Growing into their Own? Plant Molecular Farming and the Pursuit of Biotechnological Sovereignty for Lower and Middle Income Countries.

Ana Christine Zeghibe

Submitted in Partial Fulfillment of the Prerequisite for Honors In Biological Sciences Under the advisement of Martina Koniger, Ph.D.

Wellesley College May 2021

© 2021 Ana Zeghibe Acknowledgements

I am extraordinarily grateful for the support, kindness and guidance of my major and thesis advisor Professor Martina Koniger. The strong plant biology influence on an otherwise biotechnologically and medically oriented thesis was largely inspired by her own expertise in chloroplasts. When coming to this thesis, I knew I wanted to write something that was not simply a research paper, but a synthesis of ideas and observations about science and its interactions with the world beyond the laboratory that I had made over my 4 years at Wellesley. Thank you, as always, for allowing me to develop this unusual project, for keeping me sane through the many pitfalls and trials of writing, and for making me fall in love with science communication and plant biology.

Thank you to my committee member Professor Natali Valdez for supporting and guiding my analyses and thoughts on the societal implications of plant molecular pharming. Professor Valdez's classes and work in Feminist Science and Technology Studies strongly influenced the later chapters of this thesis, helping me to think about the hopeful possibilities and critical realities for humanitarian applications of this unusual biotechnology. I am extremely thankful for her critiques of my argument and writing, her insight into the existing literature on biotechnological sovereignty and her passion for this project's potential.

I am thankful for the guidance and critiques of my other committee members Professor Yui Suzuki and Professor Adam Matthews. Their suggestions for how my work might be able to fit in the extant literature and for the future of my thesis project have been essential to defining the scope of my thesis and how I chose to write it in the end.

Many deep thanks to Professor Yue Hu for being my honors visitor. I will never forget the kindness you showed me throughout my final year.

I am also deeply grateful for the aid and moral support of my loved ones and friends both in the US and abroad in the UK throughout this thesising process. Amidst truly extraordinary global circumstances, the love, cheer, pride, forgiveness and flexibility you have all shown me as I have worked on this project will always be treasured.

Finally, I would like to thank the Department of Biological Sciences at Wellesley College for supporting me in exploring and writing this unconventional piece.

I dedicate this thesis to everyone above. I would not have been able to complete this piece without all of your combined efforts to keep me sane, smiling and well fed. Table of Contents

I. Introduction 1

II. Chapter 1- Methods of Transformation 8

III. Chapter 2- Plant Molecular Pharming and Platform Diversity 33

IV. Chapter 3- Plant Molecular Pharming and Biotechnological

Sovereignty for LMICs 58

V. Chapter 4- Hurdles for Biotechnological Sovereignty Using Plant

Molecular Pharming 83

VI. Conclusion 111

VII. References Cited 116

VIII. Appendix 179 Introduction

For over 40 years, the biopharmaceutical industry has studied and used genetically modified organisms to produce desirable proteins for use in medicines, therapies and biological tests.

Today, such proteins are dominantly produced by a combination of prokaryotes like Escherichia coli , simple eukaryotes like yeast, or complex eukaryotes like mammalian cell lines. Whilst the products of living organisms have been used in medical practice since the dawn of history, it was only at the beginning of the 1980s when genetically modified organisms were first used to produce commercially sold proteins and peptides, with early products including synthetic insulin pharmaceuticals produced in genetically modified E.coli.1,2

However, using bacteria as the first commercially established genetically modified platforms, also suffered from protein production limitations that stemmed from the organisms' prokaryotic biology. Prokaryotic cells do not have the organelles nor perform all the metabolic processes needed to create proteins that originate from eukaryotes. This means that if a prokaryotic cell is genetically modified to produce a eukaryotic protein product, it may not necessarily fold the protein into the correct shape for proper function, nor add important post-translational modifications that influence the protein's stability and activities.3 Furthermore, many bacteria collect eukaryotic protein products in aggregated clumps called inclusion bodies, which require difficult and time consuming processes to extract product from.4 As such, eukaryotic platforms had to be developed to expand the portfolio of proteins and peptides that genetically modified organisms could produce commercially.

1 Numerous types of eukaryotic platform organisms have since been developed to compensate for the drawbacks of prokaryotic platforms. These eukaryotes range from simple unicellular organisms like yeast, to cultures of tissue cells derived from multicellular animals, like Chinese

Hamster Ovary (CHO) or insect ovary cell lines.5 Unlike prokaryotic platforms, these eukaryotic platforms have most of the molecular machinery and metabolic processes necessary for properly producing proteins from most eukaryotic and prokaryotic organisms. However, each type of eukaryotic platform faces its own series of challenges as well. Yeasts struggle with not being able to correctly replicate complex post-translational modifications, like glycan addition, for foreign eukaryotic proteins, which limits the products they are capable of producing.6 Cell lines derived from multicellular organisms, like CHOs and insect cells, also struggle with slower cell multiplication and the extended production time that results.7

Yet, for all of the innovation, improvement and platform diversification over 50 years of genetically modified organism research, all of the above prokaryotic and eukaryotic platforms are grown in fairly similar production facilities which have their own series of drawbacks. Most genetically modified organism production methods that dominate industry remain dependent on enclosed, sterile bioreactor facilities for growth of organisms in culture and product production.

Such facilities are expensive to set up and maintain, demand highly skilled labour to run, require extensive sterility measures to protect the cultures from pathogens, and ultimately limit the number of productive cells to what can fit within the enclosed bioreactor.8,9,10

A relatively new eukaryotic cell based production platform is currently expanding the capabilities and production methods of using genetically modified organisms. Molecular

2 pharming, or plant molecular farming, is the production and recovery of protein or peptide products from a genetically modified platform derived from plants. Unlike a eukaryotic or prokaryotic cell culture, what a molecular pharming platform looks like is far more difficult to define. Molecular pharming platforms include production methods that rely on familiar cultures of plant tissue cells, but that also can prominently involve whole plant organisms grown like agricultural crops11,12. Further complicating molecular pharming's definition are the diversity of species, target tissues, growth environments and transformation methods that the biotechnology can employ, granting genetically modified plant-based production a unique flexibility that few other platforms can match.13-16

Molecular pharming platforms have a specific and unique set of advantages. These advantages include the ability to avoid the expenses and scale limitations of bioreactor facilities through whole-plant based processes, their inherent safety against many dangerous pathogens, and their retention of most benefits of complex eukaryote-based production platforms.

Whole plant platforms, unlike enclosed bioreactor based platforms, only rely on well-established and affordable agricultural techniques for growth.17 Whole plants are also not necessarily limited by scale, with open field production easily scaled up to meet demand through sewing more seeds or planting more plants.18 Even within more enclosed growth environments, like greenhouses or underground vertical farms, meeting multi-tonne scale levels of protein production is entirely possible.19 Furthermore, the costs and maintenance efforts are typically not as extensive for whole plant platforms as those for eukaryotic mammalian cell platforms, their closest functional equivalent in protein production capability. 20-22

3 Plant platforms, whether whole or in cell culture, also require less maintenance than their animal or prokaryotic counterparts because they are less susceptible to pathogens that can harm humans or animals. Plants are not currently known to host human- or animal- infecting pathogens23. In a similar vein, plant-specific pathogens are also generally not harmful to the humans and animals that are exposed to them.24 This ultimately means that, during product extraction, the pathogen removal steps that are essential for prokaryotes, yeasts or animal cell platforms may not be necessary for plant-based platforms. A direct display of this advantage was seen in the production story of Elelyso, a therapeutic enzyme for Gaucher's disease produced using genetically modified carrot cells. When Elelyso was being developed, the most used therapeutic enzyme for Gaucher's was a CHO-produced protein called Cerenzyme. In late 2009, viruses contaminated the CHO cultures in the facilities producing Cerenzyme, which triggered an FDA warning and a shutdown of production. This contamination event led to worldwide medication shortages for patients with Gaucher's disease. Luckily, the FDA gave special permissions for the use of Elelyso to help fill in for the shortages. Production of the plant-produced protein was quickly raised, and the increase in patient demand was addressed. It was this event that factored into Elelyso being FDA approved. By virtue of being a plant platform produced protein, Elelyso would not have been vulnerable to pathogen contamination unlike its competitor Cerenzyme, which is produced in mammalian cells.25,26

Plants also are complex, multicellular eukaryotes that have most of the post-translational machinery and pathways that mammalian cell platforms like CHOs have. This means that their cells generally possess the capability of correctly folding and adding chemical modifications to

4 most proteins they produce.27 As a result, most plant proteins are similarly functional to proteins produced by CHO platforms. By contrast, other industry favoured platforms like bacteria or yeasts do not have the same breadth of protein production capabilities that CHOs or plant platforms do. In addition, plants are also capable of synthesizing and storing products that are impossible to produce efficiently in bacterial, yeast or CHO cell lines.28,29 These include therapeutic anticancer proteins like viscumin, which are otherwise too toxic and complex to produce successfully and efficiently in any other platform type.30

Molecular pharming's sheer flexibility, relative affordability, and support of large scale protein production, amongst its many other advantages, has made the biotechnology incredibly attractive for humanitarian applications. One such potential application is for low and middle income countries (LMICs) seeking to establish their own means of biotechnological production and reduce dependence on wealthier countries for products like pharmaceuticals. Functionally, molecular pharming is likely refined enough for large scale production needs like those indicated by LMICs. Since first proofs of concept were demonstrated in the 1980s, the technology has developed to the point where it has successfully been used in many pharmaceutical and non pharmaceutical contexts for protein manufacture. These include the manufacture of an

FDA-approved drug for Gaucher's disease,the achievement of phase 3 trials for a faster-to-produce flu vaccine, and the active contribution of plant-made biologics to short and long term global efforts against the ongoing COVID-19 crisis.31-33

However, despite all of their successes, molecular pharming platforms are still hardly the mainstream protein production platform that gold-standard industry choices like bacteria, yeasts

5 or mammalian CHO cells are. This is in part due to non-technological issues that have created

some hesitance around the platform from the mainstream pharmaceutical industry. Indeed,

LMICs seeking to uptake molecular pharming technology to produce proteins may also be faced

with similar difficulties that do not have to do with how well the technology works. Molecular

pharming's identity as a genetically modified-plant based biotechnology subjects it to restrictive

legislation for growth, development and export.34 This strict legislation sits alongside widespread skepticism against genetically modified agriculture based technologies from special-interest groups and general members of the public, especially after well-publicized crop contamination incidents in the early 2000s.35 Alongside the difficulties caused by molecular

pharming's intertwinement with genetically modified agriculture, issues like navigating and

negotiating the patents on important tools, methods and platforms that achieve good results can

be especially difficult for LMICs seeking to adopt the technology36.

This piece seeks to demonstrate that the factors that could ultimately harm the uptake and

success of molecular pharming in LMICs are not scientific at all, but rather due to the larger

societal context that the technology occupies. It will demonstrate this firstly by showcasing the

scientific development and success of the molecular pharming technology. In the first chapter,

standard methods of genetic plant transformation are described and evaluated, demonstrating that

they can be used towards long term stable production and short term rapid production of a given

protein product. Following this, the second chapter gives an overview of the sheer breadth of

plant-based platforms and tissues available for transformation, as well as how the field is

overcoming current challenges with protein yield and modifications. The third and fourth

chapters expand beyond the confines of the laboratory and into the real-world context for

6 molecular pharming technology. The third chapter will establish grounds for why LMICs might desire local biotechnological production in the first place, the advantages of pursuing local production and why molecular pharming technology is well suited to filling this need. By contrast, the fourth chapter points to non-technological difficulties that molecular pharming may pose for LMICs that try to adopt it, including legislature, patents and public opinion, and how such difficulties have been confronted in the past by the field.

7 Chapter 1- Methods of Transformation

1.1.- Introduction

When plant based platforms used in plant molecular farming (PMF) are made to produce a foreign protein, two general types of transformation can typically be used to express the necessary gene. The first type of transformation is transgenic expression, an umbrella term for any transformation that permanently adds a foreign, non-naturally occurring, gene to a source of

DNA within an organism. In plants, these DNA sources tend to be within the plant's nucleus

(nuclear genome) or within the plant's plastid genome (plastome)- typically that of the chloroplast1,2. Transgenic plants are those plants that successfully incorporate foreign genes into their nuclear genome or plastome, and thus obtain permanent expression of the gene and its respective protein product. As transgenic plants add foreign genes to their genetic material due to genetic engineering techniques, transgenic plants are also considered to be a type of GM organism. Depending on whether the nuclear genome or plastid genome has taken up the foreign gene, the transgenic plant's acquired gene trait can be expressed in very different ways.

The other type of transformation is transient expression, where a foreign, non-naturally occurring, gene is introduced to and expressed within the host plant's nucleus, but the gene is never incorporated into the nuclear genome. Instead, the plant typically expresses the foreign gene and produces its respective protein over a short period of time before its genetic material degrades, or the gene delivery method eventually kills or is cleared from the plant. As a result, since plants using transient expression have their cellular functions temporarily hijacked for foreign protein production, it is widely debated whether they are true GM organisms like transgenic plants. Both transgenic and transient methods have advantages and constraints

8 dictated by the current biological and technological circumstances each works with. This chapter will describe how each method is carried out in the lab, present the advantages and constraints inherent to each method, and examine how all these prior factors, in sum, influence the current practical applications each method is used for.

1.2.- Transgenic Plants

1.2.1.- Nuclear Transformation

Prior to using any method for transforming a plant, a source of DNA must be designed that will get the plant host to correctly form the desired protein and produce it at high yields. These sources of DNA are called transgene cassettes, and contain various components to ensure that the foreign gene is successfully expressed and that plants expressing the foreign gene are successfully identified. These components typically are the following: a promoter sequence to help initiate the expression of the foreign gene in the right place and circumstances, a termination sequence to indicate the end of the gene sequence, a marker gene to help identify plant tissues that successfully express the cassette, and the foreign gene itself, which encodes the desired trait for expression.

Plants going under nuclear transformation typically are transformed using one of two established methods- the biolistics method or the agrobacterium method. Each method is better for use in certain types of platforms. The biolistics method, where the transgene cassettes are coated onto tiny projectiles that are fired into plant cells, can be used in any plant but is best used in the monocots like the cereals barley (Hordeum vulgare), maize (Zea mays) and rice (Oryza sativa)3,4.

9 Transforming such monocots is advantageous since many can localise protein product to seeds for long term, stable storage5. Agrobacterium methods, where a modified version of the

Agrobacterium tumefaciens bacteria transfers transgene cassettes to plant tissue, are most useful for affordably and easily transforming dicots like the high biomass-producing tobacco (Nicotiana tabacum) and benth (Nicotiana benthamiana)6. However, agrobacterium methods are far more difficult to use efficiently in monocots due to several biological hurdles including being more difficult to regenerate after the transformation process7. Thus, the plant platforms that are preferred by the researchers or companies investigating plant-based production can often help dictate which popular nuclear transformation method is adopted. Below, the precise function of these agrobacterium-based and biolistics-based methods are described in detail.

1.2.1.1.- Agrobacterium Transformation

In nature, the bacterium Agrobacterium tumefaciens causes a tumor-inducing plant disease called crown gall disease. The disease occurs when the agrobacterium is able to enter wounds inflicted on the plant host8. The agrobacterium transfers a large loop of DNA called the Ti-plasmid, or tumor inducing plasmid, to the plant through its wounds. The Ti-plasmid has two important regions of DNA within itself - the transfer DNA (T-DNA) which contains the gene that integrates into the plant's nuclear DNA, and the vir genes, a group of genes which produce proteins that move the T-DNA off the plasmid, get it into the nucleus and help insert it into the nuclear genome9. Flanking DNA sequences, which sandwich the T-DNA, both serve as targets for some vir gene proteins and help scientists identify the genetic material that will get transferred10. Once this integration of the T-DNA into the nuclear DNA is complete, the plant is considered transformed. In the wild, this T-DNA usually codes for the formation of tumor-like swellings

10 called galls and the production of modified amino acids for the agrobacterium to subsist on11. In the lab, when designing a Ti-plasmid to carry a transgene cassette, the Ti-plasmid's tumor inducing properties are removed, and the transgene cassette is put between the T-DNA border regions so it will be inserted in the nuclear DNA. The agrobacterium itself is then put through a process of genetic alteration so it solely carries the altered Ti-plasmid for transfer into a plant host of choice.12 To create a full nuclear transgenic plant, the agrobacterium with the Ti-plasmid containing the transgene cassette are usually incubated with plant fragments for approximately 2 days.13

1.2.1.2.- Biolistics/'Gene gun' Transformation

The biolistics method is a non-biological method for delivering genetic material to a planet. The foreign DNA is delivered through being coated onto tiny metallic particles that are then fired at extremely high speeds into plant cells within fragments of plant tissue14. Little is known about the precise process or mechanism of insertion for biolistic methods, but recent research suggests the foreign DNA is violently integrated, with nuclear genome DNA appearing shattered after a biolistic event occurs. If integration is successful, diverse DNA repair methods to address the damage appear to follow.15 Biolistics is incredibly useful as it is not bound by biological constraints like agrobacterium. It can successfully transform nearly any type of plant and can transmit any DNA regardless of size, sequence or shape16. However, biolistics suffers from requiring expensive equipment to work, from possibly causing cell death from use and from inserting the gene of interest multiple times in the same DNA source in a cell (multi-site insertion)17.

11 Following a typical transformation process by bacterium or gene gun (or most other transformation methods currently existing), the treated plant tissues, which are usually from the leaf or root, then get transferred to culture plates with a selection condition. These culture plates may, for example, contain a strong antibiotic where any plant fragment that did not take up the transgene cassette with a resistance marker gene will die in the culture. Thus, only plants that have successfully integrated the transgene cassette with a resistance-conferring marker gene should survive. The surviving plant tissue fragments, at this point, are then transferred to controlled conditions in nutrient and hormone rich tissue culture. Most plant tissues are totipotent in nature, meaning that their cells can become any of the plant cell and tissue types present in a whole plant. Thus, using the right combination of growth conditions and hormonal factors, a whole transgenic plant can be generated from these transformed tissues. These newly formed transgenic plants are then allowed to mature and produce seed. Often, this first generation of transformed plants then involves another series of evaluation steps to investigate how well the desired trait passes to progeny 18.

1.2.2.- Plastid Transformation

Plastid transformation targets the plastid organelles (generally the chloroplast) for transformation. These organelles have their own separate genomes called plastomes where the

DNA integration takes place. The plastome is a transformation site with a collection of unique and desirable traits. These traits include high levels of gene expression, the plastome's ability to express several genes at once, and easy, specific integration.19,20 However, plastome transformation has a far narrower host range that mostly encompasses dicot platforms from the

Solanaceae family, like the ever-prevalent tobacco and benth.21 Microalgae like C. reinhardtii are

12 also being used in plastid transformation with success, but along with plastid transformation in monocots, transformation methods and products for these platforms are still in proof of concept stages.22

Transformations of the plastome can only occur by homologous recombination, a process where two similar molecules of DNA swap genetic information with one another. As a result, scientists must design gene constructs for plastid transformation differently to those used in nuclear transformation. A plasmid, a small ring-like DNA strand, is typically constructed with two important components- the transgene cassette containing the gene(s) intended for plastid integration, and an antibiotic resistance cassette specifically for plastids. Most importantly, these cassettes are sandwiched between fragments of the plastome called targeting regions. These targeting regions ensure that the cassettes are integrated via homologous recombination at a predetermined location within the plastome. 23

The constructs primarily get delivered to the plastids by biolistic methods after being coated onto gold or tungsten particles. It is currently not fully understood why the constructs are able to specifically integrate in the plastid alone, but empirical evidence from several studies suggests that biolistic methods are generally successful at specifically transforming the plastid24. Unlike nuclear transformation, agrobacterium transformation is difficult to do for plastids since the

Ti-plasmid's vir proteins seem to inherently direct any gene in the T-DNA region to the nucleus.25

Thus, when searching for more affordable options for plastid transformation, other methods are used. One such method includes PEG (polyethylene glycol) mediated transformation, where the plant cell wall is removed and exposed to purified DNA in the presence of PEG. This method is

13 fairly inexpensive to carry out compared to biolistics, but is also fairly difficult and has a far lower transformation efficiency. 26,27

After the plastid transformation event, plant cells that successfully integrate the foreign gene into their plastids typically become heteroplasmic- a state in which only some of their plastids have successfully transformed. The ultimate goal of plastid transformation is to achieve homoplasmy, where all of the many plastids present within a plant cell only carry the transformed plastome.

Homoplasmy is achieved by subjecting the successfully transformed plant cells to several cycles of a selection condition, This usually means placing the cells in a culture environment treated with an antibiotic. This antibiotic helps greatly reduce the number of plastids and plastome copies within the plant cells. 28 The significant reduction in numbers of plastids prompts rapid division of the remaining plastids within the plant cells during cell division. It is thought that plastids carrying the transformed and antibiotic-resistant plastome copies divide at a far faster rate than their non-transformed counterparts.29 As a result, when the plastids are sorted randomly between the newly forming cells in the division, the new cells are more likely to receive a greater proportion of the plastids carrying the transformed plastome. Eventually, the non-transformed plastids are lost through dilution over several rounds of selection and division.

When homoplasmy is finally achieved, the antibiotic resistance marker is then usually excised from the plastome using a wide variety of techniques. These include Cre-LoxP recombination, which targets recombinase enzymes to excise a marker gene flanked by certain DNA sequences, or direct-repeat-mediated excision, where identical DNA sequences around the marker gene can undergo homologous recombination with each other and cut out the marker themselves.30,31

After marker-free transplastomic plant tissue is created, it is then carefully developed and grown

14 into mature plants through tissue culture, in a similar manner to nuclear transformed transgenic plants.

1.2.3.- Advantages of Nuclear and Plastomic Transgenic Platforms.

Nucleus transformed plants have a bevy of advantages that come with their long-standing use within the plant molecular farming community. Such transformations are widely used throughout the field and are effective in a great variety of plant platforms that are grown globally like food crops, tobacco plants and scientific model plants like thale cress (Arabidopsis thaliana)32-34.

Nuclear transformation methods are also easy to carry out, with major techniques like agrobacterium transformation often being affordable as well due to minimal facility and equipment requirements35,36. When such techniques are paired with agricultural scale cultivation of host plants, nucleus transformed plants are capable of meeting large and consistent demand for a protein product. An agricultural scale growing and processing operation of nuclear transformed plants can commonly yield 100-1000kg of final pure protein product per year.37

Nucleus transformed plants also have the ability to pass their foreign gene cassette onto future generations through seeds. This is beneficial, since a plant with a consistent and high yielding gene cassette for protein production can be maintained over a multiple generation lineage, and can be stored as a seed in preservation banks for future use38,39. It also means that meeting fast increases in demand is fairly simple, requiring only that more transgenic seeds or plants are grown to increase production40. Seeds can also be transformed into storage units for the foreign protein. This is done through using seed-specific promoters and amino acid tags to localize the protein to seed tissues and storage organelles41. Such seed storage can stably maintain a protein

15 in dry, room temperature conditions for almost 3 years42. Nuclear transformation methods are also the best established out of all the transformation methods for altering the greatest variety of plant tissues. The variety of transformation methods that bring about nuclear transformation, along with the use of promoters that only express a foreign gene in certain parts of the plant or under certain conditions creates great flexibility in where, when and how a protein can be expressed.43 Such flexibilities allow for the exploitation of different tissue- and species-specific properties, like storage organelles, condition survival or using atmospheric nitrogen. One particularly useful application is that targeting the desired proteins to edible organs like fruits unlocks the possibility of creating orally administered therapeutics and vaccines44. Such orally administered pharmaceuticals have a bevy of theoretical and proven advantages for public health interventions that have been researched to significant extent. Some of the benefits and drawbacks of such oral pharmaceutical systems are discussed in further detail in Chapter 2.

Plastid transformation plants see their greatest advantages in the degree and types of proteins they are capable of expressing. If successfully transformed, plastid transformed plants often express the foreign gene within the transgene cassette at extremely high levels. This extremely high level of expression results from the sheer prevalence of plastids and their respective DNA sources, the plastomes, within plant cells. In tobacco, a common platform for plastid transformation, one leaf cell can contain 100 chloroplasts alone. Within each of these respective chloroplasts are roughly 100 copies of the plastome. This ultimately leads to a total of 10000 plastomes for every tobacco leaf cell.45,46, If all plastids only carry and express the transformed plastomes, very high expression levels usually occur, and thus more protein products can come from less plant resources overall.

16 The nature of the plastid itself as a prokaryote-like self contained organelle also has its own benefits. Control over expression is especially good, since the plastids do not appear to have epigenetic interference nor gene silencing mechanisms that would reduce expression levels.47 In addition, foreign genes inserted into the plastome are particularly non-susceptible to position effects. Position effects, when concerning transgene cassettes, mean that the transgene cassette's level of expression may be influenced by where it integrates into the genetic code. Factors already present in the genetic code, such as nearby promoter regions, can unexpectedly decrease or influence the expression levels of the transgene cassette in unwanted ways. The lack of position effects seen in plastid transformation is in part because transgene cassettes can only be integrated into the plastome by homologous recombination. For homologous recombination to integrate the transgene cassette, the targeting region DNA flanking the transgene cassette must be similar to the DNA in a pre-existing region of the plastome. This means that, as long as the targeting regions remain the same, transgene cassettes will integrate into the same predetermined place in plastid transformation. 48 Additionally useful is the unique potential for the plastomes to simultaneously express several transgenes at once in a process called co-transformation, which could also expand a plant platform's product portfolio to include adjuvants with antigens, antigens with proteins that increase immunogenicity or multivalent vaccines49.

On top of all these genetic advantages, multiple types of containment are provided through using the chloroplast as a site of genetic transformation. The self contained nature of the chloroplast has led to it successfully storing and accumulating some potentially harmful protein types like toxic proteins, cell-wall degrading enzymes and antibiotics 50-52 The use of the chloroplast also

17 provides a form of genetic containment as a plant's plastome (in most species) is transmitted to its egg cells, which do not normally leave an individual plant. By contrast, the plastome is rarely ever passed onto offspring through the sperm produced by a plant's pollen53,54. It is this pollen which is usually moved from one plant to another. This means that the transformed plastome is unlikely to leave the transformed plant through the pollen and ultimately create unwanted offspring with wild plants or crops.

1.2.4.- Disadvantages of Nuclear and Plastomic Transgenic Platforms

Despite the many advantages of nuclear transformation, there are also a significant number of disadvantages associated with this method. Possibly the greatest disadvantage posed by nuclear transformation is the risk of uncontrolled breeding between the transformed plants and other crops or wild plants. Unlike the plastome, which is only typically passed down through the plant's egg cells, the nuclear genome can get passed on through sperm produced by the pollen.

This pollen is able to travel beyond the individual transformed plant and potentially cross-breed with crops or wild plants not meant to receive the protein production trait55. The presence of such a trait in the wild or in food crops might have unexpected and harmful impacts on local wildlife and on food production for both livestock and humans48. Some scenarios might include a crop plant taking on a trait for producing a protein that is toxic to both humans and animals, or a weed plant taking on a protein producing trait that may also make it more resistant to eradication 56,57.

Even seeds produced by nuclear transformed plants could potentially contaminate other seed storage facilities or accidentally be transplanted outside of a controlled growth environment.58,59

The potential for nuclear transformed plants to crossbreed with other plants via pollen has far reaching effects. Controlling this breeding has dictated best practices for cultivating transformed

18 plants, has influenced laws around the development and growth of transformed plants and has impacted public perception of plant molecular farming technology. Such issues are discussed in greater detail in the context of Chapter 4.

Beyond this pertinent issue, nuclear transformed plants also have some production-related drawbacks. One such drawback is that of position effects when the transgene cassette is introduced to the genome. The agrobacterium and biolistics based methods used to generate nuclear transformed plants usually involve the random integration of the transgene cassette into various different areas of the nuclear genome.60 This random integration alone means that the same transgene cassette can be expressed very differently depending on the genetic environment it ends up being surrounded by. For example, some nearby genes could repress the expression of the transgene cassette or a nearby enhancer region might affect how well the gene can get expressed61,62. However, even if an ideal position was identified for the transgene cassette and precisely inserted into using a gene-editing tool like CRISPR-Cas9, epigenetic factors, like RNA and DNA structures involved in transgenic silencing, would still pose a problem to achieving high levels of expression63. Often, yields in plants that have undergone nuclear transformation are lower than those seen when using other methods like plastid transformation and transient expression.

Despite this, yields can and have been increased with additional genetic modifications to the

DNA constructs designed for insertion. Such modifications may affect different aspects of the protein production pathway- often targeting transcription, where DNA is converted to immature mRNA that is then matured through modification and editing, and translation, where the mature

19 mRNA provides instructions in a series of codons (3-member base-pair groups) that lead to the creation of a chain of amino acids that will form the final protein. Construct modifications might include using a different promoter that results in higher transcription of the transgenic cassette genes, altering base pairs in the transgenic cassette to accommodate a plant's codon bias- making translation more efficient, or modifying an untranslated region upstream from the transgenic cassette, (which alone, can account for up to a 200 x difference in translation levels)64,65.Even with these modifications in mind, creating successful and productive nuclear transformed plants for industrial use remains a lengthy process that requires numerous trial-and-error experiments.

Plastid transformed plants have their own series of disadvantages that come with producing proteins within the plastid organelle. The chloroplast (the main plastid often transformed) is capable of most post-translational modifications, which are the additional modifications and folding steps that come after the amino-acid chain is produced from translation. However, the chloroplast does not have the biological machinery to carry out a critical type of post-translational modification called glycosylation, which helps dictate the structure, function and stability of many useful proteins. Thus, scientists cannot create glycosylation-requiring proteins within the plastid without either significantly modifying the plastid itself or carrying out the glycosylation in separate steps outside of the plant66,67. Additionally, the choice of plastid matters to plastid transformation. Whilst chloroplasts (green plastids) are the primary plastid of choice and do express transgenic cassettes well when transformed, other plastid types are far less effective at expressing the cassettes when they are inserted into their own plastomes.68,69 The biological containment advantage that comes from the plastome not being transmitted through pollen is also not absolute. In a highly unlikely event, the plastome can enter the pollen grain and

20 potentially be carried away and passed onto offspring70-72. In the strict growth circumstances for

transformed plants producing pharmaceutical proteins, where no cross-breeding is desirable,

even this low chance can be too high. Thus, even plastid transformed plants must still undergo

other severe containment measures like geographic isolation or indoor growth.

Plastid transformed plants, much like nuclear transformed plants, also require a long time to

create. The time-consuming generation process for whole plastid transformed plants is in large

part due to the need to achieve homoplasmy. Homoplasmy is not achieved immediately for

newly transformed plant cells, but is gradually obtained over several long cycles of selection,

which draws out the process of development73. Other technological limitations, like the lack of efficient ways to identify homoplasmic cells in most plant species, also add to the length of the process74. On top of the production time disadvantage, successful protocols for plastid transformation mostly exist for a few dicot plants of the Solanacea family and some microalgae,

compared to the breadth of species that can successfully undergo nuclear transformation75,76.

Monocot plastid transformation, which could help modify plants like cereals, is still in a proof of concept stage77. This is mainly for two reasons: monocot cells do not respond well to existing methods for regenerating transformed plants and monocots tend to be resistant to the antibiotics used in the chloroplast selection cycles, which makes it more difficult to tell whether the plant cells were successfully transformed78-80.

1.2.5. - Transgenic Expression: How Properties and Constraints Translate to Use

All transgenic plants take substantial time to generate, usually requiring around half a year or more to develop a plant that expresses a transgenic cassette homoplasmically in the nucleus or

21 plastid.81,82 Thus, they are not typically suitable for immediate and urgent health interventions like disease outbreaks or bioterrorism events. Instead, many transgenics are good for longer term, consistent manufacture of necessary protein products that could serve as the backbone of protein product production. In the case of nuclear transformation, its diversity of potential plant platform species, its potential for accommodating large scale production, and its demonstrated ability to store and produce protein within several different tissue types means that it is a great choice for products needed reliably at large scale. Nuclear transformation's flexibility also opens up avenues for plant pharmaceutical products like oral therapeutics or topicals, which require fewer costly processing steps in manufacture to be deemed safe for use. Plastid transgenics, whilst more limited in the choice of plant platform than nuclear transformation, could also see application in edible vaccine production and seed storage. However, this form of transgenic modification is probably best poised to take advantage of niche protein-based products, where toxicity, danger to a plant's cellular structure or a need to produce multiple proteins simultaneously could all otherwise make plant-based production difficult. Thus, plastid transgenics could help expand possibilities for proteins that can be explored for future therapeutics or vaccines.

1.3.- Transient Expression

1.3.1. - Platforms and Vectors

Transient expression is typically done in dicots like members of the Nicotiana family like benth and tobacco83. This is primarily because the vectors used in transient expression are best adapted

22 for dicot use. Monocot specific techniques and vectors for transient expression have been trialled

as well with some success, but are far less efficient than dicot specific techniques84.

Two vector types have shaped the history and growth of transient expression based plant transformation- the plant infecting bacterium Agrobacterium tumefaciens and plant viruses, which have a great variety of infection hosts and strategies for replication85. Agrobacterium in transient expression is used fairly similarly to agrobacterium in nuclear transformation. A

Ti-Plasmid vector is designed with a transgene cassette where the T-DNA would normally sit, and the genetically modified agrobacterium carrying the vector Ti-Plasmid is introduced to a plant's leaf cells for infection.

This introduction can happen through many means, but popular methods include: vacuum infiltration, where plants in agrobacterium solution are exposed to a vacuum chamber that forces bacteria into the leaves via pores called stomata; syringe infiltration, where a needleless syringe containing agrobacterium solution is injected against the underside of the leaves to enter the stomata; or "wound and agrospray", where plants are wounded in the leaves then sprayed with agrobacterium solution86,87,88. Whilst most transient expression work with agrobacterium is done within plant leaves, roots can also receive vectors from Agrobacterium tumefaciens when cultured as cells89. T-DNA transfer in transient expression works identically to T-DNA in nuclear transformation, with the T-DNA processed and ferried into the nucleus. However, transient expression systems also have special differences in the Ti-Plasmid. These differences usually are alternate gene forms for some vir family genes, which ultimately prevent the

T-DNA's integration into the nuclear DNA90. As a result, the T-DNA temporarily exists on its

23 own within the nucleus over a time period that spans a week at most, until it degrades due to

cellular or other environmental factors.91

Viral vectors deliver the gene cassette to the plant as a part of their viral genome, which is made

of either one of the two nucleic acids: DNA or RNA. In the wild, viruses are only able to

replicate themselves through hijacking a host cell, since they do not inherently have the chemical

processes nor the nucleic acid replication machinery that cells do. The hijacking process

involved attaching to specific proteins on the cell surface, and releasing the viral genome into the

cell. The viral genome forces the cell to replicate the genome and to produce useful proteins for

the virus at very high levels. Such useful proteins might include parts of a protective shell for

the genome or receptor binding proteins. All of the protein and nucleic acid components are then

packaged together to make new viruses, which then leave the cell. The virus-releasing event

usually causes the cell to die: either immediately due to extensive damage or after some time due

to negative impacts left on cell processes like reproduction92.93. In plant molecular farming, this

ability of an infectious viral genome to hijack a plant cell's protein production is repurposed for

producing proteins instructed for in a gene cassette. The viral-vector gene cassette can be

presented to plants in many different forms that include: full viruses that also carry the gene

cassette within their genomes, viral genomes as nucleic acids divorced from their protein

containers or deconstructed viral genomes with some viral functions removed94,95,96. Viral vectors used for transient expression in plants tend to be derived from plant viruses that encode their genome in RNA. This means that a transgenic cassette must be converted from DNA, typically as complementary DNA (cDNA), into RNA form to be inserted into the viral genome.

