UTILIZING PLANTS FOR PHARMACEUTICAL PRODUCTION

A TECHNICAL SEMINAR REPORT

Submitted by

DHARMASASTHA V
(113020203001)

in partial fulfilment for the award of the degree of

BACHELOR OF TECHNOLOGY

in

CHEMICAL ENGINEERING

VEL TECH HIGH TECH

Dr. RANGARAJAN Dr. SAKUNTHALA ENGINEERING COLLEGE

An Autonomous Institution

NOVEMBER- 2023

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 VEL TECH HIGH TECH
Dr. RANGARAJAN Dr. SAKUNTHALA ENGINEERING COLLEGE
(An Autonomous Institution)

BONAFIDE CERTIFICATE

Certified that this is the bonafide report of Technical seminar (CH8812) done by
DHARMASASTHA V (113020203001) in “UTILIZING PLANTS FOR
PHARMACEUTICAL PRODUCTION” during the academic year of 2023 –
2024.

SIGNATURE SIGNATURE
STAFF INCHARGE HEAD OF THE DEPARTMENT

Submitted for the University Examination held on ……………. at VEL

TECH HIGH TECH Dr. RANGARAJAN Dr. SAKUNTHALA
ENGINEERING COLLEGE, AVADI, CHENNAI – 600 062.

SIGNATURE OF INTERNAL EXAMINER

II
 ACKNOWLEDGEMENT

I wish to express my sincere thanks to the people who extended their help and
support during my Technical seminar work.
First of all I would like to express my deep gratitude to our beloved and respected
Founder President and Chairman Col. Prof. Dr. Vel. Shri. R. Rangarajan,
B.E(EEE), B.E(Mech), MS(Auto)., Vice Chairman Dr. Sakunthala Rangarajan,
M.B.B.S., Chief Managing Trustee Mrs. R. Mahalakshmi Kishore Kumar, B.E.,
MBA(UK), Ph.D., and our dynamic director Mr. K. V. D. Kishore Kumar,
B.E.,MBA (US).for their kind encouragement and blessings.

My deepest gratitude and thanks to our honourable Principal Dr. E. Kamalanaban,

B.E., M.E., Ph.D., for his continuous support and motivation during this Technical
seminar .

I take this opportunity to thank our Dean, SoBCE Dr. B. Bharathiraja, M. Tech.,
Ph.D., for always encouraging and helping me by giving necessary inputs whenever
needed.

I am thankful and extremely grateful to our HoD Dr. J.B. Veeramalini, M.

Tech.,PhD., Department of Chemical Engineering for her sustained help, guidance
and inspiration in doing Technical seminar.

I would like to express special thanks to our Deputy Dean of PPP Dr. A.
Saravanaraj, M.Tech., Ph.D., for his useful advice and suggestions for my
Technical seminar completion.

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 ABSTRACT

A growing interest in sustainable, alternative pest management approaches has

resulted from growing concerns about the effects of chemical pesticides on the
environment and human health. In order to examine the efficacy of different fruit
seeds as biopesticides in managing common agricultural pests, this study investigates
their potential. Fruit seeds were extracted and their pesticidal qualities assessed.
These included citrus, papaya, and neem seeds. Chemical analysis, field testing, and
bioassays were used in the research technique to evaluate the effects on insect
populations and the safety of the chemicals for non-target organisms. The findings
suggest that fruit seed extracts have the ability to operate as a biopesticide against
various pests and are a sustainable substitute for synthetic pesticides. Insights into the
potential of fruit seeds in integrated pest management systems are gained from this
comparative investigation, which supports sustainable agricultural practices.
Biopesticides are environmentally safe insect management products derived from
naturally occurring materials that do not damage bugs. Biopesticides are less
dangerous for the environment and general public health because they are living
organisms (natural enemies) or products. Biopesticides, which are increasingly being
used in pest management, can be divided into three primary groups: substances
derived from microorganisms and plants, semi-chemicals, and plant-incorporated
protectants (PIPs). Because of their advantages for the environment, target
specificity, efficacy, biodegradability, and use in integrated pest management (IPM)
programmes, biopesticides are growing in popularity. Biopesticides continue to make
up a relatively small percentage of pest management solutions, even with significant
advancements in their market penetration. The approximately 3000 tonnes produced
globally each year are being produced at an accelerated rate.

