This document provides an overview of molecular pharming, which uses genetic engineering techniques to produce pharmaceuticals through biological systems like plants and microorganisms. It discusses the history and development of molecular pharming, strategies like tissue culture and genetic transformation used, and various transformation methods including biological, chemical, and physical means. Examples of products include vaccines, antibodies, and industrial enzymes produced in plants. The document also covers applications, outcomes like plantibodies and edible vaccines, pharmacokinetics/dynamics testing, future prospects, and biosafety challenges of molecular pharming.

1. Bahauddin Zakariya University Multan
Molecular Pharming
Submitted by:
Awais Ali: BSBTL-13-005
Asad Razzaq: BSBTL-13 -021
M. Aqil Yaqoob: BSBTL-13- 026
Rizwan Abbas: BSBTL-13 -027
Tooba Shehzad: BSBTL-13 -028
M. Ahmad Parvez: BSBTL-13 -029
Course: Diagnostics
Course no: BSBT-417
Instructor: Dr. Hamid Manzoor
Date of Submission: 15th
October 2017

2. Table of Contents
Molecular Pharming......................................................................................................................................................... 3
History......................................................................................................................................................................... 3
Development................................................................................................................................................................ 3
Strategies of molecular Pharming..................................................................................................................................... 4
Tissue culture techniques ................................................................................................................................................. 4
Uses of tissue culture in molecular Pharming ............................................................................................................... 5
Genetic transformation /Engineering................................................................................................................................ 5
Pharming /Genetic engineering by biological methods ..................................................................................................... 5
Transformation by chemical methods............................................................................................................................... 6
Liposomal gene transfer protocol ................................................................................................................................. 7
Poly ethylene glycol mediated gene transfer ................................................................................................................. 7
Physical Means of Gene Transfer..................................................................................................................................... 7
Gene gun/Golden gun/Biolistics................................................................................................................................... 8
Micro injections ........................................................................................................................................................... 8
Electroporation ............................................................................................................................................................ 9
Applications and examples............................................................................................................................................... 9
Pharmacokinetics and Pharmacodynamics........................................................................................................................ 9
Major outcomes of Molecular Pharming..........................................................................................................................12
Plantibodies ....................................................................................................................................................................12
Methods of plantibody production...............................................................................................................................12
Application of plants as transgenes for biologicals.......................................................................................................13
Advantages of using plants for antibody production.....................................................................................................14
Edible vaccines...............................................................................................................................................................14
Vaccine production systems............................................................................................................................................15
Nuclear transformation system ....................................................................................................................................15
Chloroplast transformation system .............................................................................................................................16
Products of Molecular Pharming .....................................................................................................................................17
Future Prospects and Biosafety Challenges..................................................................................................................18
Conclusion......................................................................................................................................................................20
References ......................................................................................................................................................................20

3. Molecular Pharming
Molecular pharming is self explanatory word which refers to production of pharmaceuticals using molecular
biology techniques.
Basically molecular pharming is collection of techniques which uses whole organism, organ, tissue, or cell &
cell cultures as a bioreactor for the production of commercially valuable products. Via recombinant DNA
technique
Molecular Pharming is the growing and harvesting of genetically engineered crops of transgenic plants, to
produce biopharmaceuticals. The idea is to use these genetically modified crops as biological factories to
generate valuable medical products difficult or expensive to produce in any other way. Combining advance in
molecular biology, gene engineering and immunology, the scientists take genes from other sources such as
microorganisms, and splice them into the plant's genome. These genetically engineered plants synthesize
recombinant proteins which can be vaccines, blood substitutes, enzymes or diagnostic reagents which are then
extracted from the crop. Therapeutic proteins, edible vaccines are already in clinical trials. Several antigens
from different infectious agents have been successfully produced in plants.
History
 1986: First plant derived recombinant therapeutical protein
 1989: First plant derived recombinant antibody
 1990 :First native human protein produce in plants
 1992: First plant derived vaccine candidate
 1992: First plant derived industrial enzyme
 1995: secretory IgA produced in tobacco
Development
Protein-based biopharmaceuticals are often produced in mammalian cell cultures, which are more expensive
than microbial systems but capable of authentic post-translational modifications. The costs are lower if plants
are used as an alternative platform to produce complex proteins such as monoclonal antibodies, vaccines and
enzymes. This review highlights recent advances that have been achieved in plant-based biopharmaceutical
production platforms in terms of expression strategies, product yields and process development. The first
generation of plant-derived pharmaceuticals now entering the market is also discussed. Finally, the review
considers the downstream processing of plant-derived pharmaceuticals which can account for up to 80% of the
production costs. In this context, recent improvements in clarification and integrated process methods will have
a strong impact on the economic feasibility of production, especially if supported by and combined with process
analytical technology as part of the quality-by-design initiative.
Significance
Global demand for pharmaceuticals is at unprecedented levels, and current production capacity will soon be
overwhelmed. Expanding the existing microbial systems, although feasible for some therapeutic products, is not
a satisfactory option on several grounds. First, it would be very expensive for the pharmaceutical companies.
Second, other proteins of interest are too complex to be made by microbial systems. These proteins are
currently being produced in animal cell cultures, but the resulting product is often prohibitively expensive for
many patients. Finally, although it is theoretically possible to synthesize protein molecules by machine, this
works only for very small molecules, less than 30 amino acid residue in length.