Families of virus typically used for transient expression include the RNA virus families of the

24 tobamoviruses, potexviruses, potyviruses, bromoviruses and comoviruses, alongside the geminivirus DNA virus family.97,98 Each family has characteristic advantages and disadvantages for host range, expression, or insert size. For example, the geminivirus systems can handle larger foreign gene inserts, but expression of the gene peaks early before silencing or necrosis of the leaf99. Tobamoviruses and potexviruses, by contrast, can induce very high levels of expression, but their reliance on RNA replication makes it more difficult to express large proteins or several proteins simultaneously99. As such, selecting a specific virus family to be a vector requires the consideration of the protein product desired and the plant platform used.

In the past, both viral and agrobacterium-based expression systems were seen as rival platforms with complementary strengths and weaknesses to one another. Viruses, on their own, were extremely capable at achieving high levels of protein expression in the cells they infected.

However, virus-only platforms long relied on mechanical inoculation methods, in which wounded leaves are brought in contact with a virus-containing suspension.101 Reliance on this mechanical inoculation method placed significant restrictions on a viral vector's capabilities. The method restricted usable vector candidates to a smaller subset of viruses that could be transmitted mechanically and could only typically infect a few cells within the inoculated plant leaf. The functional demands of mechanical inoculation on viral vectors also placed size constraints on the transgene cassettes they could carry. Since the virus still needed to retain its infection-related genetic functions to transmit the transgene cassette from cell to cell, larger transgenes were generally impossible to fit into viral vector-based systems102. The retention of infectious functions in order for the modified viral vectors to work also increased the risk of creating new, potentially transmissible viral particles103. Agrobacterium, on its own, was extremely capable of

25 getting plants to express a foreign gene quickly and efficiently. Standard agroinfiltration

methods of getting agrobacterium into the plant leaf, like vacuum infiltration or syringe

infiltration, flooded the intercellular space of the leaf and could reach most of the cells104,105. As such, agrobacterium methods had generally high success in infecting the majority of the leaf.

A revolution thus occurred in the field when combined agrobacterium-viral methods appeared. In such combined methods, the plant leaf is infiltrated with agrobacteria containing the DNA codes of edited viral genomes for plant viruses within their T-DNA106,107,. These combined methods are highly efficient at delivering genetic material to most of the leaf and obtaining high expression levels, combining both of the benefits of agrobacteria and viruses. As a result, this technology is now widely used for efficient transient expression. The technology also had the effect of making viral vectors far more flexible. Since the agrobacterium and its T-DNA essentially took on the role of infecting the plant cell and of entering the nucleus for expression respectively, viral constructs no longer needed genes that would help them spread amongst cells. This gave rise to the deconstructed virus where viral constructs have all genes removed that are not strictly involved with the foreign protein production process. For example, removed genes usually include CP genes, which produce proteins that protect the virus's genome in a shell structure and help it move into a cell through the cell membrane. This 'un-necessary' gene removal freed up space for replacement with the transgene cassette(s) of interest, allowing for larger gene sequences and opening up the possibility of producing larger proteins108.

1.3.2. - The Transient Expression Process

26 Any plant undergoing transient expression enters two distinct process phases, one before the initiation of transient expression with agrobacterium (pre-inoculation) and one after the plant has undergone the transformation (postinoculation)109. Both phases require consideration because the conditions endured under each phase ultimately can dictate how much protein is obtained from the leaves in the final yield event. Ideal pre-inoculation conditions prioritize plant growth- as how many leaves the plant grows and how much biomass it develops may affect final protein content and the plant's productivity110. Pre-inoculation traits are not as well researched, but preliminary studies point to protein yield-affecting factors such as leaf to stem ratios, planting density, and light conditions. Leaf to stem ratios are important, because,unlike the cells of the leaves, the cells of the stem do not get infected by the agrobacterium carrying a viral vector111.

Thus, efforts are made in the growing process to maximise the number of leaves the host plant produces. One way this is done is through managing planting density- lower planting densities are favoured since they push leaf to stem ratios in favour of leaf growth, allowing for the development of more leaf tissue and, in turn, more cells to successfully infect112. Another factor that pushes the leaf to stem ratio to produce more leaf is light conditions. Light conditions that are closer to red in the visible light spectrum also seem to encourage the growth of more leaves113. Despite these early findings, more research is needed in this area since few studies have been devoted to extensively examining preinoculation conditions.

By contrast, ideal postinoculation conditions are considerably more well understood. The goal of postinoculation is very different to that of preinoculation. Instead of prioritizing plant growth, the primary goal is now to maximise protein production and yield as much protein as possible.

Several environmental factors have been studied for postinoculation in transient expression,

27 which include temperature, light intensity and humidity. In several studies, temperatures between

15-25 C, with specific peaks depending on species and protein type, have seemed to coincide

with protein expression peaks. Any deviation of temperature equal or greater to 5C above or

below this range causing a significant drop in yield114. This yield sensitivity to temperature change is speculated to be related to T-DNA transfer efficiency and the plant host's own stress response. Too high or low temperatures make T-DNA transfer less effective, and higher temperatures seem to induce severe necrosis associated with stress in the plant.115,116 In many protein production facilities, static temperatures are thus maintained. However, there is some suggestion that fluctuating temperatures throughout different stages of the inoculation process may encourage greater yields.117 Potentially, slightly cooler temperatures could be used to encourage T-DNA transfer within the first day of inoculation, followed by an appropriate warmer temperature to encourage protein accumulation afterwards. Investigations have found that light intensity has had variable effects on protein accumulation- some studies have found that light was a significant factor for increasing recombinant protein yield, but others did not find significant difference in recombinant protein accumulated in plants that were or were not exposed to light118-121. Although not many strong conclusions could be drawn from the extant literature since so many proteins and platforms have been examined in these studies, light exposure and intensity does not appear to have a negative effect on protein yield and is obviously a positive for fundamental plant survival and growth. Humidity as a factor has only been lightly investigated in the literature, but some papers suggest that controlling this factor in post-inoculation circumstances is very important for transient expression, with the removal of the agrobacterium suspension after inoculation dramatically increasing yield122. Other factors, such

28 as nitrate-richness of the plant solutions also seem to have some effect on obtaining a high

yield123.

1.3.3.- Advantages and Disadvantages of Transient Expression Systems

Transient expression systems have several unique advantages that separate them from other

transgenic plant platforms. Perhaps the most famous advantage of transient expression is the

sheer speed with which it can express a foreign gene and produce a final protein product.

Expression can be detected within the span of 3-4 days within transformed plants, and only

weeks are needed to move from the expression of a gene cassette to a pure, testable protein

product 124,125. Popular and effective methods for transient expression, like combined agrobacterium/viral systems, are often very simple and cheap to carry out. This is because protocols for these methods typically require relatively inexpensive experimental supplies and equipment like syringes or vacuum chambers126,127. Additionally, expression and yields tend to be

high in transient expression plants for several reasons. The agrobacterium vector is able to reach

most cells in leaves treated using agroinfiltration methods, and the foreign viral DNA brought to

the nucleus never integrates into the nuclear genome, meaning it is not vulnerable to being

silenced by other genes or epigenetic factors128,129. Anti-pathogen silencing responses from the

plant can impact the yield of a transient expression event negatively if too much transcription

occurs for the foreign transgene cassette. However, effective defenses against this silencing do

exist. In many cases, this response is easily countered with a silencing suppressor gene

accompanying the transgenic cassette that stops it in its tracks130. Most strikingly, the risks of

unwanted cross-breeding or movement of transformed plant material outside of containment are

fairly small for transient expression. Since the plant undergoing transient expression never has

29 the transgene cassette enter its genome or plastome permanently, the plant itself is not technically a GM organism. Instead, the plant has its cellular functions temporarily hijacked for protein production over a short period of time.131 In addition to this, plants used in transient expression are usually single use platforms for protein production, since the plants may die after the protein is transiently expressed, and their reproductive tissues (like the seeds or pollen) are not preferred targets in production132,133. The compounded result of all these factors means that the risk of genetic material escaping into the environment or crops via the plants is very small.

Despite this, transient expression systems have their own series of disadvantages. One of the main disadvantages is that combined agrobacterium-virus vectors are not able to effectively infect most monocots. Though the general host range for transient expression is wider than that for plastid transformation, transient expression in monocot plants remains difficult. This is because Agrobacterium tumefaciens does not usually infect monocots in nature, so is not adapted to overcome differences that could impact infection success, such as cell wall structure or a lack of phenolic compounds that help induce the Ti-plasmid transfer process134. Biosafety concerns around GM organisms are also not completely removed, since the viral genomes and agrobacterium used as vectors have the potential to spread GM material themselves135. Also of concern is the single use nature of many transient expression systems. Since the foreign gene never integrates into the plant's genome, no long term seed bank can be generated for a transiently expressed protein.136 This means that a series of transiently transformed plants with reliable, high protein yield is a once-only production event. Nor can consistency of output be relied on- an unknown number of repeated cycles of expression, each time with a new batch of plants, may be required to achieve a certain protein yield, versus the consistent and trackable

30 outputs of transient crops. Yields in transient expression also appear to be especially sensitive to environmental fluctuations137. This may mean that best results can only really be achieved within a more expensive to maintain indoors environment, like a greenhouse or vertical farm, where such external factors can be controlled.

1.3.4.- Transient Expression: How Properties and Constraints Translate to Use

Though plants made to transiently express a foreign gene are only doing so in a single protein yielding event, that single event is also extremely fast to induce, high yielding, and maintains detectable expression for about a week. This means that transient expression is uniquely good in situations where proofs of concept for a pharmaceutical can be developed quickly, or an urgently needed therapeutic is required, like in a breakout disease event or bioterrorist attack138.

The pharmaceutical products that transient expression typically yields are also likely to be more purified. Whilst transient expression is possible in many plant platforms, this expression is primarily done at scale using benth or tobacco plants. Benth and tobacco contain numerous toxic chemicals within them, including endotoxins, polyphenols and alkaloids, which require many processing and purification steps to separate away from the desired protein product139-141. Even if transient expression is done in a plant that is not benth or tobacco, the use of GM pathogen-based vectors like agrobacterium and viruses may also still impose a need for more extensive purification and processing.142 Whilst nuclear transformed platforms also use agrobacterium for transformation, the plants that ultimately undergo processing for pharmaceutical production tend to be descendants of the original transformed plants, so do not carry any agrobacterium that might need to be separated out. In the case of transient expression, however, the transformed

31 plants exposed to the agrobacterium are the plants that get processed for pharmaceutical production. The situation of transient expression would certainly demand more purification to separate out the bacterium from the plant material. Ultimately, this may mean that the purification-reducing benefits of producing less refined topical or oral pharmaceuticals are not as easy to achieve with transient expression methods.

32 Chapter 2- Plant Molecular Pharming and Platform Diversity

2.1.- Introduction

Pharming, as a field, has generally convened around two main platform types. These include bioreactor based platforms, where the platform is mainly grown within a contained bioreactor environment, and whole-plant based platforms, where the platform is grown as a full plant in fields or in contained greenhouses and indoor farms.

2.2.- Bioreactor-Based Plant Platforms

Bioreactor based plant platforms use facilities and technologies that are similar to those used for the cultivation of established bacteria, yeasts and CHO platforms. Despite the superficial similarities in growth and processing to these gold-standard platforms, bioreactor-based plant platforms have similar advantages to their whole plant counterparts within the constraints of the bioreactor. Much like whole plant platforms, bioreactor-based plant platforms generally tend to be more affordable to cultivate and have a great variety of platform types to choose from.

Platform types include plant cell suspension cultures, hairy root cell cultures and even fully intact photosynthetic organisms like aquatic plants, mosses or algae1,2.

2.2.1.- Cell Suspension Culture

Plant cell suspension culture involves the collection of small groups of plant cells from plant tissue, then their culturing and growth using plant hormones into calluses, which are clumps of unorganized structural tissue. The calluses are then genetically transformed to produce a specific

33 protein product, are disaggregated in shake flasks, and then are finally placed into a sterile liquid medium for cultivation and product collection within the bioreactor 3. Calluses can technically be generated from any somatic (non-reproductive) cell in a given plant, since most plant cells can transform into any other plant cell. However, the cells that are ultimately chosen for creating the calluses are determined by external factors like the original plant's species or the inherent biological properties of the cells4,5. Despite this, many commercial plant cell suspension platforms still represent a wide range of species including everything from rice, alfalfa, tomatoes and even carrots6-9. Possibly the most well used of these platforms in cell suspension culture are two cell lines known as BY2 and NT1, which derive from tobacco plant tissues10,11. A significant advantage of cell suspension culture is that the protein product produced by the cells can be targeted for direct secretion into the culture medium. This direct secretion tends to make separating and purifying the final product easier and cheaper12-14. Whether direct secretion is possible for a protein, however, is highly dependent on its size. If a protein is too big, its secretion will be slowed or outright hindered since it will struggle to pass through the pores of the cell wall surrounding the plant cell's membrane15.

2.2.2.- Hairy Root Cell Culture

Hairy root cell culture is created through infecting sterile, and often juvenile, plant material with the bacterium Rhizobium rhizogenes16,17. The R. rhizogenes bacterium works fairly similarly to agrobacterium in the wild. Through the wound, it transfers a root inducing plasmid that carries

T-DNA which will be integrated in the plant's genome and expressed. The expression of the

T-DNA leads to the development of hairy root-like structures on the plant tissue.

34 Whilst R. rhizogenes typically targets plant roots in the wild, most plant material can successfully develop hairy root structures if infected with the bacteria. To induce hairy roots, the plant material is wounded and exposed to R. rhizogenes via inoculation, and then is transferred to a medium containing antibiotics to kill the remaining bacteria. Within a span of time that can range from over a week to a month, the hairy root structures appear on the plant material. These hairy root structures can then be transformed to produce a protein product and grown on their own as a tissue culture in medium. Hairy root structures are different to a plant's normal adventitious roots since they do not require phytohormones to grow stably, indefinitely, and quickly in culture18. Hairy root cell cultures have several platform-specific advantages: they are very easy to scale up within the bioreactor environment, they stably integrate and maintain foreign gene cassettes, and they are also fully capable of secreting proteins into their growth medium19,20.

2.2.3.- Duckweeds and Microalgae

Certain types of plants and plant-like organisms can also be grown within the bioreactor environment. These include mosses, aquatic plants like duckweeds (typically Lemna minor) or even eukaryotic microalgae (generally Chlamydomonas reinhardtii)21-23. These organisms are suitable for orally administered therapeutics since they are regarded as safe for human consumption, are fast growing, are typically easy to maintain and harvest protein from, and produce high protein content. Microalgae like C. reinhardtii have been particularly well studied, combining the fast growth and easy to culture natures of many bacterial culture platforms with the post translational machinery and photosynthetic abilities of all plant-like platforms24.

35 C.reinhardtii is able to use light as efficiently as whole plant systems and accumulate biomass quickly25. The protein products obtained from this algal system are also generally cheaper to purify since they can be separated out using similar processes to those used for bacteria or yeast.

Its single plastid is fairly easy to genetically transform, and stable transgenic lines can be produced within 1-1.5 months26.

2.2.4.- Mosses

Moss (usually Physcomitrella patens) can be maintained easily in a juvenile stage when exposed to certain pHs or ammonium tartrate, which is the life cycle stage in which the organism is most responsive to genetic transformation.27 P. patens only requires light and carbon dioxide as sources of energy, and can be cultured in a simple inorganic salt medium.28,29 This means that cultivating the moss is relatively inexpensive and that protein product recovery from the medium is a fairly simple process, since the water and salts are not likely to interfere with the protein. P. patens also tends to stably incorporate genetic modifications, and, uniquely among plants, can precisely target any new gene construct to specific parts of its nuclear genome using homologous recombination30,31.

2.2.5.- Benefits of Bioreactor-Based Plant Platforms

The bioreactor environment is particularly beneficial for condition control. It is mostly indoors and the cells or plants within are provided with nutrients and maintained in a stable, controlled environment. As a result, platforms raised in these bioreactors tend to have more consistent outputs of a protein product, so conform to standard regulations and good manufacturing processes more easily. Both this consistency and the similarities of these platforms to

36 gold-standard CHO, yeast and bacterial platforms, ultimately means that protein products from bioreactor based pharming are more likely to be approved by regulatory authorities32. It is partially for this reason that Elelyso was the first FDA approved pharming product, as it derived from a carrot-based cell suspension line.33 Biological containment issues for the genetically transformed plant platforms are also less of a concern within an indoors environment. Beyond regulation, many of the bioreactor based platforms tend to grow rapidly compared to their whole plant counterparts, have generally uniform cell size and forms which leads to greater product consistency, and have the potential to secrete the protein into the medium, which makes downstream processing far less expensive. Even when comparing plant platforms in bioreactors to other standard bioreactor platforms like CHOs, bioreactor-based plant platforms tend to have cheaper mediums than these other standard methods34. Despite the many advantages of the numerous bioreactor based plant platforms, this category of pharming platforms also loses two of the great advantages of using whole plant platforms. These two advantages include the cost benefits of not relying on bioreactor facilities, which have upfront construction and maintenance costs on the same scale as CHOs, bacteria and yeasts, and the ability to scale up production without limits, which cannot be done within the closed, fixed volume environment of the bioreactor35,36.

2.2.6.- Disadvantages of Bioreactor-Based Plant Platforms

Many of the bioreactor based platforms are also further restricted by platform-specific disadvantages. Plant suspension cells tend to be slower growing compared to microbe platforms, and fewer productive cells can fit within the bioreactor compared to CHOs. This is because plant cells tend to be larger than CHOs, due to having large vacuoles that take up space. Whilst the

37 vacuoles can be used as protein storage sites if secretion is difficult, they do not contribute to increasing protein production and can take up valuable fermenter space.37

Hairy root cultures are more difficult to create from many monocot plants and are somewhat difficult to scale-up for industrial production, even within the scale limits of the bioreactor, due to the delicate nature of the hairy root tissues.38,39 C. reinhardtii (microalgae) as a platform struggles with very low yields when its nuclear genome is transformed. Though it is entirely possible to express protein products in C.reinhardtii after transforming the nucleus, the yields from nuclear transformation are not commercially viable compared to yields obtained from using plastid transformation.40 C. reinhardtii's restriction to the plastid to achieve commercially viable expression has its own series of problems. The plastid organelles do not have the cellular machinery necessary to carry out all the post translational modifications that a protein may need.

These post translational modifications, which help fold the protein into its final shape and add various chemical modifications, give proteins characteristics that ensure they function correctly.

Since the plastid cannot do all of the post translational modifications properly, it is more difficult to create those proteins that require the modifications in C. reinhardtii.41 More generally, the algal platform concept, though promising as a whole for protein production, is fairly restricted as few algae species beyond C. reinhardtii have the reliable genetic tools needed to be made effective and consistent platforms.42

2.3.- Whole Plant Platforms

Whole plant platforms in pharming are genetically transformed plants that can be cultivated with standard agricultural techniques, then harvested to obtain the desired foreign protein product.

38 Numerous types of plants can be used as whole plant platforms including dicots (with standard species like benth or tobacco) and monocots (with standard species like the cereal crops, such as rice or maize)43,44. Whilst standard platform species such as tobacco or rice are popular choices for producing protein products, a research group's final choice of which platform plant species to work with can depend on the characteristics the plant offers them. These characteristics may include selecting a plant platform that is easier or safer to grow in a given local climate, or, more pertinently, that has organs of interest with desirable properties for the storage, purification or medical administration of the final protein product. Numerous plant organs have been harnessed for plant molecular farming, with each organ having its own characteristic set of advantages for protein production, storage and administration. Such useful plant organs include leaves, fruits, and seeds.

2.3.1.- Leaves as Target Organs

Leaves are a strong and popular choice for localizing protein because they are easy to genetically transform and they accumulate extremely high biomass45. Higher biomass means many more cells are producing the desired protein product, so yields of protein product may be higher. As one of the most frequently transformed organ types, leaves have long been a standard site of simple plant transformation in pharming, no matter whether the protein product is transgenically or transiently expressed. Leaves are also beneficial as a protein localization site because the host plant does not need to reach sexual maturity in order to harvest the desired protein product46.

The protein of choice can be harvested at any point in the plant's life cycle as long as leaves are present. This means that, if so desired, any plant life stage that produces pollen or seed can be avoided entirely during protein production. Avoiding pollen or seed producing life stages in

39 production may be beneficial in helping to provide a level of biocontainment. Spreading a

transformed plant's seeds could lead to unwanted establishment of the plants in the wild and

could expose local fauna to potentially dangerous protein products. In a similar vein, allowing

opportunities for the spread of pollen grains could lead to undesirable breeding events between

the transformed plants and other plants, such as food crops or wild flora47.

However, leaf based protein expression also suffers from a few inherent issues. Protease enzymes breaking down protein products is a general issue in all whole plant platform organs, but the leaves are a particularly active site for protein breakdown when protein expression is induced within them. This is because mature leaf cells contain many large vacuoles with numerous protease enzymes within them48,49. The vacuoles aid in the cellular process of protein breakdown, that affects both the native plant proteins and the desirable foreign protein products.

The breakdown process especially picks up during the leaf harvesting process and during the extraction and purification of the protein product from the leaf tissue50. What this ultimately

means is that the leaves must be processed immediately on harvest or stored as dry and/or frozen

material to preserve the maximum amount of protein product within them. Even this storage

cannot be maintained long-term, so the leaves must be processed quickly overall to make the

most of high protein yields in planta51. Conversely, high yields of foreign protein in the leaves

can also cause several issues for the plant. If the foreign protein product is highly expressed, and

not contained within a storage vacuole or chloroplast, it can sometimes affect the regular

development of the plant and cause cell death within the leaf tissues52,53.

40 Standard host plants for leaf-based protein harvest include the two Nicotiana genus species tobacco (Nicotiana tabacum) and benth (Nicotiana benthamiana) and the edible crops alfalfa

(Medicago sativa) and cabbage (Brassica oleracea var. capitata). The Nicotiana species are particularly loved as platform plants because of their especially high biomass yield, prolific seed production for propagation needs, and an ability to be grown and harvested year round54. The

Nicotiana plants are also not food or feed crops, so the risks of the transformed plant material accidentally contaminating or breeding with plant material intended for the food supply are lower. Additionally, the technology for working with Nicotiana species is strong. Large scale infrastructure and expertise exists for easily growing and harvesting the plants and the technology needed to transform them is simple and generally affordable55,56. However, the leaves of the Nicotiana species also contain toxic compounds like alkaloids and phenolics, which demand more intensive and expensive downstream processing and separation procedures to ensure that the final protein product is safe to use57. Despite this, new cultivars of these species exist that produce next to none of the toxic compounds of concern, which could help avoid extensive purification issues in future58. Another option is looking at edible crop platforms like alfalfa and cabbage, which avoid many of the toxic compound issues seen in the Nicotiana species and open leaves up for use in edible therapeutics. Alfalfa is particularly of interest since it yields high amounts of protein, can be harvested up to nine times per year and is a leguminous plant, which means it can convert inert nitrogen from the air into use-able forms for itself, and ultimately requires less fertilizer59.

2.3.2.- Edible Plant Organs as Target Organs

41 As many plants and their organs (like leaves, fruits or roots) are commonly eaten, scientists working in pharming have long investigated the possibility of creating oral therapeutics. These pharmaceutical proteins are delivered via edible plant parts. Numerous food crop species have been studied as potential platforms for oral therapeutics, including potatoes, tomatoes, bananas, lettuce, alfalfa, strawberries, carrots and cereals like rice,60-62. Whilst fruits are mainly targeted, many edible leaves, roots and seeds are also studied and genetically transformed with the oral delivery of proteins in mind63. The promise of orally delivered therapeutics, especially vaccines, lies in their potential to interact with and immunize the gut mucosa. The gut mucosa, or the inner lining tissue of the intestines, is often the first point of contact for many pathogens like viruses and bacteria when they enter the human body.64 This mucosa also functions as part of the mucosal immune system, the largest component of the body's immune defenses. Theoretically, priming the gut mucosa for infectious agents would likely result in a fast and powerful response from the mucosal immune system (along with the systemic immune system) against future pathogens, preventing them from proceeding further into the body65. The protein therapeutic could prime the gut mucosa more effectively by staying within plant tissue. The plant tissue, whether the flesh of a fruit or a cell's storage organelle, may act as a biological barrier between the digestive acids and enzymes of the stomach and the protein therapeutic66,67. The barrier could give enough time for the protein therapeutic to successfully reach the cells of the Peyer's patches, small clumps of lymph tissues in the small intestine that can kick off the mucosal immune response when exposed to pathogens68. Indeed, small, early clinical trials of oral vaccines for norovirus, hepatitis B and toxic strains of E. coli saw elevated production of antibodies for the pathogen-related protein contained within the plant material69. Oral immunization not only has an interesting series of benefits for an individual's immunity, but also for public health

42 interventions as well. Making an oral alternative to a parenteral (injectable) vaccine has positive effects on vaccination costs, on vaccination feasibility and safety, and on a patient's willingness to receive the treatment70,71. In addition, oral vaccines on their own do not require vaccination personnel who are trained to use needles, do not always require cold chain storage nor extensive purification of the protein from the plant material, and can reduce the overall costs of health interventions72.

Though the theoretical benefits of oral pharmaceuticals are exciting, in practice immunogenicity using the oral therapeutic alone is more difficult to ensure. Scientists have struggled with getting the mucosal immune system to recognize orally delivered pathogen proteins in plant tissue. To get the pathogen proteins to be recognized by the mucosal immune system, transmucosal carrier proteins must help ferry them across the mucosa tissue to reach the immune cells. If these transmucosal carrier proteins are not present and fused to the pathogen proteins, the pathogen proteins will not be delivered to the mucosal immune system nor enter circulation to come in contact with the systemic immune system73,74. As such, the co-production of adjuvants like CTB

(Cholera Toxin B Subunit) along with the pathogen protein desired is often necessary to get successful responses75,76. Furthermore, though oral vaccines are good at maintaining immunity in later booster vaccinations, they have trouble achieving high levels of success as the initial priming vaccination for a given pathogen77,78. Dosage consistency remains another issue for oral vaccines, with the need to ensure that the therapeutic plant tissue always contains the same known amount of protein pharmaceutical79. Happily, workarounds like lyophilization (drying) of the plant product and partial purification are trying to manage the dosage issue 80,81.

43 2.3.3.- Seeds as Target Organs

Seeds are especially good to select for protein localization if long term storage is desired for the protein product. Seeds are capable of storing protein with little degradation at room temperature for long periods of time. In a particularly notable case, dry canola seed was capable of storing the foreign protein product hirudin for over 3 years82. Seeds are also very good at storing considerable and concentrated amounts of protein within themselves, with numerous storage sites to select from within the seed, ranging from oil bodies to various types of vacuoles83,84.

However, seed production necessitates that a plant becomes sexually mature and to flower, so risks of a genetically modified plant's reproduction and escape increase when using this type of platform85. Furthermore, the time needed for seed production to occur often precludes seeds from being used for more urgent health needs, like addressing rapidly spreading or mutating diseases86. Typical platforms for seeds include cereals, legumes like soybean and peas or oilseeds plants like safflower87.

Cereal grasses (or cereals) like maize, rice, wheat and barley are popular experimental platforms for pharming because they can store proteins for long periods of time.

Other advantages exist for cereal seeds beyond long-term storage. Cereal seeds have easier downstream processing due to having fewer organic compounds and native proteins to separate out compared to leaves88. The seeds can also undergo surface washes and sterilization processes that remove unwanted microorganisms prior to processing without having their protein storing tissues damaged, which makes adhering to quality standards for pharmaceuticals far easier.

Cereals also happen to be edible and generally express recombinant proteins at high levels relative to other seed types89,90

44 Each cereal has its own characteristics, both advantageous and disadvantageous. Maize yields especially high biomass and is easy to transform using nuclear transgenic methods and to scale up for production91. Maize also has special storage organelles for proteins called zeins. These organelles can generally store any protein that is tagged with a zein, which makes locating and separating a protein product during purification far easier and cheaper92. However, maize also requires more stringent biocontainment measures because it is one of the few cereals that is adapted for cross-pollination, which is when a plant primarily reproduces using its own eggs and the sperm cells produced by the pollen of another plant. In addition, maize pollen, which is transferred by wind, can travel far, reaching up to half a mile on a 15 mile per hour gust93-95.

These factors make maize particularly predisposed to cross-breeding with other, separate maize crops. Rice, much like maize, is easy to transform and to scale up for large production needs.

However, rice is primarily self-pollinating, which means a plant mostly reproduces by using only its own sperm and eggs. This means rice can potentially avoid many of the unwanted breeding events that could come about from growing maize. Barley and wheat, though entirely possible to genetically transform, are generally less studied than rice and maize due to difficulties in cultivar transformation and yield efficiency96. All of the cereals are also monocots, which generally struggle with transformation more than dicots do. This monocot struggle is often due to incompatibilities between the monocots and commonly used transformation vectors such as

Agrobacterium tumefaciens. Such incompatibilities include the failure of the Agrobacterium to target monocot cells that can transform into any plant organ (which hinders the regeneration of

45 whole transformed plants in culture), or that many monocots do not produce the chemicals

needed to trigger Agrobacterium's DNA-integration process97.

Legumes are also a popular platform for seed localization, including crops like alfalfa, soybean and peas. Whilst yielding less leaf biomass than leafy heavyweights like tobacco or benth, alfalfa and soy can harness atmospheric nitrogen through symbiotic relationships with bacteria in their roots, so require less fertilizer input98. These legumes also are especially good at storing exceptionally high protein content in their seeds, which suggest latent potential for high recombinant protein yields. Soybean has also drawn some interest in research both as a self-pollinating and cheap to produce crop99. Though similarly advantageous to soy and alfalfa on the seed and protein storage front, peas have only been examined as a platform in a few studies, partially due to some common cultivars being more difficult to transform100.

Oil crops like safflower are also being trialled for seed-based storage in pharming due to their

unique traits. Oil crops hold promise for minimizing degradation of recombinant proteins (i.e.

making the product more stable) and for simplifying downstream extraction procedures, making

them cheaper. This is because the seeds of oil crops have storage compartments called oil bodies

within them, which any recombinant protein can be targeted to through fusion to a partner

protein called oleosin101. After the oil bodies are separated off during purification, the oleosin can

be separated off the protein product by using enzymes to digest the bond between them.

Additional benefit also potentially lies in use-able secondary products like the oils that are

separated off during production, which can be used to help reduce costs for recombinant protein

46 production102. Despite these considerable advantages, oil body fusion is low yielding at large scale production compared to other plant platforms103.

2.4. Addressing Protein Yield in Plant Molecular Pharming

For all of their flexibility and their considerable advantages, plant platforms do generally produce lower yields than industry standard protein production platforms like bacteria, yeast or

CHO cells. These industry standard platforms tend to express protein at around a maximum magnitude of 5g per litre of fermentation volume. Yet, after decades of technological optimization, yields in plant platforms have reached a similar magnitude of protein expression, producing a maximum of roughly 2g per kilogram of plant biomass (assuming 1kg of biomass is equivalent to 1L of fermentation volume)104. Whole plant platforms also have the advantage of being easier and more affordable to scale up production in. This means that the ease of supporting a particularly large group of whole plant platforms in production could make up for the less prevalent protein expression in a given amount of their biomass. By contrast, industry standard platforms are typically fairly expensive to scale up and have their biomass restricted to the volume of their bioreactor105. Nevertheless, for the other plant based platforms in bioreactors and for the continued growth and acceptance of the pharming field, improving yields in plant platforms to reach industry-standard levels is still an important goal to achieve. Recent research suggests three main areas of improvement for increasing yield and quality of product in plant-based platforms: optimizing the central dogma pathway of protein production, addressing protein degradation caused by protease enzymes and tackling the differences in glycosylation patterns between plant and mammalian cells.

47 2.4.1. The Central Dogma Pathway

The central dogma pathway, in molecular biology, is the general series of steps through which

DNA within a cell can come to produce a fully functional protein for use within or beyond the cell. Three distinct stages, with intermediate steps linking each, are recognized. The first is the transcription of the DNA, where the information within a sequence of DNA bases (i.e. a gene) is converted to an unrefined mRNA strand. The sequence of bases within the unrefined mRNA strand is then refined and stabilized through a series of modifications that occur within the nucleus. The refined mRNA contains instructions on forming an amino acid sequence for a protein, and on the situation in which that amino acid should be produced (i.e. how, when and where amino acid production should occur). The second stage is the translation of the refined mRNA into the chain of amino acids. The refined mRNA reaches and binds to a ribosome, which then reads along the mRNA's sequence of codons: groups of 3 bases that each correspond to a specific tRNA. The corresponding tRNAs each have a specific amino acid attached to themselves, and bring these amino acids together at the ribosome. There, the amino acids are bound together by peptide bonds and form a structure called a polypeptide chain. This polypeptide chain continues to grow until a special stop codon is reached by the ribosome and the chain breaks away. In the third stage, post-translational modification, the newly formed chain of amino acids then can undergo a series of folding steps and molecular modifications with the aid of molecular chaperones, enzymes and other cellular machinery in and around the organelles within the cell. This series of steps helps the protein attain its final shape and molecular capabilities before it is stored, released or used.

48 Cellular events that bring about yield loss in pharming can occur at any point along this central

dogma pathway. The sort of cellular events affecting yield may also vary depending on

environmental factors: including the species of plant used to produce the protein, the desired

protein's specific characteristics and function, or which organs the protein is expressed in. As

such, whilst general issues that do cause yield loss along the central dogma pathway are

described here, they are by no means universal to all pharming situations. In field scientists

typically develop and optimise transformations with low yields of protein products via

empirical, trial-and-error testing alongside careful study of their platform plants and growing

environments.

2.4.1.1. Yield Reducing Factors in Transcription

Transcription governs the baseline level of expression of the desired gene. In the pharming field,

high levels of nuclear expression have been achieved constitutively (i.e. in all parts of the plant)

by using the cauliflower mosaic virus 35s (CaMV 35S) promoter in dicots and the maize

ubiquitin-1 (ubi-1) promoter in monocots106, 107. Despite this, constitutive expression of the

protein product is not always desirable. This is because the protein product could have negative

effects on the growth, development and metabolic function of the various parts of the whole

plant. Organisms interacting with or consuming parts of the plant, like pollinating insects or

microbes in the soil, could also be harmfully exposed to the protein product through its cells and

tissues108. Thus, controlled expression through either inducible promoters, that only help express a gene under an applied environmental or chemical condition, or tissue specific promoters, that only express a gene within a specific plant organ or tissue, are increasingly desirable. This control of expression also ensures that protein products are present in organs with useful

49 properties like possessing many stable protein storage sites, being easy to harvest and isolate

products from, or being edible109. Even though inducible or tissue-specific promoters are

extremely helpful for biosafety and processing needs, getting them to work with a selected plant

platform species is not always easy. During empirical assessments of promoters, expression

levels of the protein must be balanced carefully. Too low expression of the foreign gene may

mean that not enough protein will be yielded from the plant. Too high expression of the foreign

gene may bring about harmful effects on the plant platform,like low seed production or stunted

growth110. Luckily, tactics for maximizing transcriptional level gene expression abound. These

include stacking multiple copies of the promoter around the gene, designing powerful synthetic

or chimeric promoters based on strong promoters found in nature, or putting regulatory DNA

sequences next to the promoters that encourage transcription machinery to interact with a

promoter111.