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 LIST OF CONTENTS

CHAPTER NO TITLE PAGE NO

1 INTRODUCTION 1

2 LITERATURE SURVEY 5

4 TOOLS AND TECHNIQUE FOR PMP 12

EXPRESION

5 NUCLEAR TRANSFORMATION 13
6 HOST PLANT FOR PMP EXPRESSION 16

7 FUTURE PERSPECTIVES 20

8 CONCLUSION 21

9 REFERENCES 23

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 CHAPTER 1

INTRODUCTION

Transgenic proteins known as recombinant therapeutic proteins are created in a

heterologous organism and are utilised in the treatment or prevention of diseases in
humans or animals. Human insulin, which is manufactured in Escherichia coli, was
the first recombinant therapeutic protein to be made accessible for purchase. Since
then, as science has advanced, further medications, vaccines, and cytokines have
been created to both prevent infectious diseases and treat diseases that were once
thought to be incurable. In contrast to conventional medications made by chemical
synthesis, recombinant proteins possess larger and more intricate structures. As an
illustration, aspirin, also known as acetylsalicylic acid, has 21 atoms, whereas a
monoclonal antibody, which is employed as an anticancer drug, has 25,000 atoms
and a complex structure. Given this complexity, systems to produce such
recombinant proteins generally use living cells, including E. coli, yeast, insect, and
mammalian cells.

The kind of medicine being manufactured determines which production system is

used. Whereas eukaryotic production systems are used to produce proteins with
complex structures (like antibodies) and post-translational modifications,
prokaryotic production systems are used to produce proteins with simple structures
and lack post-translational modifications like N-glycosylation. Because bacterial
cells are easy to handle and grow quickly, bacterial expression systems were the
first systems used to create recombinant proteins. These systems are still in use
today. But there are also serious drawbacks to bacterial systems, such as endotoxin
formation and the generation of non-glycosylated proteins. These factors, along
with the fact that the majority of biopharmaceuticals depend on post-translational
1
protein processing to operate, have led to the majority of biopharmaceutical
synthesis taking place in eukaryotic yeast and mammalian cell expression systems.
Similar to bacteria, yeast cells may be produced cheaply and expand swiftly. They
are also capable of producing glycosylated proteins. The existence of the cell wall
makes it tough to disturb yeast cells. The correct folding and post-translational
modification of recombinant proteins can occur in insect cell systems, however
using these systems is costly and time-consuming. Most recombinant protein
production platforms are based on mammalian cells since they are optimised for
human protein production and share the same advantages as insect cells. Such
systems have been approved for the production of a number of drugs. However,
these systems are highly vulnerable to infection by animal pathogens, are difficult
to scale up and require expensive culture conditions.

Plant manufacturing techniques for biopharmaceuticals have been developed to

address these drawbacks. For the most part, plants can be used to produce
recombinant proteins. Transient expression in plants is a quick way to create
recombinant proteins. Furthermore, plant-based protein manufacturing systems are
simple to scale up and resistant to infection by mammalian pathogens. It is
projected that the cost of manufacturing when employing a plant production
system is only 0.1%, while that of using a mammalian cell culture system and a
microbial fermentation system is between 2 and 10%. Additionally, the
recombinant proteins undergo post-translational modification in plants due to their
eukaryotic nature and the simplicity of their growing conditions. But compared to
animal cell systems, the glycosylation mechanism in plants is distinct. While there
are currently few licenced plant-derived recombinant proteins for use as medicinal
agents, biotechnology advancements are helping to solve these issues. Compared
to insect and mammalian cell expression methods, the manufacture of

