This document outlines a presentation on plant biopharming. It discusses the use of transgenic plants to produce therapeutic proteins and some key points: - Biopharming involves using transgenic plants to produce proteins of therapeutic value. It started about 20 years ago with the promise to produce expensive molecules cheaper. - Different production systems are discussed, including stable nuclear transformation, plastid transformation, transient transformation, and stable hydroponic transformation. Tobacco, lettuce, alfalfa, rice and maize are common plant species used. - Applications include pharmaceuticals, industrial enzymes, monoclonal antibodies, edible vaccines. Successful reports demonstrate production of measles virus protein in transgenic carrot and human papillomavirus protein

1. 1
Welcome

2. 2

3. Plant Biopharming: Importances,
Applications and Usages
Kalilu S. Donzo
2013-11-199
CPBMB
COH, Vellanikkara
KAU
3

4. Outlines
 Introduction
 General strategy in biopharming
 Different biopharming production systems
 Applications
 Successful reports
 Biosafety issues in biopharming
 Conclusion
 Future lines
4

5. Introduction
 Biopharming
Involves the use of transgenic plants to
produce proteins of therapeutic value
• Biopharming is also known as molecular farming or
molecular pharming
5
(Humphreys et al.,2000)

6. …introduction
 Biopharming started about 20 years ago with the
promise to produce therapeutic molecules
 Some therapeutic molecules are very expensive to
produce
 Falls under the category of green biotechnology
6

7. Milestones
7

8. General strategy in biopharming
• 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
8

9. …general strategy in biopharming
9
(Rehbinder et al.,2009)

10. Why plants for biopharming?
10
 Low cost of production
 Stability – storage
 Safety - free from animal virus; eukaryotic system
Disadvantages
 Environmental safety- gene flow and wildlife exposure
 Food chain contamination
 Health safety concern-case specific

11. Expression
system
Expression systems comparison
Yeast Bacteria Plant
viruses
Transgenic
plants
Transgenic
animal
Animal
cell
culture
Cost of
maintaining
inexpen
sive
inexpen
sive
inexpen
sive
inexpensive expensive Expensi
ve
Type of
storage(Celsiu
s)
-2.0 -2.0 -2.0 RT N2 N/A
Gene(protein)
size
Unknow
n
Unknow
n
limited Non limited Limited limited
Production
cost
Medium Medium Low Low High High
Protein yield High Medium Very
high
High Medium High
11
(Ma et al., 2003 )

12. Different biopharming production
systems
12
 Stable nuclear transformation
 Plastid transformation
 Transient transformation
 Stable transformation
( Nikolov and Hammes, 2002)

13. Stable nuclear transformation
 Most common
 A species with a long generation cycle
 Foreign genes are transfer via Agrobacterium
tumefaciens or particle bombardment
 Genes are taken up and incorporated in a stable
manner
13
(Boehm, 2007; Obembe et al.,2011;Tremblay et al., 2010)

14. …stable nuclear transformation
Advantages Disadvantages
 Long-term non-refrigerated
storage the
seed upto 2yrs
 Large acres can be
utilized with the lowest
cost
 Eg. Grains
 Manual labor required
 Lower yield
 Less effective genetic
 Outcrossing
14

15. Plastid transformation
 First described by Svab et al. (1990)
 Tobacco only species (Daniell et al., 2002)
 No transgenic pollen is generated
15

16. …plastid transformation
Advantages Disadvantages
 No outcrossing
 Protein – upto 70% on dry
weight
 Very high expression
levels can be achieved
 Protein unstable
 Extraction and purification
at specific time
 Edible vaccine is not
feasible since tobacco is
highly regulated
16 (Oey et al., 2009)

17. Transient transformation
17
 Depend on recombinant plant viruses to infect
tobacco plants like TMV
 Target protein is temporary express in the plant
 Protein accumulate in the interstitial spaces
 No stable transgenic plants are generated
(Boehm, 2007; Komarova et al., 2010; Pogue et al., 2010)

18. …transient transformation
Advantages Disadvantages
18
 Infection process is
rapid
 Small amounts target
protein is obtained in
weeks
 Efficient for custom
proteins needed in small
amount
 Not needed for protein
in large amount
 No long term storage
due to tissue damage
 Scalability and
expression levels