4. Virtually all proteins of therapeutic value are larger than this and require live cells to produce them. For these
reasons, science has been exploring other options for producing proteins of therapeutic value.
The use of plants offers a number of advantages over other expression systems. Plants can be used in two ways.
One way is to insert the desired gene into a virus that is normally found in plants, such as the tobacco mosaic
virus in the tobacco plant. The other way is to insert the desired gene directly into the plant DNA to produce a
transgenic plant.
In simple words
 Aims to replace very expensive bioreactors (upstream process).
 Gene expression yield better quality therapeutic proteins.
 Cheaper rather than products from downstream process.
 Opportunity /technique to produce valuable medicines on a massive scale.
Strategies of molecular Pharming
Molecular Pharming refers to the collection of technique of production of pharmaceuticals from biological
sources. Molecular Pharming is the tool which provides precise, safe and applicable platform for the production
of pharmaceutical products. It favors’ use of biological system as bioreactors/machinery in two major means,
which includes(Ritala, Häkkinen, & Schillberg, 2014)
a. Cell & tissue culture
b. Gene transfer method ( Recombinant products)
The major methodologies generally the biotechnologist adopt is by expression of desired gene in suitable host
organisms, like, bacteria, plants and other eukaryotic system. These include
A. Mass production
 By tissue culture
 Micro propagations
B. Genetic engineering
 Recombinant microorganisms
 Transgenic plants
 Transgenic plants
 Stem cell mediated gene transfer
 Targeted gene therapy
Tissue culture techniques
Plant tissue culture can be defined as the in vitro manipulation of plant cells. Advancement in plant tissue
culture brings a revolutionary stage in field of Agriculture /Plant biotechnology. Plant tissue culture serve as
back bone in such scientific revolution by introducing genetically transformed (transgenic) products. Plant
regeneration by such process is quiet easy and feasible rather than cloning and genetic engineering(Tobergte &
Curtis, 2013).
To get success in culture of plant cells, tissues /organs we have to follow precise methods and proper sterilized
use of materials and nutritional medium such as growth regulators for the specimen (explants) according to its

5. requirement. Within favorable conditions the plant cells can multiply and even can generate whole plant.
Regeneration of plant is important factor for quantitative and qualitative production of genetic desired
characteristic. A (totipotent cell) a cell having ability to regenerate is used for mass production and this
phenomenon is termed as Clonal propagation(Jelaska, Mihaljević, & Bauer, n.d.).
Uses of tissue culture in molecular Pharming
 Callus culture is used for transformation of plant cells.
 Cell suspension culture is applicable for isolation of secondary metabolites. We can say Cell
suspension culture is main factory for production of secondary metabolites. E.g. Taxols, terpenoids etc.
 Protoplast, anther cultures are transformed genetically via ballistics/microinjections to obtain desired
traits in plant.
 Stem cells are cultured in order to transform animals.
 Cell culture facilitate hybridoma technology for production of monoclonal antibodies
 Better, quality and more food production is now possible by this technology.
 Production of medicines, vaccine by genetic engineering depends on this technology.
Genetic transformation /Engineering
Gene transformation refers to the genetic change in genome. It represents the replacement of or transfer desired
gene in the cell / genome of an organism(Stevenage, Catalyst, Road, & Sciences, 2012). The major steps of
genetic transformation for Pharming a product include
 Identification and Isolation of gene of interest.
 Cloning of desired gene.
 Generation of Vectors.
 Restriction of vectors and ligation of gene.
 Integration of gene.
 Expression , product isolation and purification
 Regulation of pharmacodynamic and pharmacokinetics
 Encapsulation/preservation
 Clinical trials
 Approval from Regulatory Authorities
 Marketing
Genetic engineering or modification is done by three modes with different principles.
 Biologically
 Physical methods
 Chemical methods
Pharming /Genetic engineering by biological methods
Biological method is based on carriers. Biological agents like bacteria, plasmids and even viruses are used as
carrier of desired gene to host. Gene is integrated and expressed in order to achieve desired products(Jelaska et
al., n.d.).

6. Examples
Plasmids like (Boilver Redriguez series 322) pBR322, (plasmid synthesized in University of California)
pUC19, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are common
examples of non viral gene carriers.
Viral genomes are manipulated as replication deficient. The synthesized viral genomes including phagemids,
cosmids and SV40 etc are used as carrier of genes(Jelaska et al., n.d.).
Transformation by chemical methods
The chemical molecules are used as carrier and regulate targeted gene delivery in different systems. The
chemicals enhance or regulate transformation by targeted delivery. The principle is based on the binding affinity
of molecules with gene/DNA. The chemicals include positively charge particles. The positively charge particles
binds the DNA molecule due to opposite charge. The bind complex is transfer to target site using different
signaling agents(Torney, Trewyn, Lin, & Wang, 2007).
Techniques include
 Polyethylene glycol method
 Liposomes mediated gene transfer.
 Polycation mediated gene transfer.
 DEAE dextran method
Poly ethylene Glycol and Liposome’s mediated gene transfer protocols are preferred to transfer desired gene
in target location.
Insertion of gene in plasmid Restriction by
similar endonuclease and ligation
B. Gene transfer via TI plasmid, followed by insertion,
ligation, integration

