Master's Seminar final

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Welcome

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Plant Biopharming: Importances,

Applications and Usages

Kalilu S. Donzo

2013-11-199

CPBMB

COH, Vellanikkara

KAU

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Outlines

Introduction

General strategy in biopharming

Different biopharming production systems

Applications

Successful reports

Biosafety issues in biopharming

Conclusion

Future lines

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Introduction

Biopharming

Involves the use of transgenic plants to

produce proteins of therapeutic value

• Biopharming is also known as molecular farming or

molecular pharming

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(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

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Milestones

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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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…general strategy in biopharming

9(Rehbinder et al.,2009)

Why plants for biopharming?

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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

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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

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Plastid transformation

First described by Svab et al. (1990)

Tobacco only species (Daniell et al., 2002)

No transgenic pollen is generated

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…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

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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

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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

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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

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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

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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

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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)

Applications

Parental therapeutics

and pharmaceutical

intermediates

Industrial proteins and

enzymesMonoclonal antibodies

BiopolymersAntigens for edible

vaccines

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Plantibodies (mAb)

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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

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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)

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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

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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

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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 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

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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

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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

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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

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Outline

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Industrial enzymes

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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

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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

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Sba tagged rprotein

loaded into the column

wash to remove non

specific protein bound

then eluted

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Current biopharming companies

Production costs for antibodies

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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

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Successful reports

Neutralizing immunogenicity of transgenic

carrot (Daucus carota L.)-derived measles virus

hemagglutinin

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Report by Blouin et al. (2003)

Antigenic protein- Hemagglutinin

Crop-Carrot

Method of transformation- Agrobacterium mediated transformation

Trial - Mice

Result- Antibodies observed

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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

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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

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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

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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

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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

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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- 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

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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

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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

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• 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

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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

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Thank you!!!