2.4.1.2. Yield Reducing Factors in Translation

Translation itself is reliant on three main components- the tRNA, mRNA and the ribosome organelle. All three work together to help form the chain of amino acids that will eventually be shaped into the final protein product. As immature mRNA travels through the nucleus after transcription, many things can potentially hinder it from leaving the nucleus to reach the ribosome. Issues may lie within the untranslated region (UTR) sequences in the 3' and 5' regions on the RNA, that sandwich the RNA sequence that helps code for the amino acid112,113. The 5'

UTR region, or leader sequence, is important for making sure the ribosome can bind to the mRNA before translation starts. The 3' UTR region, or trailer sequence, follows after the end of the amino acid coding sequence, and helps stabilize the mRNA through binding to a chain of

50 several attached adenosine phosphates collectively called the poly(A) tail. Other factors that

may make the mRNA less productive at translation include cryptic splicing sites, which can get

the mRNA sequence edited by splicing machinery to be more unstable, decay motifs that shorten

the lifespan of the mRNA transcript or the GC content within the mRNA, which can affect how

efficiently the mRNA is translated at the ribosome114-116,. All of the above phenomena, and more,

are targets of specialist research to make the maturing mRNA sequences more stable and to

enhance their translation efficiency. These efforts often involve trying to optimize the 5'UTR and

3'UTR sequences. In the case of the 5' UTR, sequences may exist in nature that are known to

help with making translation more efficient. For example, the 5'UTRs of alfalfa mosaic virus or

of tobacco mosaic virus are known to help increase the translation rate of amino acids for given

proteins117. In the case of the 3' UTR, where an mRNA stabilizing process called polyadenylation occurs, modifying specific sequences called cytoplasmic polyadenylation elements can help regulate when polyadenylation occurs which in turn, can help stimulate translation and stabilize the mRNA sequence118. A good part of this research also focuses on addressing codon bias within tRNA, the RNA molecules that bring the amino acids to the ribosome. Codon bias is when organisms have different amounts of tRNAs that code for a given amino acid. So, even if two separate codons, A and B, code for the same amino acid, the organism producing the amino acid may more frequently use tRNAs that correspond to codon A. In this scenario, this would mean that if a foreign mRNA sequence is given that uses codon B for the desired amino acid, translation would occur far more inefficiently. Codon optimization is how codon bias is rectified.

This is intentionally modifying the DNA sequence to be transcribed so the mRNA reflects the codon bias of the organism. In the codon A and B scenario, this would mean changing all codon

B sequences to codon A sequences to reflect the biases of the platform organism. Codon

51 optimization has been used successfully to increase the efficiency of translation and, thus, protein

product yields as well119.

2.4.1.1. Post-Translational Yield Reducing Factors

Even after translation, where the newly-made amino acid sequence travels and what processes it is subjected to is important for its stability and function as a protein. Post translational modifications must be controlled to ensure the amino acid is processed and stored appropriately so a fully functional protein can be isolated with ease and the plant cell's regular activity can be maintained. The cellular organelles that get targeted for protein storage depend on the plant tissues that are being worked with. These storage organelles can range from seed oil bodies, which help plant leaves collect high concentrations of protein without impacting growth, to the endoplasmic reticulum, which can help proteins avoid some undesirable plant-specific glycosylation patterns120,121. Depending on the type of protein product and its function, the choice

of where to collect the protein within the cell can be variably helpful or harmful. No real solution

universally exists for all proteins, since the functions and structures of different protein types

vary so greatly from one another. Instead, finding post-translational solutions that increase

protein yield are largely dependent on trial-and-error experiments and, if successful, are usually

only a viable solution for a specific group of proteins, like a certain family of enzymes. An

example of a well-researched series of post-translational modification tactics are general

methods for producing antibodies. Such antibody proteins are usually targeted to a plant cell's

secretory pathway through adding signal peptide molecules to the N-terminus (or the start of the

polypeptide chain), which seems to increase their stability, and thus their yield.122,123 The

antibody yield can be increased even more when a signal molecule called H/KDEL is attached to

52 the C-terminus (or end of the polypeptide chain) of the protein. This H/KDEL signal is a retention signal, which keeps the antibodies within the endoplasmic reticulum organelle, and stops them from travelling through the Golgi complex organelle to the apoplast and having unwanted glycosylation patterns attached.124,125 Even though these molecules are helpful at increasing yield for the antibodies, they are not always 100% reliable. The H/KDEL signal, for example, does not always permanently keep the protein within the endoplasmic reticulum.

Indeed, H/KDEL tagged proteins can still sometimes have the unwanted glycosylation patterns and appear in the apoplast. 126

Protease related degradation has recently been recognized as an issue that can significantly affect the yield of a protein product from a plant platform. When protease enzymes act on a protein, the protein undergoes hydrolysis reactions, in which it is broken up partially or completely. As a result, when the product is extracted, a mixture of both full proteins and degraded fragments is present, which means less of the valuable fully functional proteins are obtained.127 Since protease degradation significantly reduces the amount of useful product that can be obtained from a plant platform, the pharming field's efforts to identify the precise protease enzymes involved and to minimize their activity have increased. Tactics typically used by scientists to inhibit most plant proteases include co-expressing protease-inhibitor genes with the foreign gene cassette, altering pH conditions to make the proteases less effective at their functions, and using genetic interventions to temporarily or permanently stop the production of proteases in the first place128-130. Protease inhibitor genes, usually regulatory genes from other plants like tomatoes, have been repeatedly shown to help increase the yield of protein products and broadly seem to inhibit the activity of many different families of proteases within the cell.

53 pH alteration and regulation also seem to inhibit broad-spectrum (i.e. not very protein specific) proteases like the cysteine, serine and aspartic proteases effectively.131 More recently, targetted anti-protease approaches intervening on the central dogma pathway have also been used in interventions on protease activity. These specific methods include using double stranded RNAs to deplete highly expressed protease mRNAs from the cell (RNA interference/RNAi), using plants or cell lines that only express poor or non-functioning forms of given protease genes

(knockdown and knockout plants), or even using sequence-specific nuclease systems like

TALENs or CRISPR-Cas9 to edit out the protease genes from the genome altogether132-134.

However, one singularly large limitation against all of these anti-protease interventions, both general and relatively specific, is that it currently remains unclear which parts of a given protein product actually invite protease activity. Hence, the precise functions of the proteases that are degrading the protein product remain fairly hazy. To tackle this issue, several experimental strategies are underway. These include applying mass-spectrometry based proteomics methods to the plant proteome to identify cleavage sites and the sequence specificity for given proteases.

Alongside this approach, proteome derived peptide libraries are also currently being used to determine N- and C- terminal based sites for protease cleavage, and activity based protein profiling is being applied to determine which proteases' active sites are reacting covalently and irreversibly with specified and tagged chemical probes135.

N-glycosylation is also a significant issue for plant based platforms. Though few differences exist between the cellular machinery and processes within mammalian cells and plant cells, one important difference is how the organisms each accomplish N-glycosylation, the post-translational attachment of sugar molecule structures to a specific nitrogen atom within a

54 protein. Plant N-glycosylation processes seem identical to those seen in humans (and other mammals) until the partially glycosylated protein comes to the Golgi complex. At this point, the patterns of the sugar structures become organism dependent. In humans, glycoproteins are modified with β1,4-galactose and sialic acid structures, which are not normally present in plants136. By contrast, in plants, glycoproteins are modified with β1,2-xylose and core

α1,3-fucose, which are not normally present in animals137. The differences seen between human and plant N-glycan structures has caused worries about potential harms that could arise from these differences. Such worries include plant protein products with the plant type N-glycan structures having altered functions, activities, stabilities to those proteins with the human type structures138. Some are even concerned that the proteins with plant type N-glycan structures pose a risk of setting off an immune response in someone who uses them139. Whilst how much of a risk alternative glycan structures actually pose to health is a hotly debated topic within the pharming research community, it is likely that this modification difference between plant and mammalian platforms will draw some degree of regulatory attention and concern. This means that a demonstration that the plant-specific modification can be controlled or stopped altogether is often required anyways for protein production done in a plant platform.

Strategies for changing plant glycans can largely be categorized as either preventing the plant-specific modification from happening through protein tagging and redirection, or through interfering with the creation of the glycosylation enzymes needed for the plant-specific modification to occur. In the second instance, where the plant's own enzymes are interfered with, some modification strategies may also genetically introduce human type enzymes and

55 components to help obtain human-like N-glycan structures140. These above strategies provide opportunities for customizing the N-glycan patterns on proteins, which can have far reaching effects on product quality and efficacy. CRISPR-Cas9 and TALENs also have more recently been used to edit plant glycosylation enzyme genes in planta to make plant platforms easier to use hosts for protein expression141. However, this is not to say that the different N-glycan structures provided by plants are always undesirable for protein production. Sometimes, plant specific glycan patterns grant beneficial properties to a protein product, and make the product functionally equivalent (biosimilars) or even better (biobetters) than similar protein products without the alternative modifications. Elelyso, the FDA approved enzyme-replacement therapy produced in carrot cell suspension, is an example of such a product. Elelyso is a recombinant protein replacement for the human enzyme protein glucocerebrosidase, which the Gaucher's disease patients cannot produce effectively. The enzyme must be taken up into macrophage cells to work properly, but the standard, human form of the enzyme is unable to enter the cells. This is because normal glucocerebrosidase has glucose structures on itself, like sialic acid, galactose and

N-acetylglucosamine, that stop it from being taken into the macrophages. By contrast, the carrot cells that produce Elelyso cannot produce sialic acid on their own, and the glycan residues present on the Elelyso protein are not made more complex due to targeting the plant cell vacuoles. Both of these modifications mean that Elelyso, on its own, can be taken up by macrophages with ease. An additional benefit of Elelyso's carrot cell production platform is that its competitor, the mammalian cell-produced enzyme Cerenzyme, cannot avoid putting on the sialic acid. This means that, in manufacture, Cerenzyme (unlike Elelyso) must undergo additional, costly processing steps to remove the unnecessary sialic acids and become functional

142. Examples like Elelyso suggest that the protein that is 'normal' for a human or animal to

56 produce, may not necessarily be the best working possible iteration of that protein for treating a disease or for use in other applications. Similar host engineering and modification efforts to those seen for N-glycosylation are also starting to crop up for other post-translational modification types in plants, like O-glycosylation, furin cleavages or gamma carboxylations, but discussions of these lie beyond the scope of this paper143.

2.5. Conclusion: Plant Molecular Pharming's Technological Development

Pharming is a refining, up-and-coming technology that allows flexibility of choice in what organs you can store the protein in, where you can grow the transformed plants and what types of proteins can be produced . This sheer flexibility means that pharming can be adapted for all sorts of growth and processing situations, for most proteins desired and for most production scales demanded. The technology is still only around 40 years old, but is refining a portfolio of transformation methodologies and plant species, organs and tissues that it can uniquely exploit.

Whilst yields and downstream processing issues are current bottlenecks for the field, these are beginning to be overcome, with recent and exciting developments in technology and approaches.

These include everything from engineering plants to have human or animal like post translational modification pathways, to ensuring plants cannot express toxic compounds like alkaloids that make processing more difficult, to using proteomics methodologies to search for and target interfering proteases144-146,. As pharming works through its growing pains as a rising method of production, its sheer flexibility and unique set of advantages may help it conquer difficult to reach biotechnological niches and, in future, possibly stand head-to-head with gold standard industry methods like CHOs, bacteria or yeast.

57 Chapter 3- Plant Molecular Pharming and Biotechnological Sovereignty for LMICs

3.1.- Introduction

Most low- or middle-income countries (LMICs), a category which includes countries all across

South America, Central and South Asia, and Sub-Saharan Africa, deal with a diverse disease burden that is greater in size than that of high-income countries (HICs). LMICs deal with both a wide variety of endemic diseases that persist within a given region or community, like malaria or leishmaniasis, and novel emerging diseases that suddenly are introduced to a population or area, like Ebola, Rift Valley Fever Virus or Crimean-Congo Haemorrhagic Fever Virus.1-,4 Such diseases are major contributors to high ongoing mortality (death) rates within LMICs, but also are sources of high morbidity as well, reducing quality of life, impairing physical and mental function, and often shortening the lifespan of an infected individual. 5

Alongside a large and greatly varied disease burden, the peoples and governments of LMICs must also contend with the cost of obtaining medicine. In LMICs worldwide, the financial demands of disease treatment can be detrimental for impoverished families. When a health need arises for such a family, wages that could easily be far under $1 per day are typically spent out-of-pocket on necessary but expensive pharmaceuticals for the disease.6,7 In many cases, the sheer cost of treating serious illness can help generational impoverishment of a family or community persist, because paying for the pharmaceuticals may demand the sale of household assets like livestock or the withdrawal of children from affordable schooling.8 On a larger scale,

LMIC governments invest considerable amounts of their healthcare budgets into obtaining these

58 pharmaceuticals. After hiring health personnel, pharmaceuticals are the second largest source of expenditure for LMICs, with purchases taking up to 66% of total healthcare spending for some of the poorest countries. 9

To meet high demands for pharmaceuticals, LMICs are primarily reliant on manufacture, foreign imports and health interventions. Poorer LMICs often do not have the financial nor technological capacity to support extensive local production of pharmaceuticals. As a result, many such countries have come to rely on the import or donation of pharmaceutical products from the wealthier countries that can produce them. In Sub-Saharan Africa, for example, most countries import around 70-90% of their pharmaceuticals.10 Imported and donated health materials have been helpful, affordable and, indeed, beneficial for health issues within many LMICs. However, numerous issues arise for LMICs from a sheer dependence on the biotechnological infrastructure and pharmaceutical imports of wealthier countries, and from not having their own strong health and biotechnology infrastructure. Indeed, many LMIC governments like the Brazilian and South

African governments, alongside intergovernmental organizations like the East African

Community, have made clear statements on the urgency of addressing their own local production needs. 11-14

3.2.- The Disadvantages of Import Dependence for LMICs

An LMIC primarily remaining dependent on imports to meet their pharmaceutical needs is has several disadvantages for said LMIC. One such disadvantage is that the pharmaceutical companies supplying these imports are primarily incentivized to manufacture products for their

59 lucrative, domestic, and typically HIC-based markets. Since production is biased towards

HIC-based markets, earlier stage pharmaceutical research and development also prioritizes

HIC-prevalent conditions like lung or breast cancers, chronic obstructive pulmonary disease

(COPD) or dementias like Alzheimer's, Indeed, a 2015 analysis of industry reported

pharmaceutical trials over a period of 5 years (2006-2011), suggested that the number of drugs

made for HIC-prevalent conditions, was roughly 3.46 times greater than the number of drugs

made for LMIC-prevalent conditions like TB, malaria or HIV/AIDS.15 By contrast, the markets for many undertreated diseases in LMICs typically consist of large groups of extremely poor people. These populations often cannot afford vaccines or medications considered ‘affordable’ in a HIC context (for example, $15 per dose of hepatitis vaccine). 16 Since these markets for

LMIC-prevalent diseases have little purchasing power, HIC-based pharmaceutical companies widely consider producing medication for extremely poor communities to be economically challenging and financially risky. 17-19As a result, many such companies avoid attempting de novo humanitarian research on most LMIC-prevalent diseases, and instead try to address the health disparities of communities in need through the provision or donation of pre-existing medications,. Successful examples of this intervention type include Novartis providing the chemotherapy drug Glivec for free to poorer patients with certain cancers, and Boehringer

Ingelheim donating the antiretroviral Viramune to HIV-1 positive mothers, which is estimated to have prevented roughly 2 million children from contracting HIV-1.20,21

Even if pharmaceutical donations or imports are provided to an LMIC for a certain disease, the particular pharmaceuticals provided may not be effective for an LMIC’s particular manifestation of the disease. Many pharmaceuticals imported from HIC-based producers exist to address

60 common strains or clinical presentations of a given disease within an HIC context, and not those

of a LMIC. An example of this are human papillomavirus (HPV) vaccines, pharmaceuticals that

protect against the transmission and infection of the virus through sexual contact. Preventing

HPV transmission is desirable, since contracting high risk genotypes of the virus can have

severe future consequences for an individual, like cervical cancer or other genital-related

cancers.22-24 HPV currently has existing vaccines against several of its 120 genotypes, including

a bivalent vaccine and quadrivalent vaccine that both target the highest risk genotypes 16 and 18,

and a nonavalent vaccine targeting nine separate genotypes, including the high risk types 31, 33,

45, 52, and 58, alongside 16 and 18.25 However, none of the HPV genotypes targeted by these vaccines are the most common in the populations of some African countries. Indeed, many of the high-risk genotypes that are prevalent in the populations of countries like Burkina Faso (HPV

35) or Benin (HPV 39) do not have any existing preventative vaccine for them.26-29 In addition,

HPV 16, which already has many existing vaccines, is less common in sub-Saharan Africa than in other parts of the world.30 Thus, the HPV vaccines that currently exist are specific to high risk genotypes that are not of greatest concern to the disease prevention efforts of many countries in sub-Saharan Africa. Of additional concern is that most treatments for the high-risk genotypes of

HPV are prophylactic in nature. No therapeutic vaccination exists for those who have already contracted the disease, and the current prophylactic vaccines do not appear to affect established infections.31 Most individuals who have already contracted HPV and who suffer the cervical cancers that ensue also happen to be within LMIC countries like those in sub-Saharan Africa.32

The lack of existing treatment for both LMIC-prevalent HPV genotypes and of prophylactic treatment for HPV-infected individuals suggests that the true medical needs of LMIC are neither reflected nor addressed in the production goals of HIC-based companies. Once again, the

61 financial risk of only filling the need of a large community with little purchasing power may deter HIC-based pharmaceutical companies from devoting resources to developing the most useful treatments for LMICs. This hesitancy is particularly unfortunate, since contracting HPV is one of the leading causes of cancer-related death for women in LMICs.33

A HIC-based pharmaceutical company may also only research, develop and manufacture pharmaceuticals relevant to an LMIC-prevalent disease if there are financially sound reasons to target the disease in the first place. One such pragmatic target is malaria, which is considered one of the ‘Big Three’ diseases of poverty (alongside HIV/AIDS and TB). Malaria has been actively researched and targeted for product development by many pharmaceutical companies, with the considerable publicity and support of funding initiatives like the Global Fund to Fight AIDS,

Tuberculosis, and Malaria behind them.34,35 Though the disease is certainly a source of great suffering in LMICs and though pharmaceutical companies have helpfully contributed to efforts against it, malaria impacts other markets beyond impoverished communities. Malaria is also a significant concern for the US military and international travelers, to the point where considerable information and research on the disease has derived from work carried out by the military. 36,37 Beyond these stakeholders, malaria also continues to persist in wealthier parts of the industrialized world, such as Oman or Saudi Arabia.38 As such, investing in malaria research may be considered less of a financial risk by pharmaceutical companies, since treatments for it are also demanded by a variety of wealthier markets in addition to LMIC-based communities.

Worryingly, many of the LMIC-prevalent diseases that pharmaceutical companies are reluctant to research already cause significant problems for impoverished communities, and have the

62 potential to cause more in the future. One such disease is Rift Valley Fever Virus (RVFV), which is endemic to areas of sub-Saharan Africa, is at the center of several outbreaks throughout the

African continent and in the Middle East, and is transmissible to both livestock and humans.

RVFV is of such concern that the World Health Organization (WHO) has listed it as a likely future cause of an epidemic, but few pharmaceutical defenses exist against it. Currently, the only known preventative treatment for RVFV is an expensive and highly risky veterinary vaccine. 39,40

Emerging diseases are also present in LMICs that have no known cure or vaccine at all. One such disease is Crimean-Congo Haemorrhagic Fever, which has been also singled out as a target of concern for medical research against future epidemics, and which is already spread throughout Africa, Asia, South/ Eastern Europe, and the Middle East.41Another comparatively under-researched, yet greatly impactful, group of diseases are the neglected tropical diseases

(NTDs). The NTDs are a group of chronic parasitic conditions that include leishmaniasis, dengue fever or African sleeping sickness. These diseases significantly harm physical and mental development, contribute to trapping LMIC-based communities within cyclical poverty and are speculated to perpetuate suffering on a scale comparable to malaria and HIV/AIDS. 42 Since the

NTDs are typically only suffered by subsistence farmers and the world’s poorest communities, there has long been no pragmatic incentive for pharmaceutical companies to develop the products needed to treat them. Happily, NTD-focused research and medical campaigns are increasingly supported and funded by non-profits and some pharmaceutical companies.

However, funding allocated to combatting the NTDs is still dwarfed by investments made into efforts against the ‘Big Three’ diseases. 43,44 In data tracked by the Institute for Health Metrics and Evaluation in 2009, NTDs received only 0.6% of official health development funding, versus

HIV/AIDS (36.3%), malaria (3.6%) and TB (2.2%).45,46

63 Another separate disadvantage of being import dependent as an LMIC is that this dependent

position makes an LMIC vulnerable to the harmful health, political, and economic effects of

vaccine nationalism. Vaccine nationalism is a political phenomenon observed in global health

crises where countries, typically HICs with the financial and technological means to secure

pharmaceuticals, will prioritize the immunization of their citizens. LMICs, who typically do not

have the wealth to secure a supply of pharmaceuticals nor cannot produce their own medication,

are often imperiled by the effects of health crises for longer. Often, HICs quickly deplete the first

stock offerings of vaccines, and many LMICs must wait for their replenishment. During this

wait, LMICs often are impacted greatly by rising death tolls and long-term health effects on their

populations. The large-scale harm to LMIC populations could also impact other structural

aspects of their health systems: overburdening under-resourced healthcare systems with patients

and risking the health of essential medical workers and vulnerable individuals for a longer time.

This vaccine-procurement disadvantage may also spur an LMIC government to take drastic

measurements for their country’s public health, and harm their long term interests in the process.

Such harmful measures might include an LMIC government blocking exports of critical industry

products to force negotiations on vaccine procurement, which could lead to supply chain and

trade breakdown, or entering short-term deals to secure vaccines with criteria that are

disadvantageous to the economic and political development of the country in the long term. 47

Sadly, this phenomenon is not something of the far past. Vaccine nationalism was witnessed during the H1N1 pandemic in 2009, where HICs addressed their national needs first and left

LMICs with no local production capacity open to the full impact of the pandemic.48 It also

continues to be evident throughout the current SARS-CoV-2/COVID-19 pandemic. Wealthier

64 countries have already pre-ordered vaccine doses in excessive droves. For example, from data

recorded on the 25th of January, 2021 the UK pre-ordered 5 doses per citizen, when only two are

needed for a person to be fully immunized49. Alongside this, most vaccines for the pandemic are

being manufactured within facilities in Northern America and Europe, which greatly sped up

procurement for these countries50. Whilst ongoing movements to promote equal vaccine

procurement, like the COVAX program, exist, they are struggling to achieve their goal and a

globally unequal vaccine access situation is likely.51-53 This bodes poorly for many LMICs

struggling medically, economically and socially with managing COVID-19. For example,

medically, almost half of Kenya’s general hospital beds are without oxygen supplies for

COVID-19 patients, and many of Zimbabwe's frontline health workers are under-paid,

under-protected and under-trained for working against the virus.54,55 Economically and socially,

the pandemic is expected by the World Bank to push 40-60 million people worldwide into

extreme poverty and negatively impact most aspects of life in LMICs, from critical childhood

education, to standard healthcare access, to sex equity56-58.

Yet another reason why import dependency is harmful is that LMIC governments must negotiate with pharmaceutical companies to secure necessary medicines for import, but pharmaceutical companies are under no obligation to offer their products at fair prices that account for the needs of a less wealthy buyer. 59 Negotiations with pharmaceutical companies are opaque, with discounts provided to other buyers kept highly confidential. Institutional purchasers entering the deal are thus typically unaware of whether lower prices have previously been offered for a product. Pharmaceutical companies maintain that opaque negotiations are necessary to prevent every buying country from demanding the lowest available price so they can continue to serve a

65 greater breadth of markets. Despite this assertion, there is no knowing who is receiving the

greatest discount for a pharmaceutical product. It is as likely that a wealthy country with

significant negotiation leverage would be able to secure the most favorable deal as a poorer

country that would require the discount to afford the product in useful quantities. Whilst the

opaqueness of pharmaceutical purchase negotiations makes any direct measure of pricing

practices difficult to collect, indirect indicators of unfair negotiation terms do exist. For

example, the list (suggested retail) prices provided by pharmaceutical manufacturers to buyers

are higher in LMICs than for HICs. These list prices are higher for LMICs both in terms of base

prices put forward in the lists and when the prices are compared, percentage-wise, to average

incomes within both LMICs and HICs. 60.61 Pharmaceutical manufacturers have argued that these high prices are fair to demand so returns can be made on the extremely high investments needed to develop pharmaceutical products. Yet the demand to maintain high prices becomes suspect when manufacturers also refuse to disclose their true research and development costs, and instead provide hypothetical price figures that fail to account for additional government and non-profit support, like investments or tax deductions. 62-64 Slivers of evidence such as these suggest that pharmaceutical manufacturers may not necessarily prioritize giving the best deal possible to the LMICs that are dependent on them for their medication supply.

The degree of reliance LMICs have on import to obtain necessary pharmaceuticals means that which of their diseases gets effectively addressed is often highly reliant on the intents and willingness of HIC-based external producers. Whether a LMIC-prevalent disease receives effective treatment largely depends on whether a HIC-based pharmaceutical manufacturer deems

66 it relevant to their financial interests and thus worth the risk of targeting a poorer, smaller market.

If not, medications may either not exist at all for a given disease, or may exist, but may not

necessarily be adapted for the health needs of the LMIC receiving them. Import dependence also

makes LMICs highly vulnerable to the harms of vaccine nationalism in global health crises and

to unfair price negotiation terms that are difficult to refuse or challenge. Yet, these discussed

areas only scratch the surface of the significant disadvantages that import dependence poses for

the health and development of LMICs. Important angles not discussed in this paper include the

larger scale effects of trade deficits caused by import dependence on the economies of LMICs,

the variable quality of pharmaceuticals that get imported to LMICs, and even the ethics of

treating health- and life- sustaining products as standard commercial goods. 65-69 However, a core observation throughout all of these angles is that LMICs are consistently at a public health, political and economic disadvantage when they are not in a position of sovereignty over the research, development and manufacture of the pharmaceuticals required to address their own health needs.

3.3.- Advantages of Local Biotechnological Production for LMICs

Since remaining primarily dependent on external producers for pharmaceuticals is so disadvantageous, some LMICs are at various stages of investing in internal, local pharmaceutical production. LMICs that choose to emphasize local pharmaceutical production hope to decrease their reliance on external manufacturers for vital health products and thus mitigate some of the societal, economic, and political harms that come from dependence.70.71 Most LMICs do not yet have competitive local pharmaceutical production nor direct quantitative measures of whether

67 local production beneficially affects medication access and disease prevention. As a result,

evaluating the effectiveness of local production for LMICs with concrete evidence is currently

difficult to do. 72,73 Despite this, the theoretical advantages of an LMIC’s sovereignty over pharmaceutical production are significant.

3.3.1.- Local Advantages

One such advantage is that LMICs would be able to direct pharmaceutical research and resources to prioritize local health needs. 74-76 For example, early research efforts based in LMICs have led to several proofs of concept for virus-like particles that could be used in a vaccine against livestock-harming Foot and Mouth Disease, or antigens for diagnostic tests for Rift Valley Fever

Virus. 77-79 Such locally based proofs-of-concept reflect the disease priorities of the LMICs that develop them, in a similar fashion to how HIC-based pharmaceutical research prioritizes

HIC-prevalent health troubles. Furthermore, urgent health needs could also be addressed more efficiently. Large scale, locally based manufacture of vaccines and diagnostic products could help quickly and precisely combat future pandemics or disease outbreaks. 80

Another advantage is that local production could make pharmaceutical products more affordable and, thus, more accessible in LMICs. Whilst it is known that some pharmaceuticals are cheaper to import altogether, the existence of competitive local pharmaceutical facilities could make other pharmaceuticals far more affordable than imported equivalents. If local pharmaceutical facilities within an LMIC are of comparable size and quality to an external producer, the additional cost benefits afforded to local facilities are considerable. These benefits include the reduction of transport costs, the affordability of raw manufacturing material compared to refined

68 product, and the avoidance of price markups due to freight, duty and value added taxes. All of

the above could combine to create a pharmaceutical product that is cost competitive or even

cheaper than its imported equivalent. 81

Prioritizing local production could also positively affect pharmaceutical quality regulation within

an LMIC. Currently, many LMICs struggle with receiving pharmaceuticals of highly variable

quality. Pharmaceuticals manufactured for export are often not as heavily regulated as those

manufactured for domestic use, even in HIC-based facilities. In past audits by Médecins Sans

Frontieres pharmacists, even pharmaceutical manufacturers with high compliance to quality

regulations will adjust their manufacturing standards based on the country or organization

receiving the pharmaceutical. Whilst clients with strict regulations, like HICs or international aid

agencies, may receive a carefully produced pharmaceutical, clients with less stringent regulation

standards, typically LMICs, will often receive a less carefully produced pharmaceutical instead.

82 This might mean that LMICs receive products with substandard doses of the pharmaceutical

agent or products that are of poorer quality in general. Local production could incentivize

government investment into developing stricter pharmaceutical quality regulation standards. For

example, the Ethiopian government has invested around $6.6 million into strengthening its

pharmaceutical regulatory agency in an effort to promote local pharmaceutical manufacture. This

investment was made alongside a commitment to identify counterfeit drugs circulating

in-country and to push pharmaceutical manufacturers to meet higher export standards. 83 Since external pharmaceutical manufacturers use a country’s quality control standards as a production guideline, investments into pharmaceutical regulation sparked by local production could also force external manufacturers to produce higher quality products for LMIC markets as well.

69 The development of a local pharmaceutical industry may also create an incentive for the

scientific talent of an LMIC to develop and remain in-country. Whilst predictive analyses suggest

that the number of jobs created by local pharmaceutical plants may only be in the order of a few

thousand, these jobs may be considerable opportunities for LMIC-based scientists. Scientists

from LMICs who are invested in pharmaceutical development and production, often come to

HIC-based institutes in the US and Western Europe to study. However, when training ends, these

scientists often do not return to implement their research in their own communities, because

opportunities are hard to find within the LMICs they came from. For example, national or

international research grants may be difficult to access, or few companies may exist in an LMIC

that could support their expertise. Investing in local research, development and production,

which could include funding pilot manufacturing facilities or centers of excellence for

biotechnology, might provide the necessary opportunities needed for LMICs to retain their

highly educated specialists. 84

Finally, local pharmaceutical production might also help with an LMIC’s economic

development, through the establishment of local trade regions for pharmaceuticals. Local trade

regions could help supply the needs of niche markets within and around a given LMIC, like

those for parasitic diseases or “orphan” diseases. Trade of locally produced pharmaceuticals

could also help supplement the needs of neighboring LMICs that may not necessarily be in a

financial or political position to start pursuing pharmaceutical production for themselves. 85,86 In addition, more money could be kept circulating within an LMIC’s economy through the employment of local workforces, investment into infrastructure and facilities, and reduction of

70 spending on imported pharmaceuticals that local pharmaceutical production might bring about.

In turn, the trade deficits that many LMICs are currently subjected to due to their dependence on pharmaceutical imports, could be reduced and trade balance could be improved. 87-89

3.3.2.- Global Advantages

Beyond the individual benefits that it could have for a specific LMIC, supporting pharmaceutical production sovereignty could be a critical component of global preparedness against zoonoses and against disease spread changes brought about by climate change.

Having more developed local production in LMICs could help combat zoonoses, animal-transmitted diseases which often tend to emerge or become endemic within LMICs first.

Many global health initiatives, such as the One Health Initiative, recognize that maintaining control of disease spread must involve both humans and other species of animal. In general, zoonoses are a pressing healthcare concern, typically representing 6 out of 10 infectious diseases in humans and 7 out of 10 emerging or re-emerging diseases globally. 90 A pertinent example of this is COVID-19, which is now largely believed to have originated in wild bats before mutations occurred that helped it spread to humans. 91 In LMICs, zoonoses demand very different disease control strategies to those used in HICs, and cheaper vaccines and therapeutics are often required to effectively combat their spread. 92 Of additional concern are the effects that zoonoses could have on livestock, with significant negative impact on farming and food production. Such diseases could especially harm LMICs that rely on agriculture for food security, meeting local dietary protein needs, and as a major component of their economies. 93

71 Local production in LMICs would also create a first line of defense against diseases spreading across the globe under the effects of climate change. Climate change’s progression changes the conditions of areas all around the world, impacting and altering the global distribution of animals and humans as habitable zones on the planet change along with weather, climates and ecosystems. 94 Changes in conditions and organism distribution also impacts the movement of disease. Diseases once understood as endemic to the warmer climates of the tropics may also move up into countries where no adequate protection, vaccination or therapeutic against them may currently exist. Evidence exists that such spread is already occuring. Bluetongue virus, a livestock disease transmitted by Culicoides midges, is newly emerging in northern Europe as climate change has altered the range of its midge vector.95 The transmission of difficult-to-treat viral diseases like Rift Valley Fever Virus (RVFV) and Crimean Congo Haemorrhagic Fever

Virus (CCHV) are also being impacted by the climate. For RVFV, warming temperatures are increasing mass hatching events for the virus’s mosquito vectors, and for CCHV, changing conditions are promoting the survival of its tick vectors and growing the pool of potential viral hosts. 96-100 RVFV, CCHV, and their insect vectors are also spreading to numerous parts of the world where they have not previously been seen before. Numerous other examples of zoonoses and other transmissible diseases being worsened by climate change exist, but discussing these in detail is beyond the scope of this paper. 101

Establishing local sovereignty over pharmaceutical production in LMICs could benefit both the fight against zoonoses and the development of future defenses against climate change. Local production in LMICs would open opportunities for faster on site research, anti-disease responses and preventative actions against future outbreaks that could harm animals of humans. These

72 opportunities might include the rapid development of disease diagnostic tools like PCR tests or simple ELISAs , developing vaccines for LMIC-prevalent diseases with the potential to spread, or creating therapeutics that heal or protect livestock, wild fauna and LMIC communities from illness. 102-104 In addition, original intellectual properties (IPs) for LMIC institutions and companies could originate from the research and manufacture carried out to meet these disease needs. These IPs, in turn, could be used to aid others in the global community facing challenges from novel diseases, to the financial and social benefit of the pioneering LMIC who developed them.

