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1
Welcome
2
Plant Biopharming: Importances,
Applications and Usages
Kalilu S. Donzo
2013-11-199
CPBMB
COH, Vellanikkara
KAU
3
Outlines
Introduction
General strategy in biopharming
Different biopharming production systems
Applications
Successful reports
Biosafety issues in biopharming
Conclusion
Future lines
4
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)
…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
Milestones
7
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
…general strategy in biopharming
9(Rehbinder et al.,2009)
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
Expression
system
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
Expression systems comparison
11(Ma et al., 2003 )
Different biopharming production
systems
12
Stable nuclear transformation
Plastid transformation
Transient transformation
Stable transformation
( Nikolov and Hammes, 2002)
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)
…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
Plastid transformation
First described by Svab et al. (1990)
Tobacco only species (Daniell et al., 2002)
No transgenic pollen is generated
15
…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)
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)
…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
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)
…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
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)
…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)
Applications
Parental therapeutics
and pharmaceutical
intermediates
Industrial proteins and
enzymesMonoclonal antibodies
BiopolymersAntigens for edible
vaccines
23
Plantibodies (mAb)
24
Antibody that is produced by geneticallyengineered Plant i.e. insertion of antibodies into atransgenic plant
The term is the trademark of Biolex(NorthCarolina)
Have no risk of spreading diseases to humans
Hiatt. et al (1989) were the first to demonstrate theproduction of antibodies in tobacco plants
25
Produce as therapeutic protein and plant protection against diseases
Traditional system of production is mammalian cell culture
All current therapeutic antibodies are of the IgGclass
Purification is done through processes such as filtration, immunofluorescence, and chromatography
…plantibodies (mAb)
26
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)
…plantibodies (mAb)
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)
Production of mAb using mammalian cell
28 (Biotech, 1989)
Antibodies from transgenic plants
29
Plant Antibody type Purpose References
Tobacco IgG Catalytic
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
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
…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
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)
…edible vaccine production methods
33(Mason and Arntzen, 1995)
Examples of plant edible subunit
vaccines
34Mason et al.,1992
35
Outline
35
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
…industrial enzymes
37 (Seon et al., 2002,Hood et al.,1997)
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
One step purification method
39
Sba tagged rprotein
loaded into the column
wash to remove non
specific protein bound
then eluted
40
Current biopharming companies
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
Successful reports
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
Genetic analysis of 10
independent transgenic plants
transformed with pBIN19-MVH
plasmid
Transcriptional (A) and
translational (B) activity of
transgenic clones
…neutralizing immunogenicity of transgenic carrot
(Daucus carota L.)-derived measles virus hemagglutinin
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
PCR analysis of transgenic tobacco
plants for the HPV16 L1 gene
Western blot analysis of HPV16 L1
expression in transgenic tobacco plants
Hemagglutination assay
…expression of Human Papillomavirus Type 16 L1 Protein
in Transgenic Tobacco Plants
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
Serum antibody response in mice immunized orally with transgenic lupin
callus containing HBsAg.
…plant derived edible vaccines against hepatitis B virus
Biosafety issues on biopharming
49
…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
…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
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
...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
Future lines
54
• Engineering challenges like maximization of expressionlevels
• Environmental safety
• Stability of product under storage
• Evaluation of dosage requirement
• Regulatory considerations and legal standards
Roadmap of plants for the future
55
2005
Efficient
agriculture
-Bt technology
-Herbicide
resistance
Health food and quality-Amino acids
-Oil
-StarchPlant 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
Thank you!!!
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