7. Liposomal gene transfer protocol
Liposomes are smallest spherical structure and lipid in nature. Artificially designed liposomes of 5µm-10 µm
are used for this purpose. Liposomes are designed as carriers by encapsulation the gene and different signals in
it. Certain bind peptides like homing peptide enhance target bind and gene transfer(Tobergte & Curtis, 2013).
Poly ethylene glycol mediated gene transfer
Polyethylene glycol is a chemical molecule which used as a fusicogenic agent. This molecule helps in fusion of
certain materials. Polyethylene glycol gene transformation is based on principle of inducing pore and act as
transporting agent.
Polyethylene glycol mediated gene delivery protocol is designed for protoplast transformation(Stevenage et al.,
2012).
Mechanism
Mechanical or enzymatic action is used for Protoplast preparation by removing cell membrane.
The desired DNA is isolated or collected from libraries and used in transformation via 40% PEG-4000 (w/v)
dissolved in mannitol and calcium nitrate solutions used as medium. These mixtures are incubated, which
enhance protoplasts and chloroplast transformation(Torney et al., 2007).
Physical Means of Gene Transfer
Physical apparatuses and instruments are used as vehicle /tool for transfer of gene in order to transform the host
cell. This method is based on the direct transformation of the host cell or tissue by transferring DNA present on
or within the apparatus. These techniques provide us sufficient as well as rapid transformation of target cells
within optimized conditions and handling. Another benefit is that unlike biological means such methods need
less number of molecular steps to perform and minimize the work burden and save time also(Moshelion &
Altman, 2015).
The well known physical means of gene transformation techniques include,
 Gene Gun , Biolistic / Ballistic or Golden Gun method
 Microinjections
 Electropration.
 Laser beam technique
 Ultrasound / Sonication etc
 Stem cell mediated
A. Liposome construct
Signaling agent like NLS
Nuclear localization signals
and targeting peptides like
homing peptides enhance
integration and specific
delivery of gene

8. Gene gun/Golden gun/Biolistics
Gene gun is an instrument used to shoot the DNA on target sites. It is like a pistol and also known as Biolistics.
(Ballistics) and Golden gun because it uses gold particles for transformation process. This amazing and
successful invention was done by John C Sanford and coworkers of Cornell University in 1985. Gene gun is
carrier free i.e. direct transformation tool and usually used in both in-vitro as well as in-vivo gene transfer. Gene
gun mediated transfer is used for stable integration of gene(Gangrade, Waghmare, & Lad, 2016).
Micro injections
Microinjection technique is the physical mean of gene transfer. This technique is rooted in the work of Dr
Marshall Barber. It is the most efficient technique of transformation of animal cell as well as plant protoplast.
It is based on principle of direct transformation. The transformation is done by injection of gene of interest
through glass syringe i.e. pipette in target cells under observing lenses. The plates containing target cells as well
as injecting pipettes are hold by holders known as micromanipulators(Wilmut & Campbell, 2001).
Gene gun
shooting of
gene
Construct & gene delivery
Microinjection
 Microinjector uses for injecting Gene
 Holding plate holds cells target

9. Electroporation
Electroporation is another physical mean of gene transfer. This technique is also known as electro-
permeabilization which refers to induce permeability in cells. Electro-poration uses electric pulse to increase the
permeability of plant cells which allow or permit the entrance of foreign particle. The target cells are placed in
optimized medium already containing the gene to be inserted. The temperatures and other parameters are
optimized. Usually temperature is maintained up to 60-75 Celsius. Voltage of 150-160 volt is passed by power
source which enables the gene transfer(Tobergte & Curtis, 2013).
Applications and examples
 Transgenic organism and gene therapy are the two major outcomes.
 BT product like BT cotton and BT rice are produced by genetic transformation.
 Ripening of fruits is delayed, shelf life of fruits and vegetables are increased.
 Golden rice is produced to treat eye diseases.
 Herbicide resistant onions are produced by transformation through Agro bacterium mediated
transformation(Zambryski et al., 1983).
 Productivity of Barely (Hordeum vulgare) is enhancing by electroporation methods.
 Sorghum species productivity was enhanced by Biolistics or gene gun.
 Sunflower oilseed plants productivity is increased by PEG mediated gene transfer.
 Insulin was produced in 1972 for first time by plasmid /vector mediated transformation.
 Gene therapy is kind of treatment of diseases using gene transfer.
Pharmacokinetics and Pharmacodynamics
Pre-clinical trials held on animals model to check that whether a drug is good or bad or it has some side effect
or not. If pre-clinical trials are best then it is held on human. And it is called clinical trials. Some tests applied
on animal model to check its accuracy. Tests are given below(Tobergte & Curtis, 2013).
1. Pharmacokinetic profile
2. Pharmacodynamic profile
3. Bioequivalence and bioavailability
4. Acute toxicity
5. Chronic toxicity
6. Reproductive toxicity and teratogenicity
Electroporation