3.4.- Advantages of Plant Molecular Pharming for Local Production in LMICs

3.4.1.- Advantages for Research and Development

There are numerous viable platforms for producing protein-based pharmaceuticals, but plant-based platforms are especially attractive for establishing pharmaceutical research and development within LMICs. Plant platforms, particularly whole-plant platforms, offer a diverse array of characteristic advantages that few other platforms can replicate. The combined benefits of whole plant-based platforms could make research and development far more affordable and easier to carry out for many LMICs.

Plant platforms, particularly whole plant-based platforms, are extremely simple, accessible and affordable to maintain. Plant cultivation requires only globally practiced basic agricultural techniques and low technological input to grow. 105 Many of the standard species used to create platforms are widely available and established agricultural crops in many LMICs around the

73 world, such as cereal grasses or cash crops like tobacco. 106-108 Compared to fermenter-based mammalian cell platforms, whole plant platforms are also relatively affordable to cultivate.

Instead of requiring costly cell culture medium, whole plant systems only demand that standard plant needs like light, carbon dioxide, minerals, water and nitrogen are fulfilled to ensure growth and protein production109-112.

Plant platforms are also beneficial with regards to biosafety, due to their insusceptibility to pathogens and their ability to be modified to prevent unwanted spread of genetic material. Plants are not susceptible to human or animal pathogens, so present far lower risks of pathogenic contamination than other protein producing platforms. In addition, few plant pathogens are known to be capable of harming humans or other animals. 113,114 By contrast, mammalian cell, yeast and bacterial cultures often must be maintained in sterile conditions and demand additional purification steps during product refinement to remove viruses and pathogens from raw material.

115 If biosafety is of particular concern for a whole plant platform, and it cannot be grown in a standard agricultural field, large scale growing operations are entirely possible to carry out indoors in greenhouses or vertical farming facilities. 116 Even though indoors operations for growing a plant platform may be more expensive, significant benefits come with contained growth, including the opportunity to maintain standard growth conditions that could promote higher and more consistent yields of product. 117 However, if outdoors growth is ultimately desired, biosafety barriers within the genome of and around the agricultural field for the plant platform can be implemented. These barriers include co-expressing male gamete sterility genes, targeting genetic modifications to plastid organelles to prevent gene spread through pollen, and selecting a growth site that is relatively isolated from other agricultural activity. 118

74 Whole plant platforms also have the potential to meet large scale production needs through simple scale up procedures and biomass production. Scaling up plant production for large scale testing is simple and incredibly inexpensive. If large volumes of protein are suddenly required, more individual transformed plants can simply be grown or planted to meet need. 119 Whilst more land would be required to accommodate such scale up, this scale up ability is comparatively far more flexible than that of other cell-based pharmaceutical production platforms, which are ultimately limited by the capacity of their bioreactor.120 The other scale-related benefit of using whole plant platforms is that many standard platform species, like tobacco or benth, produce extremely high amounts of biomass. 121 High amounts of biomass translate to more cells and tissue producing protein product, and thus potentially higher expression and yields of product.

The productivity of plants used in molecular pharming has refined to the point where whole plant platforms are currently estimated to express around 2g of protein per kilo of biomass. This level of production is in the same order of magnitude as the highest expressing mammalian cell platforms, which produce 5g of protein per liter of fermenter volume. 122

Plants are also strong platforms for quickly testing a wide range of candidates for a protein pharmaceutical. Whole plant platforms can use transient expression to express large-scale amounts of a protein product in a period spanning a few days to a few weeks. 123,124 This especially quick form of protein expression could allow the rapid development of proofs-of-concept for a drug or of candidate proteins that could then undergo testing. Plants are not only capable of quickly producing candidate pharmaceuticals but also are capable of producing a wider range of protein products that could be used to create a pharmaceutical.

75 Plants can produce most of the protein products that mammalian cell, bacteria, or yeast-based

platforms can, and also tackle toxic or difficult-to-assemble proteins beyond their capabilities. 125

Plants also can create complex proteins with most of the appropriate mammalian

post-translational modifications, which helps protein products avoid in-vitro “correction” steps

that could add to production expenses. 126 The most major post-translational modification that plants carry out differently is mammalian-type glycosylation, due to glycoform differences between mammals and plants. However, this glycosylation difference is not always an issue requiring correction. Sometimes, plant glycosylation differences lead to biosimilar plant proteins which work identically to counterparts with mammalian glycoforms. Occasionally, the glycosylation differences can even lead to biobetters like Elelyso, whose plant-type glycoforms help it carry out its therapeutic function better than its mammalian protein counterpart. 127 Even if the glycoform differences ultimately turn out to be undesirable, plant platforms have existing methods that can help correct glycoforms to a mammalian type, such as glycoform engineering or genetic modifications to the plant. 128,129

3.4.2.- Advantages for Manufacture

LMIC based companies or institutions planning later steps of pharmaceutical production beyond research and development, such as implementing large scale manufacturing processes, can also benefit from the advantages of whole plant platforms. Many of the advantages of plant platforms that are useful to research and development, such as easier scale-up or reduced susceptibility to pathogens, are also applicable to large scale production operations as well. Yet plants integrate

76 these shared benefits with other benefits that are specific to this later stage of pharmaceutical production, such as the set-up costs, the achievable scale of production and the speed with which demand can be met.

Plant platform-based facilities for larger scale manufacture of pharmaceuticals tend to have more affordable setup costs, due to how plants are grown and the inherent qualities of plants themselves. Compared to the costs of mammalian cell culture facilities with equivalent capacity, molecular pharming facilities tend to be more affordable to establish. 130 This is, in part, because a lot of the technology needed to grow and process the plant products are already widely used in other agricultural industries, which means that machinery and material can easily be repurposed for molecular pharming. 131,132 By virtue of using plants, molecular pharming facilities also tend to avoid many of the additional costs that come with using large scale mammalian cell, bacteria and yeast systems. These additional costs that plant platforms can avoid include the stringent facility sterility requirements needed to maintain cell cultures and the price of having to build, run and maintain a large fermenter or bioreactor. 133-135 Indeed, the manufacturing cost reductions that come from using plant platforms can even have positive impacts on the final price tag of a pharmaceutical they produce. At its time of release in 2012, a year’s worth of doses of Elelyso were estimated to be around 25% cheaper than the equivalent dosage of its main mammalian-cell produced competitor, Cerezyme. 136

The ease with which whole-plant platforms can be scaled up may also mean that meeting extremely large demand is more feasible. A biopharmaceutical that is relevant to an

LMIC-prevalent disease is likely to be required in great quantities if it is to effectively aid large

77 populations of sick people. Meeting such demand using fermenter-based protein production platforms is difficult for LMICs, since it is extremely expensive to maintain a large facility that would be capable of filling the need. 137 By contrast, whole plant platforms require only that more productive plants be sewn to increase production scale. This may mean that flexibly and affordably addressing great demand may be more feasible with a plant platform, provided that a large enough space is provided for growth.

Plant platforms are also capable of meeting needs for both rapid and reliable pharmaceutical production. In growth and manufacture, plant platforms can be tailored to two types of production speed: rapid, immediate production and reliable, steady production. For faster production requirements, transient expression can be harnessed to yield high amounts of protein within a few days. 138 Transient expression is thus an excellent choice for developing quick proofs of concept for a pharmaceutical, for producing products rapidly in crisis situations like pandemics or bioterrorism events, and for shortening time-to-market, which can help an innovative pharmaceutical launch faster and take advantage of market changes before competitor products. 139 Transgenic plants, whilst slower to produce protein in than transient expression-using systems, can be relied on for consistent and steady supplies of pharmaceutical protein. Transgenic plants are well suited to the production of seeds, which can be used to create more transgenic plants and to stably store the pharmaceutical protein in dry, room temperature conditions. 140 Seed-based storage thus provides an affordable option around using the expensive cold-chain storage methods necessary to keep other pharmaceutical products stable. 141

Transgenic plants are also suited to producing oral and topical pharmaceuticals. Since transgenic plants are permanently genetically altered, they can reasonably be expected to consistently

78 produce edible fruits and leaves containing a desirable protein product. Oral and topical products

have several theoretical benefits of their own, providing yet another alternative to cold-chain

stored products and not requiring both the medical training and potential risk of use

needle-administered pharmaceuticals. 142-144 The benefits and drawbacks of oral products themselves are illustrated in more detail in Chapter 2.

3.5- Solving Downstream Processing- Molecular Pharming's Costliest Step

Downstream processing is potentially the greatest source of costs in plant-platform based pharmaceutical production, and is a major disadvantage for plant based methods. Downstream processing refers to the series of manufacturing steps needed to recover and purify protein products created in a biological platform like a plant or a mammal cell line. Whilst the downstream processing steps used in plant molecular pharming are not radically different to those used in standard fermenter-based cell platforms, these steps also represent about 80% of total pharmaceutical production costs. 145

.Plant molecular pharming’s particularly expensive downstream processing is due to factors that include a high particle burden, the presence of host cell proteins and the release of numerous secondary metabolites during extraction. With plant based platforms, a high particle burden refers to the unique particulate contaminants that plants produce, like fibres and oils. These contaminants have the potential to foul downstream chromatography media and make later product separation steps far more difficult. To reduce the particle burden, a series of clarification steps is required, using techniques such as centrifugation, depth filtration or microfiltration to separate desired product from undesirable particulates of all sizes. 146,147 Another factor that

79 causes considerable issues for all biological production platforms is the presence of host cell proteins. The targeted protein product typically represents only a minor fraction of all the proteins within a given cell, tissue or organ. The host cell proteins, proteins that are not desired in the final product, must be removed to reduce the risk of unwanted immunogenic effects and to reduce effects on the potency and stability of the desired pharmaceutical protein product. 148 A final factor that plant platforms contend with is the presence of numerous secondary metabolites within plant tissue, like pigments and phenols. These metabolites can permanently bind to the target protein product and alter its function in unpredictable ways. 149 When a plant used in protein production has its tissue broken down and processed to extract the pharmaceutical product, the particulate contaminants, the host cell proteins and secondary metabolites all get released as well. As a result, numerous purification and clarification steps must be used to obtain the protein product in a pure, safe and use-able form.

Plant molecular pharming’s downstream processing issues have not gone unaddressed in research. One method of reducing downstream processing is by designing a plant-made protein pharmaceutical for an oral vaccination or therapeutic. Oral plant protein products still require processing, like dose consistency checks or lyophilization of the edible plant material, but far fewer processing steps are required overall, since plant tissues selected for oral use are entirely safe to eat. 150 Legal downstream processing requirements can also be reduced if the protein product is used in a veterinary or non-medical context (e.g. reagents or assay proteins)151,152.

Whilst these types of protein products are not human-targeted pharmaceuticals, they are all still extremely useful for public health interventions- helping to combat zoonoses or helping to develop effective assessments for a disease.

80 If the protein pharmaceutical desired does require extensive downstream processing, research has

developed numerous improvements and refinements on processing steps to make them both more

affordable and more efficient. . These refinements include improving established purification

techniques and introducing new, more effective, methods to the purification arsenal.

Choosing extraction methods like centrifugal extraction and screw presses has helped reduce

both host cell protein content and the particle burden of primary extracts. 153-155Effective clarification and filtration methods for other established platforms like mammalian cell cultures have also been adapted for the specific needs of plant-based platforms. These methods are accompanied by cost-reducing technologies and techniques like flocculation (the use of charged polymers to aggregate unwanted particulates together) or filter aids (particles of a pre-defined length and shape that collect the particulates collect as a film on a filter). 156 Even the environmental conditions of downstream processing have been modified to better suit plant production platforms. An acidic environment (pH ˜5.5) at moderate temperatures (~65C) and the prior use of ultrafiltration for molecular masses between 100-300kDa have both been effectively used to reduce both host cell protein content and total purification costs.157-159

Additionally, new separation methods reduce the purification steps needed, and make obtaining the target protein easier and cheaper. These newer methods include using fusion partners like oleosins or zeins that localize the protein product in easy to separate plant tissues or that get the plant to secrete the product into solution160,161. Whilst plant cell cultures are best known for being

able to secrete protein into solution, whole plants can be made to secrete as well when infected

with Agrobacterium rhizogenes. As long as the protein in question can enter the secretory

81 pathway and can stand the conditions of the fertilizer or hydroponic solution it is secreted to, opportunities for easier and cheaper protein separation exist.162.163

3.6- Conclusion: Plant Molecular Pharming as an Affordable Production Method

Overall, data suggest that the advantages of plant-based production platforms can lower costs of production to levels that are far cheaper than in standard fermenter based cell platforms.

Examples include the production of IL-10 in plants at 10-50x lower cost compared to E.coli fermentation. IL-10 is far more expensive to produce in E.coli as the protein must be refolded after purification and tests must be carried out to ensure the final product is endotoxin free. The additional costs of E.coli based production has led to a higher market price for the microbe-produced version of IL-10, and has made using it in research or therapy unsustainable.

164 Another example is seen in comparing the production of the diabetes autoantigen hGAD65 in plant-based platforms and conventional baculovirus-insect cell platforms. With similar development costs assumed and personnel costs ignored, production costs for the whole plant system were 0.7% of the costs for the insect cell platform. The greater cost of the insect cell platform was due to the need to maintain sterile cell culture and the constraints that the bioreactor environment placed on production scalability. 165 These two examples suggest that the cost cutting advantages of using plant-based platforms may lead to a pharmaceutical product that is affordable to develop and manufacture. For LMICs seeking a production method to establish their pharmaceutical production sovereignty with, the affordable, flexible and scalable plant-based platform may provide an interesting and exciting option.

82 Chapter 4- Hurdles for Biotechnological Sovereignty Using Plant Molecular

Pharming

4.1.- Introduction

Though molecular pharming has numerous theoretical advantages for LMICs seeking biotechnological sovereignty, efforts to actually implement the technology come with challenges that are often entangled with the need to secure funding. Funding is required to build facilities, to employ and train scientists and workers, and to cover the costs for setting up and maintaining the various stages of pharmaceutical development and manufacture. In LMICs, funding for molecular pharming projects has ranged considerably. On one end of the scale, South Africa’s government has provided small 3 year grants that fund research institutions to develop concepts for molecular pharming products. 1 On the other end, the Bio-Manguinhos unit of the Brazilian government’s Oswaldo Cruz Foundation ( a public health research institution) has invested around $180 million into developing a molecular pharming manufacturing facility. 2 Still more investments into molecular pharming efforts, LMIC-based or otherwise, have been secured from a wide variety of sources that range from international consortiums like the Newcotiana Project or Pharma-Planta, to private-sector funding. 3,4

Even though plant-based platforms are more affordable than other protein production platforms, their set-up, development and manufacture costs could still be prohibitive for many LMICs with little spending power. Large scale molecular pharming manufacturing facility projects, like

Brazil’s Centro Technologico de Plataformas Vegetais, Canada’s Medicago Inc. facility, and

83 Japan’s “METI Project” have had several tens of millions of dollars invested into them. .5,6

Additonally, these project costs do not account for other potential sources of production expenses

such as licensure. 7 South African efforts to fund plant molecular pharming research demonstrates some of the struggles LMICs may encounter in efforts to fund plant molecular farming research efforts. Though the South African government helped cover early research and development for molecular pharming products, the duration of grant provision and the amount of money provided per year were not enough to help cover later product development stages nor critical components of manufacture like downstream processing equipment. 8 On a larger scale,

the South African government's inability to cultivate extensive funding for local production

means that South Africa is presently unable to develop cGMP (good manufacturing practice)

compliant large-scale pilot facilities for plant molecular pharming, with decently sized facilities

typically costing around $20 million.9,10

As such, successfully establishing molecular pharming within LMICs will likely require external

financial support from public and private investors. Garnering financial support for LMIC-based

molecular pharming facilities and projects demands collaboration with other groups within the

biotechnology community. Partnerships between LMIC-based molecular pharming projects and

more established biotechnology groups are important to build throughout product development.

These established partners, whether pioneering molecular pharming companies or leading

academic institutions, can help mitigate the costs and financial risks of facility and product

development, alongside contributing to personnel training, technology transfer and research

efforts. 11,12 A concrete example of such an effort is Kentucky BioProcessing LLC and Icon

Genetics GmbH collaborating with the South African Council for Scientific and Industrial

84 Research (CSIR) to help develop two plant-expressed antibodies that could neutralize rabies .

Together, they developed a program that involved designing a pilot facility, transferring licensed

cutting-edge technologies, and collaboratively researching and refining the antibody products. As

a result, CSIR now has plans for developing good manufacturing practice (GMP) compliant pilot

facilities based in South Africa that could be scaled up to meet future commercial demand.13

Obtaining this financial support in the first place is dependent on whether potential partners

perceive a proposed molecular pharming effort to be reliable enough to risk an investment on.

Many investors, especially wealthy partners like larger pharmaceutical companies, are reluctant

to finance molecular pharming-based production efforts without proofs of reliability14. For a proposed molecular pharming effort, such proofs might include compliance to GMP requirements, which demonstrates that a facility or method reliably produces high quality products, or the creation and development of patents for products, methodologies and technologies, which might attract funding from investors interested in supporting promising innovations. 15,16

Investors’ perceptions of whether molecular pharming proposals in LMICs are reliable enough to receive funding are also often greatly influenced by sociopolitical factors beyond the laboratory.

As such, when evaluating molecular pharming’s potential for success in LMICs, an awareness of the sociopolitical factors that could impact both the technology itself and the willingness of investors like large pharmaceutical companies to fund it is critical. 17 Whilst numerous sociopolitical phenomena could affect plant molecular pharming’s success in an LMIC, significant themes repeatedly arise as cause of concern. This chapter will investigate three major

85 sociopolitical areas that warrant special consideration for proposed plant molecular pharming

projects: how regulation in LMICs may affect the success of the technology, how pre-existing

patents and patent laws may cause additional struggles during the development of new

technologies and products, and how public perceptions of GM crops may impact acceptance and

support for the biotechnology.

4.2.- Regulations

Establishing regulations for plant molecular pharming is necessary for a country to ensure the

quality of the biotechnology and its resulting protein products. Regulation often sets and defines

quality standards, like good manufacturing practice (GMP), that a manufacturing process must

meet if it is to be considered capable of producing products that are high quality, consistent and

safe-to-use. 18,19

Developing high standards of regulation for novel biotechnologies like molecular pharming is

important to making a case for their reliability and quality to potential investors. This is because

regulation acts as a safeguard against some of the risks of investing in innovation by providing a

standardized framework for manufacture and quality control.20 ,21 These established frameworks are especially important for conservative investors like large pharmaceutical companies, who will typically not support unregulated innovative technologies if an equivalent technology with regulation already exists. Yet, since creating these regulations is an arduous, long and expensive process, large pharmaceutical companies are also unwilling to invest in establishing regulation for new biotechnologies. Since smaller molecular pharming companies neither have the funds

86 nor the regulatory expertise of larger pharmaceutical companies, molecular pharming has struggled in the past with a catch-22 where both a lack of regulation and a lack of will to establish hindered the technology’s acceptance into the mainstream.22,23 However, in general, establishing regulation is a critical component in arguments to potential investors or investment in an innovative biotechnology.

Developing strong regulation standards is also critical to build the groundwork for international qualifications that could secure financial grants for public health projects. One such qualification is WHO prequalification, which involves the rigorous inspection of products and manufacturing facilities according to GMP standards. 24,25 Such qualifications are often essential to secure or compete for international funding from agencies that provide external grants for setting up public health interventions, like the Global Fund to Fight Against AIDS, Tuberculosis and

Malaria (GFATM). 26 In addition, quality qualifications like WHO prequalification are also essential for arguing the competitivity and reliability of an facility and biotechnology to private investors and large pharmaceutical industry companies. 27.28

4.2.1- LMICs and Establishing Molecular Pharming Specific Regulation

LMICs around the globe are at varying stages of establishing their own specific regulation for molecular pharming biotechnologies. Some LMICs, like South Africa, have more established and extensive regulation for molecular pharming whilst others, like Thailand, have little specific and defined regulation of their own. 29 As such, when assessing whether a country might be able to support molecular pharming successfully, each country’s progress with establishing appropriate regulation must be understood on a case-by-case basis.

87 Some LMICs are not in a good political or financial position to commit to developing regulation for molecular pharming biotechnology. For example, Tanzania has no coherent policy strategy for industry development, poor patent enforcement and stagnant investment in biotechnology.

This is in part due to a lack of political support from the country's political leaders for investing in local biotechnological development or intellectual property protection. 30 Similarly, Thailand, as a country with limited financial resources, is reluctant to risk investing in molecular pharming and to develop the associated regulation around the biotechnology. Instead, Thailand is hesitating until other countries with more established pharmaceutical manufacturing and regulatory systems approve of molecular pharming and use it more widely. 31 In short, some LMICs see an investment into molecular pharming as too large or too risky for them to carry out in their present circumstances.

Other LMICs that are investing in molecular pharming may define regulation for the biotechnology using pre-established guidelines in other countries as inspiration.

In Thailand’s case, for example, future drug approval systems for molecular pharming will likely take inspiration from the regulatory guidelines for the biotechnology that already exist in the US and EU. What this ultimately means is that the HICs that establish the first regulations for molecular pharming could greatly influence how the biotechnology is legally defined and treated in LMICs. Indeed, if a hypothetical molecular pharming product or production platform were approved first in an HIC like the US, its approval might also be accelerated in a given LMIC as well.32

88 However, the influence of existing HICs’ molecular pharming regulations means that their definitions and assertions about the biotechnology also should be considered carefully. Indeed, the currently existing regulations and guidelines established for molecular pharming are not without issue themselves. Thus, critically examining such issues and conflicts within established molecular pharming guidelines will be crucial for LMICs that want to develop their own laws for this biotechnology and that want to support GMP compliant and WHO-prequalified technologies in the future.

4.2.2- Existing Molecular Pharming Regulation

4.2.2.1- Adapting Prior Regulation for Molecular Pharming

Pre-existing regulation for molecular pharming, especially that of the EU, is largely based off of regulation developed for fermenter-based protein-producing platforms like microbes, yeast and animal cell systems. 33 The cell-culture based scaffold for molecular pharming regulation has had considerable effects on how plant-based production platforms have been defined in law and where the molecular pharming field has developed in response to regulation. For example, many molecular pharming companies have elected to focus on fermenter-based plant platforms for an easier path to regulatory approval, since they are similar to standard fermenter-based cell platforms.34 Despite this, the contained conditions of fermenter based plant platforms also take away many of the unique advantages that molecular pharming could have for LMICs, like the ease of production scale up or the relative affordability of early stage production.35,36

89 Whole plant systems, which are more affordable and flexible than fermenter-based platforms,

required the adaptation of existing regulations to accommodate them. However, existing

regulation only reflected the conditions and usage of fermenter-based platforms, and now needed

alteration to reflect the vastly different conditions and techniques for whole-plant based protein

production. 37,38

One example of how regulations were adapted from fermenter platforms is the regulatory

concept of the master and working seed banks for whole-plant systems, which has not been

adapted consistently across country lines. Fermenter platforms had the concept of master and

working cell banks, which respectively represent a preserved population of the original modified

cell line and the original line-derived cells that are actually used in manufacture. 39 This cell bank concept was the framework for the molecular pharming concept of the master and working seed bank, where master seeds and working seeds are used in a similar fashion to master and working cell lines. Master and working seed bank concepts translated well to transgenic and transplastomic plants, which each respectively maintain their introduced genes in the nuclear genomes and plastomes of their seeds. However, countries with molecular pharming regulation have variably adapted the master and working bank concept to transient expression, which does not permanently integrate the introduced foreign gene. In some countries, like the US and

Canada, both the bacterial or viral transformation vectors and the host plants used are maintained in master-working bank arrangements. 40.41 In other countries, like those of the EU, no adapted master-working bank arrangements specific to transient expression exist. This lack of specificity has caused numerous issues for molecular pharming projects in the EU, and has made transient expression notoriously difficult to pursue in this region. 42

90 Alongside regulatory inconsistencies are guidelines like strong demands for consistency that

make adopting certain platforms and methods of molecular pharming production more desirable.

Good manufacturing practice guidelines demand evidence that a production method is capable of

producing consistent amounts of quality protein product. This is harder to do in whole plant

systems, since they are complex organisms susceptible to a wide variety of biotic and abiotic

conditions that are not always easy to document and monitor.43 Conditions that could affect

consistency include risk of contact with insects and pesticides, nutrition available within the soil,

or light available to the plants at given times of day.44-46 To minimize the factors that could

impact protein product yield consistency, whole plant platforms are increasingly moving indoors.

In indoor facilities like greenhouses or growth chambers, growth conditions like light or mineral

nutrients can be tightly controlled, potentially increasing the consistency of plant growth and

protein production.47 However, indoors facilities are also more expensive to maintain when

compared to field growth and, though capable of meeting high demand, do place restrictions on

the extent to which production can be scaled up. 48, 49 Consistency regulations also assume that the utilities and resources needed to maintain constant conditions indoors, like electricity or water, are reliable and available. In many LMICs, the reliability of these utilities are not a given, and thus meeting the requirements of GMP with an ideal, completely indoors facility could be far harder than anticipated. 50,51

4.2.2.2- The GM Organism Identity of Whole Plant Platforms

Whole plant systems are also legally recognized as GM organisms in most existing molecular pharming regulations, which means that GM crop regulations must be considered on top of all

91 other regulations for a given product. This makes the plant-based production of pharmaceuticals especially difficult, since it warrants the simultaneous consideration of both stringent pharmaceutical and GM crop regulation. 52 Avoiding this regulatory burden is a significant reason why some pioneering molecular pharming companies have focused their attention on non-pharmaceutical applications of the biotechnology. 53

The GM identity of molecular pharming plants also has further ramifications for how they are grown and treated. Transgenic plants are, once again, strongly urged into contained conditions as their modified DNA can enter their gametes. The transgenic plants and their gametes, in turn, can pose risks to the surrounding environment if kept outside, such as crossing with food or livestock feed crops, or exposing local wildlife with product proteins. 54 If open field growth is insisted upon for a transgenic plant, existing legislation often demands extensive biosafety measures.

Methods to meet these biosafety demands may include both physical containment, such as isolation distances around growing fields, and biological containment, like genetically engineered sterility or cleistogamy (the self fertilization of closed flowers). 55 Mitigation strategies, like identifiable marker traits or genes conferring survival disadvantages outside of the contained field, are also included to help prevent the establishment of transgenic plants outside of the designated area. 56 However, some of these field-based containment and mitigation methods are not infallible. Geographic isolation, for example, is fairly unreliable at keeping transgenic plants from establishing themselves beyond the designated isolation perimeter.57,58 In addition, the mitigation strategies using survival inhibiting genes require that the genes do not stop working due to mutation and that they do not become genetically unlinked from the protein

92 producing transgene.59 By contrast, GM crops grown within the greenhouse or the laboratory have no documented cases of physical containment failure, and can reduce potentially harmful interactions with other organisms.60 Therefore, it is often far easier to grow such transgenic plants indoors to avoid the additional biosafety uncertainty that comes with outdoor growth.

In the case of transient plants, the bacterial or viral lines needed for agroinfiltration are defined and regulated as GM organisms, though the plants themselves never integrate the foreign DNA61

Thus, transient plants are also subject to stringent GM biosafety regulations to stop the release of the microbes or viruses to the environment. Transient expression is also subject to additional regulatory demands, including procedures for plant material disposal or the exclusion of potential vector organisms.62 Overall containment guidelines developed for HIC-based production also may not acknowledge the additional impacts that might be had if a containment breach of a GM plant, or other associated biological material, occurred in an LMIC. If the LMIC in question had fewer resources to rapidly and effectively cope with an escape, such an event may have downstream impacts on agricultural livelihoods or public and environmental health. 63

Whole plant platforms legally being deemed GM organisms can lead to difficulties in the international commercialization and trade of molecular pharming products. Countries across the world have variably strict legislation around whether GM products can be imported across their borders. 64,65 Whilst organic molecular pharming material like seeds or whole plants certainly comes under GM regulations, the question of whether purified products derived from plant molecular pharming also do remains unanswered. As such, a plant molecular pharming-derived product may be acceptable for import to one country but may be a regulation breaking issue in another.

93 Ultimately, the outlook for the international commercialization of molecular pharming materials

is far more challenging , especially for pharmaceutically-related products, because of their dual

legal identity as both pharmaceutical and GM products.,66,67,68

4.2.3.- Proposed Solutions to Regulatory Issues

Ideas have been proposed to help LMICs develop and navigate regulations for the molecular

pharming biotechnology. One such idea is selecting a product type that is inherently less

restrained by regulation, like a non-pharmaceutical product or a niche target disease. Whilst

non-pharmaceutical products cannot prevent or cure disease, they are not divorced from

supporting the health needs of an LMIC. Medically useful non-pharmaceutical protein products

include research reagents or immunological assays for diagnosis, which all could contribute to

efforts to affordably track, research and control spreading disease. 69 Non-pharmaceutical protein

production has also helped build industry confidence in molecular farming as a technology, with

a greater portfolio of products having reached the market. 70 If pharmaceutical production is desired, selecting niche disease targets like rare “orphan” diseases or seasonal diseases like the flu can also be beneficial. Niche disease target selection has sometimes been incentivized through authorities easing regulations in countries with established guidelines for production.71

Another strategy for developing regulation might be forming a regulatory consortium for an

LMIC, like the EU-funded Pharma-Planta Consortium. Pharma-Planta recruited the expertise of

academics, molecular pharming biotech companies and legislators to create a plant-produced

HIV-neutralizing antibody. Along the way, the molecular pharming process would undergo risk

assessments and, in collaboration with EU legislators, attempt to establish appropriate regulatory

94 oversight for the technology that would meet GMP, toxicity-testing and general regulatory

requirements. In 2011, the consortium successfully brought the antibody product to phase 1 and

provided the EU regulatory bodies with more information and better guidelines on how to

approach molecular pharming in legislation. ,72,73,74

Even the vague and relatively undefined nature of molecular pharming in regulation can be seen as an advantage that could be exploited. Whilst the lack of definition around molecular pharming methods and products means dealing with more case-by-case regulatory negotiations, it also allows molecular pharming proponents to choose from a wider variety of platforms and product types. 75 This gives molecular pharming groups the opportunity to legally authenticate a wider diversity of platforms and products under the PMF umbrella, and to fully exploit the technology’s characteristic flexibility.

4.3.- Patenting

Many of plant molecular pharming’s supporting techniques, technologies and materials are patented by the companies and research institutions that helped establish the field. Several important components of the molecular pharming production process have existing intellectual property (IP) patents attached to them. These include extremely successful vectors for transient expression (e.g. Icon Genetics' magnICON® vector), methods for chloroplast transformation

(e.g. the IP portfolio of Chlorogen, a now defunct startup), downstream processing technologies

(e.g. SemBioSys's oleosin fusion proteins), affordable large-scale bioreactor designs (e.g. a

95 plastic lined disposable culture chamber developed by Pennsylvania State University), or even

plant platforms themselves (e.g. Biolex, Inc.'s duckweed-based LEX system).76- 80

Filing intellectual property patents is beneficial for the HIC-based biotechnology companies who

file them, but less so for humanitarian programs and LMIC-based research institutions and

companies. Patents help stimulate greater investment into a biotechnology company’s new

property from other local and international companies, and help recoup costs from the expensive

development process. 81,82,83 However, the creation of numerous patents within a given biotechnological field also creates additional difficulties and expenses for humanitarian programs and for LMIC based research institutions and companies looking to introduce and use the biotechnology.

4.3.1- Problems Posed by Patenting to LMIC Producers

An aspect of patenting that is particularly troublesome for potential LMIC producers is achieving freedom to operate (FTO). Freedom to operate is the ability of a company or institution to develop, make and market a product in a given country without any legal obligations to or infringements on other existing patents. When a new biotechnology is adopted to help produce a new product, it is essential to ensure that freedom to operate exists at all stages of development and production. This is because infringing on the intellectual properties of other patent-holders can lead to expensive liability fees which can slow down or even stop product development.84 To use the existing intellectual properties legally, negotiations must be made between the innovators and the patent holders. An example of the extent to which patenting can impact humanitarian technological development is seen in the development of Golden Rice, a beta-carotene

96 expressing GM rice crop to be used in impoverished communities suffering vitamin A deficiencies. Golden Rice's production timeline was severely delayed by a tangle of intellectual property negotiations, including 16 patents and around 72 potential IP barriers. These IPs covered everything from which genes were used in the beta-carotene production pathway, to methods for isolating and cloning the DNA, to the techniques for regenerating the transgenic plants from calluses.85 Ultimately, the IP negotiations were resolved and Golden Rice was successfully created without the imposition of licensing fees by the patent holders. However, similar situations to that of Golden Rice could arise for other humanitarian biotechnology interventions, with far less forgiveness shown in terms of licensing fees.

Whilst determining freedom to operate is critical to successful production, it is also expensive, time consuming and more difficult to do with the resources available in many LMICs. The determination process for freedom to operate often requires extensive, lengthy search consultations and analyses to make sure a proposed product does not infringe on patents that exist in the countries in which it will be produced and marketed. On top of this, research methods and tools for determining freedom to operate are not equally use-able or available. The most financially accessible patent search engines are difficult to navigate and often can only provide limited information on listed patents due to agreements with commercial database providers. By contrast, the expensive-to-purchase commercial patent databases are easier to use and provide useful features and information for freedom to operate determination. In the case of many LMICs, such as India, an additional concern is that many existing patents within the country are not even electronically available for review, which makes determination an even lengthier and more arduous process. 86,87

97 Intellectual property patents might also not be fairly distributed, with the creation of ‘patent

thickets’ potentially preventing the entry of new participants into the biotechnological market.

Two primary goals behind patent creation are to spark investment and to maximize financial

return from licensing. The drive of numerous companies to obtain and, then, to maintain a patent

for a biotechnological process can lead to the creation of patent thickets. Patent thickets are

created when a particular technology is subject to numerous, overlapping patent claims from a

variety of industry and academic applicants. Navigating these intellectual property overlaps is an

arduous process, requiring many steps of consultation that slows the development of newer

technologies or protein products. 88 For LMIC-based companies, carving through these thickets

for their own biotechnological properties could be prohibitively expensive, especially when

accounting for increased difficulty in determining freedom to operate and the unlikelihood of

LMICs making the first patents in a given market. 89 Indeed, there is some suggestion that the raised costs of navigating and negotiating patent thickets hinders the entry of those seeking to create new patents for a technological field. 90

Patents might also be artificially extended through the practice of “patent evergreening”, which could harm technology transfer initiatives targetting LMIC producers. Patent evergreening artificially extends a company’s period of having exclusive rights to a patent by obtaining related protections for the product or method that was patented. Companies that practice evergreening on their intellectual property tend to repeatedly use the technique, creating an effectively permanent barrier to others using certain technologies or producing certain pharmaceuticals. 91,92

98 This bodes poorly for LMICs who intend to produce pharmaceuticals using novel biotechnology, since they could be financially and legally prohibited from selecting many intellectual properties.

This ultimately limits the choice of material, technique and product available to a given LMIC.

