Molecular Pharming with case study

1. 1/7/2017 1Dept. of Plant Biotechnology

2. Upasana Mohapatra
PALB 6290
Jr Msc. Plant Biotechnology
UAS,GKVK,Bengaluru
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3. CONTENTS
• Definition
• History
• Molecular farming strategy
• Molecular farming host
• Plant molecular pharming
• Antibiotics, enzymes and vaccines produced from
microbes and plant
• Transgene pollution
• Case study
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4. DEFINITION
• The use of whole organisms, organs, tissues
or cells, or cell cultures, as bio-reactors for
the production of commercially valuable
products like recombinant proteins,
antibodies, vaccines via recombinant DNA
techniques.
• It is also known as Molecular farming or
Bio pharming.
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5. HISTORY
• 1986 - First plant -derived recombinant therapeutic protein-
human GH in tobacco & sunflower. (A. Barta, D. Thompson etal.)
• 1989 - First plant -derived recombinant antibody – full-sized IgG in
tobacco. (A. Hiatt, K. Bowdish)
• 1990 - First native human protein produced in plants –
human serum albumin in tobacco & potato. (P. C. Sijmons et al.)
• 1995 - First plant derived industrial enzyme – α-amylase in tobacco. (J.Pen,
L. Molendijk et al.)
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6. HISTORY
• 1986 First plant -derived recombinant therapeutic protein-
human GH in tobacco & sunflower. (A. Barta, D. Thompson et al.)
• 1997 First clinical trial using recombinant bacterial antigen
delivered in a transgenic potato. (C. O. Tacket et al.)
• 1997 Commercial production of avidin in maize.(E. E. Hood et al.)
• 2000 Human GH produced in tobacco chloroplast.(J. M. Staub et al.)
• 2003 Human GH produced in tobacco chloroplast.(J. M. Staub et al.).
Expression and assembly of a functional antibody in algae
Commercial production of bovine trypsin in maize.(S. L.
Woodard )
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7. 1. Clone a gene of interest
2. Transform the host platform species
3. Grow the host species, recover biomass
4. Process biomass
5. Purify product of interest
6. Deliver product of interest
Molecular Farming Strategy
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9. Downstream processing & analysis of
recombinant proteins from plants
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10. 1. Bacteria
2. Yeasts, (single celled fungi)
3. Unicellular algae
4. Mammalian, insect, plant, and filamentous
fungal cell cultures
5. Whole plants, ( corn, barley, rice, duckweed,
moss protonema)
6. Whole animals, (insects, birds, fish, mammals)
Molecular Farming Hosts
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11. BACTERIA:
1. Do not produce glycosylated full –sized
antibodies.
2. Contaminating endotoxin difficult to remove.
3. Recombinant proteins often form inclusion
bodies.
4. Labour-and cost –intensive refolding in vitro
necessary.
5. Lower scalability
6. Preferred for the production of small,
aglycosylated proteins like Insulin, interferon-
β.
1. Limited by legal and ethical restriction
2. Require expensive equipment & media
3. Delicate nature of mammalian cells
4. Human pathogens and oncogenes
5. Scaling up problems
ANIMAL BASED SYSTEMS
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13. S. Biemelt;U. Sonnewald (2004)
Comparison Of Different Production Systems For Expression Of
Recombinant Proteins
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14. Cost of Production: Antibodies
HOST COST
Animal cell culture $ 333/g
Transgenic milk $ 100/g
Yeast cell culture $ 100/g
Milled corn endosperm $ 0.2/g
Enriched corn fraction $ 0.6/g
Extracted corn fraction $ 2.1/g
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15. Plant Molecular Farming
1. Significantly lower production cost than with
transgenic animals, fermentation or bioreactors.
2. Infrastructure & expertise already exists for the
planting, harvesting & processing of plant material.
3. Plants contain no known human pathogens (such as
prions, virions,etc.) that could contaminate the final
