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Welcome

Production of

antibodies from

Cost per gram (USD)

Mammalian cell 1000.00

Transgenic plants 200.00

According to Monsanto’s Integrated

Protein Technologies

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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)

21(Fischer et al., 2004)

…downstream processing and recovery

Isolation

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

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Method of production

harvested and downstream processing

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)

…edible vaccine production methods

30(Mishra et al., 2008)

Examples of plant edible subunit

vaccines

31(Mason et al. 2008)

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Outline

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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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…industrial enzymes

35(Seon et al., 2002)

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)

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Current biopharming companies

(Horn et al., 2008)

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Case study

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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Construct design

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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 on biopharming

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…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

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