For example, many of the patented technologies of plant molecular pharming are core platforms, transformation techniques or research tools that would make research, development and manufacture easier to carry out. Patenting to maintain exclusivity of use for their creators could additionally harm technology transfer between HIC-based and LMIC-based institutions, compromising the ability of LMICs to develop the best biotechnological solutions to their healthcare needs. 93 A worrying trend in molecular farming suggests that pioneering companies

involved with plant molecular pharming may not necessarily prioritize humanitarian interests for

the technology. In a series of interviews conducted by Matthew Paul in 2015, with

representatives of 16 current and former molecular pharming companies, most of the

representatives recognized the potential of the biotechnology to benefit humanitarian use.

However, most of the representatives also indicated that meeting humanitarian obligations was

not a core part of their business practice, generally because humanitarian use was not compelling

to venture capital investors nor part of the conditions for the financial support that they received.

This series of interviews ultimately suggests that the majority of the molecular farming field,

which is primarily based in HIC companies and institutions, may not necessarily keep

humanitarian applications of the biotechnology in mind when developing patents and obtaining

protections for intellectual properties. 94 Ultimately, patenting which prioritizes the personal

interest of a company could undermine important collaborative relationships between public

sector institutions in HIC and LMIC countries.

99 4.3.2- Proposed Solutions to Patenting Issues

Proposals for making patent navigation easier and more accessible for LMICs seeking to implement cutting-edge biotechnologies generally involve approaches to licensing.

In many cases, free licensing of material and technologies out to LMIC producers is a known strategy. Existing humanitarian projects have implemented this core concept in various ways.

The Grand Challenges in Global Health grants demand that successful applicants ensure affordable access to their proposed research and the Pharma Planta Consortium explicitly stated that the processes and products it developed would be donated to LMICs free of charge. 95,96.97

Another strategy is the concept of socially responsible licensing, which negotiates intellectual properties in a way that keeps underserved communities at the forefront of the discussion, helping to ensure access to life-enhancing medicine or innovation.98,99There are several ways to practice socially responsible licensing. One proposed method is practicing market segmentation, where different prices or accessibilities are provided for different markets of varying wealth.

This might look like tiered pricing for a given product where the most affordable tiers are provided to LMICs, not asserting intellectual property rights in given LMICs, or granting further licenses to LMIC-based manufacturers on the basis of addressing humanitarian needs. A related concept includes placing conditions on patents for technologies or products with multiple uses in different markets. Socially responsible licensing might require that the intellectual property holder must cater to multiple markets, with the most preferential terms, access, or pricing treatment for the poorest users.100 More specific details on the economic and legal problems of patenting for biotechnology and how they could be resolved for LMICs are out of the scope of this paper. However, the topic warrants significant consideration when wrangling with the global humanitarian and trade difficulties a developing biotechnology may face.

100 4.4.- Perceptions of Plant Molecular Pharming

Public perception of plant molecular pharming is critical to consider as it impacts how successful the platform is perceived to be by the research community, the biotechnology industry and potential investors. Early attempts at commercial molecular pharming have experienced popular backlash in the past. This backlash became prominent during the early 2000s because of two crop contamination incidents on the part of Prodigene, Inc, a US-based molecular pharming company. In 2002, Prodigene was trialing transgenic maize crops that produced avidin in

Nebraska. After trials had been completed and the maize was harvested, soybeans were then sown. Unknowingly, volunteer plants from the previous transgenic maize trials had grown alongside them. Some of this transgenic maize eventually became co-mingled with the soybeans, and was eventually detected. The USDA, which has strict regulations against transgenic crop co-mingling, hit Prodigene with fines of $250,000 for failing to prevent this incident along with fees totaling $3 million to destroy the tainted soybean crop and clean all facilities and equipment.101,102 In the second incident, which took place in Iowa, the avidin-producing maize line cross-pollinated with non-transgenic maize in an adjacent field, which led to the destruction of 150 acres of potentially contaminated maize.103 The accumulation of so many costs and fines ultimately led to Prodigene undergoing liquidation.104,105

Unfortunately, this series of incidents had coincided with a growing and increasingly public anti-GM movement in the West. Environmental groups like Greenpeace and food safety groups like the Center for Food Safety saw Prodigene’s contamination incidents as evidence that GM

101 plant technology was not good or safe enough for use. 106,107 The influence of this growing anti-GM sentiment in the public was considerably harmful to plant molecular pharming’s early development as a field. For example, other early molecular pharming companies like Ventria

Bioscience faced backlash from rice growers and the Anheuser Busch beer company when proposing field trials for a GM rice line in Missouri. 108 Critically, anti-GM attitudes also started to globally influence law concerning GM products, increasing pharmaceutical industry hesitancy around plant molecular pharming and placing more restrictions on the biotechnology’s development. 109

Anti-GM sentiment continues to be influential within political spheres all across the world, and has influenced legislation, debate and government funding. For example, the vocal concern and uncertainty of the European public and anti-GM lobbying groups has influenced the EU to take a

"precautionary approach" in legislation regarding upcoming GM plant-based technologies. The precautionary approach regards GM plant technologies as inherently hazardous in nature.110,111

Such an approach has meant that EU-based GM plant biotechnologies need stringent safety testing, are subject to extremely strict biosafety thresholds, and are more difficult to trade with other countries, amongst many other restrictions. Many high-income EU member states go even further with restrictive GM legislation. The Austrian government, for example, has been known to be vocally anti-GM and has had provincial authorities approach the EU to establish GM-free zones within the country.112

Anti-GM political attitudes are also prevalent within many LMIC governments as well. For example, in Mexico, the current president, Andrés Manuel López Obrador, and the director of

102 the national science budget, Dr Elena Álvarez-Buylla, are both opposed to GM research in

field-grown food crops like maize.113,114 GM-based biotechnologies are passionately debated in

other LMICs like Ghana, where anti- and pro- GM parties and activist groups wage legal battles

over whether the introduction of GM to the local market should occur.115 Still other LMICs, like

Venezuela or Algeria, have banned the import and cultivation of GM plant material or seeds entirely116,117 However, this is not to suggest that all countries, especially LMICs, have an inherently prohibitive political and legal relationship with GM plant technologies. LMICs like

Brazil, Argentina and South Africa have considerably more permissive GM food and crop legislation, especially as all three produce GM crops for agricultural purposes.118.119.120 Indeed,

Brazil and South Africa are already pursuing molecular pharming technology as part of their public health infrastructure.121

4.4.1. - Public Opinion on Molecular Pharming

The variety of global political stances and debates around GM are suggestive of a diverse range of public attitudes that could exist towards molecular pharming and related biotechnologies. The attitudes of the general public are extremely important to consider, since they will ultimately use and be affected by a protein product created using plant molecular pharming. Some evidence suggests that the presence of stricter GM regulations in a country make the general public view stricter regulations towards similar biotechnologies more favorably. 122 Other influences on public attitudes towards GM plant-based biotechnologies may draw on historical experiences of

GM introduction to or usage within a given country. For example, in 2002, many Southern

African countries experienced a food crisis brought about by a complex combination of weather patterns, health issues like HIV/AIDS, poor governance and poorly regulated agricultural sectors.

103 Other countries, including the US, provided food aid to address the crisis. However, the US included GM maize in their provisions and did not provide non-GM alternatives to this product for recipient countries, despite demonstrating that they could segregate GM and non-GM maize products. This was met with great public and political concern by countries like Malawi,

Mozambique, Zambia and Zimbabwe, who worried about the GM crops' effects on health, biodiversity, economic strength and burgeoning trade relationships. These Southern African countries were particularly concerned about the impact of receiving GM crops on their political relationships with the EU, which had recently taken a strict legislative approach to this biotechnology. As a result of this incident, some of the Southern African community perceived the US’s unwillingness to back down on donating GM maize as a foreign policy ploy that used their crisis to conveniently isolate the EU as a prominent GM-critical power.123 Past experiences such as these can lead LMICs to adopt an inherently cautious approach towards GM-based biotech like molecular pharming, especially if the technology is being introduced to them by companies or institutions from HICs like the US.

The social attitudes of the public towards GM-plant based biotechnologies are critical to evaluate as they can inform how willingly public and private investors may support plant molecular pharming. It is far harder to make a case for supporting a novel biotechnology like plant molecular pharming if the general public greatly opposes it. During the early 2000s, when anti-GM attitudes and the Prodigene incidents were prominent in the public consciousness, many large multinational agricultural companies like Monsanto or Syngenta ended their developing molecular pharming departments and withdrew from pursuing the biotechnology altogether. This

104 withdrawal from the biotechnology was partially attributed to potential backlash from the media and the public, alongside the risks of anti-GM activists destroying trial fields and sites.125,126

Whilst public attitudes vary greatly across the globe and from circumstance-to-circumstance, some themes and concerns in studies of sentiment towards molecular pharming and GM plant crops are consistently found. Knowing these attitudes is critical for any effort to introduce or establish plant molecular pharming for local biotechnological production in an LMIC. As such, themes of public concern and understanding around plant molecular pharming and GM-plant technologies are discussed below, drawn from numerous studies and surveys from communities across the world. Emphasis must be made that the themes discussed are fairly general, and that different countries, communities and groups will almost certainly have different perspectives based on current and past relationships with GM plant biotechnologies and the institutions that try to implement them.

Numerous themes on how people draw conclusions about or choose to trust GM technologies like molecular pharming are present within the literature. Often, a person's opinions on GM biotechnology are based on perceptions of how much they know about the technology versus factual knowledge of the technology.127 How much an individual tends to trust molecular pharming or GM plant biotechnology also draws from their own perceptions and experiences.

For example, the level of trust people feel towards the political, scientific or business institutions involved in research and production often plays a significant role in how accepting they are of a new biotechnology.128 In a similar vein, people often weigh up their stances on novel biotechnologies along personal cost-benefit analyses. These cost-benefit analyses often lead to

105 more positive views of medically-oriented biotechnology like plant molecular pharming as compared to agricultural biotechnology like GM food crops.129 In general, when the public is asked to consider plant-based biotechnology, medical and humanitarian applications like those of plant molecular pharming tend to be more favoured.130-133

The public has had consistent worries about plant molecular pharming and related GM-plant technologies which tend to concern the strength of containment, the quality of technological regulation and the stakeholders benefitting off of the biotechnology’s development. Fears around biological containment of GM plant biotechnologies are common. With prominent incidents like

Prodigene's maize contamination event in the popular consciousness, members of the public frequently express that they are concerned about the effects of a GM plant's escape on the environment, on the food supply and on their community.134-138 Public distrust is also often vocalized towards the companies developing GM plant biotechnologies, alongside the regulatory bodies who manage them. Many people, particularly those in countries with strict GM regulations, believe that their regulatory authorities are incompetent, and will not manage biotechnologies like molecular pharming with an appropriate degree of strictness and caution.139,140,141Finally, the public is also generally concerned about who ultimately benefits off of the mainstream adoption of a biotechnology like plant molecular pharming. Many members of the public worry whether their interests and concerns will be appropriately represented and prioritized over those of powerful corporate stakeholders as a GM plant biotechnology is developed.142-144 Though the above public concerns tend to be expressed across a variety of countries and age groups, a person’s own background can further dictate how much trust they show molecular pharming and related GM plant biotechnologies. For example, scientists

106 involved in GM biotechnology and non-organic farmers tend to view molecular pharming more optimistically, whereas environmental and anti-GM activists, organic farmers, and regulatory personnel tend to be far more skeptical of its promise.145

4.4.2- Proposed Solutions to Public Perception Issues

Several solutions have been proposed by the scientific community to ensure that biotechnologies like plant molecular pharming are received well by the general public. One such proposal is using educational interventions to help build understanding and trust. Experts in the plant molecular pharming field believe that public resistance stems from either a lack of general scientific knowledge about the biotechnology or from active misinformation by the biotechnology’s opponents, like consumer or environmental organizations. 146,147 Empirical evidence exists to support these in-field beliefs. Studies indicate that members of the general public do not seem to be aware of plant molecular pharming as a biotechnology. In addition, independent data collection conducted by the author suggests that public information on molecular pharming is either factually sparse or written for a specialist scientific or investing audience. 148,149 There is also some suggestion that a higher level educational background also raises support for plant molecular pharming. 150 Altogether, these pieces of evidence suggest science-education based interventions may be helpful for creating an informed public awareness of molecular pharming.

However, assuming that the public may only be acting on either an informational deficit or misinformation when resisting a biotechnology is also reductive. This assumption fails to realize that public trust of the technology and its creators is also a significant component of acceptance.

107 In many cases, providing factual information on plant molecular pharming and related GM

agricultural technologies may not be enough to sway public opinion. An example of this was

seen in the early 2000s and 2010s, when the German Federal Ministry of Education and

Research attempted to hold several educational and round-table discussions on GM crops with

the public. Despite the provision of a free-to-access online directory of GM crop hazard

detection results, the implementation of public discussions with organizations opposed to GM,

and the organization of public educational seminars, widespread opposition to GM remained. 151

Germany’s example strongly suggests that education-based interventions alone are insufficient for building support for a biotechnology. Other studies suggest that members of the public may be well educated on a biotechnology but oppose it based on social distrust of the technology or those developing it. 152 Furthermore, studies also suggest that people may develop their opinion based on their own perceived knowledge of a biotechnology, perhaps including its riskiness, versus empirical facts about it. 153 As such, social interventions that help build public trust in the scientific and commercial institutions developing molecular pharming should be considered a critical component to ensuring its success.

One method for building public trust in biotechnologies is the concept of upstream public engagement, proposed by James Wilsdon and Rebecca Willis in “See Through Science”. The authors argue that for biotechnologies, education-based interventions or even 'downstream' round-table discussions are not good enough for building public trust. This is because these standard public trust interventions take place after the technology is already fully realized and prepared for exploitation. With the public largely unable to give input on a technology's development and research priorities, downstream discussions and education methods only allow

108 the public to give commentary on how they would want to manage the risks of a pre-formed technology. As a result, public distrust towards the biotechnology and its creators remains because public concerns about the upstream development process, such as who the technology benefits, what impacts the technology could have on the environment, or why the new technology is a necessary solution, remain unaccounted for. By moving public interactions upstream into the actual development process, the public is allowed to effectively voice these questions to scientific and industrial developers, to influence the direction of research, and to give input on the technology's political, economic and institutional commitments.

Alongside upstreaming, Wilsdon and Willis also advocate for a policy of field transparency on the part of biotechnological developers. To practice transparency effectively, developers must articulate their intents and social visions for the biotechnology and demonstrate the incorporation of public critiques into the development process. 154 Upstream engagement and field transparency would help build public sentiments of agency over and accountability for the biotechnological development process, which could result in greater overall public trust.

Upstream engagement could be particularly beneficial for constructively engaging opposed activist NGOs and the popular media, two groups often considered to be sources of misinformation in the scientific community. In the case of NGOs, actively involving them in the technological development process may get them to clearly articulate their positions, concerns and interests from the onset of development. Using this approach has actually been successful in practice for NGOs concerned about nanotechnology. Recognizing that nanotechnology researchers were considering using upstream engagement during the early stages of their technological development process, the NGO Greenpeace took action to make their views and

109 concerns known. After discussions with nanotechnology scientists, businesses and policymakers,

Greenpeace created a 72 page document outlining subtly considered risk assessments, societal and environmental concerns and a cautious, but not prohibitive, stance on the new technology.

155,156 If dealt with appropriately throughout development, plant molecular pharming could hope for similar results when engaging opposed NGOs. In a similar fashion, instead of mischaracterizing journalists and the media as sources of public hysteria and misinformation, scientists and manufacturers must also engage with the media in earnest about their novel biotechnology. One manner of doing this might be driving public engagement in the media through connecting public interests and concerns to the novel biotechnology versus writing purely factual scientific pieces about it. 157 Other recommendations and approaches to developing public trust are articulated both in See Through Science and in many other excellent pieces not discussed here, but a core message about the necessity of trust-building remains. In short, transparency of intent and serious engagement with the concerns, needs and questions of the public are probably best for kindling trust between biotechnological innovators and general society.

110 Conclusion

This piece has demonstrated that factors that may create significant barriers to the success of molecular pharming in low and middle income countries (LMICs) are not scientific but rather societal in nature. Molecular pharming brings a series of uniquely advantageous traits to protein production, including flexibility, affordability and an ease of scale-up, alongside most of the protein production abilities of a mammalian cell platform. Plant platforms can be transformed using transgenic and transient expression techniques that allow them to respectively meet sustained and sudden demand for a given protein product. Such platforms also provide a wide choice of cultivation methods, species and tissue types to express a protein within, which can confer additional beneficial properties to the product including edibility, stable dry storage, or ease of separation. Most of the technological barriers that have faced plant-based protein production platforms, like plant material processing or product yield, are gradually being overcome as the field evolves and grows. Indeed, it could be said that the scientific progress for plant platforms is going through the standard 'growing pains' of developing biotechnologies, much like those once experienced by predecessor platforms like bacteria or mammalian cells.

This is not to say that the scientific and technological challenges faced by molecular farming are trivial. Remaining challenges to overcome include refining aspects of large scale production, such as the the development of high throughput screening methods or indoor facilities that support numerous organisms, dealing with the absence of mammal-specific post translational modifications in plants (like tyrosine sulfation or gamma-carboxylation), or combatting proteolysis-based loss of product during purification1,2,3. However, promising technologies that

111 are capable of facing these issues do exist and are already being implemented. Newer technologies like plant cell packs and farming methods like hydroponics and vertical farming facilities hold promise for enabling larger scale plant-based production.4,5. Highly refined genetic modification technologies, like CRISPR-Cas9 mediated gene alteration, are also currently being applied to alter the suite of post translational modifications that plants are capable of performing.6 In addition, newer forms of analysis and proteolysis prevention, like proteomics surveys and targeted gene knockouts respectively, could be used to identify and tackle enzymes involved in the proteolysis of a given protein product7,8.

Molecular pharming's flexibility, ease of technology transfer and relative affordability, amongst many other reasons, makes it a prime candidate for humanitarian applications of biotechnologies.

One particularly promising application for molecular pharming is establishing biotechnological sovereignty in low and middle income countries (LMICs), who continue to be socially, politically and economically harmed by a necessary dependence on exports from pharmaceutical companies based in higher income countries. Molecular pharming has the potential to relieve this dependence burden, bringing the focus of research, development and production decisions to the local markets and needs of these poorer countries. Although the theoretical benefits of local protein production using plant-based platforms are considerable, significant risk to the technology's social acceptance, uptake and rapid development also exists. As molecular pharming occupies a boundary between medicinal and agricultural biotechnologies, it also takes on the legal and social baggage of using genetically modified plant material, which is a contentious topic in many LMICs. On top of its ambiguous biotechnological identity, the patenting of many molecular pharming techniques, platforms and processing methods makes

112 navigating and developing new intellectual properties a more difficult task for LMICs hoping to use the technology.

Highlighting the complexity and seriousness of these beyond-the-laboratory challenges for molecular pharming is essential to ensure the technology is properly able to address the societal problems it has the potential to help with. Not anticipating and preparing for factors like public concerns or extensive legal barriers can create problems for the technology at later stages of implementation or development. For example, it might mean that obtaining funding for a molecular pharming protein production project is more difficult, as investors stay away from products that are not as easy to move through the regulatory process or that bring about cautious or negative public responses.

In addition, the three main issues discussed as significant roadblocks for molecular farming are far from the only societal problems that could impact the development and success of the biotechnology in LMICs. Other issues that could impact the success of the biotechnology include the strength of existing health, agricultural and technological infrastructure, the ability of a country to support and retain a community of highly-skilled scientists and the existence of collaborative relationships between LMIC partners and international industrial, academic and not-for-profit groups involved in molecular pharming,9,10,11. It must also be noted that whilst innovative technologies like molecular pharming will certainly help with pharmaceutical supply and public health issues in LMICs, it is unlikely to be a silver bullet solution for the numerous complex health and healthcare problems faced by their populations. Efforts that could make significant impact, in tandem with the introduction of new biotechnologies, also target

113 addressing and improving 'unglamorous' basic factors. In 'See-Through Science', such looking

beyond the immediate vicinity of the biotechnology to larger background issues is termed

whole-system innovation by Wilsdon and Willis. In the case of micro-power plant technology

Wilsdon and Willis discuss, whole system innovation would not just examine financial

incentives and technological policy that directly affects in- home energy generation technologies,

but would also account for planning, land use and building design.12 Similarly, with molecular pharming, simultaneously bolstering efforts towards disease spread prevention and increasing a country's ability to support healthcare and biotechnology would also benefit the technology's success. Such support might include helping to increase the availability and reliability of utilities like water and electricity, strengthening local healthcare and hospital infrastructure, or building strong educational resources.

In short, for emerging technologies with significant potential in humanitarian spheres, like molecular pharming, looking beyond the laboratory for factors that could impact the technology is a must. An awareness of these factors and developing tactics for addressing them, like upstream involvement of the public or developing the support and groundwork needed for building appropriate regulations and legislation for the technology is essential for ensuring the success of the technology in its humanitarian purpose. It is hoped that this piece will help bring attention, thought and consideration to large areas of concern and inspire similar analyses in other areas and efforts for biotechnologies like molecular pharming and beyond. When this happens, anticipatory action can be taken in future to ensure a technology is implemented well and best represents the interests of the people it will affect.

114 115 Intro- Citations

1. Jozala AF, Geraldes DC, Tundisi LL, Feitosa VA, Breyer CA, Cardoso SL, Mazzola PG, Oliveira-Nascimento L, Rangel-Yagui CO, Magalhães PO, Oliveira MA, Pessoa A Jr. Biopharmaceuticals from microorganisms: from production to purification. Braz J Microbiol. 2016 Dec;47 Suppl 1(Suppl 1):51-63. doi: 10.1016/j.bjm.2016.10.007. Epub 2016 Oct 26. PMID: 27838289; PMCID: PMC5156500.

2. Walsh G. Biopharmaceutical benchmarks 2014. Nat Biotechnol. 2014 Oct;32(10):992-1000. doi: 10.1038/nbt.3040. PMID: 25299917.

3. Rai M, Harish P. Expression Systems for Production of Heterologous Proteins. Current Science.c2001; 80(9): 1121–1128. JSTOR, www.jstor.org/stable/24105768. Accessed 6 Apr. 2021.

4. Bhatwa A, Wang W, Hassan YI, Abraham N, Li XZ, Zhou T. Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications. Front Bioeng Biotechnol. 2021 Feb 10;9:630551. doi: 10.3389/fbioe.2021.630551. PMID: 33644021; PMCID: PMC7902521.

5. Legastelois I, Buffin S, Peubez I, Mignon C, Sodoyer R, Werle B. Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules. Hum Vaccin Immunother. 2017 Apr 3;13(4):947-961. doi: 10.1080/21645515.2016.1260795. Epub 2016 Dec 1. PMID: 27905833; PMCID: PMC5404623.

6. Wildt S, Gerngross TU. The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol. 2005 Feb;3(2):119-28. doi: 10.1038/nrmicro1087. PMID: 15685223.

7. Du, C. (2011). Comprehensive Biotechnology || Cellular Systems. , (), 11–23. doi:10.1016/B978-0-08-088504-9.00080-5

8. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

9. Buyel JF, Twyman RM, Fischer R. Very-large-scale production of antibodies in plants: The biologization of manufacturing. Biotechnol Adv. 2017 Jul;35(4):458-465. doi: 10.1016/j.biotechadv.2017.03.011. Epub 2017 Mar 25. PMID: 28347720

10. Schillberg S, Finnern R. Plant molecular farming for the production of valuable proteins - Critical evaluation of achievements and future challenges. J Plant Physiol. 2021 Mar-Apr;258-259:153359. doi: 10.1016/j.jplph.2020.153359. Epub 2021 Jan 5. PMID: 33460995.

11. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

116 12. Fischer R, Emans N. Molecular farming of pharmaceutical proteins. Transgenic Res. 2000;9(4-5):279-99; discussion 277. doi: 10.1023/a:1008975123362. PMID: 11131007.

13. Schillberg S, Finnern R. Plant molecular farming for the production of valuable proteins - Critical evaluation of achievements and future challenges. J Plant Physiol. 2021 Mar-Apr;258-259:153359. doi: 10.1016/j.jplph.2020.153359. Epub 2021 Jan 5. PMID: 33460995.

14. Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 2001 May;6(5):219-26. doi: 10.1016/s1360-1385(01)01922-7. PMID: 11335175; PMCID: PMC5496653.

15. Juarez P, Virdi V, Depicker A, Orzaez D. Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications. Plant Biotechnol J. 2016 Sep;14(9):1791-9. doi: 10.1111/pbi.12541. Epub 2016 Feb 13. PMID: 26873071; PMCID: PMC5067594.

16. Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM. Plant-based production of biopharmaceuticals. Curr Opin Plant Biol. 2004 Apr;7(2):152-8. doi: 10.1016/j.pbi.2004.01.007. PMID: 15003215.

17. Ma JK, Christou P, Chikwamba R, Haydon H, Paul M, Ferrer MP, Ramalingam S, Rech E, Rybicki E, Wigdorovitz A, Yang DC, Thangaraj H. Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies. Plant Biotechnol J. 2013 Dec;11(9):1029-33. doi: 10.1111/pbi.12127. Epub 2013 Oct 14. Erratum in: Plant Biotechnol J. 2017 Jan;15(1):144. PMID: 24119183.

18. Buyel JF, Twyman RM, Fischer R. Very-large-scale production of antibodies in plants: The biologization of manufacturing. Biotechnol Adv. 2017 Jul;35(4):458-465. doi: 10.1016/j.biotechadv.2017.03.011. Epub 2017 Mar 25. PMID: 28347720

19. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

20. Buyel JF, Fischer R. Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs. Biotechnol Bioeng. 2012 Oct;109(10):2575-88. doi: 10.1002/bit.24523. Epub 2012 May 1. PMID: 22511291.

21. Xu S, Gavin J, Jiang R, Chen H. Bioreactor productivity and media cost comparison for different intensified cell culture processes. Biotechnol Prog. 2017 Jul;33(4):867-878. doi: 10.1002/btpr.2415. Epub 2017 Jan 28. PMID: 27977910.

117 22. Sack M, Rademacher T, Spiegel H, Boes A, Hellwig S, Drossard J, Stoger E, Fischer R. From gene to harvest: insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants. Plant Biotechnol J. 2015 Oct;13(8):1094-105. doi: 10.1111/pbi.12438. Epub 2015 Jul 27. PMID: 26214282.

23. Commandeur U, Twyman RM, Fischer R. The biosafety of molecular farming in plants. Ag. Biotech. 2003; Net 5: 1-9.

24. Kim JS, Yoon SJ, Park YJ, Kim SY, Ryu CM. Crossing the kingdom border: Human diseases caused by plant pathogens. Environ Microbiol. 2020 Jul;22(7):2485-2495. doi: 10.1111/1462-2920.15028. Epub 2020 May 4. PMID: 32307848.

25. Mor TS. Molecular pharming's foot in the FDA's door: Protalix's trailblazing story. Biotechnol Lett. 2015 Nov;37(11):2147-50. doi: 10.1007/s10529-015-1908-z. Epub 2015 Jul 7. PMID: 26149580; PMCID: PMC4583803.

26. Aggarwal S. What's fueling the biotech engine--2008. Nat Biotechnol. 2009 Nov;27(11):987-93. doi: 10.1038/nbt1109-987. PMID: 19898448.

27.Chen Q, Davis KR. The potential of plants as a system for the development and production of human biologics [version 1; peer review: 3 approved]. F1000Research 2016, 5(F1000 Faculty Rev):912 (https://doi.org/10.12688/f1000research.8010.1)

28. Gengenbach BB, Müschen CR, Buyel JF. Expression and purification of human phosphatase and actin regulator 1 (PHACTR1) in plant-based systems. Protein Expression and Purification. 2018 Nov;151:46-55. DOI: 10.1016/j.pep.2018.06.003.

29. Covarrubias AA, Cuevas-Velazquez CL, Romero-Pérez PS, Rendón-Luna DF, Chater CCC. Structural disorder in plant proteins: where plasticity meets sessility. Cell Mol Life Sci. 2017 Sep;74(17):3119-3147. doi: 10.1007/s00018-017-2557-2. Epub 2017 Jun 22. PMID: 28643166.

30. Gengenbach BB, Keil LL, Opdensteinen P, Müschen CR, Melmer G, Lentzen H, Bührmann J, Buyel JF. Comparison of microbial and transient expression (tobacco plants and plant-cell packs) for the production and purification of the anticancer mistletoe lectin viscumin. Biotechnol Bioeng. 2019

31. Mor TS. Molecular pharming's foot in the FDA's door: Protalix's trailblazing story. Biotechnol Lett. 2015 Nov;37(11):2147-50. doi: 10.1007/s10529-015-1908-z. Epub 2015 Jul 7. PMID: 26149580; PMCID: PMC4583803.

32. Ward BJ, Makarkov A, Séguin A, Pillet S, Trépanier S, Dhaliwall J, Libman MD, Vesikari T, Landry N. Efficacy, immunogenicity, and safety of a plant-derived, quadrivalent, virus-like particle influenza vaccine in adults (18-64 years) and older adults (≥65 years): two multicentre, randomised phase 3 trials. Lancet. 2020 Nov 7;396(10261):1491-1503. doi: 10.1016/S0140-6736(20)32014-6. Epub 2020 Oct 13. PMID: 33065035.

118 33. Dhama K, Natesan S, Iqbal Yatoo M, Patel SK, Tiwari R, Saxena SK, Harapan H. Plant-based vaccines and antibodies to combat COVID-19: current status and prospects. Hum Vaccin Immunother. 2020 Dec 1;16(12):2913-2920. doi: 10.1080/21645515.2020.1842034. Epub 2020 Dec 3. PMID: 33270484; PMCID: PMC7754927.

34. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

35. Case Studies In Agricultural Biosecurity [Internet]. Federation of American Scientists;c2011. The Prodigene Incident. [Cited 2021 April 28]. Available from: https://biosecurity.fas.org/education/dualuse-agriculture/2.-agricultural-biotechnology/ prodigene-incident.html.

36. Drake PM, Thangaraj H. Molecular farming, patents and access to medicines. Expert Rev Vaccines. 2010 Aug;9(8):811-9. doi: 10.1586/erv.10.72. PMID: 20673006.

119 Chapter 1-Citations

1. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

2. Burnett, MJB, Burnett, AC. Therapeutic recombinant protein production in plants: Challenges and opportunities. Plants, People, Planet. 2020; 2: 121– 132. https://doi.org/10.1002/ppp3.10073

3. Lacroix B, Citovsky V. Biolistic Approach for Transient Gene Expression Studies in Plants. Methods Mol Biol. 2020;2124:125-139. doi: 10.1007/978-1-0716-0356-7_6. PMID: 32277451; PMCID: PMC7217558.

4. Xu J, Dolan M, Medrano G, Cramer C, Weathers, P. Green factory: Plants as bioproduction platforms for recombinant proteins. Biotechnology Advances.2011; 30: 1171-84. 10.1016/j.biotechadv.2011.08.020.

5. Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM. Seed-based expression systems for plant molecular farming. Plant Biotechnol J. 2010 Jun;8(5):588-606. doi: 10.1111/j.1467-7652.2010.00511.x. PMID: 20500681.

6. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

7. Peyret H, Lomonossoff GP. When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol J. 2015 Oct;13(8):1121-35. doi: 10.1111/pbi.12412. Epub 2015 Jun 12. PMID: 26073158; PMCID: PMC4744784.

8. Gelvin SB. Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiol Mol Biol Rev. 2003 Mar;67(1):16-37, table of contents. doi: 10.1128/mmbr.67.1.16-37.2003. PMID: 12626681; PMCID: PMC150518.

9. Hwang HH, Yu M, Lai EM. Agrobacterium-mediated plant transformation: biology and applications. Arabidopsis Book. 2017 Oct 20;15:e0186. doi: 10.1199/tab.0186. PMID: 31068763; PMCID: PMC6501860.

10. Nester EW. Agrobacterium: nature's genetic engineer. Front Plant Sci. 2015 Jan 6;5:730. doi: 10.3389/fpls.2014.00730. PMID: 25610442; PMCID: PMC4285021.

11. Gordon JE, Christie PJ. The Agrobacterium Ti Plasmids. Microbiol Spectr. 2014 Dec;2(6):10.1128/microbiolspec.PLAS-0010-2013. doi: 10.1128/microbiolspec.PLAS-0010-2013. PMID: 25593788; PMCID: PMC4292801.

12. OpenLearn: Free Learning from the Open University [Internet]. UK: The Open University. Using A. tumefaciens to Genetically Modify Plant Cells [cited 2021 April

120 05]. Available from: https://www.open.edu/openlearn/science-maths-technology/science/biology/gene-man ipulation-plants/content-section-2.3.

13. OpenLearn: Free Learning from the Open University [Internet]. UK: The Open University. From Infected Cells to Transgenic Plants [cited 2021 April 05]. Available from: https://www.open.edu/openlearn/science-maths-technology/science/biology/gene-man ipulation-plants/content-section-2.4.

14. Lacroix B, Citovsky V. Biolistic Approach for Transient Gene Expression Studies in Plants. Methods Mol Biol. 2020;2124:125-139. doi: 10.1007/978-1-0716-0356-7_6. PMID: 32277451; PMCID: PMC7217558.

15. Liu J, Nannas NJ, Fu FF, Shi J, Aspinwall B, Parrott WA, Dawe RK. Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize. Plant Cell. 2019 Feb;31(2):368-383. doi: 10.1105/tpc.18.00613. Epub 2019 Jan 16. PMID: 30651345; PMCID: PMC6447018.

16. Lacroix B, Citovsky V. Biolistic Approach for Transient Gene Expression Studies in Plants. Methods Mol Biol. 2020;2124:125-139. doi: 10.1007/978-1-0716-0356-7_6. PMID: 32277451; PMCID: PMC7217558.

17. Rivera AL, Gómez-Lim M, Fernández F, Loske AM. Physical methods for genetic plant transformation. Phys Life Rev. 2012 Sep;9(3):308-45. doi: 10.1016/j.plrev.2012.06.002. Epub 2012 Jun 6. PMID: 22704230.

18. Transgenic Crops: An Introduction and Resource Guide [Internet]. Colorado: Dept. of Soil and Crop Sciences at Colorado State University; c2015. [cited 05 April 2021]. Available from:https://www.uni-weimar.de/kunst-und-gestaltung/wiki/images/Transgenic_Crops .pdf

19. Ahmad N, Michoux F, Lössl AG, Nixon PJ. Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot. 2016 Nov;67(21):5945-5960. doi: 10.1093/jxb/erw360. Epub 2016 Oct 3. PMID: 27697788.

20. Waheed MT, Ismail H, Gottschamel J, Mirza B, Lössl AG. Plastids: The Green Frontiers for Vaccine Production. Front Plant Sci. 2015 Nov 17;6:1005. doi: 10.3389/fpls.2015.01005. PMID: 26635832; PMCID: PMC4646963.

21. Ahmad N, Michoux F, Lössl AG, Nixon PJ. Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot. 2016 Nov;67(21):5945-5960. doi: 10.1093/jxb/erw360. Epub 2016 Oct 3. PMID: 27697788.

121 22. Adem M, Beyene D, Feyissa T. Recent achievements obtained by chloroplast transformation. Plant Methods. 2017 Apr 19;13:30. doi: 10.1186/s13007-017-0179-1. PMID: 28428810; PMCID: PMC5395794.

