Trends in Biotechnology

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appliedbiotech.org

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THE CHALLENGES OF COMMERCIALIZING PLANT
BIOTECHNOLOGY PRODUCTS 

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PROTEINS USED TODAY

• Therapeutics
• Vaccines
• Specialty chemicals
• Detergents
• Food processing
• Pulp and paper processing
• Feed enhancers

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LIMITATIONS FOR PROTEIN PRODUCTS

• Specificity
• Cost of production
• Energy to change status quo

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POTENTIAL FOR OTHER USES

• Protein therapeutics/antibodies
• Oral vaccines
• Replace chlorinated compounds for pulp, paper and
  textile industries
• Replace caustic agents used in cellulose
  degradation and adhesive agents

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PRODUCTION PLATFORMS

• Synthetic
• Size-limited to small peptides
• Cost-proportional to size
• Microbial
• Very desirable cost
• Downstream processing limitations
• Animals
• Human pathogens raise concern
• Expensive for cell cultures
• Plants ?

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POTENTIAL BENFITS FROM PLANTS PRODUCTION

• Cost
• Low input cost
• Volume easily scaled
• Low capital equipment requirements
• Added value
• Many options to address protein specific
  expression problems
• Cellular processing
• Toxicity

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PROCESS

• Concept
• Proof of Principle
• Commercialization
• Sustainability

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CHALLENGES

• Technical
• Regulatory
• Economic
• Industry acceptance
• Customer acceptance
• Public acceptance

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PROOF OF PRINCIPLE

• Can plants express protein of interest?
• Can plants make functionally equivalent proteins?
• Can proteins be extracted/purified from plants?
• Will they work in the application of interest?

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BASIC VECTOR
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PLANT TISSUES

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PLANT CELL
 

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PROTEIN SPECIFIC EXPRESSION

• Factors
• Protein stability
• Interaction within cellular compartment
• Interaction with host tissue
• Interference with non-target tissue

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OPTIMIZATION FOR PROTEIN ACCUMULATION
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EXPRESSION IMPROVEMENTS

• Establishing Germline Importance
  
• Cellular targeting High
• Tissue targeting High
• Event High
• Promoter Strength High
• Codon preferences Medium/High
• Leader Sequence Standard
• Termination Sequence Standard

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CORN TRANSFORMATION PROCESS
Timeline 0 months 2 months 5 months 7-9
months
embryos
callus
embryo germination
T0 plants
selection
shoots
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EXPRESSION IMPROVEMENTS

• Optimizing Germplasm Individual
• Improvements
• Line selection 50X
• Copy number 5X
• Multiple alleles 4X
• Maize variants 5X
• Hybrid Production 4X
• Cumulative Total Observed UP TO 100X

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APROTININ

• Protease Inhibitor
• Derived from Bovine Pancreas and Lung
• M.W. 6511 Da
• Applications
• Therapeutic
• Animal Cell Culture
• Research Reagent
• Reduces blood loss and hospital stay
• Plant source could give an ample supply with
  reduced fears of human pathogens (e.g.. Mad Cow
  Disease)

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SDS-PAGE OF CORN-PRODUCED AND NATIVE APROTININ
Native
Corn-produced
1 ug
1 ug
2.5 ug
2.5 ug
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APROTININ FROM CORN COMPARED TO NATIVE SOURCE
Commercial Aprotinin 6573.28
Corn-produced Aprotinin 6574.33
6000
7000
Mass (Da)
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APROTININ SEQUENCE
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APROTININ CHARACTERISTICS
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TRYPSIN

• Protease
• Native Source Bovine pancreas
• Applications
• Cell culture reagent
• Research reagent
• Bioprocessing (e.g. insulin)
• Digestive aid
• Detergent
• Food processing
• Plant source may provide a supply
  with reduced fears of human pathogens

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TRYPZEAN KM DETERMINATION
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LACCASE

• Redox enzyme
• Native Source white rot fungi
• Applications
• Adhesives
• Bleaching agent
• Detoxification agent
• Problem- not easily expressed in other systems
• Plant source may provide a supply
  with reduced fears of caustic agents

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EXPRESSION OF LACCASE IN MAIZE EMBRYO
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AVIDIN - PROPERTIES

• Native Source Chicken egg whites
• Homotetramer molecular weight 67,200
• Subunit molecular weight 16,800
• Isoelectric point 10
• Glycoprotein
• Assay Binding of 1 biotin per subunit

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PHYSICAL CHARACTERISTICS
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TOXICITY

• Avidin - sequestered in cell wall
• Aprotinin localized in seed tissue rich in
  protease inhibitors
• Trypsin - expressed as pro-enzyme and localized
  in seed tissue rich in protease inhibitors
• Laccase High oil lines and embryo expression

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PLANT-BASED ORAL PRODUCT
Cost of Traditional Cost Utilizing Plant
Orally Injectable Product Delivered Product
Raw material
Misc.
Misc.
Lower cost of raw material
Labor for injections
Purification
Cost savings
Low temperature storage and transport
Needles, syringes
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TRAVELERS DISEASELT-B (B subunit of the
heat-labile toxin of ETEC)

• Enterotoxigenic strains of E. coli
• Similar to cholera causes diarrhea
• Endemic to developing countries
• 650 million cases annually
• 800 thousand deaths annually
• 20 of travelers contract disease
• Current vaccine inactivated bacteria Ct-B
• Can plant source be used for oral delivery?