product.
4. Higher plants generally synthesize proteins from
eukaryotes with correct folding, glycosylation &post
translational activity.
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16. 1. Plant cells can direct proteins to environments that
reduce degradation and therefore increase stability.
2. Low ethical concerns.
3. Easier purification (homologs don’t pose any
purification challenge, e.g.serum proteins or
antibodies).
4. Versatile(production of a broad diversity of proteins).
5. Take more time to develop.
6. Transgene & protein pollution.
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17. Expression systems for PMF
1. Transgenic plants
2. Plant -cell -suspension culture
3. Transplastomic plants
4. Transient expression system
5. Hydroponic cultures
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18. 1.Transgenic plants:
• Foreign DNA incorporated into the nuclear
genome using-
-Agrobacterium tumefaciens
-Particle bombardment
• Most common
• Long term non-refrigerated storage
• Scalability
• More ‘gene to protein’ time
• Biosafety concerns
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19. 2.Plant Cell Suspension Culture
1. Culture derived from
-transgenic explants
-Transformation after desegregation
2. Recombinant protein localization depends on –
-presence of targeting / leader peptides in the
-recombinant protein. Permeability of plant cell
wall for macromolecules
3. Containment & production under GMP procedure
4. Low scale up capacity
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21. 3.Transplastomic Plants:
1. DNA introduced into chloroplast genome
2. High transgene copy number
3. No gene silencing
4. Recombinant protein accumulate in chloroplast
5. Natural transgene containment
6. Long term storage not possible
7. Long development time
8. Limited use for production of therapeutic
glycoproteins
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22. 4.Transient expression system
1. Biolistic delivery of ‘naked DNA
• Usually reaches only a few cells
• Can be used for a rapid test for protein expression
2. Agroinfiltraion
•Delivery of Agrobacterium in intact leaf tissue by vacuum
infiltration
•Targets many more cells in a leaf
3. Infection with modified viralvector
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23. Virus Infected Plants
• Gene of interest is cloned into the genome of a
viral plant pathogen
• Infectious recombinant viral transcripts are
used to infect plants
• Rapid & systemic infection
• High level production soon after inoculation
• Genetic modification of plant is entirely
avoided
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25. 5.Hydroponic culture
• A signal peptide is attached to the recombinant protein
directing it to the secretory pathway
• Protein can be recovered from the root exudates
(Rhizosecretion) or leaf guttation fluid (Phylosecretion)
• Technology being developed by the US biotechnology
company Phytomedics Inc.
• Purification is easier
• Reduced fear of unintentional environmental release
• Expensive to operate hydroponic facilities
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26. Choice Of Host Species
Depends On:
• Protein To Be Produced & Its Desired
Application
• Transformation Efficiency
• Overall Production Cost
• Containment
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27. Comparison Of Various Plant
Expression Host Species
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28. Antibiotics And Enzymes Produced
From Microbes
Antibiotic Microbes Enzymes Microbes
Griseofulvin Penicillium
griseofulvum
Glucanase Aspergillus niger
Bacillus subtilis
Kanamycin Streptomyces
kanamyceticus
Cellulase Aspergillus niger
Trichoderma reesei
Rhizopus spp
Neomycin Streptomyces fradiae Lipase Aspergillus niger
Pimaricin Streptomyces
natalensis
Lactase Aspergillus niger
Penicilin G Penicillium
chrysogenum
Polymixin B Bacillus polymixa
Streptomycin Streptomyces griseus
Tetracyclin Streptomyces spp.
Trichomycin Streptomyces
hachijoensis1/7/2017 28Dept. of Plant Biotechnology