10. 7. Mutagenicity
8. Carcinogenicity
9. Immunotoxicity
10. Local tolerance
Bioavailability relates to the proportion of a drug that actually reaches its site of action after administration.
Bioequivalence studies come into play if any change in product production/delivery systems was being
contemplated.
Acute toxicity is usually assessed by administration of a single high dose of the test drug to rodents. Both rats
and mice (male and female) are usually employed. The test material is administered by two means, one of
which should represent the proposed therapeutic method of administration. The animals are then monitored for
7–14 days, with all fatalities undergoing extensive post-mortem analysis. Earlier studies demanded calculation
of an LD50 value (i.e. the quantity of the drug required to cause death of 50 per cent of the test animals). Such
studies required large quantities of animals, were expensive, and attracted much attention from animal welfare
groups. Its physiological relevance to humans was often also questioned. Nowadays, in most world regions,
calculation of the approximate lethal dose is sufficient.
Chronic toxicity studies also require large numbers of animals and, in some instances, can last for up to 2
years. Most chronic toxicity studies demand daily administration of the test drug (parentally for most
biopharmaceuticals). Studies lasting 1–4 weeks are initially carried out in order to, for example, assess drug
levels required to induce an observable toxic effect. The main studies are then initiated and generally involve
administration of the drug at three different dosage levels. The highest level should ideally induce a mild but
observable toxic effect, whereas the lowest level should not induce any ill effects. The studies are normally
carried out in two different species, usually rats and dogs, and using both males and females. All animals are
subjected to routine clinical examination, and periodic analyses of, for example, blood and urine are undertaken.
Reproductive toxicity studies complement teratogenicity studies, which aim to assess whether the drug
promotes any developmental abnormalities in the fetus. (A teratogen is any substance/agent that can induce
feotal developmental abnormalities. Examples include alcohol, radiation and some viruses.) Daily doses of the
drug are administered to pregnant females of at least two species (usually rats and rabbits). The animals are
sacrificed close to term and a full autopsy on the mother and fetus ensues.
Mutagenicity tests aim to determine whether the proposed drug is capable of inducing DNA damage, either by
inducing alterations in chromosomal structure or by promoting changes in nucleotide base sequence. Although
mutagenicity tests are prudent and necessary in the case of chemical-based drugs, they are less so for most
biopharmaceutical substances. In many cases, biopharmaceutical mutagenicity testing is likely to focus more so
on any novel excipients added to the final product, rather than the biopharmaceutical itself. (Excipients refers to
any substance other than the active ingredient that is present in the final drug formulation). Mutagenicity tests
are usually carried out in vitro and in vivo, often using both prokaryotic and eukaryotic organisms. A well-
known example is the Ames test, which assesses the ability of a drug to induce mutation reversions in E. coli
and Salmonella typhimurium.
Longer-term carcinogenicity tests are undertaken, particularly if (a) the product’s likely therapeutic indication
will necessitate its administration over prolonged periods (a few weeks or more) or (b) if there is any reason to
suspect that the active ingredient or other constituents could be carcinogenic. These tests normally entail

11. ongoing administration of the product to rodents at various dosage levels for periods of up to (or above) 2
years(Tobergte & Curtis, 2013).
Pharmacokinetics and Pharmacodynamics
Pharmacokinetics relates to the fate of a drug in the body, particularly its ADME, i.e. its absorption into the
body, its distribution within the body, its metabolism by the body, and its excretion from the body. The results
of such studies not only help to identify any toxic effects, but also point to the most appropriate method of drug
administration, as well as the most likely effective dosage regime to employ. Generally, ADME studies are
undertaken in two species, usually rats and dogs, and studies are repeated at various different dosage levels. All
studies are undertaken in both males and females. If initial clinical trials reveal differences in human versus
animal model pharmacokinetic profiles, additional pharmacokinetic studies may be necessary using primates.
Pharmacodynamics studies deal more specifically with how the drug brings about its characteristic effects.
Proteins Pharmacokinetics
After initial filtration many proteins are actively reabsorbed (endocytosed) by the proximal tubules and
subjected to lysosomal degradation, with subsequent amino acid reabsorption. Thus, very little intact protein
actually enters the urine.
Uptake of protein by hepatocytes can occur via one of two mechanisms: (a) receptor-mediated endocytosis or
(b) non-selective pinocytosis, again with subsequent protein proteolysis. Similarly, a proportion of some
proteins are likely degraded within the target tissue, as binding to their functional cell surface receptors triggers
endocytotic internalization of the receptor ligand complex. Cellular uptake of some glycosylated therapeutic
proteins occurs via specific sugar-binding cell surface receptors. Cell surface mannose receptors, for example,
are capable of binding glycoproteins whose sugar side chains terminate in mannose, fucose, N-acetyl
glucosamine or N-acetyl galactosamine. Evidence suggests that a liver-specific form of the mannose receptor
mediates clearance of luteinizing hormone. The sugar side chains of many glycoproteins exhibit terminal sialic
acid residues (sialic acid caps). The hepatic asialoglycoprotein receptor binds glycoproteins whose sialic acid
caps have been removed, likely mediating their removal from general circulation.
Pharmacokinetic and indeed pharmacodynamic characteristics of therapeutic proteins can be rendered (even
more) complicated by a number of factors, including:
The presence of serum-binding proteins
Some biopharmaceuticals (including insulin-like growth factor (IGF), GH and certain cytokines) are notable in
that the blood contains proteins that specifically bind them. Such binding proteins can function naturally as
transporters or activators, and binding can affect characteristics such as serum elimination rates.
Immunogenicity
Many, if not most, therapeutic proteins are potentially immunogenic when administered to humans. The
likelihood that non-human proteins are immunogenic in humans is an obvious one. However, human proteins
can also be potentially immunogenic. Antibodies raised in this way can bind the therapeutic protein,
neutralizing its activity and/or affecting its serum half-life.