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HUMAN CLINICAL TRIAL
IgA IgG
Ig Titer
Lt-B Oral Inoculations

Pre Post
Pre Post
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TRANSMISSIBLE GASTROENTERITIS VIRUS
Swine disease in young pigs
which can lead to mortality Make transgenic
corn with S protein
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DISEASE SEVERITY INDEX
Clinical Severity Index total clinical score
values divided by the total number of pig days.
TGEV-2 Oragen Technologies Veterinary Resources
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All sows were vaccinated initially with
commercial TGEV vaccine 35 and 14 days before
farrowing they were boosted
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INDUSTRIAL FEEDSTOCK ADDED VALUE

• Direct delivery reduces downstream processing
• Add value beyond protein
• Collect by-product credits

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BIOMASS CONVERSION Maize Crop
Grain Endosperm
Germ
Stover Chemical Pretreatment
Cellulose suitable for enzyme degradation
Containing Cellulases
Currently used For ethanol production
Carbohydrates
Carbohydrates
Fermentation

ETHANOL
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COMMERCIALIZATION

• Can the product meet specifications for
• Commercial Efficacy
• Cost of production
• Capital cost
• Within an acceptable time frame
• Regulatory guidelines
• Beyond what currently exist
• Can the product provide a reasonable ROI?

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CAN THIS BE ECONOMICALLY COMPETITIVE?

• Raw materials cost lt100 times that of eggs
• Amount of mass needed for purification lt10 times
  that of eggs
• Grain can store protein for years without loss of
  activity
• No threat of contamination

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TECHNOLOGY CURVE
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WHICH PLANTS ?

• Growing and Harvesting
• Recombinant Protein Yield
• Confinement Requirements
• Geographic Limitations
• Seasonal Limitations
• Mechanical or hand labor
• Temperature Sensitivity
• Time Sensitivity
• Transport and Storage
• Temperature Sensitivity
• Protein Stability in Tissue

• Tissue Processing
• Stability
• Biomass Quantity
• Extraction
• Purification
• Interfering agents
• cGMP
• Final Product Specifications
• Allergens
• Toxins
• Antinutritionals

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DIFFERENT PLANT CHARACTERISTICS

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ADDED VALUE

• Plant provides additional product safety features
• Plant material adds value as a feedstock, food or
  feed independent of the transgenic protein
• Plant material offers applications not otherwise
  possible

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BEST PLANT PLATFORM

• No one perfect solution
• Depends on application and protein
• When cost and volume are major factors the only
  current option is a modified commodity crop

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COMMERCIALIZED PRODUCTS

• Avidin
• Gus
• Trypsin
• Aprotinin

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SUSTAINABILITY

• Scientific Community
• Industry
• Public

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SCIENTIFIC COMMUNITY

• Much of the basic science still unknown
• Recent work in genomics, proteomics and
  metabolomics has just begun to be applied to gene
  expression
• Potential to raise expression levels more than
  10-fold from best cases today
• Potential to control gene expression in new ways

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INDUSTRY ACCEPTANCE

• ROI
• Products meet customer expectations
• Cost benefit is realized
• Demonstrated reliable supply
• Demonstrated regulatory path
• Demonstrated products on the market

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PUBLIC ACCEPTANCE

• Customer Acceptance
• Benefits gt Cost
• Public Acceptance
• Benefits gt Risks

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SAFETY/RISK ASSESSMENT

• Science based
• Similar to what is used for other regulated
  products
• Accepted by industry, regulatory agencies,
  special interest groups and public

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REGULATIONS FOR MAIZE

• GRAS status for maize
• Confinement for transgenic maize
• Physical isolation
• Temporal isolation
• Genetic sterility
• Handling
• Identification markers
• Completely separated from agricultural products

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Hazard Quotient
Cumulative Intake Reference Dose
Hazard Quotient

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SAFETY/RISK ASSESSMENT OF APROTININ PRODUCED IN
MAIZE

• Worst case - no containment
• Adjacent farmer segregates crop which goes
  directly to food
• Hazard Quotient
• 1 million times below no effect level
• Amount needed for 1 dose as an antigen
• 350 tacos
• 350 bowls of cereal
• Best case more than 1 million times this amount
  with confinement practices

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PERCEPTIONS
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ACCEPTANCE OF ANY NEW TECHNOLOGY

• Do we get a choice?
• Do we understand the technology?
• Do we see direct benefits?

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BIOLOGICS FROM PLANTS

• What is the new concept ?
• Obtaining drugs from plants?
• De Materia Medica by Discorides in 78 A.D.
  described 600 plants to have medicinal properties

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• Obtaining drugs from an uncontained
  non-controlled source?
• 12 million units of blood used for human health
  products
• Use of transgenic plants?
• 167 million acres of transgenic plants grown last
  year

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• Use of food organisms to produce biologics?
• 100 million eggs used to make vaccines
• Yeast for bread, beer and biologics
• Regulatory guidelines?
• NIH, FDA and USDA guidelines

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CONCLUSIONS

• Protein products are an environmentally friendly
  and safe alternative to many current practices
  used today
• Cost is a limiting factor for implementing
  specific products
• Plants offer options over other systems
• Technology and applications are growing rapidly
• Many plant platforms to choose from - each with
  different characteristics that can contribute to
  final product
• First commercial protein products now on the
  market
• Public perceives biologics from plants as a new
  concept

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CONCLUSIONS

• Public perceives biologics from plants as a new
  concept
• General concept is not new

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EVOLUTION OF PHARMACEUTICAL PRODUCTION
USE ALL METHODS
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Acknowledgments

• Stephen Streatfield
• Kamesh Pappu
• Carol Drees
• Elena Delgado
• Brain Vu
• Elizabeth Hood
• Ian Tizzard
• K.C. Donnely
• Bruce Lawhorn

• Mike Horn
• Donna Delaney
• Susan Woodard
• Joseph Jilka
• Michele Bailey
• Barry Lamphear
• Richard Clough
• John Clemmons
• Carol Tacket
• Mark Welter