molecular pharming

05/01/2023 Dept. of Plant Biotechnology 1


MOLECULAR PHARMING

Upasana MohapatraPALB 6290Jr Msc. Plant BiotechnologyUAS,GKVK,Bengaluru


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


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.


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.)


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 )


1. Clone a gene of interest2. Transform the host platform species3. Grow the host species, recover biomass4. Process biomass5. Purify product of interest6. Deliver product of interest

Molecular Farming Strategy


Downstream processing & analysis of recombinant proteins from plants


1. Bacteria2. Yeasts, (single celled fungi)3. Unicellular algae4. Mammalian, insect, plant, and filamentous

fungal cell cultures5. Whole plants, ( corn, barley, rice, duckweed,

moss protonema)6. Whole animals, (insects, birds, fish, mammals)

Molecular Farming Hosts


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 scalability6. Preferred for the production of small,

aglycosylated proteins like Insulin, interferon-β.

1. Limited by legal and ethical restriction2. Require expensive equipment & media3. Delicate nature of mammalian cells4. Human pathogens and oncogenes 5. Scaling up problems

ANIMAL BASED SYSTEMS

Dept. of Plant Biotechnology12

05/01/2023 Dept. of Plant Biotechnology 13S. Biemelt;U. Sonnewald (2004)

Comparison Of Different Production Systems For Expression Of Recombinant Proteins


Cost of Production: Antibodies

HOST COSTAnimal 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


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.


Plant Molecular Farming

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.


Expression systems for PMF

1. Transgenic plants

2. Plant -cell -suspension culture

3. Transplastomic plants

4. Transient expression system

5. Hydroponic cultures


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


2.Plant Cell Suspension Culture1. Culture derived from -transgenic explants -Transformation after desegregation2. Recombinant protein localization depends on – -presence of targeting / leader peptides in the -recombinant protein. Permeability of plant

cell wall for macromolecules3. Containment & production under GMP procedure4. Low scale up capacity


3.Transplastomic Plants:

1. DNA introduced into chloroplast genome2. High transgene copy number 3. No gene silencing4. Recombinant protein accumulate in chloroplast5. Natural transgene containment6. Long term storage not possible7. Long development time8. Limited use for production of therapeutic

glycoproteins


4.Transient expression system1. 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


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


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


Choice Of Host Species

Depends On:• Protein To Be Produced & Its Desired

Application• Transformation Efficiency• Overall Production Cost• Containment


Comparison Of Various Plant Expression Host Species


Antibiotics And Enzymes Produced From Microbes

Antibiotic Microbes Enzymes Microbes

Griseofulvin Penicillium griseofulvum

Glucanase Aspergillus nigerBacillus subtilis

Kanamycin Streptomyces kanamyceticus

Cellulase Aspergillus nigerTrichoderma reeseiRhizopus spp

Neomycin Streptomyces fradiae Lipase Aspergillus nigerPimaricin Streptomyces

natalensisLactase Aspergillus niger

Penicilin G Penicillium chrysogenum

Polymixin B Bacillus polymixaStreptomycin Streptomyces griseusTetracyclin Streptomyces spp.Trichomycin Streptomyces

hachijoensis


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 licheniformisBacillus subtilis

Fungimycin Streptomyces coelicolor

Neutral protease Bacillus amyloliquifaciens

Gentamycin Micromonospora purpurea

Acid protease Aspergillus niger

Gramicidin Bacillus brevis Pectinase Aspergillus nigerBacillus subtilis


Therapeutic Proteins Produced In Different Plant Hosts System


Industrial Enzymes & Proteins Produced In Different Plant Host System


Antibodies Produced In Different Plant Host System


Vaccines Produced In Different Plant Host Systems


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.


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).


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.


MATERIALS AND METHODS1) 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 vectors3) 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.


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 assay6) Protein Purification-• Anion exchange chromatography.


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


2.EXPRESSION OF NVCP IN LETTUCE LEAVES

Leaf protein extracts were separated on a 10% SDS-PAGE gel andtransferred ontoPVDF 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


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 leavesb) Sucrose gradient sedimentation profile of purified NVCP. reference standard (I-NVCP)c) Electron microscopy of lettuce-derived NVCP


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 goatanti-human-gamma chain antibody to detect HC (a) or a goat anti-human-kappa chainantibody 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


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).


6. CHARECTERIZATION OF MONOCLONAL ANTIBODIES

(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. 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


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


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)

molecular pharming

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