19. Stable transformation
19
 Transgenic plants are grown hydroponically
 Hydroponics is a technology for growing plants in
nutrient solutions (water and fertilizers) for high-density
maximum crop yield, crop production
where no suitable soil exists
 Desired products are released as part of root fluid
into a hydroponic medium
(Raskin, 2000)

20. …stable transformation
Advantages Disadvantages
20
 Plants are contained in
green house
 Reduced fears of
environmental release
 Easier purification
 Expensive to operate
 Not suitable for large
scale production

21. Plants most often used
21
 Tobacco
 Most popular used
 High biomass yield
 Rapid scalability
 Break the barrier of biosafety-Non food
 Leafy crops- lettuce & alfalfa
 Immediately process
 Rapid degeneration of proteins in leaves-Less stable
 Clonal propagation
(Fischer et al., 2003)

22. …plants most often used
22
 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)

23. Applications
Parental therapeutics
and pharmaceutical
intermediates
Industrial proteins and
enzymes
Monoclonal antibodies
Biopolymers
Antigens for edible
vaccines
23

24. Plantibodies (mAb)
24
 Antibody that is produced by genetically
engineered Plant i.e. insertion of antibodies into a
transgenic plant
 The term is the trademark of Biolex(North
Carolina)
 Have no risk of spreading diseases to humans
 Hiatt. et al (1989) were the first to demonstrate the
production of antibodies in tobacco plants

25. 25
…plantibodies (mAb)
 Produce as therapeutic protein and plant protection
against diseases
 Traditional system of production is mammalian
cell culture
 All current therapeutic antibodies are of the IgG
class
 Purification is done through processes such as
filtration, immunofluorescence, and
chromatography

26. 26
…plantibodies (mAb)
 Chloroplast transformation ideal for single chain
fragment(scFv) due to the lack of glycosylation
(Daniel,2002a)
 Agrofiltration is ideal for transient expression of
heavy and light chain genes
 Assembling of the full-size mAb in tobacco report
by Scholthof et al.,(1996)

27. Two main approaches to produce
plantibodies in plants
27
 Cross-pollination - transformed plants expressing
light or heavy chains (Hiatt et al.,1989; Ma et
al.,1994)
 Co-transformation of the heavy and light chain
genes on a single two or more expression vectors
to produce full-size mAb (Nicholson et al., 2005)

28. Production of mAb using mammalian cell
28 (Biotech, 1989)

29. Antibodies from transgenic plants
Plant Antibody type Purpose References
Tobacco IgG Catalytic
29
antibodies
Hiatt et al., 1989
Tobacco IgG-nematode Plant pathogen
resistance
Baum et al., 1996
Tobacco sIgA/G-s.mutans Therapeutic Ma et al., 1998
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

30. Edible vaccines
30
 A vaccine developed by engineering a gene for an
antigenic protein into a plant
 Expressed in the edible portion
 Due to ingestion, it releases the protein and get
recognized by the immune system

31. …edible vaccines
31
 The concept of edible vaccine got incentive after
Arntzen et al. (1992) expressed hepatitis B antigen
in tobacco
 Stimulate both humoral and mucosal immunity
 It is Feasible to administer unlike injection
 Heat stable - no need of refrigeration

32. Edible vaccine production methods
32
 Expression of foreign antigens in plant via
stable transformation- agrobacterium mediated
Delivery of vaccine epitopes via plant virus
(Mason and Arntzen, 1995)

33. …edible vaccine production methods
33
(Mason and Arntzen, 1995)

34. Examples of plant edible subunit
vaccines
34 Mason et al.,1992

35. 35
Outline
35

36. Industrial enzymes
36
 Avidin and β- glucuronidase first commercialized
industrial proteins from Maize
 ProdiGene Inc. company produce trypsin
(proteolytic enzyme) on large scale using maize
 Avidin was the first commercial transgenic protein
produced via transgenic maize

37. …industrial enzymes
37 (Seon et al., 2002,Hood et al.,1997)

