics ( Nikolov and Hammes, 2002)
- 16. Stable nuclear transformation 16 Most common method Foreign genes are transferred via Agrobacterium tumefaciens or particle bombardment Large acres can be utilized with the lowest cost- grains Long-term non-refrigerated storage of the seed upto 2yrs Manual labor required Lower yield and outcrossing
- 17. Plastid transformation Firstdescribed by Svab et al. in 1990 No transgenic pollen is generated Very high expression levels can be achieved Protein – upto 70% on dry weight but relatively stable No outcrossing 17
- 18. Transient transformation 18 Dependon recombinant plant viruses to infect tobacco plants like TMV Small amounts target protein is obtained in weeks Infection process is rapid No long term storage Target protein is temporarily expressed in the plant No stable transgenic plants are generated
- 19. Stable transformation forhydroponics 19 Transgenic plants are grown on hydroponic medium Desired products are released as part of root fluid into a hydroponic medium Plants are contained in greenhouse Easier purification but expensive to operate Not suitable for large scale production
- 20. Downstream processing andrecovery 20 Concerned with the isolation and purification of the product from the raw biomass Regardless of the production system, downstream processing represents up to 80% of overall production costs Basically, there are 4 stages: Removal of insolubles Product isolation Product purification Product Polishing (Fischer et al., 2004)
- 21. 21(Fischer et al.,2004) …downstream processing and recovery Isolation
- 22. Applications Parental therapeutics intermediates-collagen Industrial enzymesMonoclonal antibodies Antigens for edible vaccines 22
- 23. Monoclonal antibody 23 Antibodythat is produced by genetically engineered plant is referred to as plantibody All current therapeutic antibodies produced are of the IgG class Hiatt et al. were the first to demonstrate the production of antibodies in tobacco plants in 1989 The plantibody is the trademark of Biolex (North Carolina)
- 24. Two main approachesto produce mAb in plants 24 Cross-pollination - transformed plants expressing light or heavy chains Co-transformation of the heavy and light chain genes on two or more expression vectors to produce full-size mAb
- 25. 25 Method of production harvestedand downstream processing
- 26. Antibodies from transgenicplants 26 Plant Antibody type Purpose References Tobacco IgG Catalytic antibodies Hiatt et al., 1989 Tobacco IgG-nematode Plant pathogen resistance Baum et al., 1996 Tobacco &maize IgG-HIV gp120 (2G12) Therapeutic Rademacher et al., 2008 Soybean, rice IgG-herpes virus Therapeutic Zeitlin et al., 1998 Tobacco IgG-colon cancer Systemic injection Verch et al., 1998; Ko et al., 2004 Alfalfa IgG-human Dianostic Khoudi et al., 1999 Tobacco IgG-rabies virus Therapeutic Ko et al., 2003 Tobacco IgG-hepatitis B virus Immunopurificati -on of hepatitis B surface antigen Valdes et al., 2003
- 27. Edible vaccines 27 Theconcept of edible vaccine got incentive after hepatitis B antigen was expressed in tobacco by Arntzen et al. in 1992 Developed by engineering a gene for an antigenic protein into a plant
- 28. …edible vaccines 28 Expressedin the edible portion like tubers, fruits etc Due to ingestion, it releases the protein and get recognized by the immune system
- 29. Edible vaccine productionmethods 29 Expression of foreign antigens in plant via stable transformation Delivery of vaccine epitopes via plant virus (Mishra et al., 2008)
- 30. …edible vaccine productionmethods 30(Mishra et al., 2008)
- 31. Examples of plantedible subunit vaccines 31 (Mason et al. 2008)
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- 33. Industrial enzymes 33 Avidinand β- glucuronidase first commercialized industrial proteins from transgenic maize Trypsin produced prodiGene company (proteolytic enzyme) on large scale using maize Avidin was the first commercial transgenic protein produced via transgenic maize
- 34. Industrial enzymes fromtransgenic plants Proteins Plants Reference ά Amylase Tobacco Seon et al.,2002 Avidin Corn/maize Hood et al.,1997 β glucuronidase Brassica Seon et al.,2002 Xylanase Brassica Seon et al.,2002 Hirudin Brassica Seon et al., 2002 34
- 35. …industrial enzymes 35(Seon etal., 2002)
- 36. Plant-derived pharmaceuticals inclinical stages of development 36 Vaccines: Product Disease Plants Clinical trial status Company Hepatitis B antigen (HBsAg) Hepatitis B Potato Phase II Arizona State University Fusion proteins Rabies Spinach Phase II T.J.University Cancer vaccine Non-Hodgkin’s lymphoma Tobacco Phase II Large Scale Biology, USA Vibrio Cholerae Cholera Potato Phase I Arizona State University DoxoRX Side-effects of cancer therapy Tobacco Phase I completed Planet Biotechnology IgG (ICAM1) Common cold Tobacco Phase I Planet Biotechnology Lactoferon™ (α- interferon) Hepatitis B & C Duckweed Phase III Biolex (Obembe, 2010)
- 37. 37 Current biopharming companies (Hornet al., 2008)
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- 39. Production of highlyconcentrated, heat stable hepatitis B surface antigen in maize 39(Hayden et al., 2012)
- 40. 40 Construct design Transformation intomaize and propagation of seeds Oil extraction Experimental procedures
- 41.
- 42. 42 Immunoblotting of maize material Antigendetection by ELISA Confocal microscopy …experimental procedures
- 43. 43 Results HBsAg accumulation insingle seeds from the first generation (Hayden et al., 2012) 0.12% 0.31% 0.41% 0.51% 0.15%
- 44. 44 …results HBsAg concentration insecond generation (T2) ears with highest antigen accumulation, as determined by ELISA (Hayden et al., 2012) 0.05% 0.17% 0.27% 0.26%
- 45. …results Effect of oilextraction and temperature on maize-produced HBsAg, as determined by immunoblot. 45
- 46. 46 …results Confocal microscopy :presenceof protein(fast Green) and lipids(Nile Red) A- full fat B- hexane C- SFE (Hayden et al., 2012)
- 47. …results 47 Total soluble proteinand HBsAg protein content in HBsAg maize seed stored at −20°C, 55°C, and 80°C for one week Hayden et al., 2012
- 48. Biosafety issues onbiopharming 48
- 49. …biosafety issues inbiopharming Gene and protein pollutions Vertical gene transfer- most prevalent form via pollen/seed dispersal among partially compatible plant Horizontal gene transfer- between very different taxonomic groups; and common in bacteria 49
- 50. …biosafety issues inbiopharming 50 Product safety- public concern about the potential health and environmental risks associated with the transgenic plants Economic risks to farmers and food industry as result of co-mingling and contamination of MP plants with food/feed chain
- 51. Conclusion 51 Provides safe,economical and large-scale production of pharmaceuticals, industrial enzymes and technical proteins PMPs have already achieved preclinical validation in a range of disease models like hepatitis B, rabies etc. We must ensure that the potential benefits are not outweighed by risks to human health
- 52. Future lines 52 Maximizationof expression level Improvement of downstream processing Evaluation of dosage requirement Improve and establish a more reliable biosafety system
- 53.