23. Ahmad N, Michoux F, Lössl AG, Nixon PJ. Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot. 2016 Nov;67(21):5945-5960. doi: 10.1093/jxb/erw360. Epub 2016 Oct 3. PMID: 27697788.

24. Yu Y, Yu PC, Chang WJ, Yu K, Lin CS. Plastid Transformation: How Does it Work? Can it Be Applied to Crops? What Can it Offer? Int J Mol Sci. 2020 Jul 9;21(14):4854. doi: 10.3390/ijms21144854. PMID: 32659946; PMCID: PMC7402345.

25. Breeding Genetics and Biotechnology M.J. Wilkinson, R.M. Twyman, in Encyclopedia of Applied Plant Sciences (Second Edition), 2017 https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/transplast omic-plant

26. Waheed MT, Ismail H, Gottschamel J, Mirza B, Lössl AG. Plastids: The Green Frontiers for Vaccine Production. Front Plant Sci. 2015 Nov 17;6:1005. doi: 10.3389/fpls.2015.01005. PMID: 26635832; PMCID: PMC4646963.

27. Wani SH, Haider N, Kumar H, Singh NB. Plant plastid engineering. Curr Genomics. 2010 Nov;11(7):500-12. doi: 10.2174/138920210793175912. PMID: 21532834; PMCID: PMC3048312.

28. Ahmad N, Michoux F, Lössl AG, Nixon PJ. Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot. 2016 Nov;67(21):5945-5960. doi: 10.1093/jxb/erw360. Epub 2016 Oct 3. PMID: 27697788.

29. Maliga P, Bock R. Plastid biotechnology: food, fuel, and medicine for the 21st century. Plant Physiol. 2011 Apr;155(4):1501-10. doi: 10.1104/pp.110.170969. Epub 2011 Jan 14. PMID: 21239622; PMCID: PMC3091108.

30. Jin S, Daniell H. The Engineered Chloroplast Genome Just Got Smarter. Trends Plant Sci. 2015 Oct;20(10):622-640. doi: 10.1016/j.tplants.2015.07.004. PMID: 26440432; PMCID: PMC4606472.

31. Day, A. and Goldschmidt‐Clermont, M. (2011), The chloroplast transformation toolbox: selectable markers and marker removal. Plant Biotechnology Journal, 9: 540-553. https://doi.org/10.1111/j.1467-7652.2011.00604.x

32. Obembe OO, Popoola JO, Leelavathi S, Reddy SV. Advances in plant molecular farming. Biotechnol Adv. 2011 Mar-Apr;29(2):210-22. doi: 10.1016/j.biotechadv.2010.11.004. Epub 2010 Nov 27. PMID: 21115109.

122 33. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

34. Shanmugaraj B, I Bulaon CJ, Phoolcharoen W. Plant Molecular Farming: A Viable Platform for Recombinant Biopharmaceutical Production. Plants (Basel). 2020 Jul 4;9(7):842. doi: 10.3390/plants9070842. PMID: 32635427; PMCID: PMC7411908.

35. Komarova TV, Baschieri S, Donini M, Marusic C, Benvenuto E, Dorokhov YL. Transient expression systems for plant-derived biopharmaceuticals. Expert Rev Vaccines. 2010 Aug;9(8):859-76. doi: 10.1586/erv.10.85. PMID: 20673010.

36. Hwang HH, Yu M, Lai EM. Agrobacterium-mediated plant transformation: biology and applications. Arabidopsis Book. 2017 Oct 20;15:e0186. doi: 10.1199/tab.0186. PMID: 31068763; PMCID: PMC6501860

37. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

38. Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM. Seed-based expression systems for plant molecular farming. Plant Biotechnol J. 2010 Jun;8(5):588-606. doi: 10.1111/j.1467-7652.2010.00511.x. PMID: 20500681.

39. Geigert J. The Challenge of CMC Regulatory Compliance for Biopharmaceuticals. 1st.ed. Boston: Springer; c2004.Chapter 5, Recombinant Source Material: Master/Working Banks; p 83-114

40. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

41. Peters J, Stoger E. Transgenic crops for the production of recombinant vaccines and anti-microbial antibodies. Hum Vaccin. 2011 Mar;7(3):367-74. doi: 10.4161/hv.7.3.14303. Epub 2011 Mar 1. PMID: 21346415.

42. Boothe, J.G., Saponja, J.A. and Parmenter, D.L. (1997) Molecular farming in plants: oilseeds as vehicles for the production of pharmaceutical proteins. Drug Dev. Res. 42, 172–181

43. Obembe OO, Popoola JO, Leelavathi S, Reddy SV. Advances in plant molecular farming. Biotechnol Adv. 2011 Mar-Apr;29(2):210-22. doi: 10.1016/j.biotechadv.2010.11.004. Epub 2010 Nov 27. PMID: 21115109.

44. Merlin M, Pezzotti M, Avesani L. Edible plants for oral delivery of biopharmaceuticals. Br J Clin Pharmacol. 2017 Jan;83(1):71-81. doi: 10.1111/bcp.12949. Epub 2016 May 9. PMID: 27037892; PMCID: PMC5338148..

123 45. Koop HU, Herz S, Golds TJ, Nickelsen J. Cell and Molecular Biology of Plastids. Heidelberg: Springer; 15 May 2007; Chapter 13, The Genetic Transformation of Plastids; p 57-410. doi:10.1007/4735_2007_0225

46. Maliga P. Engineering the plastid genome of higher plants. Curr Opin Plant Biol. 2002 Apr;5(2):164-72. doi: 10.1016/s1369-5266(02)00248-0. PMID: 11856614.

47. Waheed MT, Ismail H, Gottschamel J, Mirza B, Lössl AG. Plastids: The Green Frontiers for Vaccine Production. Front Plant Sci. 2015 Nov 17;6:1005. doi: 10.3389/fpls.2015.01005. PMID: 26635832; PMCID: PMC4646963.

48. Scotti N, Rigano MM, Cardi T. Production of foreign proteins using plastid transformation. Biotechnol Adv. 2012 Mar-Apr;30(2):387-97. doi: 10.1016/j.biotechadv.2011.07.019. Epub 2011 Aug 6. PMID: 21843626.

49. Waheed MT, Ismail H, Gottschamel J, Mirza B, Lössl AG. Plastids: The Green Frontiers for Vaccine Production. Front Plant Sci. 2015 Nov 17;6:1005. doi: 10.3389/fpls.2015.01005. PMID: 26635832; PMCID: PMC4646963.

50. Jin S, Daniell H. The Engineered Chloroplast Genome Just Got Smarter. Trends Plant Sci. 2015 Oct;20(10):622-640. doi: 10.1016/j.tplants.2015.07.004. PMID: 26440432; PMCID: PMC4606472.

51. Petersen K, Bock R. High-level expression of a suite of thermostable cell wall-degrading enzymes from the chloroplast genome. Plant Mol Biol. 2011 Jul;76(3-5):311-21. doi: 10.1007/s11103-011-9742-8. Epub 2011 Feb 6. PMID: 21298465.

52. Oey M, Lohse M, Kreikemeyer B, Bock R. Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J. 2009 Feb;57(3):436-45. doi: 10.1111/j.1365-313X.2008.03702.x. Epub 2008 Oct 30. PMID: 18939966.

53. Ahmad N, Michoux F, Lössl AG, Nixon PJ. Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot. 2016 Nov;67(21):5945-5960. doi: 10.1093/jxb/erw360. Epub 2016 Oct 3. PMID: 27697788.

54. Clark M, Maselko M. Transgene Biocontainment Strategies for Molecular Farming. Front Plant Sci. 2020 Mar 3;11:210. doi: 10.3389/fpls.2020.00210. PMID: 32194598; PMCID: PMC7063990.

55. Clark M, Maselko M. Transgene Biocontainment Strategies for Molecular Farming. Front Plant Sci. 2020 Mar 3;11:210. doi: 10.3389/fpls.2020.00210. PMID: 32194598; PMCID: PMC7063990.

56. Breyer D, De Schrijver A, Goossens M, Pauwels K, Herman P. Molecular Farming in Plants: Recent Advances and Future Prospects. Wang A, S. Ma, editors. Dordrecht:Springer;2012. Chapter 12 ,Biosafety of Molecular Farming in Genetically Modified plants p259-274. doi: 10.1007/978-94-007-2217-0_12

124 57. Clark M, Maselko M. Transgene Biocontainment Strategies for Molecular Farming. Front Plant Sci. 2020 Mar 3;11:210. doi: 10.3389/fpls.2020.00210. PMID: 32194598; PMCID: PMC7063990.

58. Mallory-Smith CA, Sanchez Olguin E. Gene flow from herbicide-resistant crops: it's not just for transgenes. J Agric Food Chem. 2011 Jun 8;59(11):5813-8. doi: 10.1021/jf103389v. Epub 2010 Nov 8. PMID: 21058724.

59. Gressel J. Dealing with transgene flow of crop protection traits from crops to their relatives. Pest Manag Sci. 2015 May;71(5):658-67. doi: 10.1002/ps.3850. Epub 2014 Aug 15. PMID: 24977384.

60. Meyers B, Zaltsman A, Lacroix B, Kozlovsky SV, Krichevsky A. Nuclear and plastid genetic engineering of plants: comparison of opportunities and challenges. Biotechnol Adv. 2010 Nov-Dec;28(6):747-56. doi: 10.1016/j.biotechadv.2010.05.022. Epub 2010 Jun 4. PMID: 20685387.

61. Fischer U, Kuhlmann M, Pecinka A, Schmidt R, Mette MF. Local DNA features affect RNAdirected transcriptional gene silencing and DNA methylation. Plant J 2008;53:1-10.

62. Eike MC, Mercy IS, Aalen RB. Transgene silencing may be mediated by aberrant sense promoter sequence transcripts generated from cryptic promoters. Cell Mol Life Sci 2005;62:3080–91.

63. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

64. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

65. Ullrich KK, Hiss M, Rensing SA. Means to optimize protein expression in transgenic plants. Curr Opin Biotechnol. 2015 Apr;32:61-67. doi: 10.1016/j.copbio.2014.11.011. Epub 2014 Nov 25. PMID: 25448234.

66. Rigano, M Manuela et al. “Unsolved problems in plastid transformation.” Bioengineered vol. 3,6 (2012): 329-33. doi:10.4161/bioe.21452

67. Ahmad N, Michoux F, Lössl AG, Nixon PJ. Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot. 2016 Nov;67(21):5945-5960. doi: 10.1093/jxb/erw360. Epub 2016 Oct 3. PMID: 27697788.

68. Ahmad N, Michoux F, Lössl AG, Nixon PJ. Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot. 2016

125 Nov;67(21):5945-5960. doi: 10.1093/jxb/erw360. Epub 2016 Oct 3. PMID: 27697788.

69. Zhang J, Ruf S, Hasse C, Childs L, Scharff LB, Bock R. 2012. Identification of cis-elements conferring high levels of gene expression in non-green plastids. The Plant Journal 72, 115–128

70. Ruf S, Karcher D, Bock R. 2007. Determining the transgene containment level provided by chloroplast transformation. Proceedings of the National Academy of Sciences of the United States of America 104, 6998–7002.

71. Svab Z, Maliga P. 2007. Exceptional transmission of plastids and mitochondria from the transplastomic pollen parent and its impact on transgene containment. Proceedings of the National Academy of Sciences of the United States of America 104, 7003–7008.

72. Azhagiri AK, Maliga P. 2007. Exceptional paternal inheritance of plastids in Arabidopsis suggests that low-frequency leakage of plastids via pollen may be universal in plants. The Plant Journal 52, 817–823.

73. Ahmad N, Michoux F, Lössl AG, Nixon PJ. Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot. 2016 Nov;67(21):5945-5960. doi: 10.1093/jxb/erw360. Epub 2016 Oct 3. PMID: 27697788.

74. Yu Y, Yu PC, Chang WJ, Yu K, Lin CS. Plastid Transformation: How Does it Work? Can it Be Applied to Crops? What Can it Offer? Int J Mol Sci. 2020 Jul 9;21(14):4854. doi: 10.3390/ijms21144854. PMID: 32659946; PMCID: PMC7402345.

75. Ahmad N, Michoux F, Lössl AG, Nixon PJ. Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot. 2016 Nov;67(21):5945-5960. doi: 10.1093/jxb/erw360. Epub 2016 Oct 3. PMID: 27697788.

76. Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein TM, Shark KB, et al. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science. 1988 Jun 10;240(4858):1534-8. doi: 10.1126/science.2897716. PMID: 2897716.

77. Lee SM, Kang K, Chung H, Yoo SH, Xu XM, Lee SB, Cheong JJ, Daniell H, Kim M. Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny. Mol Cells. 2006 Jun 30;21(3):401-10. PMID: 16819304; PMCID: PMC3481850.

78. Ahmadabadi M, Ruf S, Bock R. A leaf-based regeneration and transformation system for maize (Zea mays L.). Transgenic Res. 2007 Aug;16(4):437-48. doi: 10.1007/s11248-006-9046-y. Epub 2006 Nov 14. PMID: 17103238.

126 79. Li W, Ruf S, Bock R. Chloramphenicol acetyltransferase as selectable marker for plastid transformation. Plant Mol Biol. 2011 Jul;76(3-5):443-51. doi: 10.1007/s11103-010-9678-4. Epub 2010 Aug 19. PMID: 20721602.

80. Fromm H, Edelman M, Aviv D, Galun E. The molecular basis for rRNA-dependent spectinomycin resistance in Nicotiana chloroplasts. EMBO J. 1987 Nov;6(11):3233-7. PMID: 16453804; PMCID: PMC553774.

81. Ashida, H. (2011). Comprehensive Biotechnology || Increasing Photosynthesis/RuBisCO and CO2-Concentrating Mechanisms. , (), 165–176. doi:10.1016/B978-0-08-088504-9.00243-9

82. Yao J, Weng Y, Dickey A, Wang KY. Plants as Factories for Human Pharmaceuticals: Applications and Challenges. Int J Mol Sci. 2015 Dec 2;16(12):28549-65. doi: 10.3390/ijms161226122. PMID: 26633378; PMCID: PMC4691069.

83. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

84. Peyret H, Lomonossoff GP. When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol J. 2015 Oct;13(8):1121-35. doi: 10.1111/pbi.12412. Epub 2015 Jun 12. PMID: 26073158; PMCID: PMC4744784.

85. Obembe OO, Popoola JO, Leelavathi S, Reddy SV. Advances in plant molecular farming. Biotechnol Adv. 2011 Mar-Apr;29(2):210-22. doi: 10.1016/j.biotechadv.2010.11.004. Epub 2010 Nov 27. PMID: 21115109.

86. Chen Q, Lai H, Hurtado J, Stahnke J, Leuzinger K, Dent M. Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins. Adv Tech Biol Med. 2013 Jun;1(1):103. doi: 10.4172/atbm.1000103. PMID: 25077181; PMCID: PMC4113218.

87. Hahn S, Giritch A, Bartels D, Bortesi L, Gleba Y. A novel and fully scalable Agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants. Plant Biotechnol J. 2015 Jun;13(5):708-16. doi: 10.1111/pbi.12299. Epub 2014 Dec 2. PMID: 25470212.

88. Komarova TV, Baschieri S, Donini M, Marusic C, Benvenuto E, Dorokhov YL. Transient expression systems for plant-derived biopharmaceuticals. Expert Rev Vaccines. 2010 Aug;9(8):859-76. doi: 10.1586/erv.10.85. PMID: 20673010.

89. Yang L, Wang H, Liu Jet al. A simple and effective system for foreign gene expression in plants via root absorption of agrobacterial suspension.J.Biotechnol.134, 320–324(2008)

90. Gelvin SB. Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiol Mol Biol Rev. 2003 Mar;67(1):16-37, table of

127 contents. doi: 10.1128/mmbr.67.1.16-37.2003. PMID: 12626681; PMCID: PMC150518.

91. Types of Transfection [Internet].ThermoFisher Scientific. [Cited 05 April 2021] Available from:https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/ transfection-basics/types-of-transfection.html

92. Virus [Internet]. Nature SciTable: c2014. [Cited 05 April 2021]. Available from: https://www.nature.com/scitable/definition/virus-308/

93. Suchman E., Blair C. Cytopathic Effects of Viruses Protocols [Internet]. Microbe Library:c2007. [Cited 05 April 2021].Available from: https://web.archive.org/web/20120602171845/http://microbelibrary.org/component/r esource/laboratory-test/2875-cytopathic-effects-of-viruses-protocols

94. Chen, Qiang et al. “Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins.” Advanced techniques in biology & medicine vol. 1,1 (2013): 103. doi:10.4172/atbm.1000103

95. Naseri Z, Khezri G, Davarpanah S, Ofoghi H. Virus-Based Vectors: A New Approach for the Production of Recombinant Proteins. Journal of Applied Biotechnology Reports. c2009; 6(1):6-14.

96. Ibrahim A, Odon V, Kormelink R. Plant Viruses in Plant Molecular Pharming: Toward the Use of Enveloped Viruses. Front Plant Sci. 2019 Jun 19;10:803. doi: 10.3389/fpls.2019.00803. PMID: 31275344; PMCID: PMC6594412.

97. Peyret H, Lomonossoff GP. When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol J. 2015 Oct;13(8):1121-35. doi: 10.1111/pbi.12412. Epub 2015 Jun 12. PMID: 26073158; PMCID: PMC4744784.

98. Komarova TV, Baschieri S, Donini M, Marusic C, Benvenuto E, Dorokhov YL. Transient expression systems for plant-derived biopharmaceuticals. Expert Rev Vaccines. 2010 Aug;9(8):859-76. doi: 10.1586/erv.10.85. PMID: 20673010.

99. Peyret H, Lomonossoff GP. When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol J. 2015 Oct;13(8):1121-35. doi: 10.1111/pbi.12412. Epub 2015 Jun 12. PMID: 26073158; PMCID: PMC4744784.

100. Peyret H, Lomonossoff GP. When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol J. 2015 Oct;13(8):1121-35. doi: 10.1111/pbi.12412. Epub 2015 Jun 12. PMID: 26073158; PMCID: PMC4744784.

101. Djikstra J, De Jager C. Practical Plant Virology. 1st ed. Berlin, Heidelberg: Springer; 1998. Chapter 1, "Introcution I-Virus Inoculation"; p3-4.

102. Peyret H, Lomonossoff GP. When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol J. 2015 Oct;13(8):1121-35. doi: 10.1111/pbi.12412. Epub 2015 Jun 12. PMID: 26073158; PMCID: PMC4744784.

128 103. Brewer, H.C., Hird, D.L., Bailey, A.M., Seal, S.E. and Foster, G.D. (2018), A guide to the contained use of plant virus infectious clones. Plant Biotechnol J, 16: 832-843. https://doi.org/10.1111/pbi.12876

104. Peyret H, Lomonossoff GP. When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol J. 2015 Oct;13(8):1121-35. doi: 10.1111/pbi.12412. Epub 2015 Jun 12. PMID: 26073158; PMCID: PMC4744784.

105. Naseri Z, Khezri G, Davarpanah S, Ofoghi H. Virus-Based Vectors: A New Approach for the Production of Recombinant Proteins. Journal of Applied Biotechnology Reports. c2009; 6(1):6-14.

106. Gleba Y, Klimyuk V, Marillonnet S. Magnifection--a new platform for expressing recombinant vaccines in plants. Vaccine. 2005 Mar 18;23(17-18):2042-8. doi: 10.1016/j.vaccine.2005.01.006. PMID: 15755568.

107. Gleba Y, Klimyuk V, Marillonnet S. Viral vectors for the expression of proteins in plants. Curr Opin Biotechnol. 2007 Apr;18(2):134-41. doi: 10.1016/j.copbio.2007.03.002. Epub 2007 Mar 23. PMID: 17368018.

108. Chen Q, Lai H, Hurtado J, Stahnke J, Leuzinger K, Dent M. Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins. Adv Tech Biol Med. 2013 Jun;1(1):103. doi: 10.4172/atbm.1000103. PMID: 25077181; PMCID: PMC4113218.

109. Fujiuchi N, Matoba N, Matsuda R. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression. Front Bioeng Biotechnol. 2016 Mar 8;4:23. doi: 10.3389/fbioe.2016.00023. PMID: 27014686; PMCID: PMC4781840.

110. Fujiuchi N, Matoba N, Matsuda R. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression. Front Bioeng Biotechnol. 2016 Mar 8;4:23. doi: 10.3389/fbioe.2016.00023. PMID: 27014686; PMCID: PMC4781840.

111. Gleba YY, Tusé D, Giritch A. Plant Viral Vectors. 1st ed. Palmer KE, Gleba YY, editors. Springer: Berlin, Heidelberg; 2014. Chapter 8, Plant Viral Vectors for Delivery by Agrobacterium; p155-92.

112. Poorter H, Niklas KJ, Reich PB, Oleksyn J, Poot P, Mommer L. Biomass allocation to leaves, stems and roots: meta-analyses of interspecific variation and environmental control. New Phytol. 2012 Jan;193(1):30-50. doi: 10.1111/j.1469-8137.2011.03952.x. Epub 2011 Nov 15. PMID: 22085245.

113. Norikane, J.H. (2015). The potential of LEDs in plant-based bio-pharmaceutical production.HortScience50, 1289–1292

114. Fujiuchi N, Matoba N, Matsuda R. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene

129 Expression. Front Bioeng Biotechnol. 2016 Mar 8;4:23. doi: 10.3389/fbioe.2016.00023. PMID: 27014686; PMCID: PMC4781840.

115. Atmakuri K, Christie PJ. Agrobacterium: From Biology to Biotechnology. 1st ed. Tzifira T, Citovsky V, editors. Springer-Verlag:New York; 2008. Chapter 9, Translocation of Oncogenic T-DNA and Effector Proteins to Plant Cells; p315-364.

116. Fullner, K. J., and Nester, E. W. (1996). Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens.J.Bacteriol.178, 1498–150

117. Jung, S.-K., McDonald, K. A., and Dandekar, A. M. (2015). Effect of leaf incubation temperature profiles on Agrobacterium tumefaciens-mediated transient expression. Biotechnol. Prog.31, 783–790. doi:10.1002/btpr.2077

118. Cazzonelli CI, Velten J. An in vivo, luciferase-based, Agrobacterium-infiltration assay system: implications for post-transcriptional gene silencing. Planta. 2006 Aug;224(3):582-97. doi: 10.1007/s00425-006-0250-z. Epub 2006 Mar 8. PMID: 16523348.

119. De Clercq J, Zambre M, Van Montagu M, Dillen W, Angenon G. An optimized Agrobacterium-mediated transformation procedure for Phaseolus acutifolius A. Gray.Plant Cell Rep. 2002 Nov; 21: 333-340. https://doi.org/10.1007/s00299-002-0518-0

120. Joh LD, Wroblewski T, Ewing NN, VanderGheynst JS. High-level transient expression of recombinant protein in lettuce. Biotechnol Bioeng. 2005 Sep 30;91(7):861-71. doi: 10.1002/bit.20557. PMID: 15937952.

121. Escudero J, Hohn, B.Transfer and Integration of T-DNA without Cell Injury in the Host Plant. The Plant Cell. 1997; 9(12): 2135–2142. www.jstor.org/stable/3870574. Accessed 5 Apr. 2021.

122. Fujiuchi N, Matsuda R, Matoba N, Fujiwara K. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system. Biotechnol Bioeng. 2016 Apr;113(4):901-6. doi: 10.1002/bit.25854. Epub 2015 Oct 26. PMID: 26461274.

123. Fujiuchi N, Matsuda R, Matoba N, Fujiwara K. Effect of nitrate concentration in nutrient solution on hemagglutinin content of Nicotiana benthamiana leaves in a viral vector-mediated transient gene expression system. Plant Biotechnology. 2004; 31(3): 207–211. doi:10.5511/plantbiotechnology.14.0403a

124. Ramalingam, S., Iyappan, G., Priya, S., Kadirvelu, K., Kingston, J., Gopalan, N., & Sharma, R. (2017). Rapid production of therapeutic proteins using plant system. Defence Life Science Journal, 2(2), 95-102. https://doi.org/10.14429/dlsj.2.11372

130 125. Naseri Z, Khezri G, Davarpanah S, Ofoghi H. Virus-Based Vectors: A New Approach for the Production of Recombinant Proteins. Journal of Applied Biotechnology Reports. c2009; 6(1):6-14.

126. Negrouk V, Eisner G, Lee H, Han K, Taylor D, W Hing. . Highly efficient transient expression of functional recombinant antibodies in lettuce. Plant Science. 2005; 169: 433-438. 10.1016/j.plantsci.2005.03.031.

127. Komarova TV, Baschieri S, Donini M, Marusic C, Benvenuto E, Dorokhov YL. Transient expression systems for plant-derived biopharmaceuticals. Expert Rev Vaccines. 2010 Aug;9(8):859-76. doi: 10.1586/erv.10.85. PMID: 20673010.

128. Chen Q. Expression and purification of pharmaceutical proteins in plants biological engineering. Biological Engineering Transactions. c2008;1(4):291–321. doi: 10.13031/2013.26854

129. Chen Q, Lai H, Hurtado J, Stahnke J, Leuzinger K, Dent M. Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins. Adv Tech Biol Med. 2013 Jun;1(1):103. doi: 10.4172/atbm.1000103. PMID: 25077181; PMCID: PMC4113218.

130. Johansen LK, Carrington JC. Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol. 2001 Jul;126(3):930-8. doi: 10.1104/pp.126.3.930. PMID: 11457942; PMCID: PMC1540124.

131. Breyer D, Goossens M, Herman P, & Sneyers M. Biosafety considerations associated with molecular farming in genetically modified plants. Journal of Medicinal Plants Research. c2009: 3; 825-838.

132. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

133. Clark M, Maselko M. Transgene Biocontainment Strategies for Molecular Farming. Front Plant Sci. 2020 Mar 3;11:210. doi: 10.3389/fpls.2020.00210. PMID: 32194598; PMCID: PMC7063990.

134. Sood P, Bhattacharya A, Sood A. Problems and possibilities of monocot transformation.Biologia Plantarum. 2011;55:1-15doi:10.1007/s10535-011-0001-2

135. Brewer HC, Hird DL, Bailey AM, Seal SE, Foster GD. A guide to the contained use of plant virus infectious clones. Plant Biotechnol J. 2018 Apr;16(4):832-843. doi: 10.1111/pbi.12876. Epub 2018 Feb 6. PMID: 29271098; PMCID: PMC5867029.

136. Rosales-Mendoza S, Márquez-Escobar VA, González-Ortega O, Nieto-Gómez R, Arévalo-Villalobos JI. What Does Plant-Based Vaccine Technology Offer to the Fight against COVID-19? Vaccines (Basel). 2020 Apr 14;8(2):183. doi: 10.3390/vaccines8020183. PMID: 32295153; PMCID: PMC7349371

131 137. Fujiuchi N, Matoba N, Matsuda R. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression. Front Bioeng Biotechnol. 2016 Mar 8;4:23. doi: 10.3389/fbioe.2016.00023. PMID: 27014686; PMCID: PMC4781840.

138. Streatfield SJ, Kushnir N, Yusibov V. Plant-produced candidate countermeasures against emerging and reemerging infections and bioterror agents. Plant Biotechnol J. 2015 Oct;13(8):1136-59. doi: 10.1111/pbi.12475. PMID: 26387510; PMCID: PMC7167919.

139. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

140. Marsian J, Lomonossoff GP. Molecular pharming - VLPs made in plants. Curr Opin Biotechnol. 2016 Feb;37:201-206. doi: 10.1016/j.copbio.2015.12.007. Epub 2016 Jan 14. PMID: 26773389.

141. Rybicki EP. Plant-produced vaccines: promise and reality. Drug Discov Today. 2009 Jan;14(1-2):16-24. doi: 10.1016/j.drudis.2008.10.002. Epub 2008 Nov 18. PMID: 18983932.

142. Rosales-Mendoza S, Nieto-Gómez R, Angulo C. A Perspective on the Development of Plant-Made Vaccines in the Fight against Ebola Virus. Front Immunol. 2017 Mar 10;8:252. doi: 10.3389/fimmu.2017.00252. PMID: 28344580; PMCID: PMC5344899.

132 Chapter 2- Citations

1. Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des. 2013;19(31):5531-42. doi: 10.2174/1381612811319310008. PMID: 23394569.

2. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

3. Mustafa NR, de Winter W, van Iren F, Verpoorte R. Initiation, growth and cryopreservation of plant cell suspension cultures. Nat Protoc. 2011 Jun;6(6):715-42. doi: 10.1038/nprot.2010.144. Epub 2011 May 5. PMID: 21637194.

4. Bhatia, S, Sharma K, Dahiya R. and Bera T. Modern Applications of Plant Biotechnology in Pharmaceutical Sciences. 1st ed. Academic Press; c2015. Chapter 2, Plant Tissue Culture;p31-107. doi:10.1016/B978-0-12-802221-4.00002-9

5. Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des. 2013;19(31):5531-42. doi: 10.2174/1381612811319310008. PMID: 23394569.

6. Torres E, Vaquero C, Nicholson L, et al. Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies. Transgenic Res 1999; 8: 441-9

7. Kwon T, Kim Y, Lee J, Yang M. Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures. Biotechnol Lett 2003; 25: 1571-4.

8. Pires AS, Rosa S, Castanheira S, Fevereiro P, Abranches R. Expression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago. Plant Cell Tiss Organ Cult 2012; 110: 171-81

9. Mikschofsky H, Hammer M, Schmidtke J, et al. Optimization of growth performance of freshly induced carrot suspensions concerning PMP production. In vitro Cell Dev-Pl 2009; 45: 740-9.

10. Nagata T, Sakamoto K, Shimizu T.Tobacco BY-2 cells: The present and beyond. In Vitro Cell Developmental Biology-Plant. Mar-April 2004;40(2):163-166. https://doi-org.ezproxy.wellesley.edu/10.1079/IVP2003526

11. Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des. 2013;19(31):5531-42. doi: 10.2174/1381612811319310008. PMID: 23394569.

133 12. Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des. 2013;19(31):5531-42. doi: 10.2174/1381612811319310008. PMID: 23394569.

13. Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM. Plant-based production of biopharmaceuticals. Curr Opin Plant Biol. 2004 Apr;7(2):152-8. doi: 10.1016/j.pbi.2004.01.007. PMID: 15003215.

14. Obembe OO, Popoola JO, Leelavathi S, Reddy SV. Advances in plant molecular farming. Biotechnol Adv. 2011;29(2):210–22

15. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

16.Hu, Z.‐B. and Du, M. (2006), Hairy Root and Its Application in Plant Genetic Engineering. Journal of Integrative Plant Biology, 48: 121-127. https://doi.org/10.1111/j.1744-7909.2006.00121.x

17. Doran, PM. Hairy Root and Its Application in Plant Genetic Engineering. Heidelberg:Springer, c2013. p159. (Springer series on Advances in Biochemical Engineering/Biotechnology). https://doi.org/10.1007/978-3-642-39019-7

18.Hu ZB, Du M. Hairy Root and Its Application in Plant Genetic Engineering. Journal of Integrative Plant Biology. 2006; 48: 121-127. https://doi.org/10.1111/j.1744-7909.2006.00121.x

19. Häkkinen ST, Raven N, Henquet M, Laukkanen ML, Anderlei T, Pitkänen JP, Twyman RM, Bosch D, Oksman-Caldentey KM, Schillberg S, Ritala A. Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody. Biotechnol Bioeng. 2014 Feb;111(2):336-46. doi: 10.1002/bit.25113. Epub 2013 Sep 24. PMID: 24030771.

20. Guillon S, Trémouillaux-Guiller J, Kumar Pati P, Rideau M, Gantet P. 2006. Hairy root research: Recent scenario and exciting prospects. Curr Opin Plant Biol 9:341–346.

21. Decker EL, Reski R. The moss bioreactor. Curr Opin Plant Biol. 2004 Apr;7(2):166-70. doi: 10.1016/j.pbi.2004.01.002. PMID: 15003217.

22. Xu J, Dolan MC, Medrano G, Cramer CL, Weathers PJ. Green factory: plants as bioproduction platforms for recombinant proteins. Biotechnol Adv. 2012 Sep-Oct;30(5):1171-84. doi: 10.1016/j.biotechadv.2011.08.020. Epub 2011 Sep 8. PMID: 21924345.

134 23. Rasala BA, Mayfield SP. The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics. Bioeng Bugs. 2011 Jan-Feb;2(1):50-4. doi: 10.4161/bbug.2.1.13423. PMID: 21636988; PMCID: PMC3127021.

24. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

25. Rosales-Mendoza S, Paz-Maldonado LM, Soria-Guerra RE. Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives. Plant Cell Rep. 2012 Mar;31(3):479-94. doi: 10.1007/s00299-011-1186-8. Epub 2011 Nov 12. PMID: 22080228.

26. Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des. 2013;19(31):5531-42. doi: 10.2174/1381612811319310008. PMID: 23394569.

27. Decker EL, Reski R. The moss bioreactor. Curr Opin Plant Biol. 2004 Apr;7(2):166-70. doi: 10.1016/j.pbi.2004.01.002. PMID: 15003217.

28. Reski R, Abel WO: Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine. Planta 1985, 165:354-358 . 29. Decker, E. L., & Reski, R. (2007). Moss bioreactors producing improved biopharmaceuticals. Current Opinion in Biotechnology, 18(5), 393–398. doi:10.1016/j.copbio.2007.07.012

30. Decker, E. L., & Reski, R. (2004). The moss bioreactor. Current Opinion in Plant Biology, 7(2), 166–170. doi:10.1016/j.pbi.2004.01.002

31. Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des. 2013;19(31):5531-42. doi: 10.2174/1381612811319310008. PMID: 23394569.

32. Santos RB, Abranches R, Fischer R, Sack M, Holland T. Putting the Spotlight Back on Plant Suspension Cultures. Front Plant Sci. 2016 Mar 11;7:297. doi: 10.3389/fpls.2016.00297. PMID: 27014320; PMCID: PMC4786539.

33. Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des. 2013;19(31):5531-42. doi: 10.2174/1381612811319310008. PMID: 23394569.

34. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

135 35. Buyel JF, Twyman RM, Fischer R. Very-large-scale production of antibodies in plants: The biologization of manufacturing. Biotechnol Adv. 2017 Jul;35(4):458-465. doi: 10.1016/j.biotechadv.2017.03.011. Epub 2017 Mar 25. PMID: 28347720.

36. Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des. 2013;19(31):5531-42. doi: 10.2174/1381612811319310008. PMID: 23394569.

37. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

38. Shanks JV, Morgan J. Plant 'hairy root' culture. Curr Opin Biotechnol. 1999 Apr;10(2):151-5. doi: 10.1016/s0958-1669(99)80026-3. PMID: 10209145.

39. Gutierrez-Valdes N, Häkkinen ST, Lemasson C, Guillet M, Oksman-Caldentey KM, Ritala A, Cardon F. Hairy Root Cultures-A Versatile Tool With Multiple Applications. Front Plant Sci. 2020 Mar 3;11:33. doi: 10.3389/fpls.2020.00033. PMID: 32194578; PMCID: PMC7064051.