38. Industrial products close to market
38
Product Company Uses References
Trypsin ProdiGene Immediate in
pharmaceutical
Woodard et
al.,2003
GUS ProdiGene Reagent for
diagnostics
Kusnadi et
al.,1998
Avidin ProdiGene Immunological
reagent
Hood et al.,1997
Aprotinin Large scale
Biology
Wound closure Zhong et al.,1999
Collagen ProdiGene,Medica
go
Gel cap Ruggiero et
al.,2000
Lipase Meristem
therapeutics
Exocrine
pancreatic
insufficiency
Gruber et al.,2001
Lactoferrin Ventria Natural defense Samyn-petit et
al.,2001
TGEV edible
vaccine
ProdiGene Swine Lamphear et
al.2002

39. One step purification method
39
Sba tagged rprotein
loaded into the column
wash to remove non
specific protein bound
then eluted

40. 40
Current biopharming companies

41. Production costs for antibodies
41
Production cost Cost in $ per gram
Hybridomas 1000
Transgenic animals 100
Transgenic plants 10
(Daniell et al., 2001)
E. coli & yeast Tr. animals and
animal cells
Transgenic
plants

42. 42
Successful reports

43. Neutralizing immunogenicity of transgenic
carrot (Daucus carota L.)-derived measles virus
hemagglutinin
43
 Report by Blouin et al. (2003)
 Antigenic protein- Hemagglutinin
 Crop-Carrot
 Method of transformation- Agrobacterium mediated
transformation
 Trial - Mice
 Result- Antibodies observed

44. 44
…neutralizing immunogenicity of transgenic carrot
(Daucus carota L.)-derived measles virus hemagglutinin
Genetic analysis of 10
independent transgenic plants
transformed with pBIN19-MVH
plasmid
Transcriptional (A) and
translational (B) activity of
transgenic clones

45. Expression of Human Papillomavirus Type 16 L1
Protein in Transgenic Tobacco Plants
45
 Report by Liu Hong et al., (2005)
 Antigenic protein- HPV type 16 L1 protein
 Crop- Tobacco
 Method of transformation- Agrobacterium
mediated transformation
 Trial - Mice
 Result- Antibodies developed in mice

46. …expression of Human Papillomavirus Type 16 L1 Protein
in Transgenic Tobacco Plants
PCR analysis of transgenic tobacco
46
plants for the HPV16 L1 gene
Western blot analysis of HPV16 L1
expression in transgenic tobacco plants
Hemagglutination assay

47. Plant derived edible vaccines against
hepatitis B virus
47
 Report by Kapusta J., et al.(1999)
 Crops- lettuce
 Antigenic protein- HBsAg Protein
 Method of transformation- Agrobacterium
mediated transformation
 Trial- In Mice
 Result-Mice developed HB virus specific
antibodies

48. …plant derived edible vaccines against hepatitis B virus
48
Serum antibody response in mice immunized orally with transgenic lupin
callus containing HBsAg.

49. Biosafety issues on biopharming
49

50. …biosafety issues in biopharming
 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
50

51. …biosafety issues in biopharming
51
 Product safety- toxic metabolites (such as the
alkaloids produced in many tobacco cultivars),
allergens and field chemicals such as pesticides
and herbicides
 Accidental contamination of food and feed chain

52. Conclusion
52
 Plant biopharming has potential to become a major
new method for low-cost, mass and safe
production of biopharmaceutical
 It has translated into rapid growth in the number of
plant- made biopharmaceutical
 There are several plant-based expression systems
that are currently being explored to serve as
production platforms, each offering specific
benefits

53. ...conclusion
53
 PMPs have already achieved preclinical validation in
a range of disease models with some plant-made
vaccines in Phase II and Phase III clinical trials
 The potential benefit of plant-made pharmaceuticals
to human health should not be underestimated though
they have allergic and regulatory concerns

54. Future lines
54
• Engineering challenges like maximization of expression
levels
• Environmental safety
• Stability of product under storage
• Evaluation of dosage requirement
• Regulatory considerations and legal standards

55. Roadmap of plants for the future
55
Efficient
agriculture
-Bt technology
-Herbicide
resistance
2005
Health food and quality
-Amino acids
-Oil
-Starch
Plant protection
-Viruses
-Nematodes
-Fungi
-Insects
2015
Plant production platforms
-Vitamins
-Fatty acids/fibers
-Enzymes/Pigments
-Bio-polymers
-Pharmaceutical products
Stress resistance
-Cold
-Drought
-Salinization
2025

56. 56
Thank you!!! 