2
biopharmaceuticals in plants—dubbed "molecular farming," a phrase coined by
Fischer et al.—offers advantages such scalability, rapidity, and enhanced safety.
These characteristics could be useful for the quick manufacture of vaccinations
against pandemic disease outbreaks or biological terrorism, and they are desirable
when resources are few. The US Food and Drug Administration approved the first
vaccine produced in Nicotiana tabacum cell suspension culture expression system
in 2006; this vaccine protects Newcastle disease virus in poultry. In 2012, the first
plant-made pharmaceutical (PMP) for humans was approved: a recombinant
human glucocerebrosidase produced in a carrot (Daucus carota) cell suspension
culture system. This enzyme is used to treat Gaucher’s disease, a hereditary
lysosomal storage disorder caused by a mutation of the β-glucocerebrosidase gene.
The disease is characterized by enlargement of the liver and spleen, fatigue, and
anaemia. Since then, a variety of technologies have been used to produce PMPs,
and demand for these goods is rising. Plant-derived vaccine production technology
has attracted the interest of the US Defence Advanced Research Projects Agency
(DARPA), which has invested in both proof-of-concept operations and the
construction of commercial-scale facilities for the manufacturing of plant-derived
vaccines. Specifically, DARPA disclosed the planning and execution of a plant-
derived vaccine production facility capable of producing tens of millions of doses
using transient expression in Nicotiana Benthamian. After the plants are treated
with the proper vector, these vaccines can be used within a month, demonstrating
how quickly transient expression can be used to initiate the production of "green
vaccines.". By emphasizing how easily the upstream process could be expanded,
the authors demonstrated the suitability of plant-derived vaccine production as a
means to rapidly protect the population against new infectious diseases.

3
We review plant-produced therapeutic proteins in this review. We start by
examining the several kinds of recombinant pharmaceutical proteins that have been
created thus far, such as therapeutic proteins, antibodies, and vaccines. Next, we go
into the technologies and instruments used to produce these proteins in plants, as
well as the different plant systems that can be used for production. In conclusion,
we go over the drawbacks of utilising plant systems for recombinant protein
production in contrast to mammalian, insect, yeast, and E. coli systems, as well as
the present status of research and development targeted at resolving these issues.

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 CHAPTER 2
REFERENCE

1.Title: " Particle bombardment and the genetic enhancement of crops: myths and
realities." Altpeter F, Baisakh N, Beachy R, Bock R, Capell T, Christou P, Daniell H,
Datta K, Datta S, Dix PJ, Fauquet C , 2005
Summary: DNA transfer by particle bombardment makes use of physical processes to
achieve the transformation of crop plants. There is no dependence on bacteria, so the
limitations inherent in organisms such as Agrobacterium tumefaciens do not apply.
The absence of biological constraints, at least until DNA has entered the plant cell,
means that particle bombardment is a versatile and effective transformation method,
not limited by cell type, species or genotype.

2.Title: "Efficacy of a BVDV subunit vaccine produced in alfalfa transgenic plants.

Vet Immunol Immunopathol"
Aguirreburualde MSP, Gómez MC, Ostachuk A, Wolman F, Albanesi G, Pecora A,
Odeon A, Ardila F, Escribano JM, Santos MJD,2013 .
Summary: Bovine viral diarrhea virus (BVDV) is considered an important cause of
economic loss within bovine herds worldwide. In Argentina, only the use of
inactivated vaccines is allowed, however, the efficacy of inactivated BVDV vaccines
is variable due to its low immunogenicity. The use of recombinant subunit vaccines
has been proposed as an alternative to overcome this difficulty. Different studies on
protection against BVDV infection have focused the E2 protein, supporting its
putative use in subunit vaccines.

5
3.Title: ‘’coordinators of immune and inflammatory responses " Arai K-i, Lee F,
Miyajima A, Miyatake S, Arai N, Yokota T 1990
Summary: Proliferation and differentiation of mammalian cells are generally
controlled by extracellular signals. Among these signals are a group of polypeptide
growth factors and differentiation factors such as EGF, NGF, PDGF, and FGF. More
recently, a battery of lymphokines and monokines produced by activated T cells or
macrophages have been added to this catalogue.