40. Rasala BA, Mayfield SP. The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics. Bioeng Bugs. 2011 Jan-Feb;2(1):50-4. doi: 10.4161/bbug.2.1.13423. PMID: 21636988; PMCID: PMC3127021.

41. Rosales-Mendoza S, Paz-Maldonado LM, Soria-Guerra RE. Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives. Plant Cell Rep. 2012 Mar;31(3):479-94. doi: 10.1007/s00299-011-1186-8. Epub 2011 Nov 12. PMID: 22080228.

42. Gangl D, Zedler JA, Rajakumar PD, Martinez EM, Riseley A, Włodarczyk A, Purton S, Sakuragi Y, Howe CJ, Jensen PE, Robinson C. Biotechnological exploitation of microalgae. J Exp Bot. 2015 Dec;66(22):6975-90. doi: 10.1093/jxb/erv426. Epub 2015 Sep 22. PMID: 26400987.

43.Ramessar K, Capell T, Christou P. Molecular pharming in cereal crops. Phytochem Rev. 2008; 7; 579–592. https://doi-org.ezproxy.wellesley.edu/10.1007/s11101-008-9087-3

44. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

45. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

136 46. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

47. Clark M, Maselko M. Transgene Biocontainment Strategies for Molecular Farming. Front Plant Sci. 2020 Mar 3;11:210. doi: 10.3389/fpls.2020.00210. PMID: 32194598; PMCID: PMC7063990.

48. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

49. Müntz K. Protein dynamics and proteolysis in plant vacuoles. J Exp Bot. 2007;58(10):2391-407. doi: 10.1093/jxb/erm089. Epub 2007 Jun 1. PMID: 17545219.

50. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

51. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

52. Yao J, Weng Y, Dickey A, Wang KY. Plants as Factories for Human Pharmaceuticals: Applications and Challenges. Int J Mol Sci. 2015 Dec 2;16(12):28549-65. doi: 10.3390/ijms161226122. PMID: 26633378; PMCID: PMC4691069.

53. Phoolcharoen W, Bhoo SH, Lai H, Ma J, Arntzen CJ, Chen Q, Mason HS. Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana. Plant Biotechnol J. 2011 Sep;9(7):807-16. doi: 10.1111/j.1467-7652.2011.00593.x. Epub 2011 Feb 1. PMID: 21281425; PMCID: PMC4022790.

54. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

55. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

56. Conley AJ, Joensuu JJ, Jevnikar AM, Menassa R, Brandle JE. Optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants. Biotechnol Bioeng. 2009 Jun 15;103(3):562-73. doi: 10.1002/bit.22278. PMID: 19266472.

57. Tremblay R, Wang D, Jevnikar AM, Ma S. Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnol Adv. 2010

137 Mar-Apr;28(2):214-21. doi: 10.1016/j.biotechadv.2009.11.008. Epub 2009 Dec 2. PMID: 19961918; PMCID: PMC7132750.

58. D’Aoust, M.A. Perennial plants as a production system for pharmaceuticals. In Handbook of Plant Biotechnology (Christou, P. and Klee, H., eds), Wiley-VCH (in press)

59. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

60. Ahmad, P., Ashraf, M., Younis, M., Hu, X., Kumar, A., Akram, N.A. and Al-Qurainy, F. (2012) Role of transgenic plants in agriculture and biopharming.Biotechnol. Adv.30, 524–540

61. Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 2001 May;6(5):219-26. doi: 10.1016/s1360-1385(01)01922-7. PMID: 11335175; PMCID: PMC5496653.

62. Kurup VM, Thomas J. Edible Vaccines: Promises and Challenges. Mol Biotechnol. 2020 Feb;62(2):79-90. doi: 10.1007/s12033-019-00222-1. PMID: 31758488; PMCID: PMC7090473.

63. Merlin M, Pezzotti M, Avesani L. Edible plants for oral delivery of biopharmaceuticals. Br J Clin Pharmacol. 2017 Jan;83(1):71-81. doi: 10.1111/bcp.12949. Epub 2016 May 9. PMID: 27037892; PMCID: PMC5338148.

64. Mason HS, Herbst-Kralovetz MM. Plant-derived antigens as mucosal vaccines. Curr Top Microbiol Immunol. 2012;354:101-20. doi: 10.1007/82_2011_158. PMID: 21811930; PMCID: PMC7122597.

65. Specht EA, Mayfield SP. Algae-based oral recombinant vaccines. Front Microbiol. 2014 Feb 17;5:60. doi: 10.3389/fmicb.2014.00060. PMID: 24596570; PMCID: PMC3925837.

66. Kwon KC, Verma D, Singh ND, Herzog R, Daniell H. Oral delivery of human biopharmaceuticals, autoantigens and vaccineantigens bioencapsulated in plant cells. Adv Drug Deliv Rev 2013;65: 782–99.

67. Arcalis E, Stadlmann J, Rademacher T, Marcel S, Sack M, Altmann F,et al.Plant species and organ influence the structure and subcellular localization of recombinant glycoproteins. Plant MolBiol 2013; 83: 105–17

68. Chan HT, Daniell H. Plant-made oral vaccines against human infectious diseases-Are we there yet? Plant Biotechnol J. 2015 Oct;13(8):1056-70. doi: 10.1111/pbi.12471. Epub 2015 Sep 7. PMID: 26387509; PMCID: PMC4769796.

138 69. Kurup VM, Thomas J. Edible Vaccines: Promises and Challenges. Mol Biotechnol. 2020 Feb;62(2):79-90. doi: 10.1007/s12033-019-00222-1. PMID: 31758488; PMCID: PMC7090473.

70. Merlin M, Pezzotti M, Avesani L. Edible plants for oral delivery of biopharmaceuticals. Br J Clin Pharmacol. 2017 Jan;83(1):71-81. doi: 10.1111/bcp.12949. Epub 2016 May 9. PMID: 27037892; PMCID: PMC5338148.

71. Specht EA, Mayfield SP. Algae-based oral recombinant vaccines. Front Microbiol. 2014 Feb 17;5:60. doi: 10.3389/fmicb.2014.00060. PMID: 24596570; PMCID: PMC3925837.

72. Kwon, K.C., Verma, D., Singh, N.D., Herzog, R. and Daniell, H. (2013b) Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells.Adv. Drug Deliv. Rev.65, 782–799

73. Merlin M, Pezzotti M, Avesani L. Edible plants for oral delivery of biopharmaceuticals. Br J Clin Pharmacol. 2017 Jan;83(1):71-81. doi: 10.1111/bcp.12949. Epub 2016 May 9. PMID: 27037892; PMCID: PMC5338148.

74. Chan HT, Daniell H. Plant-made oral vaccines against human infectious diseases-Are we there yet? Plant Biotechnol J. 2015 Oct;13(8):1056-70. doi: 10.1111/pbi.12471. Epub 2015 Sep 7. PMID: 26387509; PMCID: PMC4769796.

75. Specht EA, Mayfield SP. Algae-based oral recombinant vaccines. Front Microbiol. 2014 Feb 17;5:60. doi: 10.3389/fmicb.2014.00060. PMID: 24596570; PMCID: PMC3925837.

76. Chan HT, Daniell H. Plant-made oral vaccines against human infectious diseases-Are we there yet? Plant Biotechnol J. 2015 Oct;13(8):1056-70. doi: 10.1111/pbi.12471. Epub 2015 Sep 7. PMID: 26387509; PMCID: PMC4769796.

77. Merlin M, Pezzotti M, Avesani L. Edible plants for oral delivery of biopharmaceuticals. Br J Clin Pharmacol. 2017 Jan;83(1):71-81. doi: 10.1111/bcp.12949. Epub 2016 May 9. PMID: 27037892; PMCID: PMC5338148.

78. Chan HT, Daniell H. Plant-made oral vaccines against human infectious diseases-Are we there yet? Plant Biotechnol J. 2015 Oct;13(8):1056-70. doi: 10.1111/pbi.12471. Epub 2015 Sep 7. PMID: 26387509; PMCID: PMC4769796.

79. Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 2001 May;6(5):219-26. doi: 10.1016/s1360-1385(01)01922-7. PMID: 11335175; PMCID: PMC5496653.

80. Daniell H, Singh ND, Mason H, Streatfield SJ. Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci. 2009 Dec;14(12):669-79. doi:

139 10.1016/j.tplants.2009.09.009. Epub 2009 Oct 14. PMID: 19836291; PMCID: PMC2787751.

81. Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 2001 May;6(5):219-26. doi: 10.1016/s1360-1385(01)01922-7. PMID: 11335175; PMCID: PMC5496653.

82. Boothe, J.G., Saponja, J.A. and Parmenter, D.L. (1997) Molecular farming in plants: oilseeds as vehicles for the production of pharmaceutical proteins. Drug Dev. Res. 42, 172–181.

83. Shewry PR, Halford NG. Cereal seed storage proteins: structures, properties and role in grain utilization. J Exp Bot. 2002 Apr;53(370):947-58. doi: 10.1093/jexbot/53.370.947. PMID: 11912237.

84. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

85. Lau OS, Sun SMS. Plant seeds as bioreactors for recombinant protein production. Biotechnol Adv. 2009;27:1015–22

86. Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM. Seed-based expression systems for plant molecular farming. Plant Biotechnol J. 2010 Jun;8(5):588-606. doi: 10.1111/j.1467-7652.2010.00511.x. PMID: 20500681.

87. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

88.Ramessar K, Capell T, Christou P. Molecular pharming in cereal crops. Phytochem Rev. 2008; 7; 579–592. https://doi-org.ezproxy.wellesley.edu/10.1007/s11101-008-9087-3

89. Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM. Seed-based expression systems for plant molecular farming. Plant Biotechnol J. 2010 Jun;8(5):588-606. doi: 10.1111/j.1467-7652.2010.00511.x. PMID: 20500681.

90. Lau OS, Sun SMS. Plant seeds as bioreactors for recombinant protein production. Biotechnol Adv. 2009;27:1015–22

140 91. Ramessar, K., Sabalza, M., Capell, Teresa and Christou, P. (2008). Maize plants: An ideal production platform for effective and safe molecular pharming. Plant Science. 174. 409-419. 10.1016/j.plantsci.2008.02.002.

92. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

93. Spök A. Molecular farming on the rise—GMO regulators still walking a tightrope. Trends Biotechnol 2007;25:74–82.

94. Lau OS, Sun SMS. Plant seeds as bioreactors for recombinant protein production. Biotechnol Adv. 2009;27:1015–22

95. Nielsen, R.L. Purdue University Online [Internet[. Indiana; c1995-2021. Tassel Emergence & Pollen Shed; 2020 July [cited 2 May 2021]. Available from: https://www.agry.purdue.edu/ext/corn/news/timeless/Tassels.html

96. Lau OS, Sun SMS. Plant seeds as bioreactors for recombinant protein production. Biotechnol Adv. 2009;27:1015–22

97. Sood, P. , Bhattacharya, A., and Sood, A. (2011). Problems and possibilities of monocot transformation. Biologia Plantarum. 55. 1-15. 10.1007/s10535-011-0001-2.

98. Obembe OO, Popoola JO, Leelavathi S, Reddy SV. Advances in plant molecular farming. Biotechnol Adv. 2011;29(2):210–22.

99. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

100. Mikschofsky H, Broer I. Feasibility of Pisum sativum as an expression system for pharmaceuticals. Transgenic Res. 2012 Aug;21(4):715-24. doi: 10.1007/s11248-011-9573-z. Epub 2011 Nov 6. PMID: 22057506.

101. Lau OS, Sun SMS. Plant seeds as bioreactors for recombinant protein production. Biotechnol Adv. 2009;27:1015–22

102. Bhatla SC, Kaushik V, Yadav MK. Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications. Biotechnol Adv. 2010 May-Jun;28(3):293-300. doi: 10.1016/j.biotechadv.2010.01.001. Epub 2010 Jan 11. PMID: 20067829.

103. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

141 104. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

105. Buyel JF, Twyman RM, Fischer R. Very-large-scale production of antibodies in plants: The biologization of manufacturing. Biotechnol Adv. 2017 Jul;35(4):458-465. doi: 10.1016/j.biotechadv.2017.03.011. Epub 2017 Mar 25. PMID: 28347720.

106. Tyagi, A.K. (2001) Plant genes and their expression. Curr. Sci. 80, 161–169

107. Christensen, A.H. and Quail, P.H. (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5, 213–218

108. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

109. Obembe OO, Popoola JO, Leelavathi S, Reddy SV. Advances in plant molecular farming. Biotechnol Adv. 2011;29(2):210–22.

110. Nahampun, HN. Plant molecular farming for the production of industrial enzyme and vaccines [dissertation].[Iowa]:Iowa State University;2015. 220 p.https://lib.dr.iastate.edu/etd/14962

111. Streatfield SJ. Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J. 2007 Jan;5(1):2-15. doi: 10.1111/j.1467-7652.2006.00216.x. PMID: 17207252.

112. Habibi P, Prado GS, Pelegrini PB, Hefferon KL, Soccol CR, Grossi-de-Sa MF. Optimization of inside and outside factors to improve recombinant protein yield in plant. Plant Cell Tiss. Organ Cult. 2017;130;449-467. https://doi-org.ezproxy.wellesley.edu/10.1007/s11240-017-1240-5

113. Nahampun, HN. Plant molecular farming for the production of industrial enzyme and vaccines [dissertation].[Iowa]:Iowa State University;2015. 220 p.https://lib.dr.iastate.edu/etd/14962

114. Zhao D, Hamilton JP, Hardigan M, Yin D, He T, Vaillancourt B, Reynoso M, Pauluzzi G, Funkhouser S, Cui Y, Bailey-Serres J, Jiang J, Buell CR, Jiang N. Analysis of Ribosome-Associated mRNAs in Rice Reveals the Importance of Transcript Size and GC Content in Translation. G3 (Bethesda). 2017 Jan 5;7(1):203-219. doi: 10.1534/g3.116.036020. PMID: 27852012; PMCID: PMC5217110.

115. Koziel, M.G. et al. (1996) Optimizing expression of transgenes with an emphasis on post-transcriptional events. Plant Mol. Biol. 32, 393–405

116. Chiba Y, Green PJ. mRNA Degradation Machinery in Plants. J. Plant Biol. 2009; 52;114-124.https://doi-org.ezproxy.wellesley.edu/10.1007/s12374-009-9021-2

142 117. Sharma AK, Sharma MK. Plants as bioreactors: Recent developments and emerging opportunities. Biotechnol Adv. 2009 Nov-Dec;27(6):811-832. doi: 10.1016/j.biotechadv.2009.06.004. Epub 2009 Jun 30. PMID: 19576278; PMCID: PMC7125752.

118. Abiri R, Valdiani A, Maziah M, Shaharuddin NA, Sahebi M, Yusof ZN, Atabaki N, Talei D. A Critical Review of the Concept of Transgenic Plants: Insights into Pharmaceutical Biotechnology and Molecular Farming. Curr Issues Mol Biol. 2016;18:21-42. Epub 2015 May 6. PMID: 25944541.

119. Webster GR, Teh AY, Ma JK. Synthetic gene design-The rationale for codon optimization and implications for molecular pharming in plants. Biotechnol Bioeng. 2017 Mar;114(3):492-502. doi: 10.1002/bit.26183. Epub 2016 Dec 15. PMID: 27618314.

120. Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM. Seed-based expression systems for plant molecular farming. Plant Biotechnol J. 2010 Jun;8(5):588-606. doi: 10.1111/j.1467-7652.2010.00511.x. PMID: 20500681. . 121. Bosch D, Castilho A, Loos A, Schots A, Steinkellner H. N-glycosylation of plant-produced recombinant proteins. Curr Pharm Des. 2013;19(31):5503-12. doi: 10.2174/1381612811319310006. PMID: 23394562.

122. Schillberg S, Fischer R, Emans N. 'Molecular farming' of antibodies in plants. Naturwissenschaften. 2003 Apr;90(4):145-55. doi: 10.1007/s00114-002-0400-5. Epub 2003 Feb 18. PMID: 12712248.

123. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

124. Saint-Jore-Dupas C, Faye L, Gomord V. From planta to pharma with glycosylation in the toolbox. Trends Biotechnol. 2007 Jul;25(7):317-23. doi: 10.1016/j.tibtech.2007.04.008. Epub 2007 May 10. PMID: 17493697.

125. Makhzoum A, Benyammi R, Moustafa K, Trémouillaux-Guiller J. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming. BioDrugs. 2014 Apr;28(2):145-59. doi: 10.1007/s40259-013-0062-1. PMID: 23959796; PMCID: PMC7100180.

126. De Meyer T, Depicker A. Trafficking of endoplasmic reticulum-retained recombinant proteins is unpredictable in Arabidopsis thaliana. Front Plant Sci. 2014 Sep 15;5:473. doi: 10.3389/fpls.2014.00473. PMID: 25309564; PMCID: PMC4163989.

127. Mandal MK, Ahvari H, Schillberg S, Schiermeyer A. Tackling Unwanted Proteolysis in Plant Production Hosts Used for Molecular Farming. Front Plant Sci.

143 2016 Mar 8;7:267. doi: 10.3389/fpls.2016.00267. PMID: 27014293; PMCID: PMC4782010.

128. Margolin EA, Strasser R, Chapman R, Williamson AL, Rybicki EP, Meyers AE. Engineering the Plant Secretory Pathway for the Production of Next-Generation Pharmaceuticals. Trends Biotechnol. 2020 Sep;38(9):1034-1044. doi: 10.1016/j.tibtech.2020.03.004. Epub 2020 Apr 1. PMID: 32818443.

129. Clemente M, Corigliano MG, Pariani SA, Sánchez-López EF, Sander VA, Ramos-Duarte VA. Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming. Int J Mol Sci. 2019 Mar 17;20(6):1345. doi: 10.3390/ijms20061345. PMID: 30884891; PMCID: PMC6471620.

130. Mandal MK, Ahvari H, Schillberg S, Schiermeyer A. Tackling Unwanted Proteolysis in Plant Production Hosts Used for Molecular Farming. Front Plant Sci. 2016 Mar 8;7:267. doi: 10.3389/fpls.2016.00267. PMID: 27014293; PMCID: PMC4782010.

131. Clemente M, Corigliano MG, Pariani SA, Sánchez-López EF, Sander VA, Ramos-Duarte VA. Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming. Int J Mol Sci. 2019 Mar 17;20(6):1345. doi: 10.3390/ijms20061345. PMID: 30884891; PMCID: PMC6471620.

132. Margolin EA, Strasser R, Chapman R, Williamson AL, Rybicki EP, Meyers AE. Engineering the Plant Secretory Pathway for the Production of Next-Generation Pharmaceuticals. Trends Biotechnol. 2020 Sep;38(9):1034-1044. doi: 10.1016/j.tibtech.2020.03.004. Epub 2020 Apr 1. PMID: 32818443.

133. Clemente M, Corigliano MG, Pariani SA, Sánchez-López EF, Sander VA, Ramos-Duarte VA. Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming. Int J Mol Sci. 2019 Mar 17;20(6):1345. doi: 10.3390/ijms20061345. PMID: 30884891; PMCID: PMC6471620.

134. Mandal MK, Ahvari H, Schillberg S, Schiermeyer A. Tackling Unwanted Proteolysis in Plant Production Hosts Used for Molecular Farming. Front Plant Sci. 2016 Mar 8;7:267. doi: 10.3389/fpls.2016.00267. PMID: 27014293; PMCID: PMC4782010.

135. Jutras PV, Dodds I, van der Hoorn RA. Proteases of Nicotiana benthamiana: an emerging battle for molecular farming. Curr Opin Biotechnol. 2020 Feb;61:60-65. doi: 10.1016/j.copbio.2019.10.006. Epub 2019 Nov 22. PMID: 31765962.

136. Obembe OO, Popoola JO, Leelavathi S, Reddy SV. Advances in plant molecular farming. Biotechnol Adv. 2011 Mar-Apr;29(2):210-22. doi: 10.1016/j.biotechadv.2010.11.004. Epub 2010 Nov 27. PMID: 21115109.

137. Wilson IB. Glycosylation of proteins in plants and invertebrates. Curr Opin Struct Biol. 2002 Oct;12(5):569-77. doi: 10.1016/s0959-440x(02)00367-6. Erratum in: Curr Opin Struct Biol. 2003 Feb;13(1):141. PMID: 12464307.

144 138. Margolin E, Crispin M, Meyers A, Chapman R, Rybicki EP. A Roadmap for the Molecular Farming of Viral Glycoprotein Vaccines: Engineering Glycosylation and Glycosylation-Directed Folding. Front Plant Sci. 2020 Dec 3;11:609207. doi: 10.3389/fpls.2020.609207. PMID: 33343609; PMCID: PMC7744475.

139. Bosch D, Schots A. Plant glycans: friend or foe in vaccine development? Expert Rev Vaccines. 2010 Aug;9(8):835-42. doi: 10.1586/erv.10.83. PMID: 20673008.

140. Margolin E, Crispin M, Meyers A, Chapman R, Rybicki EP. A Roadmap for the Molecular Farming of Viral Glycoprotein Vaccines: Engineering Glycosylation and Glycosylation-Directed Folding. Front Plant Sci. 2020 Dec 3;11:609207. doi: 10.3389/fpls.2020.609207. PMID: 33343609; PMCID: PMC7744475.

141. Jansing, J., Sack, M., Augustine, S.M., Fischer, R. and Bortesi, L. (2019), CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose. Plant Biotechnol J, 17: 350-361. https://doi.org/10.1111/pbi.12981

142. Mor TS. Molecular pharming's foot in the FDA's door: Protalix's trailblazing story. Biotechnol Lett. 2015 Nov;37(11):2147-50. doi: 10.1007/s10529-015-1908-z. Epub 2015 Jul 7. PMID: 26149580; PMCID: PMC4583803.

143. Margolin EA, Strasser R, Chapman R, Williamson AL, Rybicki EP, Meyers AE. Engineering the Plant Secretory Pathway for the Production of Next-Generation Pharmaceuticals. Trends Biotechnol. 2020 Sep;38(9):1034-1044. doi: 10.1016/j.tibtech.2020.03.004. Epub 2020 Apr 1. PMID: 32818443.

144. Tremblay R, Wang D, Jevnikar AM, Ma S. Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnol Adv. 2010 Mar-Apr;28(2):214-21. doi: 10.1016/j.biotechadv.2009.11.008. Epub 2009 Dec 2. PMID: 19961918; PMCID: PMC7132750.

145. Margolin EA, Strasser R, Chapman R, Williamson AL, Rybicki EP, Meyers AE. Engineering the Plant Secretory Pathway for the Production of Next-Generation Pharmaceuticals. Trends Biotechnol. 2020 Sep;38(9):1034-1044. doi: 10.1016/j.tibtech.2020.03.004. Epub 2020 Apr 1. PMID: 32818443.

146. Jutras PV, Dodds I, van der Hoorn RA. Proteases of Nicotiana benthamiana: an emerging battle for molecular farming. Curr Opin Biotechnol. 2020 Feb;61:60-65. doi: 10.1016/j.copbio.2019.10.006. Epub 2019 Nov 22. PMID: 31765962.

145 Chapter 3-Citations

1. Bamogo PKA, Brugidou C, Sérémé D, Tiendrébéogo F, Djigma FW, Simpore J, Lacombe S. Virus-based pharmaceutical production in plants: an opportunity to reduce health problems in Africa. Virol J. 2019 Dec 30;16(1):167. doi: 10.1186/s12985-019-1263-0. PMID: 31888686; PMCID: PMC6937724.

2. Rosales-Mendoza S, Nieto-Gómez R, Angulo C. A Perspective on the Development of Plant-Made Vaccines in the Fight against Ebola Virus. Front Immunol. 2017 Mar 10;8:252. doi: 10.3389/fimmu.2017.00252. PMID: 28344580; PMCID: PMC5344899.

3. Rybicki EP. Plant-based vaccines against viruses. Virol J. 2014 Dec 3;11:205. doi: 10.1186/s12985-014-0205-0. PMID: 25465382; PMCID: PMC4264547.

4. Tsekoa TL, Singh AA, Buthelezi SG. Molecular farming for therapies and vaccines in Africa. Curr Opin Biotechnol. 2020 Feb;61:89-95. doi: 10.1016/j.copbio.2019.11.005. Epub 2019 Nov 28. PMID: 31786432.

5. Mackey TK, Liang BA, Cuomo R, Hafen R, Brouwer KC, Lee DE. Emerging and reemerging neglected tropical diseases: a review of key characteristics, risk factors, and the policy and innovation environment. Clin Microbiol Rev. 2014 Oct;27(4):949-79. doi: 10.1128/CMR.00045-14. PMID: 25278579; PMCID: PMC4187634.

6. Daniell H, Singh ND, Mason H, Streatfield SJ. Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci. 2009 Dec;14(12):669-79. doi: 10.1016/j.tplants.2009.09.009. Epub 2009 Oct 14. PMID: 19836291; PMCID: PMC2787751.

7. WHO [Internet]. c2021. Essential Medicines and Health Products. [Accessed 2021 April 28]. Available from: https://www.who.int/medicines/services/essmedicines_def/en/

8. WHO [Internet]. c2021. Access to Medicines: Making Market Forces Serve the Poor. [Accessed 2021 April 28]. Available from:https://www.who.int/publications/10-year-review/chapter-medicines.pdf.

9. WHO [Internet]. c2021. Essential Medicines and Health Products. [Accessed 2021 April 28]. Available from: https://www.who.int/medicines/services/essmedicines_def/en/

10. Conway M, Holt T, Sabow A, Sun IY. McKinsey & Company:Public and Social Sector [Internet].c 1996-2021. Should Sub-Saharan Africa Make Its Own Drugs?; 2019 January 10 [cited 2021 April 28] Available from: https://www.mckinsey.com/industries/public-and-social-sector/our-insights/should-su b-saharan-africa-make-its-own-drugs

146 11. EA Health [Internet] 2nd EAC Regional Pharmaceutical Manufacturing Plan of Action 2017-2027;2018 March [cited 2021 April 28].Available from: https://www.eahealth.org/policy-publications/2nd-eac-regional-pharmaceutical-manu facturing-plan-of-action-2017%E2%80%932027

12. Murad S, Fuller S, Menary J, Moore C, Pinneh E, Szeto T, Hitzeroth I, Freire M, Taychakhoonavudh S, Phoolcharoen W, Ma JK. Molecular Pharming for low and middle income countries. Curr Opin Biotechnol. 2020 Feb;61:53-59. doi: 10.1016/j.copbio.2019.10.005. Epub 2019 Nov 18. PMID: 31751895.

13. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

14. Mortimer E, Maclean JM, Mbewana S, Buys A, Williamson AL, Hitzeroth II, Rybicki EP. Setting up a platform for plant-based influenza virus vaccine production in South Africa. BMC Biotechnol. 2012 Apr 26;12:14. doi: 10.1186/1472-6750-12-14. PMID: 22536810; PMCID: PMC3358240.

15. Fisher JA, Cottingham MD, Kalbaugh CA. Peering into the pharmaceutical "pipeline": investigational drugs, clinical trials, and industry priorities. Soc Sci Med. 2015 Apr;131:322-30. doi: 10.1016/j.socscimed.2014.08.023. Epub 2014 Aug 19. PMID: 25159693; PMCID: PMC4334744.

16. Bamogo PKA, Brugidou C, Sérémé D, Tiendrébéogo F, Djigma FW, Simpore J, Lacombe S. Virus-based pharmaceutical production in plants: an opportunity to reduce health problems in Africa. Virol J. 2019 Dec 30;16(1):167. doi: 10.1186/s12985-019-1263-0. PMID: 31888686; PMCID: PMC6937724.

17. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

18. Rappuoli R, Miller HI, Falkow S. Medicine. The intangible value of vaccination. Science. 2002 Aug 9;297(5583):937-9. doi: 10.1126/science.1075173. PMID: 12169712.

19. Bethony JM, Cole RN, Guo X, Kamhawi S, Lightowlers MW, Loukas A, Petri W, Reed S, Valenzuela JG, Hotez PJ. Vaccines to combat the neglected tropical diseases. Immunol Rev. 2011 Jan;239(1):237-70. doi: 10.1111/j.1600-065X.2010.00976.x. PMID: 21198676; PMCID: PMC3438653.

20. Garcia-Gonzalez P, Boultbee P, Epstein D. Novel Humanitarian Aid Program: The Glivec International Patient Assistance Program-Lessons Learned From Providing Access to Breakthrough Targeted Oncology Treatment in Low- and Middle-Income Countries. J Glob Oncol. 2015 Sep 23;1(1):37-45. doi: 10.1200/JGO.2015.000570. PMID: 28804770; PMCID: PMC5551649.

147 21. Ladner J, Besson MH, Rodrigues M, Sams K, Audureau E, Saba J. Prevention of mother-to-child HIV transmission in resource-limited settings: assessment of 99 Viramune Donation Programmes in 34 countries, 2000-2011. BMC Public Health. 2013 May 14;13:470. doi: 10.1186/1471-2458-13-470. PMID: 23672811; PMCID: PMC3660172.

22. Mayo Clinic Laboratories [Internet]. c1995-2021. Human Papillomavirus (HPV) DNA Detection with Genotyping , High Risk Types by PCR, ThinPrep, Varies; [cited 2021 April 28]. Available from: https://www.mayocliniclabs.com/test-catalog/Clinical%2Band%2BInterpretive/62598

23. Steben M, Duarte-Franco E. Human papillomavirus infection: epidemiology and pathophysiology. Gynecol Oncol. 2007;107(2 Suppl 1):S2–5

24. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV,et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999;189:12

25. Bamogo PKA, Brugidou C, Sérémé D, Tiendrébéogo F, Djigma FW, Simpore J, Lacombe S. Virus-based pharmaceutical production in plants: an opportunity to reduce health problems in Africa. Virol J. 2019 Dec 30;16(1):167. doi: 10.1186/s12985-019-1263-0. PMID: 31888686; PMCID: PMC6937724.

26. Zohoncon TM, Ouedraogo TC, Brun LVC, Obiri-Yeboah D, Djigma WF,Kabibou S, et al. Molecular epidemiology of high-risk human papillomavirus in high-grade cervical intraepithelial Neoplasia and in cervical Cancer inParakou, Republic of Benin. Pak J Biol Sci PJBS. 2016;19:4

27. Piras F, Piga M, De Montis A, Zannou AR, Minerba L, Perra MT, et al.Prevalence of human papillomavirus infection in women in Benin. West Africa Virol J. 2011;8:514

28. Ouedraogo RA, Zohoncon TM, Guigma SP, Angèle Traore IM, Ouattara AK,Ouedraogo M, et al. Oncogenic human papillomavirus infection and genotypes characterization among sexually active women in Tenkodogo at Burkina Faso. West Africa Papillomavirus Res. 2018;6:22

29. Bamogo PKA, Brugidou C, Sérémé D, Tiendrébéogo F, Djigma FW, Simpore J, Lacombe S. Virus-based pharmaceutical production in plants: an opportunity to reduce health problems in Africa. Virol J. 2019 Dec 30;16(1):167. doi: 10.1186/s12985-019-1263-0. PMID: 31888686; PMCID: PMC6937724.

30. Ndiaye C, Alemany L, Ndiaye N, Kamaté B, Diop Y, Odida M, et al. Humanpapillomavirus distribution in invasive cervical carcinoma in sub-SaharanAfrica: could HIV explain the differences? Trop Med Int Health TM IH. 2012;17:1432

31. Rybicki EP. Plant-based vaccines against viruses. Virol J. 2014 Dec 3;11:205. doi: 10.1186/s12985-014-0205-0. PMID: 25465382; PMCID: PMC4264547.

148 32. Bosch FX, Broker TR, Forman D, Moscicki AB, Gillison ML, Doorbar J, SternPL, Stanley M, Arbyn M, Poljak M, Cuzick J, Castle PE, Schiller JT, Markowitz LE, Fisher WA, Canfell K, Denny LA, Franco EL, Steben M, Kane MA,Schiffman M, Meijer CJ, Sankaranarayanan R, Castellsague X, Kim JJ, Brotons M, Alemany L, Albero G, Diaz M, de Sanjose S,et al:Comprehensive control of human papillomavirus infections and related diseases. Vaccine 2013,31(Suppl 7):H1–H31

33. Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25major cancers in 1990. Int J Cancer. 1999;80:82

34. Hotez P, Ottesen E, Fenwick A, Molyneux D. The neglected tropical diseases: the ancient afflictions of stigma and poverty and the prospects for their control and elimination. Adv Exp Med Biol. 2006;582:23-33. doi: 10.1007/0-387-33026-7_3. PMID: 16802616.

35. Molyneux DH. "Neglected" diseases but unrecognised successes--challenges and opportunities for infectious disease control. Lancet. 2004 Jul 24-30;364(9431):380-3. doi: 10.1016/S0140-6736(04)16728-7. PMID: 15276399.

36. Ockenhouse CF, Magill A, Smith D, Milhous W. History of U.S. military contributions to the study of malaria. Mil Med. 2005 Apr;170(4 Suppl):12-6. doi: 10.7205/milmed.170.4s.12. PMID: 15916279.

37. Hotez P, Ottesen E, Fenwick A, Molyneux D. The neglected tropical diseases: the ancient afflictions of stigma and poverty and the prospects for their control and elimination. Adv Exp Med Biol. 2006;582:23-33. doi: 10.1007/0-387-33026-7_3. PMID: 16802616.

38. Gallup JL, Sachs JD. The economic burden of malaria. Am J Trop Med Hyg. 2001 Jan-Feb;64(1-2 Suppl):85-96. doi: 10.4269/ajtmh.2001.64.85. PMID: 11425181.

39. Rybicki EP. Plant-based vaccines against viruses. Virol J. 2014 Dec 3;11:205. doi: 10.1186/s12985-014-0205-0. PMID: 25465382; PMCID: PMC4264547.

40. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

41. Rybicki EP. Plant-based vaccines against viruses. Virol J. 2014 Dec 3;11:205. doi: 10.1186/s12985-014-0205-0. PMID: 25465382; PMCID: PMC4264547.

42. Bethony JM, Cole RN, Guo X, Kamhawi S, Lightowlers MW, Loukas A, Petri W, Reed S, Valenzuela JG, Hotez PJ. Vaccines to combat the neglected tropical diseases. Immunol Rev. 2011 Jan;239(1):237-70. doi: 10.1111/j.1600-065X.2010.00976.x. PMID: 21198676; PMCID: PMC3438653.

43. Molyneux DH. Combating the "other diseases" of MDG 6: changing the paradigm to achieve equity and poverty reduction? Trans R Soc Trop Med Hyg. 2008

149 Jun;102(6):509-19. doi: 10.1016/j.trstmh.2008.02.024. Epub 2008 Apr 14. PMID: 18413278.

44. Bethony JM, Cole RN, Guo X, Kamhawi S, Lightowlers MW, Loukas A, Petri W, Reed S, Valenzuela JG, Hotez PJ. Vaccines to combat the neglected tropical diseases. Immunol Rev. 2011 Jan;239(1):237-70. doi: 10.1111/j.1600-065X.2010.00976.x. PMID: 21198676; PMCID: PMC3438653.