29. Antibiotics And Enzymes Produced
From Microbes
Antibiotic Microbes Enzymes Microbes
Amphotericin B Streptomyces nodosus a-amylase Bacillus
licheniformis
Bacillus
amyloliquifacieens
Bacitracin Bacillus subtilis Glucoamylase Aspergillus niger
Cephalosporin C Cephalosporium
acremonium
Xylose isomerase Bacillus coagulans
Cycloheximide Streptomyces griseus Alkalineprotease Bacilus licheniformis
Bacillus subtilis
Fungimycin Streptomyces
coelicolor
Neutral protease Bacillus
amyloliquifaciens
Gentamycin Micromonospora
purpurea
Acid protease Aspergillus niger
Gramicidin Bacillus brevis Pectinase Aspergillus niger
Bacillus subtilis1/7/2017 29Dept. of Plant Biotechnology

30. Therapeutic Proteins Produced In
Different Plant Hosts System
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31. Industrial Enzymes & Proteins Produced
In Different Plant Host System
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32. Antibodies Produced In Different
Plant Host System
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33. Vaccines Produced In Different
Plant Host Systems
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35. Transgene Pollution –The Problems
•Transgene pollution is the spread of
transgenes beyond the intended genetically-
modified species by natural gene flow
mechanisms.
•Two classes of transgene pollution:
-The possible spread of primary
transgenes.
-The possible spread of superfluous DNA
sequences.
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36. Transgene Pollution –Possible Solutions
•Minimum required genetic modification.
•Elimination of non-essential genetic
information.
•Containment of essential transgenes.
•Alternative production systems transient
expression.
•Plant suspension cultures in sealed, sterile
reactor vessels
(Fischer et al., 1999a; Doran, 2000).
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37. 1. Use of lettuce, and viral vector-based transient expression systems to
develop a robust PMP production platform biological pharmaceutical agents
that is effective, safe, low-cost, and amenable to large-scale manufacturing
2. Geminiviral replicon system based on the bean yellow dwarf virus permits
high-level expression in lettuce of virus-like particles (VLP) derived from
the Norwalk virus capsid protein and therapeutic monoclonal antibodies
(mAbs) against Ebola and West Nile viruses.
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38. MATERIALS AND METHODS
1) Construction of expression vectors
• The construction of geminiviral vectors, pREP110,
pBYGFP, pBYNVCP, pBY-HL(6D8) Replicon and non-
replicon vector pP19 dual-replicon vector pBY-
HL(hE16).R
2) Lettuce agroinfiltration-
• Lettuce heads were vacuum infiltrated with GV3101
strains containing the targeted expression vectors
3) Protein extraction
• The crude leaf extract was processed by centrifugation at
to yield “lettuce extract”.
• Lettuce extract” was further clarified by filtration
through a 0.2 micron filter.1/7/2017 38Dept. of Plant Biotechnology

39. MATERIALS AND METHODS
4) Protein analysis-
• SDS-PAGE, Western blot, and ELISA analysis for
NVCP, 6D8 mAb and hE16 mAb,sucrose gradient
centrifugation and electron microscopy for NVCP VLP,
antigen binding assays for 6D8 and hE16 mAbs, and
GFP visualization were all performed.
5) WNV neutralization-
• The neutralizing activity of hE16 against WNV was
assessed using a focus reduction neutralization assay
6) Protein Purification-
• Anion exchange chromatography.
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40. RESULTS AND DISCUSSIONS
1. VISUALISATION OF GFP EXPRESSIONIN LETTUCE
Commercially produced lettuce heads were infiltrated with a single Agrobacterium culture, or co-
infiltrated with two or three cultures containing the indicated expression vector(s).
Leaves were examined and photographed 4 days post infiltration under UV (a–e) or regular light
(f).
Lettuce infiltrated with the infiltration buffer (a) was used as a negative control.
N. benthamiana was used as a positivecontrol (d). MagnICON vectors were described in
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41. 2.EXPRESSION OF NVCP IN LETTUCE LEAVES
Leaf protein extracts were separated on a 10% SDS-PAGE gel andtransferred onto
PVDF membranes probed with a rabbit polyclonal antibody against NVCP.
Lane 1: insect cell-derived NVCP standard;
lane 2: protein extract from uninfiltrated lettuce leaves (negative control); lane 3: extract from
pBYNVCP/pREP110 infiltrated lettuce leaves.
(b) Time course of NVCP expression-Total proteins from lettuce leaves infiltrated with
pBYNVCP/pREP110 or pBYNVCP/pREP110 + pP19
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42. 3.PURIFICATION AND CHARECTERISATION OF NVCP
Lane 1: Molecular weight marker;
Lane 2: insect cell-derived NVCP reference standard;
Lanes 3 and 4: crude protein extract and purified NVCP from N. benthamiana leaves as a
comparison;
Lane 5: crude extract from pBYNVCP/pREP110 infiltrated lettuce leaves; lane 6: purified NVCP
from lettuce leaves
b) Sucrose gradient sedimentation profile of purified NVCP. reference standard (I-NVCP)
c) Electron microscopy of lettuce-derived NVCP
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43. 4. EXPRESSION OF MAbS AGAINST EBV AND WNV
Total protein extracts of lettuce leaf were separated on 4–20% SDS-PAGE gradient gelstransferred to
PVDF membranes. The membranes were incubated with a goat
anti-human-gamma chain antibody to detect HC (a) or a goat anti-human-kappa chain
antibody to detect LC (b and c).
Lane 1: extract from uninfiltrated lettuce leaves;
lanes 2 and3: protein samples from lettuce infiltrated with pBY-HL(6D8).R or pBY-HL(hE16).R
construct;
lane 4: human IgG reference standard.
(d) ELISA analysis of 6D8 or hE16 mAb expression. Goat anti-human gamma and kappa chain antibodies
were used as capture and detection reagents, respectively to confirm the assembled forms of 6D8 or hE16
mAb
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44. 5. PURIFICATION OF MONOCLONAL ANTIBODIES
Lane 1: Molecular weight marker;
Lane 2: total leaf proteins from uninfiltrated lettuce leaves;
Lane 3: total leaf protein from lettuce leaves infiltrated with pBY HL(6D8).R;
Lane 4: purified 6D8 mAb
Lane 5: hE16 mAb purified from pBY-HL(hE16).1/7/2017 44Dept. of Plant Biotechnology

