kalilu seminar
TRANSCRIPT
Production of
antibodies from
Cost per gram (USD)
Mammalian cell 1000.00
Transgenic plants 200.00
According to Monsanto’s Integrated
Protein Technologies
2
Plant biopharming: applications,
importance and its usage
Kalilu S. Donzo
2013-11-199
CPBMB
COH, Vellanikkara
KAU
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Outline
Concept
General strategy in biopharming
Different production systems
Downstream processing
Applications
Case study
Biosafety issues
Conclusion
Future lines
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Concept
Biopharming
Large scale production of recombinant proteins,
including therapeutics and industrial proteins, in
transgenic plants
• Biopharming is also known as molecular pharming
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(Humphreys et al., 2000)
…concept
Biopharming started about 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
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Biopharming vs. Biofarming
Biopharming
PRODUCTION of 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
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1986
1989
1997
The first plant-derived recombinant
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
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2000
2003
2009
Human growth hormone produced 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
2006Antibody against Hepatitis B – the first
commercialized plant-derived antibody
(marketed in Cuba)
Vermin and
Waltz, 2006
…milestones
Why plants for biopharming?
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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
Expression
system
Yeast Bacteria Plant
viruses
Transgenic
plants
Transgenic
animal
Animal
cell
culture
Cost of
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?
Plants often used
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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)
…plants often used
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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)
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
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Different production systems
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Stable nuclear transformation
Plastid transformation
Transient transformation
Stable transformation for hydroponics
( Nikolov and Hammes, 2002)
Stable nuclear transformation
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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
Plastid transformation
First described 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
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Transient transformation
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Depend on 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
Stable transformation for hydroponics
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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
Downstream processing and recovery
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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)
Applications
Parental therapeutics
intermediates-collagenIndustrial enzymes Monoclonal antibodies
Antigens for edible
vaccines
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Monoclonal antibody
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Antibody that 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)
Two main approaches to produce mAb in
plants
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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
Antibodies from transgenic plants
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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
Edible vaccines
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The concept 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
…edible vaccines
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Expressed in the edible portion like tubers, fruits
etc
Due to ingestion, it releases the protein and get
recognized by the immune system
Edible vaccine production methods
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Expression of foreign antigens in plant via
stable transformation
Delivery of vaccine epitopes via plant virus
(Mishra et al., 2008)
Industrial enzymes
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Avidin and 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
Industrial enzymes from transgenic
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
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Plant-derived pharmaceuticals in clinical stages of
development
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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)
Production of highly concentrated, heat
stable hepatitis B surface antigen in maize
39(Hayden et al., 2012)
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Construct design
Transformation into maize and
propagation of seeds
Oil extraction
Experimental procedures
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Immunoblotting of maize
material
Antigen detection by ELISA
Confocal microscopy
…experimental procedures
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Results
HBsAg accumulation in single seeds from the first generation
(Hayden et al., 2012)
0.12%
0.31%
0.41%
0.51%
0.15%
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…results
HBsAg concentration in second generation (T2) ears with highest antigen
accumulation, as determined by ELISA
(Hayden et al., 2012)
0.05%
0.17%
0.27%0.26%
…results
Effect of oil extraction and temperature on maize-produced HBsAg, as
determined by immunoblot.
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…results
Confocal microscopy :presence of protein(fast
Green) and lipids(Nile Red)
A- full fat
B- hexane
C- SFE (Hayden et al., 2012)
…results
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Total soluble protein and HBsAg protein content in HBsAg maize seed stored at
−20°C, 55°C, and 80°C for one week
Hayden et al., 2012
…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
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…biosafety issues in biopharming
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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
Conclusion
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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
Future lines
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Maximization of expression level
Improvement of downstream processing
Evaluation of dosage requirement
Improve and establish a more reliable biosafety
system