4.Title: " Use of plant viruses and virus-like particles for the creation of novel
vaccines."
Balke I, Zeltins A ,2019
Summary: In recent decades, the development of plant virology and genetic
engineering techniques has resulted in the construction of plant virus-based vaccines
for protection against different infectious agents, cancers and autoimmune diseases in
both humans and animals. Interaction studies between plant viruses and mammalian
organisms have suggested that plant viruses and virus-like particles (VLPs) are safe
and noninfectious to humans and animals.

5.Title: " Plant tramsformation: problems and strategies for practical application."
Birch RG (1997)
Summary: Plant transformation is now a core research tool in plant biology and a
practical tool for cultivar improvement. There are verified methods for stable
introduction of novel genes into the nuclear genomes of over 120 diverse plant
species. This review examines the criteria to verify plant transformation; the
biological and practical requirements for transformation systems.

6
6.Title: " N-glycosylation of plant-produced recombinant proteins. "
Bosch D, Castilho A, Loos A, Schots A, Steinkellner H (2013)
Summary: Plants are gaining increasingly acceptance as a production platform for
recombinant proteins. One reason for this is their ability to carry out posttranslational
protein modifications in a similar if not identical way as mammalian cells. The
capability of plants to carry out human-like complex glycosylation is well known.
Moreover, the targeted manipulation of the plant N-glycosylation pathway allows the
production of proteins carrying largely homogeneous, human-type oligosaccharides.

7.Title: " Novel surface display system for proteins on non-genetically modified
gram-positive bacteria. Appl Environ Microbiol "
Bosma T, Kanninga R, Neef J, Audouy SA, van Roosmalen ML, Steen A, Buist G,
Kok J, Kuipers OP, Robillard G ,2006
Summary: A novel display system is described that allows highly efficient
immobilization of heterologous proteins on bacterial surfaces in applications for
which the use of genetically modified bacteria is less desirable.

8.Title: " Animal cell culture and technology."

Butler M ,2003.
Summary: Animal cell culture is an important laboratory technique in the biological
and medical sciences. It has become an essential tool for the study of most
biochemical and physiological processes and the use of large-scale animal cell culture
has become increasingly important to the commercial production of specific
compounds for the pharmaceutical industry.

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9.Title: " Production of recombinant proteins by yeast cells. Biotechnol Adv" Çelik
E, Çalık P ,2012 .

Summary: Yeasts are widely used in production of recombinant proteins of medical

or industrial interest. For each individual product, the most suitable expression
system has to be identified and optimized; both on the genetic and fermentative level,
by taking into account the properties of the product, the organism and the expression
cassette. There is a wide range of important yeast expression hosts.

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 CHAPTER 3
TYPES OF PMPs

High-value-added PMPs produced from plants include vaccines, antibodies, and

Therapeutic proteins.
Vaccines:

Vacccines confer acquired immunity against specific diseases or pathogens in

animals, including humans. Vaccines are similar in structure to the antigen
recognition sites of microbial pathogens that cause disease, but unlike pathogens,
they are not pathogenic. Thus, vaccines activate the immune system, allowing it to
quickly respond to subsequent infections with the same pathogen. The demand for
plant-based vaccines has continuously increasing since the development of a
vaccine against Newcastle virus in 2006 Research on various pathogens is being
actively conducted, and some vaccines are currently being evaluated for safety and
efficacy. In addition to general vaccines, which have attenuated pathogenicity,
other types of vaccines are available, such as those made from inactivated viruses,
recombinant protein subunits, and nucleic acids. In particular, a vaccine made from
virus-like particles (VLPs; a type of recombinant protein subunit) is attracting
attention Microscopic aquatic organisms that can be cultivated for biomass
production due to their high oil or protein content. Recently, two types of plant-
made COVID-19 vaccines were rapidly developed. Medicago's coronavirus VLP
(CoVLP) is a vaccine that mimics the surface structure of the SARS-CoV-2 virus.
Using a transient expression system in N. benthamiana, after the nucleotide

9
sequence of the virus is obtained, a vaccine can be successfully produced within
8 weeks. This process is four- to eightfold faster than the currently used egg .