12. Sugar profile of glycoproteins
Expression of a therapeutic glycoprotein in different eukaryotic expression systems results in a product
displaying differences in exact glycosylation. The exact glycosylation pattern can influence protein activity and
stability in vivo, and some sugar motifs characteristic of yeast-, insect- and plant-based expression systems are
immunogenic in man.
Major outcomes of Molecular Pharming
With the advent of genetic engineering, scientists are able to engineer living organisms, from the simple yeasts
to the more complex plants, to produce specific pharmaceuticals. Biopharmaceuticals are drug products
(proteins, including antibodies) produced in living systems and used for therapeutic or diagnostic purposes or as
dietary supplements.
The major outcomes include
 Plantibodies
 Edible vaccines
 Pharmaceutical products
 Neutraceuticals
 Invitro diagnostics
Plantibodies
A plantibody is an antibody that is produced by plants that have been genetically engineered with animal DNA.
An antibody (also known as an immunoglobulin) is a complex protein within the body that recognizes antigens
on viruses and other dangerous compounds in order to alert the immune system that there are pathogens within
the body.
Around 1990, plants were first considered as a potential host for producing antibodies and the word
“plantibody” was coined. The term “plantibodies” describes the products of plants that have been genetically
engineered to express antibodies and antibody fragments. With this technology, plants are being used as
antibody factories (bioreactors), utilizing their endomembrane and secretory systems to produce copious
amounts of clinically viable proteins which can later be purified from the plant tissue(Ou et al., 2014).
Antibodies can be expressed in plants as either full-length molecules or as smaller fragments. In essence, a
plantibody is an antibody produced by genetically modified plants. Antibodies, originally derived from animals,
are produced in plants by transforming the latter with animal antibody genes. Although plants do not naturally
make antibodies, plantibodies have been shown to function in the same way as normal antibodies10. This
concept of using plants as heterologous expression system for recombinant antibodies (plantibodies) is now
more than two decades old(Gangrade et al., 2016).
Methods of plantibody production
Various techniques have been developed to exploit plants as bioreactors to produce Pharmaceutical antibodies.
One of the several methods for synthesizing plantibody is conventional method which uses transformation and
transient expression vector to introduce new genes into a host cell. The transform ant cell is then introduced into
the plant embryo and propagation of the plant in the open field allows large-scale production of antibodies.
Plant tissue culture is the most economic and time-saving method for production of antibodies from plants. To
achieve this, plant cells in differentiated states are grown in bioreactors with foreign proteins harvested from
either the biomass or culture liquid. Cell cultures contain fewer biological proteins or molecules (along with
herbicides and pesticides) than open field plants or bacterial/yeast cell cultures, which may contaminate the
product.

13. An experiment on tobacco plant established its breeding and sexual crossing as a method for production of
plantibody. In this experiment, transformation was used to introduce kappa type of light chain into tobacco
plants. The same was done with gamma heavy chains. Upon crossing one plant with kappa-chains and another
plant with gamma-chains, an antibody was produced that expressed both chains.
Some researchers suggest use of transgenic seeds in place of green plant tissue as plants cannot store antibodies
for an extended period. Seeds contain a low level of proteases that allows proteins to be stored without
degradation.
Application of plants as transgenes for biologicals
The use of transgenic plants for the expression of molecules with therapeutic, diagnostic or veterinary
applications has been documented in the last decade. This technology represents a fantastic opportunity for the
pharmaceutical industry, since biological products now account for a large percentage of all pharmaceutical
compounds. Several plant-produced antibodies are presently undergoing clinical trials.
i. Therapeutic applications
The first plantibody created from tobacco was called CaroRx®. It is a clinically advanced anti-Streptococcus
mutants secretory immunoglobulin A plantibody that binds specifically to the bacterium, thus protecting
humans from dental caries that the organism causes23. Another plantibody with human medical applications is
a humanized antibody against herpes simplex virus glycoprotein B which was expressed in soybean.
In a study conducted by Hull et al.25, antibodies engineered to bind to Bacillus anthracis were extracted from
transgenic strains of tobacco and tested in mice. The result showed that the antibodies were effective in fighting
the B. anthracis strain and bodes well for the future if ever there is an anthrax epidemic, as there will be a cheap
and effective prevention of the disease.
In a similar study, tobacco-derived plantibodies were experimentally administered in mice against the Lewis Y
antigen, which is found on tumor cells in mice and in colorectal, breast, lung and ovarian cancer. The results
showed that the plantibodies had a definitive positive effect on the cancer-stricken mice by preventing tumor
formation in them. Also, treatment or cure for rabies through plantibodies has been investigated. A plantibody-
based rabies vaccine produced in tobacco was experimentally administered in hamsters to identify whether it
could effectively target rabies. According to Ko et al.26, the plantibody proved to be a safe and economically
feasible alternative to the current methods of antibody production in animal systems.
ii. Vaccination
The production of proteins in plants is a major task in producing pharmaceutical polypeptides. Potential proteins
produced include cytokines, hormones, enzymes, epidermal growth factors, interferons, human protein C, and
pharmaceutical food stuff which are considered for oral immunization. Transgenic plants that express antigens
in their edible tissue might be used as an inexpensive oral vaccine production and delivery system. Thus,
immunization might be possible through consumption of an “edible vaccine” to provide active immunization.
Also, plants produce different classes of proteins which are inexpensive and have increased pharmaceutical