45. 6. CHARECTERIZATION OF MONOCLONALANTIBODIES
(a) Specific binding of 6D8 mAb to EBV. Tobaccoderived 6D8 (EBV T-6D8, positive
control), or a negative control generic human IgG.
(b) Binding of lettuce-derived hE16 to domain III of WNV E displayed on the cell surface of
yeast. Lettuce-produced hE16 mAb (L-hE16), mammalian cell-derived hE16 (M-hE16,
positive control), or a generic human IgG (h-IgG, negative control)
(c) Neutralization of WNV by lettuce-produced hE16 mAb. WNV was incubated with serial
dilutions of hE16 derived from lettuce (L-hE16) or mammalian cells (M-hE16) (positive
control) and used to infect Vero cells. Cells were then fixed, permeabilized, analyzed by
focus reduction assay and quantitated by Biospot analysis.1/7/2017 45Dept. of Plant Biotechnology

46. 1. BeYDV-based geminiviral replicon system can efficiently promote high-level
expression of NVCP VLP vaccine and anti-EBV or WNV mAb therapeutic
candidates in lettuce.
2. Using the geminiviral-lettuce system, the VLP andthe two therapeutic mAbs
accumulated to levels that were comparable to that observed in tobacco (Huang et
al., 2010; Lai et al., 2010), but higher than previously reported in lettuce using
non-viral vectors (Kapusta, 1999; Rosales-Mendoza et al., 2010; Webster et al.,
2006).
3. This procedures can efficiently isolate the NVCP vaccine candidate and the two
therapeutic mAbs to high (>95%) purity, in a scalable and cGMP compatible
format.
ANALYSIS AND CONCLUSION
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47. Perspectives on Molecular Pharming
• Use of virus infected plants is best approach for
molecular farming
• Molecular farming provides an opportunity for
the economical and large-scale production of
pharmaceuticals, industrial enzymes and technical
proteins that are currently produced at great
expense and in small quantities.
• We must ensure that these benefits are not
outweighed by risks to human health and the
environment
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48. References
• Robust production of virus-like particles and monoclonal
antibodies with geminiviral replicon vectors in
lettuce.Huafang Lai1, Junyun He1, Michael Engle2, Michael
S. Diamond2, and Qiang Chen1Plant Biotechnol J. 2012
January ; 10(1): 95–104. doi:10.1111/j.1467-
7652.2011.00649.x.
• Wikipedia
• (Rainer Fischer; Stefan Schillberg)
• Su-May Yu; Institute of Molecular Biology Academia Sinica
Nankang, Taipei
• S. Biemelt;U. Sonnewald (2004)
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