Antibodies:

Antibodies are substances produced by the immune system in response to

stimulation by antigens. Their basic structure comprises two light chains and two
heavy chains, which form a Y shape through disulfide bonds. The amino acid
sequences of antibodies vary depending on the type of antigen, allowing countless
types of antibodies to be produced. In 1989, the production of single gamma or
kappa immunoglobulin chains in tobacco was made possible for the first time
since then, numerous plant-made antibody therapeutics have been researched and
developed. The plant-produced antibody drug ZMapp was developed by Mapp
Pharmaceutical to treat Ebola virus .In addition, antibodies against cancer, human
immunodeficiency virus, Zika, herpes, and dengue are currently being developed.

Therapeutic proteins:

Cytokines are small glycoproteins involved in a variety of processes in the cell,

including cell proliferation, differentiation, and migration. As cytokines are
components of the human immune system, their most widely known role is to
mediate signal transduction between adjacent or distant cells, causing positive or
negative feedback cascades. The more than 100 cytokines identified to date are
divided into several families, including hematopoietins, interferons, platelet-derived
growth factors, tumor necrosis factors, tumor growth factors, and
chemokines .Cytokines can also be produced in animal cells, but due to the potential
for contamination, efforts are focused on producing these molecules in plant
systems. Using a coat protein (CP)-deficient bamboo mosaic virus vector, the
10
expression level of human interferon-γ was higher compared to that of other viral
vectors in N. benthamiana . Moreover, the efficient removal of the affinity tag after
purification during the production of human interleukin-6 reduced production costs.
The bleeding disorder hemophilia affects a small percentage of the world’s
population, but it imposes a great burden on those who have it. In particular, more
than half of hemophilia patients do not receive adequate treatment. Hemophilia is
caused by a deficiency of factor VIII (FVIII), a blood coagulation factor. Because
FVIII is known to undergo complex post-translational modification, research has
focused on producing a therapeutic agent in eukaryotes. In plants, the FVIII domain
was produced in N. tabacum chloroplasts and bioencapsulated in plant cells up to
0.4 mg/g in fresh leaves . Furthermore, the entire FVIII factor was produced in the
chloroplasts of lettuce (Lactuca sativa) at a level of 852 µg/g . Another blood
coagulation factor, a serine protease a-thrombin precursor (pFIIa) involved in fibrin
formation, was co-expressed in N. benthamiana with Turnip Crinkle Virus coat
protein (TCV-CP), which is known to interfere with post-transcriptional silencing.

11
 CHAPTER 4

Tools and Technique for PMP Expression

Two major methods are used to produce recombinant proteins in plant systems
stable genetic transformation and transient gene expression. The method chosen
depends on the plant species, target genome (nuclear or plastid), and gene
characteristics. Stable genetic transformation involves the stable production of
proteins via the insertion of recombinant genes into the plant cell genomes .This
method has several advantages. In particular, it enables the stable, large-scale
production of recombinant proteins, and allows for the production of multiple
recombinant proteins. However, this method also has disadvantages: it is time-
consuming and must be modified to ensure mass expression. Moreover, in nuclear
transformation, since genes are randomly inserted into the nucleus, unstable
expression or silencing of genes could occur due to positional effects.
Transient gene expression can be used to produce a target protein more rapidly than
stable genetic transformation. There are two major methods for transient gene
expression: Agrobacterium-mediated infiltration and viral vector-based transient
expression. In both cases, stable integration of the transgenic is not required.

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 CHAPTER 5

Nuclear transformation

Among several methods for nuclear transformation, the most widely used nuclear
transformation method is Agrobacterium-mediated plant transformation. In the case
of plants which cannot be transformed with Agrobacterium, polyethylene glycol-
mediated protoplast transformation method or biolistic transformation method can
be used Most recombinant proteins in plants generated to date have been produced
through nuclear transformation, in which a gene of interest (GOI) is stably
integrated into a chromosome in the nucleus of a plant cell. The natural plant
pathogens Agrobacterium tumefaciens and Agrobacterium rhizogenic, which
produce crown gall disease and hairy root disease, respectively, mediate indirect
gene transfer during nuclear transformation. Agrobacterium tumefaciens carries a
tumor-inducing (Ti) plasmid comprising a transfer DNA (T-DNA) region, a
virulence (vir) gene, and an origin of replication..