14. value. Due to these reasons, transgenic plants are better alternatives. Oral vaccines offer convenient
immunization strategies for implementing universal vaccination programs throughout the world. However,
compared to vaccines, plantibodies have one major demerit - the introduced antibodies are flushed through a
person's system relatively quickly, in a matter of hours or days, before the host's immune system has adapted to
producing antibodies. Furthermore, vaccines elicit antibody production so that one or a few doses can protect
the individual for year(s). By contrast, if a plantibody is being used to prevent a disease, the patient would need
to take doses indefinitely. Other disadvantages of adoption of antibody expression in plants include gene
silencing in some instances, different patterns of glycosylation, insufficient expression in some plants, allergies
or allergic reactions to plant glycoproteins and other plant antigens.
Advantages of using plants for antibody production
Plantibodies work in a similar fashion to mammalian antibodies; however, compared to conventional methods
using mammalian cells, the use of plants for antibody production offers several unique advantages. Firstly,
plants are widespread, abundant, and grow quickly; they usually mature after one season of growth and it is
possible to bring the product to the market within a short time. Therefore, the cost of antibodies produced by
plants is substantially less than that from their animal counterparts. Secondly, plants are less likely to introduce
adventitious human or animal pathogens compared to mammalian cells or transgenic animals, thus reducing
screening costs for viruses, prions and bacterial toxins. Unlike bacterial and other prokaryotic systems, plants
share a similar endomembrane system and secretory pathway with human cells. They do not trigger immune
responses which animal antibodies are prone to doing when exposed to foreign/non-self agents and they also
produce a relatively high yield of antibodies in a comparatively shorter time. Additionally, plants are capable of
synthesizing and assembling virtually any kind of antibody molecule, ranging from the smallest antigen-binding
domains and fragments to full-length and even, multimeric antibodies. Plants can be engineered to produce
proteins efficiently, with significantly lower manufacturing costs than mammalian cell cultures. Moreover,
large-scale processing infrastructure is already in place for most crops. Hence, scale-up is rapid and efficient,
requiring only the cultivation of additional land. Also, plants that generate large biomass like corn and tobacco
can produce large amounts of genetically engineered products while proteins can be indefinitely stored on seeds
with little reduction in biological activity.
Edible vaccines
“Vaccines that triggers immune system and are formed from transgenic plants”
Why we need edible vaccines and Role in molecular Pharma
Plant-made subunit vaccines are heat stable, lack animal pathogen contamination and can be engineered to
contain multiple antigens, such as those that are combined with subunits of cholera toxin (CT), for the
protection of humans and animals against multiple infectious diseases. It is possible to harvest and process plant
material on a large scale. When plants expressing a recombinant antigen are used as feed, they eliminate the
purification requirement. Plants offer general advantages for large-scale economic production, product safety
and ease of storage and distribution. Plant-based oral vaccines could revolutionize the vaccine industry by
reducing the cost of complex production systems, such as fermentation, purification, cold storage and
transportation. In addition, the use of plants to express pathogen subunit vaccine proteins allows the rapid
production of diverse antigens that contain disulphide bonds, are glycosylated or require other post-translational
modifications to achieve their desired biological function. The use of transgenic plants to produce subunit
vaccine proteins has been developed as an alternative platform for the large-scale production and delivery of
vaccines to induce protective immune responses via the mucosal immune system. The first plant-based oral
vaccine, which used tobacco and potato to produce recombinant LTB from E. coli, induced low levels of both
serum IgG and secretory IgA (sIgA) antibodies in mice after oral administration. Vegetable and fruit crops are

15. ideal host systems for oral vaccine production. Potential plant species used for pharmaceutical protein
production include alfalfa, carrot, lettuce, tomato, potato, maize, soya bean, rice and banana. Plant-based
antigens can be fed directly to animals or humans without purification or processing. Transgenic plants are ideal
for producing oral vaccines because the antigenic proteins are protected from the acidic environment in the
stomach by the plant cell wall, enabling antigens to reach the gut-associated lymphoid tissue (GALT).
Vaccine production systems
Nuclear transformation system
Stable nuclear transformation involves transgene integration into the plant nuclear genome, leading to the
expression of therapeutic proteins and Mendelian inheritance of the introduced trait. Stable integration into the
nuclear genome allows for continual production of recombinant proteins, simultaneously reducing costs and
simplifying production. Nuclear-expressed recombinant proteins undergo typical eukaryotic post-translational
modifications and can be stored in subcellular organelles or secreted, depending on the fused signaling peptides.
Modulation of gene expression in plants to enhance accumulation of target proteins could be achieved by using
efficient promoters, adding specific signal sequences and optimizing several molecular factors like GC content,

16. codon bias, incorporation of 5′ and 3′ regulatory sequences and elimination of cryptic splicing sites, putative
polyadenylation signals, and mRNA-destabilizing sequences. Although the first nuclear genome engineering
was accomplished in 1995, two decades of research and development have not yet resulted in a single approved
vaccine worldwide.
Chloroplast transformation system
Chloroplast transformation has been developed into a highly efficient expression system for recombinant
protein production. In the chloroplast technology, site-specific integration of foreign genes into the chloroplast
genome occurs by homologous recombination, eliminating the variation in expression caused by gene silencing,
positional effects and pleiotropic effects among independent transgenic lines. Moreover, the prokaryotic nature
of the chloroplast makes multigene engineering via chloroplast transformation possible. Foreign gene products
regulated by the endogenous psbA promoter and 5′-untranslated region (UTR) and the psbA3′-UTR express up
to 72% of the total soluble protein (TSP) of trans-plastomic plants. The species specificity of the regulatory
sequences dramatically affects transgene expression levels.
Although chloroplast transformation protocols have been developed for a few edible crops like potato, carrot
and tomato, the expression level of the foreign gene in the edible parts of these plants is not adequate for using
such systems to produce vaccines or biopharmaceuticals. Compared with chloroplasts in photo synthetically
active tissues, non-green plastids like chromoplasts generally have much lower gene expression activity due to
the suppression of plastid gene expression through the interplay between transcriptional and translational
control in non-green tissues. Therefore, edible leafy vegetables are ideal for biopharmaceutical applications.
The lettuce chloroplast system has been successfully used to express a number of vaccines and
biopharmaceuticals.
Gastroenteritis and hepatitis Diarrhoeal infectious diseases:
Gastroenteritis and hepatitis Diarrhoeal infectious diseases (DID) are a major problem in developing countries,
Traveller’s diarrhoea and cholera, caused by enterotoxigenic strains of Escherichia coli (ETEC) and Vibrio
cholerae, respectively, are two enteric diseases resulting in high mortality, especially in young children in
developing countries.
CTB was expressed in maize seeds driven by a γ-zein promoter and accumulated in the endosperm of transgenic
maize kernels with an expression level of 0.0014% of the total aqueous soluble protein (TASP) in the T1
generation and significantly increased to 0.0197% of TASP in the T2 generation.
Anti-CTB IgG and IgA were detected in the sera and in fecal samples from orally administered mice, and the
mice were protected against CT holotoxin challenge. Inclusion of a heat-stable (ST) toxin into vaccine
formulations is required, as most ETEC strains can produce both LT and ST enterotoxins.
Transgenic tobacco plants carrying the LTB:ST gene accumulated up to 0.05% of TSP, and oral dosing with
transgenic tobacco leaves elicited specific mucosal and systemic humoral responses in mice.
Hepatitis B:
Hepatitis B virus attacks the liver and results in both acute and chronic disease. The expression level of the
major surface antigen of hepatitis B virus (P-HBsAg) reached 0.003–0.09% of TSP in transgenic potato. Mice
produced specific faecal IgA and serum IgG antibodies against P-HBsAg after oral administration. Herbicide-
resistant lettuce was engineered to stably express the small surface antigen of hepatitis B virus (S-HBsAg)

17. Rabies:
Rabies virus is an enveloped, negative-sense, single-stranded RNA virus of the genus Lyssavirus in the family
Rhabdoviridae. The expression level of the rabies virus glycoprotein protein (G protein) in transgenic maize
kernels reached 25 μg/g FW.
Neutralizing antibodies in sheep were induced after oral immunization with maize-derived G protein. Further,
the degree of protection achieved with 2 mg of maize-based G protein was comparable to that of a commercial
vaccine. Transgenic hairy roots of Solanum lycopersicum were engineered to express the rabies glycoprotein
fused with ricin toxin B chain (rgp-rtxB) antigen driven by a constitutive CaMV35S promoter. The expression
level of protein in different tomato hairy root lines ranged from 1.4 to 8 μg/g of tissue. A partially purified
protein was able to induce an immune response in mice after intramucosal immunization, but the IgG titres were
low.
Malaria:
Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites. Plasmodium falciparumis is
responsible for the majority of the over half a million malaria deaths per year, which are predominantly children
under the age of five that live in indigent African nations. A chloroplast derived dual cholera and malaria
vaccine expressing CTB fused with the malarial vaccine antigens apical membrane antigen 1 (AMA1) and
merozoite surface protein 1 (MSP1) accumulated up to 13.17% and 10.11% of TSP in tobacco and up to 7.3%
and 6.1% of TSP in lettuce, respectively. Significant levels of antigen specific antibody titers in orally
immunized mice not only cross-reacted with the native parasite proteins in immunofluorescence studies and
immunoblots, but also completely inhibited the proliferation of the malarial parasite.
Additionally:
 the Th1-related cytokines interleukin 12 (IL-12, a cytokine involved in the differentiation of naive T
cells into Th1 cells)
 TNF (tumor necrosis factor, a cytokine involved in the inflammatory process and apoptosis)
 IFN-γ were significantly increased in the spleens of immunized mice.
Products of Molecular Pharming
The use of plants to express proteins can be more practical, safe and economical compared to other biological
systems. Plant systems allow production with low start-up costs because the expensive equipment used in
microbial systems are not required. The production of these compounds in plants is sometimes called molecular
pharming.
The first full size native protein expressed in plants was human serum albumin, produced in 1990 in transgenic
tobacco and potato plants. Years after this pioneering work, two plant-derived pharmaceuticals (PDPs) or plant-
made pharmaceuticals (PMPs) have been commercialized (one in Cuba and one in the US). Europe is expected
to commercialize PDPs in 2009. A wide array of PDPs is now in the pipeline for commercialization to treat
diseases such as cystic fibrosis and non-Hodgkin’s lymphoma, among others (see Table 1).