The T-DNA region contains auxin, cytokinin, and opine biosynthesis genes between
the left border (LB) and right border (RB). Auxin and cytokinin are phytohormones
that increase the size of plant cells and promote division. Opines, such as octopine
and nopaline, are derivatives of various amino acids or sugar phosphates produced
in host plants that serve as nutrients for Agrobacterium after infection. When an
infection begins, the T-DNA region of the Ti-plasmid is transferred to the nucleus of
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 the plant cell and then fuses with the plant genome. At this time, the vir genes are
expressed to facilitate the transfer and integration of the T-DNA. Based on these
mechanisms by which Agrobacterium tumefaciens mediates plant transformation, a
T-DNA binary vector system was developed consisting of two autonomous-
replicating disarmed Ti-plasmids working as helpers for T-DNA processing and
transfer and a T-region-containing binary vector for cloning . The binary vector also
contains multiple cloning sites, origins of replication that operate in both E.
coli and Agrobacterium, and antibiotic selection markers that function in plants
and E. coli. The helper plasmid contains vir genes that promote gene transfer and
integration into plant cells. After Agrobacterium infection of plants, callus
formation occurs, callus differentiation is induced, and shoots and roots are
generated. The resulting transgenic plant thus becomes a host for recombinant
protein production. In many cases, the GOI is expressed under the control of a
constitutive strong promoter such as the cauliflower mosaic virus. In some cases, the
GOI is expressed under the control of a seed-specific promoter, and the target
protein accumulates in mature seeds. The advantage of this feature is that it is
possible to obtain transgenic plants immediately by planting seeds that stably
express the target gene. The protein of interest could also be targeted to subcellular
organelles such as plastids, the endoplasmic reticulum, or the vacuole. There are
also various choices for post-translational modifications, as the proteins are retained
in the endoplasmic reticulum or secreted into the apoplast.

Chloroplast transformation:
In addition to inserting a GOI into the nucleus, the GOI can also be introduced into
chloroplast chromosomes to express the recombinant protein in leaf tissue. The
chloroplast is a membrane-bound organelle whose genetic characteristics closely
resemble those of prokaryotes since chloroplasts are thought to have originated from
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cyanobacteria. Each mature leaf cell contains up to 100 chloroplasts, and each
chloroplast contains ~ 100 copies of chloroplast genomic DNA. Thus, it provides a
high gene copy number and leads to result in high expression of recombinant protein
from integrated GOI in the chloroplast genome. Chloroplast transformation is
achieved by bombarding the leaf with gold particles coated with plastid DNA
fragments containing the GOI and allowing the DNA to be integrated into the plastid
genome via homologous recombination .Homologous recombination allows the GOI
to be inserted into a specific position in the chloroplast genome, and no post-
transcriptional gene silencing has been observed .In addition to bombardment,
polyethylene glycol can be used to insert a GOI into the chloroplast genome. This
simple, efficient method allows the simultaneous transformation of many samples,
and the plant tissue shows a high survival rate, but the success rate is low compared
to other methods. Although transgenes are expressed at high levels in the chloroplast,
a chloroplast-specific promoter is often used for optimization. For example,
the psbA promoter exhibits higher translational activity than other promoters. Codon
optimization of the target gene is also performed using the sequence of psbA to
increase the translation rate .However, plastids do not provide post-translational
modification pathways such as glycosylation, making them unsuitable for the
production

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 CHAPTER 6

Host Plant for PMP expression

Arabidopsis is an excellent model for plant science research due to its short
generation time, high-density growth, and easy transformation.
However, Arabidopsis is not suitable for protein production due to its low biomass.
Higher biomass and protein content of plants is advantageous for recombinant
protein production since the relatively lower content of growth inhibitory factors
and secondary metabolites. Representative plants include tobacco, cereals, legumes,
fruits, and vegetables.