18. There are also various veterinary applications of plant-derived vaccines and therapeutic proteins but these will
not be discussed here.
Table 1: Plant-derived pharmaceuticals for the treatment of human diseases that are in the
pipeline for commercialization.
Product Class Indication Crop
Various single-chain
Fv antibody
fragments
Antibody
Non-Hodgkin’s
lymphoma
Viral vectors in
tobacco
CaroRx Antibody Dental caries Transgenic tobacco
E. coli heat-labile
toxin
Vaccine Diarrhea
Transgenic maize
Transgenic potato
Gastric lipase Therapeutic enzyme
Cystic fibrosis,
pancreatitis
Transgenic maize
Hepatitis B virus
surface antigen
Vaccine Hepatitis B
Transgenic potato
Transgenic lettuce
Human intrinsic
factor
Dietary
Vitamin B12
deficiency
Transgenic Arabidopsis
Lactoferrin Dietary
Gastrointestinal
infection
Transgenic maize
Future Prospects and Biosafety Challenges
Plant Molecular Farming (PMF), using genetically engineered plants as platforms for production of
recombinant pharmaceutical or industrial compounds, offers attractive perspectives to
produce recombinant pharmaceuticals or industrially important proteins on a large scale at low costs. The
feasibility of precise plant genetic manipulation, high-scale expression of recombinant proteins, rapid and easy
scaling up, convenient storage of raw material and less concern of contamination with human or animal
pathogens during downstream processing has attracted biotechnologists to PMF, especially plastid and
chloroplast engineering for this purpose. During the last two decades a diverse upstream (production) and
downstream (purification) technologies, such as table nuclear transformation, stable plastid transformation,
plant cell-suspension cultures and transient expression systems (Agro infiltration method, gene gun technology,
virus infection method and magnification technology)were developed in PMF, and thousands of plant-derived
biopharmaceutical proteins including antibodies, vaccines, human blood products, hormones and growth
regulators were produced at laboratory and pilot levels, and some of them reached the late stages of commercial
and are expected to be marketed soon. Also some of them, such as Caro RX previously have been
commercialized. After about two decades production of recombinant proteins in plants, only recently the focus
has shifted away from technical and principle studies to a serious consideration of the requirements for
sustainable productivity and the biosafety regulatory approval of pharmaceutical products. The manufacturing

19. and clinical development of the plant-derived pharmaceutical proteins fall under the same safety and good
manufacturing practice (GMP) regulations covering drugs from all other sources. Only recombinant proteins
produced by plant cell suspensions in the bioreactor systems may practically observe the GMP guidelines, so
for other plant systems are needed to improve new GMP and biosafety standards and regulations. Plants genetic
engineering is a new departure from conventional breeding to modern technology, so it raises some safety
concerns. Genetically modified plants are generally evaluated critically to ensure that they do not possess any
harmful characteristics for environment and human health before field trials or commercialization and release.
This risk assessment is a fascinating and challenging work involving many disciplines such as ecology,
agronomy, and molecular biology which mainly focus on food and environmental safety. The objective of risk
assessment is to identify and evaluate on a case-by-case basis potential adverse effects of a GM plant on the
environment(s) and human health. Through this approach, the GM plant is compared with its non-GM parent
(substantial equivalence) having safe use history and familiarity for the environment, in order to identify
differences. Risk assessment is performed principally according to the following steps, including problem
formulation and hazard identification, hazard characterization, exposure assessment, risk characterization,
identification of risk management and communication strategies, and finally, overall risk evaluation and
conclusions. The risk assessment finally leads to a conclusion as to whether the overall health and
environmental impact of the GM plant can be accepted or not. Similar to all genetically modified plants, those
intended for molecular farming must go through a complete risk assessment before they can be used in the field.
However, in addition to the risk assessment framework of GM plants used as food/feed or processing (FFPs),
PMF raises new questions and concerns that might trigger a need for specific biosafety considerations due to the
nature of the used recombinant genes. The public concern about the potential health and environmental risks
associated with the transgenic plants used as molecular farming sources, include the possible risks of very high
concentration of recombinant proteins on the morphology and physiology of host plants, possible physiological
responses in humans and in animals caused by the plant biologically active products, economic risks to farmers
and food industry as result of co-mingling and contamination of MF plants with food/feed chain, possible
vertical transgenes flow and spread by pollen, seed or fruit dispersal, unintended effects on nontarget organisms,
particularly birds, insects and soil microorganisms, and horizontal gene transfer by asexual means. The risk of
co-mingling and contamination of transgenic plants used as source of PMF with other agriculturally important
crops could be reduced by use of non-food/feed crops as source of PMF, production of recombinant proteins by
cell suspension cultures in bioreactors, strict physical agronomic confinement and containment strategies for
food/feed crops, post-harvest field monitoring and cleaning, use of late maturing or early maturing cultivars or
planting at different periods to ensure harvesting at different periods from other crops intended for food/feed
and processing (FFPs). Vertical gene flow or gene flow by plant sexual reproduction is the most important form
of transgenes pollution and occurs commonly via the dispersal of transgenic pollen. Plants for molecular
farming should be chosen with the minimum possible gene flow and minimum seed production. The biosafety
strategies to prevent vertical gene flow include the use of closed isolated physical containment facilities
(greenhouses, glasshouses, hydroponics and plant cell suspension cultures), biological containment (self-
pollinating species (cleistogamous lines), chloroplast transformation, cytoplasmic male-sterile transgenic plants,
sexually incompatible crop with wild relatives, non-germinating seeds or non sprouting tubers/bulbs, engineered
parthenocarpy and apomixes, transgenes excision, tissue-specific expression of the transgenes and use of
inducible promoters)(Moshelion & Altman, 2015).

20. Conclusion
Molecular pharming is the collection of techniques applicable in pharmaceutical productions. It adopts different
mechanism like bioprospecting and genetic manipulation for production of pharmaceutical products. It has wide
variety of applications and improved the life style of human beings.
Molecular pharming now serve as factory of drug production, food production in bulk using genetic
modification systems.
 The major outcomes include therapeutic harmones, drugs and antibodies which serve as for medical
purpose.
 Neutraceuticals production favors growth and neutrition ans eliminate risk of disease.
 Edible vaccine facilitates humans in several ways.
 Diagnostic analysis is quite easy by such processes.
 In Short Molecular Pharming and genetic engineering tools turn the impossible thought to possible and
implementable.
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