Nicotiana spp:
N. benthamiana and N. tabacum have many desirable characteristics for
the production of recombinant proteins, such as a fast growth rate, high biomass,
and easy acceptance of foreign genes. Thus, the levels of recombinant proteins
produced in tobacco are higher than in other crops. For example, the expression
level of green fluorescent protein in transformed tobacco exceeded 50% of total
soluble proteins when a viral vector was used. Also, because tobacco is not a food or
feed crop, it is free from food- or feed-related contamination problems. Of course,
many tobacco varieties contain high levels of toxic alkaloids, but low-alkaloid
varieties exist that are used to produce pharmaceutical proteins. When
52 Nicotiana varieties were evaluated, N. tabacum cv. I 64 showed the highest
protein concentration, large amount of biomass, and small amount of alkaloids.

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Cereals:
Unlike leaf crops, protein expression in seeds has the advantage that it
can be stored for a long time even at room temperature because proteins are stably
accumulated. In addition, since cereal seeds contain almost no phenolic compounds,
the efficiency of downstream processes such as refining and analysis is high. These
properties allow cereals containing therapeutics or vaccines to be administered
orally with minimal processing. However, it is difficult to develop a suitable
recombinant protein production system in the seeds of cereal plants because it is
time-consuming to obtain seeds.

Legumes:
Legumes such as alfalfa and soybeans have an advantage for protein
production because their ability to fix atmospheric nitrogen lowers the need for
chemical fertilizer. Many studies have been conducted to produce recombinant
proteins in alfalfa because it has a large dry biomass yield per area and can be
harvested up to nine times per year. For example, lactoferrin has been produced in
alfalfa In addition, soybean has a high protein content in seeds (> 40%) as compared
to other crops, which is beneficial for recombinant protein production In addition,
soybeans have an excellent ability to stably store protein. For example, the
recombinant protein remained stable in soybean seeds at room temperature even
after 7 years. However, since soybean seeds contain large amounts of oil, the
efficiency of downstream processes such as refining and analysis is not high.

Fruits and vegetables:

The greatest advantage of using fruits and vegetables for protein
expression is that the tissue can be consumed uncooked, unprocessed, or partially
17
processed. This is very important to produce recombinant proteins such as vaccines,
antibodies, and nutraceuticals. Potato (Solanum tuberosum) represents a major
system for vaccine production; studies have been conducted on vaccine production
for infectious bronchitis virus and hepatitis B virus in potatoes. Tomatoes, bananas,
and strawberries could also be used for recombinant protein production; unlike
potatoes, tomatoes, bananas, and strawberries that produce recombinant protein can
be eaten raw and used as 'edible vaccines'. Tomatoes also are delicious and have a
higher biomass yield than potatoes. A recent study highlighted the possibility of
generating a colorectal cancer vaccine by producing an rGAA-733 antigen in
tomatoes. Bananas are another attractive edible vaccine production platform because
they can be grown in wide areas and are enjoyed by most people. For example,
bananas producing hepatitis B surface antigen and cholera toxin B subunit have
been reported. In addition, strawberries that produce canine interferon-α were
developed in Japan and commercialized in 2014, proving their effectiveness in
preventing periodontal disease in dogs.

Duckweed:
Duckweed is a perennial monocotyledonous plant belonging to the
Lemnaceae that lives on the water surface in rice fields or ponds. A small oval
winter bud from the mother plant sinks into the water in the autumn, stays dormant
in the water in the winter, and rises to the water surface in the spring of the
following year to undergo asexual reproduction. Duckweed grows rapidly and can
double in one day, and its culture and propagation are simple. In addition, up to 45%
of the plant body is made up of protein, and it is relatively resistant to
contamination. Duckweed has several advantages as a PMP platform: First, scale-up
is easy due to the low cost of culture medium for duckweed. Second, since it does
not produce pollen, duckweed is safe from concerns about gene transfer into the
18
ecosystem. Finally, duckweed is edible.
Moss:
Physcomitrium patens (spreading earth moss) can grow rapidly in an
inorganic medium without phytohormones or vitamins. Genetic studies have
revealed that most mosses exist as haploids during the growing season, pointing to
their ease of transformation P. patens shows high stability as a protein production
platform because it undergoes a complex post-translational modification process
during protein expression. This can be an advantage to produce exogenous
recombinant proteins because it allows stable protein production.

19
 CHAPTER 7

FUTURE PERSPECTIVES

Plant expression systems for recombinant protein production have several specific
advantages. However, their application in recombinant protein production must
overcome major barriers such as a lack of regulatory approval. PMPs are currently
used primarily for diagnostic purposes and as veterinary medicines. Although it is
difficult to obtain approval for PMP as a human medical protein, it will be necessary
to secure a basis for approval to produce recombinant human medical protein in the
future. These systems also have some disadvantages, such as plant-specific
glycosylation patterns differing from those in animal cells, low yields for
recombinant protein production, and complex transgenic plant management
practices, but solutions to these problems are possible with the development of new
technologies.
Protein glycosylation is an essential post-translational modification that occurs in all
eukaryotes. The early steps of this process are well conserved in plants, animals, and
yeast, but late N-glycan maturation in the Golgi apparatus varies considerably.
Therefore, it will be important to control the glycosylation of target proteins in
plants. Indeed, Protalix BioTherapeutics increased the therapeutic efficacy of
ELELYSO™ by adding mannose to N-glycan. A recent study demonstrated a
technique for removing xylose and fucose from N-glycan in plant. In the future, we
expect that this technology will be further developed to freely control whether N-
glycan residues are attached to target proteins in plants.

20
 CHAPTER 8

Conclusion

Plant-Made Pharmaceuticals (PMPs) represent a promising and innovative approach

to the production of therapeutic proteins and pharmaceuticals. As we conclude our
discussion on this topic, several key points can be highlighted:

Versatility and Scalability: The use of plants as biofactories offers a versatile

and scalable platform to produce a wide range of pharmaceuticals. Plants can be
engineered to synthesize complex proteins, antibodies, and vaccines efficiently.
Cost-Effectiveness: PMPs have the potential to be more cost-effective compared
to traditional manufacturing methods. The low-cost inputs, scalability, and
reduced infrastructure requirements contribute to the economic viability of plant-
based production.
Rapid Response to Emerging Threats: One of the notable advantages of PMPs
is their ability to provide a rapid response to emerging infectious diseases or
biosecurity threats. The technology allows for quick development and production
of vaccines in response to evolving health challenges.
Safety and Regulatory Considerations: While PMPs offer numerous
advantages, adherence to rigorous safety and regulatory standards is paramount.
Ensuring that plant-derived pharmaceuticals meet the required quality, safety, and
efficacy standards is essential for gaining regulatory approval and public trust.
Environmental Impact: PMPs have the potential to reduce the environmental
impact associated with traditional pharmaceutical production. The use of plants
eliminates the need for large-scale bioreactors and reduces energy consumption,
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 contributing to a more sustainable and eco-friendly approach.
Challenges and Future Developments: Despite the promising aspects of PMPs,
challenges such as downstream processing, glycosylation patterns, and public
perception need to be addressed. Ongoing research and technological
advancements aim to overcome these challenges and further improve the
efficiency and reliability of plant-based pharmaceutical production.
Collaboration and Integration: Successful implementation of PMPs often
involves collaboration between biotechnology companies, academia, and
regulatory bodies. Integrating plant-based production into the broader
pharmaceutical industry requires collaborative efforts to streamline processes and
establish standards.
Global Health Impact: Plant-made pharmaceuticals have the potential to
significantly impact global health by increasing access to essential medicines.
Their cost-effectiveness and rapid production capabilities can be particularly
beneficial for addressing health challenges in resource-limited settings.

In conclusion, Plant-Made Pharmaceuticals represent a dynamic and evolving field

with the potential to revolutionize the pharmaceutical industry. As research and
development continue, PMPs have the capacity to provide cost-effective, sustainable,
and rapid solutions to global health challenges, making them a significant player in
the future of medicine production.

22
 CHAPTER 9
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