45. Liese BH, Schubert L. Official development assistance for health—how neglected are neglected tropical diseases? An analysis of health financing. Int. Health 2009; 1:141–147. 10.1016/j.inhe.2009.08.004

46. Musgrove P, Hotez PJ. 2009. Turning neglected tropical diseases into forgotten maladies. Health Affairs 28:1691–1706. 10.1377/hlthaff.28.6.1691

47. Bollyky TJ. The tragedy of vaccine nationalism: Only cooperation can end the pandemic. Foreign Affairs. 2021; 99: 96.

48. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

49. Launch & Scale Speedometer [Internet]. Durham, North Carolina: Duke University; c2020. Vaccine Procurement; 2021 January 25 [cited 2021 April 28]. Available from https://launchandscalefaster.org/covid-19/vaccineprocurement.

50. Launch & Scale Speedometer [Internet]. Durham, North Carolina: Duke University; c2020. Vaccine Procurement; 2021 April 28 [cited 2021 April 28]. Available from https://launchandscalefaster.org/covid-19/vaccineprocurement.

51. Callaway E. The unequal scramble for coronavirus vaccines - by the numbers. Nature. 2020 Aug;584(7822):506-507. doi: 10.1038/d41586-020-02450-x. PMID: 32839593.

52. Fidler DP. Vaccine nationalism's politics. Science. 2020 Aug 14;369(6505):749. doi: 10.1126/science.abe2275. PMID: 32792371.

53. Chohan UW. Coronavirus & Vaccine Nationalism (January 16, 2021). CASS Working Papers on Economics & National Affairs 2021 , Available at SSRN: https://ssrn.com/abstract=3767610 or http://dx.doi.org/10.2139/ssrn.3767610

54. Barasa E, Ouma PO, Okiro EA. Assessing the hospital surge capacity of the Kenyan health system in the face of the COVID-19 pandemic. medRxiv 2020; published online April 11. DOI:10.1101/2020.04.08.20057984v1

55. Mackworth-Young CRS, Chingono R, Mavodza C, et al. ‘Here, we cannot practice what is preached’: early qualitative learning from community perspectives on Zimbabwe’s response to COVID-19. Bull World Health Organ 2020; published online April 20. DOI:10.2471/BLT.20.260224

150 56. Mahler DG, Lakner C Aguilar RAC, Wu H. World Bank Blogs [Internet]. c2021. The Impact of COVID-19 (Coronavirus) on Global Poverty: Why Sub-Saharan Africa might be the Region Hardest Hit; 2020 April 20 [cited 2021 April 28]. Available from:https://blogs.worldbank.org/opendata/impact-covid-19-coronavirus-global-pover ty-why-sub-saharan-africa-might-be-region-hardest

57. Akseer N, Kandru G, Keats EC., Bhutta ZA. COVID-19 pandemic and mitigation strategies: implications for maternal and child health and nutrition. The American Journal of Clinical Nutrition. 2020 August; 112(2): 251–256, https://doi.org/10.1093/ajcn/nqaa171

58. Jain R, Budlender J, Zizzamia R, Bassier I. The Labor Market and Poverty Impacts of Covid-19 in South Africa. Cape Town: SALDRU, UCT. 2020. (SALDRU Working Paper No. 264)

59. Morgan SG, Bathula HS, Moon S. Pricing of pharmaceuticals is becoming a major challenge for health systems. BMJ. 2020 Jan 13;368:l4627. doi: 10.1136/bmj.l4627. PMID: 31932289.

60. Health Action International [Internet]. Life-Saving Insulin Largely Unaffordable-a One Day Snapshot of the Price of Insulin across 60 Countries. 2010 [cited 2021 April 28]. Available from: https://www.haiweb.org/medicineprices/07072010/Global_briefing_note_FINAL.pdf

61. Goldstein DA, Clark J, Tu Y, et al. A global comparison of the cost of patented cancer drugs in relation to global differences in wealth. Oncotarget 2017;8:71548-55. . doi:10.18632/oncotarget.17742

62. Morgan S, Grootendorst P, Lexchin J, CunninghamC, Greyson D. The cost of drug development: asystematic review.Health Policy 2011;100:4-17.doi:10.1016/j.healthpol.2010.12.002

63. Avorn J. The $2.6 billion pill—methodologic and policy considerations.N Engl J Med 2015;372:1877-9. doi:10.1056/NEJMp1500848

64. Giannuzzi V, Conte R, Landi A, et al. Orphan medicinal products in Europe and United States to cover needs of patients with rare diseases: an increased common effort is to be foreseen. Orphanet J Rare Dis 2017;12:64. doi:10.1186/s13023-017-0617-1

65. Ofori-Adjei D, Lartey, P. Challenges of Local Production of Pharmaceuticals in Improving Access to Medicines. In: Parker R, Sommer M, editors. Routledge Handbook of Global Public Health. Abingdon:Routledge; 2010 Dec 17.

66. Morgan SG, Bathula HS, Moon S. Pricing of pharmaceuticals is becoming a major challenge for health systems. BMJ. 2020 Jan 13;368:l4627. doi: 10.1136/bmj.l4627. PMID: 31932289.

151 67. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

68. Tsekoa TL, Singh AA, Buthelezi SG. Molecular farming for therapies and vaccines in Africa. Curr Opin Biotechnol. 2020 Feb;61:89-95. doi: 10.1016/j.copbio.2019.11.005. Epub 2019 Nov 28. PMID: 31786432.

69. Caudron JM, Ford N, Henkens M, Macé C, Kiddle-Monroe R, Pinel J. Substandard medicines in resource-poor settings: a problem that can no longer be ignored. Trop Med Int Health. 2008 Aug;13(8):1062-72. doi: 10.1111/j.1365-3156.2008.02106.x. Epub 2008 Jul 8. PMID: 18631318.

70. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

71. Murad S, Fuller S, Menary J, Moore C, Pinneh E, Szeto T, Hitzeroth I, Freire M, Taychakhoonavudh S, Phoolcharoen W, Ma JK. Molecular Pharming for low and middle income countries. Curr Opin Biotechnol. 2020 Feb;61:53-59. doi: 10.1016/j.copbio.2019.10.005. Epub 2019 Nov 18. PMID: 31751895.

72. Conway M, Holt T, Sabow A, Sun IY. McKinsey & Company:Public and Social Sector [Internet].c 1996-2021. Should Sub-Saharan Africa Make Its Own Drugs?; 2019 January 10 [cited 2021 April 28] Available from: https://www.mckinsey.com/industries/public-and-social-sector/our-insights/should-su b-saharan-africa-make-its-own-drugs .

73. Kaplan WA, Ritz LS, Vitello M. Local production of medical technologies and its effect on access in low and middle income countries: a systematic review of the literature. South Med Rev. 2011 Dec;4(2):51-61. doi: 10.5655/smr.v4i2.1002. Epub 2011 Dec 2. PMID: 23093883; PMCID: PMC3471180.

74. Ofori-Adjei D, Lartey, P. Challenges of Local Production of Pharmaceuticals in Improving Access to Medicines. In: Parker R, Sommer M, editors. Routledge Handbook of Global Public Health. Abingdon:Routledge; 2010 Dec 17.

75. Murad S, Fuller S, Menary J, Moore C, Pinneh E, Szeto T, Hitzeroth I, Freire M, Taychakhoonavudh S, Phoolcharoen W, Ma JK. Molecular Pharming for low and middle income countries. Curr Opin Biotechnol. 2020 Feb;61:53-59. doi: 10.1016/j.copbio.2019.10.005. Epub 2019 Nov 18. PMID: 31751895.

76. Tsekoa TL, Singh AA, Buthelezi SG. Molecular farming for therapies and vaccines in Africa. Curr Opin Biotechnol. 2020 Feb;61:89-95. doi: 10.1016/j.copbio.2019.11.005. Epub 2019 Nov 28. PMID: 31786432.

152 77. Bamogo PKA, Brugidou C, Sérémé D, Tiendrébéogo F, Djigma FW, Simpore J, Lacombe S. Virus-based pharmaceutical production in plants: an opportunity to reduce health problems in Africa. Virol J. 2019 Dec 30;16(1):167. doi: 10.1186/s12985-019-1263-0. PMID: 31888686; PMCID: PMC6937724.

78. Rybicki EP. Plant-made vaccines and reagents for the One Health initiative. Hum Vaccin Immunother. 2017 Dec 2;13(12):2912-2917. doi: 10.1080/21645515.2017.1356497. Epub 2017 Aug 28. PMID: 28846485; PMCID: PMC5718809.

79. Rybicki EP. Plant-based vaccines against viruses. Virol J. 2014 Dec 3;11:205. doi: 10.1186/s12985-014-0205-0. PMID: 25465382; PMCID: PMC4264547.

80. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

81. Conway M, Holt T, Sabow A, Sun IY. McKinsey & Company:Public and Social Sector [Internet].c 1996-2021. Should Sub-Saharan Africa Make Its Own Drugs?; 2019 January 10 [cited 2021 April 28] Available from: https://www.mckinsey.com/industries/public-and-social-sector/our-insights/should-su b-saharan-africa-make-its-own-drugs

82. Caudron JM, Ford N, Henkens M, Macé C, Kiddle-Monroe R, Pinel J. Substandard medicines in resource-poor settings: a problem that can no longer be ignored. Trop Med Int Health. 2008 Aug;13(8):1062-72. doi: 10.1111/j.1365-3156.2008.02106.x. Epub 2008 Jul 8. PMID: 18631318.

83. Conway M, Holt T, Sabow A, Sun IY. McKinsey & Company:Public and Social Sector [Internet].c 1996-2021. Should Sub-Saharan Africa Make Its Own Drugs?; 2019 January 10 [cited 2021 April 28] Available from: https://www.mckinsey.com/industries/public-and-social-sector/our-insights/should-su b-saharan-africa-make-its-own-drugs

84. Ma JK, Christou P, Chikwamba R, Haydon H, Paul M, Ferrer MP, Ramalingam S, Rech E, Rybicki E, Wigdorovitz A, Yang DC, Thangaraj H. Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies. Plant Biotechnol J. 2013 Dec;11(9):1029-33. doi: 10.1111/pbi.12127. Epub 2013 Oct 14. Erratum in: Plant Biotechnol J. 2017 Jan;15(1):144. PMID: 24119183.

85. Rybicki EP. Plant-based vaccines against viruses. Virol J. 2014 Dec 3;11:205. doi: 10.1186/s12985-014-0205-0. PMID: 25465382; PMCID: PMC4264547.

86. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

153 87. Wilson KR, Kohler JC, Ovtcharenko N. The make or buy debate: considering the limitations of domestic production in Tanzania. Global Health. 2012 Jun 29;8:20. doi: 10.1186/1744-8603-8-20. PMID: 22747578; PMCID: PMC3413540.

88. Conway M, Holt T, Sabow A, Sun IY. McKinsey & Company:Public and Social Sector [Internet].c 1996-2021. Should Sub-Saharan Africa Make Its Own Drugs?; 2019 January 10 [cited 2021 April 28] Available from: https://www.mckinsey.com/industries/public-and-social-sector/our-insights/should-su b-saharan-africa-make-its-own-drugs

89. Tsekoa TL, Singh AA, Buthelezi SG. Molecular farming for therapies and vaccines in Africa. Curr Opin Biotechnol. 2020 Feb;61:89-95. doi: 10.1016/j.copbio.2019.11.005. Epub 2019 Nov 28. PMID: 31786432.

90. Rybicki EP. Plant-made vaccines and reagents for the One Health initiative. Hum Vaccin Immunother. 2017 Dec 2;13(12):2912-2917. doi: 10.1080/21645515.2017.1356497. Epub 2017 Aug 28. PMID: 28846485; PMCID: PMC5718809.

91.Latinne A, Hu B, Olival KJ. et al. Origin and cross-species transmission of bat coronaviruses in China. Nat Commun 11, 4235 (2020). https://doi.org/10.1038/s41467-020-17687-3 92. Rybicki EP. Plant-made vaccines and reagents for the One Health initiative. Hum Vaccin Immunother. 2017 Dec 2;13(12):2912-2917. doi: 10.1080/21645515.2017.1356497. Epub 2017 Aug 28. PMID: 28846485; PMCID: PMC5718809.

93. Shahid N, Daniell H. Plant-based oral vaccines against zoonotic and non-zoonotic diseases. Plant Biotechnol J. 2016 Nov;14(11):2079-2099. doi: 10.1111/pbi.12604. Epub 2016 Aug 23. PMID: 27442628; PMCID: PMC5095797.

94. Yeh KB, Fair JM, Smith W, Martinez Torres T, Lucas J, Monagin C, Winegar R, Fletcher J. Assessing Climate Change Impact on Ecosystems and Infectious Disease: Important Roles for Genomic Sequencing and a One Health Perspective. Trop Med Infect Dis. 2020 Jun 3;5(2):90. doi:

95. Rybicki EP. Plant-based vaccines against viruses. Virol J. 2014 Dec 3;11:205. doi: 10.1186/s12985-014-0205-0. PMID: 25465382; PMCID: PMC4264547.

96. Sorvillo TE, Rodriguez SE, Hudson P, Carey M, Rodriguez LL, Spiropoulou CF, Bird BH, Spengler JR, Bente DA. Towards a Sustainable One Health Approach to Crimean-Congo Hemorrhagic Fever Prevention: Focus Areas and Gaps in Knowledge. Trop Med Infect Dis. 2020 Jul 7;5(3):113. doi:

97. Estrada-Peña A, Ruiz-Fons F, Acevedo P, Gortazar C, de la Fuente J. Factors driving the circulation and possible expansion of Crimean-Congo haemorrhagic fever virus in the western Palearctic. J Appl Microbiol. 2013 Jan;114(1):278-86. doi: 10.1111/jam.12039. Epub 2012 Nov 7. PMID: 23061817.

154 98. Duygu F, Sari T, Kaya T, Tavsan O, Naci M. The Relationship Between Crimean-Congo Hemmorhagic Fever and Climate: Does Climate Affect the Number of Patients? Acta Clin Croat. 2018 Sep;57(3):443-448. doi: 10.20471/acc.2018.57.03.06. PMID: 31168176; PMCID: PMC6536269.

99. Ansari H, Shahbaz B, Izadi S, Zeinali M, Tabatabaee SM, Mahmoodi M, Holakouie Naieni K, Mansournia MA. Crimean-Congo hemorrhagic fever and its relationship with climate factors in southeast Iran: a 13-year experience. J Infect Dev Ctries. 2014 Jun 11;8(6):749-57. doi: 10.3855/jidc.4020. PMID: 24916874.

100. Anyamba A, Linthicum KJ, Tucker CJ. Climate-disease connections: Rift Valley Fever in Kenya. Cad Saude Publica. 2001;17 Suppl:133-40. doi: 10.1590/s0102-311x2001000700022. PMID: 11426274.

101. Anyamba A, Small JL, Britch SC, Tucker CJ, Pak EW, Reynolds CA, Crutchfield J, Linthicum KJ. Recent weather extremes and impacts on agricultural production and vector-borne disease outbreak patterns. PLoS One. 2014 Mar 21;9(3):e92538. doi: 10.1371/journal.pone.0092538. PMID: 24658301; PMCID: PMC3962414.

102. Tschofen M, Knopp D, Hood E, Stöger E. Plant Molecular Farming: Much More than Medicines. Annu Rev Anal Chem (Palo Alto Calif). 2016 Jun 12;9(1):271-94. doi: 10.1146/annurev-anchem-071015-041706. Epub 2016 Mar 30. PMID: 27049632.

103. Capell T, Twyman RM, Armario-Najera V, Ma JK, Schillberg S, Christou P. Potential Applications of Plant Biotechnology against SARS-CoV-2. Trends Plant Sci. 2020 Jul;25(7):635-643. doi: 10.1016/j.tplants.2020.04.009. Epub 2020 Apr 24. PMID: 32371057; PMCID: PMC7181989.

104. Topp E, Irwin R, McAllister T, Lessard M, Joensuu JJ, Kolotilin I, Conrad U, Stöger E, Mor T, Warzecha H, Hall JC, McLean MD, Cox E, Devriendt B, Potter A, Depicker A, Virdi V, Holbrook L, Doshi K, Dussault M, Friendship R, Yarosh O, Yoo HS, MacDonald J, Menassa R. The case for plant-made veterinary immunotherapeutics. Biotechnol Adv. 2016 Sep-Oct;34(5):597-604. doi: 10.1016/j.biotechadv.2016.02.007. Epub 2016 Feb 12. PMID: 26875776.

105. Ma JK, Christou P, Chikwamba R, Haydon H, Paul M, Ferrer MP, Ramalingam S, Rech E, Rybicki E, Wigdorovitz A, Yang DC, Thangaraj H. Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies. Plant Biotechnol J. 2013 Dec;11(9):1029-33. doi: 10.1111/pbi.12127. Epub 2013 Oct 14. Erratum in: Plant Biotechnol J. 2017 Jan;15(1):144. PMID: 24119183.

106. Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 2001 May;6(5):219-26. doi: 10.1016/s1360-1385(01)01922-7. PMID: 11335175; PMCID: PMC5496653.

107. Juarez P, Virdi V, Depicker A, Orzaez D. Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications. Plant

155 Biotechnol J. 2016 Sep;14(9):1791-9. doi: 10.1111/pbi.12541. Epub 2016 Feb 13. PMID: 26873071; PMCID: PMC5067594.

108. Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM. Plant-based production of biopharmaceuticals. Curr Opin Plant Biol. 2004 Apr;7(2):152-8. doi: 10.1016/j.pbi.2004.01.007. PMID: 15003215.

109. Xu S, Gavin J, Jiang R, Chen H. Bioreactor productivity and media cost comparison for different intensified cell culture processes. Biotechnol Prog. 2017 Jul;33(4):867-878. doi: 10.1002/btpr.2415. Epub 2017 Jan 28. PMID: 27977910.

110. Fujiuchi N, Matoba N, Matsuda R. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression. Front Bioeng Biotechnol. 2016 Mar 8;4:23. doi: 10.3389/fbioe.2016.00023. PMID: 27014686; PMCID: PMC4781840.

111. Jamal A, Ko K, Kim HS, Choo YK, Joung H, Ko K. Role of genetic factors and environmental conditions in recombinant protein production for molecular farming. Biotechnol Adv. 2009 Nov-Dec;27(6):914-923. doi: 10.1016/j.biotechadv.2009.07.004. Epub 2009 Aug 19. PMID: 19698776.

112. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

113. Commandeur U, Twyman RM, and Fischer R. The biosafety of molecular farming in plants. AgBiotechNet. 2003; 5, 1–9

114. Kim JS, Yoon SJ, Park YJ, Kim SY, Ryu CM. Crossing the kingdom border: Human diseases caused by plant pathogens. Environ Microbiol. 2020 Jul;22(7):2485-2495. doi: 10.1111/1462-2920.15028. Epub 2020 May 4. PMID: 32307848.

115. Tripathi NK, Shrivastava A. Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development. Front Bioeng Biotechnol. 2019 Dec 20;7:420. doi: 10.3389/fbioe.2019.00420. PMID: 31921823; PMCID: PMC6932962.

116. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

117. Fujiuchi N, Matoba N, Matsuda R. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression. Front Bioeng Biotechnol. 2016 Mar 8;4:23. doi: 10.3389/fbioe.2016.00023. PMID: 27014686; PMCID: PMC4781840.

118. Clark M, Maselko M. Transgene Biocontainment Strategies for Molecular Farming. Front Plant Sci. 2020 Mar 3;11:210. doi: 10.3389/fpls.2020.00210. PMID: 32194598; PMCID: PMC7063990.

156 119. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

120. Buyel JF, Twyman RM, Fischer R. Very-large-scale production of antibodies in plants: The biologization of manufacturing. Biotechnol Adv. 2017 Jul;35(4):458-465. doi: 10.1016/j.biotechadv.2017.03.011. Epub 2017 Mar 25. PMID: 28347720.

121. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

122. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

123. Ramalingam S, Iyappan G, Priya S, Kadirvelu K, Kingston J, Gopalan N, Sharma R. Rapid Production of Therapeutic Proteins Using Plant System. Defence Life Science Journal. 2017; 2(2), 95-102. https://doi.org/10.14429/dlsj.2.11372

124. Naseri Z, Khezri G, Davarpanah S, Ofoghi H. Virus-Based Vectors: A New Approach for the Production of Recombinant Proteins. Journal of Applied Biotechnology Reports. c2009; 6(1):6-14.

125. Gengenbach BB, Keil LL, Opdensteinen P, Müschen CR, Melmer G, Lentzen H, Bührmann J, Buyel JF. Comparison of microbial and transient expression (tobacco plants and plant-cell packs) for the production and purification of the anticancer mistletoe lectin viscumin. Biotechnol Bioeng. 2019

126. Chen Q, Davis KR. The potential of plants as a system for the development and production of human biologics. F1000Res. 2016 May 19;5:F1000 Faculty Rev-912. doi: 10.12688/f1000research.8010.1. PMID: 27274814; PMCID: PMC4876878.

127. Mor TS. Molecular pharming's foot in the FDA's door: Protalix's trailblazing story. Biotechnol Lett. 2015 Nov;37(11):2147-50. doi: 10.1007/s10529-015-1908-z. Epub 2015 Jul 7. PMID: 26149580; PMCID: PMC4583803.

128. Jansing J, Sack M, Augustine SM, Fischer R, Bortesi L. CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β-1,2-xylose and core α-1,3-fucose. Plant Biotechnol J. 2019 Feb;17(2):350-361. doi: 10.1111/pbi.12981. Epub 2018 Aug 11. PMID: 29969180; PMCID: PMC6335070.

129. Margolin E, Crispin M, Meyers A, Chapman R, Rybicki EP. A Roadmap for the Molecular Farming of Viral Glycoprotein Vaccines: Engineering Glycosylation and Glycosylation-Directed Folding. Front Plant Sci. 2020 Dec 3;11:609207. doi: 10.3389/fpls.2020.609207. PMID: 33343609; PMCID: PMC7744475.

157 130. Penney CA, Thomas DR, Deen SS, Walmsley AM. Plant-made vaccines in support of the Millennium Development Goals. Plant Cell Rep. 2011 May;30(5):789-98. doi: 10.1007/s00299-010-0995-5. Epub 2011 Jan 18. PMID: 21243362; PMCID: PMC3075396.

131. Ma JK, Christou P, Chikwamba R, Haydon H, Paul M, Ferrer MP, Ramalingam S, Rech E, Rybicki E, Wigdorovitz A, Yang DC, Thangaraj H. Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies. Plant Biotechnol J. 2013 Dec;11(9):1029-33. doi: 10.1111/pbi.12127. Epub 2013 Oct 14. Erratum in: Plant Biotechnol J. 2017 Jan;15(1):144. PMID: 24119183.

132. Menary J, Amato M, Sanchez AC, Hobbs M, Pacho A, Fuller SS. New Hope for a "Cursed" Crop? Understanding Stakeholder Attitudes to Plant Molecular Farming With Modified Tobacco in Europe. Front Plant Sci. 2020 Jun 12;11:791. doi: 10.3389/fpls.2020.00791. PMID: 32595677; PMCID: PMC7304234.

133. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003 Dec;21(12):570-8. doi: 10.1016/j.tibtech.2003.10.002. PMID: 14624867.

134. Buyel JF, Twyman RM, Fischer R. Very-large-scale production of antibodies in plants: The biologization of manufacturing. Biotechnol Adv. 2017 Jul;35(4):458-465. doi: 10.1016/j.biotechadv.2017.03.011. Epub 2017 Mar 25. PMID: 28347720.

135. Schillberg S, Finnern R. Plant molecular farming for the production of valuable proteins - Critical evaluation of achievements and future challenges. J Plant Physiol. 2021 Mar-Apr;258-259:153359. doi: 10.1016/j.jplph.2020.153359. Epub 2021 Jan 5. PMID: 33460995.

136. The Medical Letter: Because the Source Matters. c2021. In Brief: Taliglucerase (Elelyso) for Gaucher Disease; 2012 July 9 [Cited April 28 2021]. Available from: https://secure.medicalletter.org/w1394d#refs_d

137. Buyel JF, Twyman RM, Fischer R. Very-large-scale production of antibodies in plants: The biologization of manufacturing. Biotechnol Adv. 2017 Jul;35(4):458-465. doi: 10.1016/j.biotechadv.2017.03.011. Epub 2017 Mar 25. PMID: 28347720.

138. Ramalingam S, Iyappan G, Priya S, Kadirvelu K, Kingston J, Gopalan N, Sharma R. Rapid Production of Therapeutic Proteins Using Plant System. Defence Life Science Journal. 2017; 2(2), 95-102. https://doi.org/10.14429/dlsj.2.11372

139. Whaley KJ, Morton J, Hume S, Hiatt E, Bratcher B, Klimyuk V, Hiatt A, Pauly M, Zeitlin L. Emerging antibody-based products. Curr Top Microbiol Immunol. 2014;375:107-26. doi: 10.1007/82_2012_240. PMID: 22772797.

158 140. Boothe JG., Saponja JA. and Parmenter DL. Molecular farming in plants: oilseeds as vehicles for the production of pharmaceutical proteins. Drug Dev. Res. 1997; 42, 172–181

141. Kwon KC, Verma D, Singh ND, Herzog R, Daniell H. Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells. Adv Drug Deliv Rev. 2013 Jun 15;65(6):782-99. doi: 10.1016/j.addr.2012.10.005. Epub 2012 Oct 23. PMID: 23099275; PMCID: PMC3582797.

142. Merlin M, Pezzotti M, Avesani L. Edible plants for oral delivery of biopharmaceuticals. Br J Clin Pharmacol. 2017 Jan;83(1):71-81. doi: 10.1111/bcp.12949. Epub 2016 May 9. PMID: 27037892; PMCID: PMC5338148.

143. Specht EA, Mayfield SP. Algae-based oral recombinant vaccines. Front Microbiol. 2014 Feb 17;5:60. doi: 10.3389/fmicb.2014.00060. PMID: 24596570; PMCID: PMC3925837.

144. Kwon KC, Verma D, Singh ND, Herzog R, Daniell H. Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells. Adv Drug Deliv Rev. 2013 Jun 15;65(6):782-99. doi: 10.1016/j.addr.2012.10.005. Epub 2012 Oct 23. PMID: 23099275; PMCID: PMC3582797.

145. Drossard J, editor. Downstream Processing of Plant-Derived Recombinant Therapeutic Proteins. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co; 2004. 315p.

146. Buyel, J.F., Opdensteinen, P. and Fischer, R. (2015), Cellulose‐based filter aids increase the capacity of depth filters during the downstream processing of plant‐derived biopharmaceutical proteins. Biotechnology Journal, 10: 584-591. https://doi.org/10.1002/biot.201400611

147. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

148. Wang X, Hunter AK, Mozier NM. Host cell proteins in biologics development: Identification, quantitation and risk assessment. Biotechnol Bioeng. 2009 Jun 15;103(3):446-58. doi: 10.1002/bit.22304. PMID: 19388135.

149. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

150. Kwon KC, Verma D, Singh ND, Herzog R, Daniell H. Oral delivery of human biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in plant cells. Adv Drug Deliv Rev. 2013 Jun 15;65(6):782-99. doi: 10.1016/j.addr.2012.10.005. Epub 2012 Oct 23. PMID: 23099275; PMCID: PMC3582797.

151. Schillberg, S. & Finnern, R. (2021). Plant molecular farming for the production of valuable proteins – Critical evaluation of achievements and future

159 challenges. Journal of Plant Physiology. 258-259. 153359. 10.1016/j.jplph.2020.153359.

152. Tschofen M, Knopp D, Hood E, Stöger E. Plant Molecular Farming: Much More than Medicines. Annu Rev Anal Chem (Palo Alto Calif). 2016 Jun 12;9(1):271-94. doi: 10.1146/annurev-anchem-071015-041706. Epub 2016 Mar 30. PMID: 27049632.

153. Buyel JF. Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing. Front Plant Sci. 2019 Jan 18;9:1893. doi: 10.3389/fpls.2018.01893. PMID: 30713542; PMCID: PMC6345721.

154. McCormick AA, Kumagai MH, Hanley K, Turpen TH, Hakim I, Grill LK, Tusé D, Levy S, Levy R. Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants. Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):703-8. doi: 10.1073/pnas.96.2.703. PMID: 9892697; PMCID: PMC15200.

155. Buyel JF, Fischer R. Downstream processing of biopharmaceutical proteins produced in plants: the pros and cons of flocculants. Bioengineered. 2014 Mar-Apr;5(2):138-42. doi: 10.4161/bioe.28061. Epub 2014 Feb 3. PMID: 24637706; PMCID: PMC4049906.

156. Buyel JF, Opdensteinen P, Fischer R. Cellulose-based filter aids increase the capacity of depth filters during the downstream processing of plant-derived biopharmaceutical proteins. Biotechnol J. 2015 Apr;10(4):584-91. doi: 10.1002/biot.201400611. Epub 2015 Feb 18. PMID: 25611947.

157. Hassan S, van Dolleweerd CJ, Ioakeimidis F, Keshavarz-Moore E, Ma JK. Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants. Plant Biotechnol J. 2008 Sep;6(7):733-48. doi: 10.1111/j.1467-7652.2008.00354.x. Epub 2008 May 29. PMID: 18513238.

158. Lightfoot EN, Root TW, O'Dell JL. Emergence of ideal membrane cascades for downstream processing. Biotechnol Prog. 2008 May-Jun;24(3):599-605. doi: 10.1021/bp070335l. Epub 2008 Apr 15. PMID: 18410154.

159. Buyel JF, Fischer, R. A juice extractor can simplify the downstream processing of plant-derived biopharmaceutical proteins compared to blade-based homogenizers.Process Biochem. 2014; 50: 859–866.doi: 10.1016/j.procbio.2015.02.017

160. Hofbauer A, Melnik S, Tschofen M, Arcalis E, Phan HT, Gresch U, Lampel J, Conrad U, Stoger E. The Encapsulation of Hemagglutinin in Protein Bodies Achieves a Stronger Immune Response in Mice than the Soluble Antigen. Front Plant Sci. 2016 Feb 16;7:142. doi: 10.3389/fpls.2016.00142. PMID: 26909090; PMCID: PMC4754457.

161. Laibach N, Post J, Twyman RM, Gronover CS, Prüfer D. The characteristics and potential applications of structural lipid droplet proteins in plants. J Biotechnol. 2015

160 May 10;201:15-27. doi: 10.1016/j.jbiotec.2014.08.020. Epub 2014 Aug 23. PMID: 25160916.

162. Madeira LM, Szeto TH, Henquet M, Raven N, Runions J, Huddleston J, Garrard I, Drake PM, Ma JK. High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures. Plant Biotechnol J. 2016 Feb;14(2):615-24. doi: 10.1111/pbi.12407. Epub 2015 Jun 3. PMID: 26038982.

163. Madeira LM, Szeto TH, Ma JK, Drake PMW. Rhizosecretion improves the production of Cyanovirin-N in Nicotiana tabacum through simplified downstream processing. Biotechnol J. 2016 Jul;11(7):910-919. doi: 10.1002/biot.201500371. Epub 2016 Mar 9. PMID: 26901579; PMCID: PMC4929045.

164. Avesani L, Bortesi L, Santi L, Falorni A, Pezzotti M. Plant-made pharmaceuticals for the prevention and treatment of autoimmune diseases: where are we? Expert Rev Vaccines. 2010 Aug;9(8):957-69. doi: 10.1586/erv.10.82. PMID: 20673017.

165. Avesani L, Merlin M, Gecchele E, Capaldi S, Brozzetti A, Falorni A, Pezzotti M. Comparative analysis of different biofactories for the production of a major diabetes autoantigen. Transgenic Res. 2014 Apr;23(2):281-91. doi: 10.1007/s11248-013-9749-9. Epub 2013 Oct 20. PMID: 24142387; PMCID: PMC3951962.

161 Chapter 4-Citations

1. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

2. Murad S, Fuller S, Menary J, Moore C, Pinneh E, Szeto T, Hitzeroth I, Freire M, Taychakhoonavudh S, Phoolcharoen W, Ma JK. Molecular Pharming for low and middle income countries. Curr Opin Biotechnol. 2020 Feb;61:53-59. doi: 10.1016/j.copbio.2019.10.005. Epub 2019 Nov 18. PMID: 31751895.

3. Newcotiana [Internet]. Newcotiana Project; c2020. Stakeholders [cited 2021 April 28]. Available from: https://newcotiana.webs.upv.es/stakeholders.

4. Sparrow PA, Irwin JA, Dale PJ, Twyman RM, Ma JK. Pharma-Planta: road testing the developing regulatory guidelines for plant-made pharmaceuticals. Transgenic Res. 2007 Apr;16(2):147-61. doi: 10.1007/s11248-007-9074-2. Epub 2007 Feb 7. PMID: 17285266.

5. Murad S, Fuller S, Menary J, Moore C, Pinneh E, Szeto T, Hitzeroth I, Freire M, Taychakhoonavudh S, Phoolcharoen W, Ma JK. Molecular Pharming for low and middle income countries. Curr Opin Biotechnol. 2020 Feb;61:53-59. doi: 10.1016/j.copbio.2019.10.005. Epub 2019 Nov 18. PMID: 31751895.

6. Medicago [Internet]. Medicago Inc. Medicago to Build $245m Production Facility in Quebec City; 2015 May 10 [cited 2021 April 28]. Available from: https://www.medicago.com/en/media-room/medicago-to-build-245m-production-facil ity-in-quebec-city/

7. Rybicki EP. Third International Conference on Plant-Based Vaccines and Antibodies. Expert Rev Vaccines. 2009 Sep;8(9):1151-5. doi: 10.1586/erv.09.85. PMID: 19722888.

8. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

9. Murad S, Fuller S, Menary J, Moore C, Pinneh E, Szeto T, Hitzeroth I, Freire M, Taychakhoonavudh S, Phoolcharoen W, Ma JK. Molecular Pharming for low and middle income countries. Curr Opin Biotechnol. 2020 Feb;61:53-59. doi: 10.1016/j.copbio.2019.10.005. Epub 2019 Nov 18. PMID: 31751895.

10. Rybicki EP, Chikwamba R, Koch M, Rhodes JI, Groenewald JH. Plant-made therapeutics: an emerging platform in South Africa. Biotechnol Adv. 2012 Mar-Apr;30(2):449-59. doi: 10.1016/j.biotechadv.2011.07.014. Epub 2011 Aug 3. PMID: 21839824.

162 11. Ma JK, Christou P, Chikwamba R, Haydon H, Paul M, Ferrer MP, Ramalingam S, Rech E, Rybicki E, Wigdorovitz A, Yang DC, Thangaraj H. Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies. Plant Biotechnol J. 2013 Dec;11(9):1029-33. doi: 10.1111/pbi.12127. Epub 2013 Oct 14. Erratum in: Plant Biotechnol J. 2017 Jan;15(1):144. PMID: 24119183.

12. Paul MJ, Thangaraj H, Ma JK. Commercialization of new biotechnology: a systematic review of 16 commercial case studies in a novel manufacturing sector. Plant Biotechnol J. 2015 Oct;13(8):1209-20. doi: 10.1111/pbi.12426. Epub 2015 Jul 3. PMID: 26140440.

13. Ma JK, Christou P, Chikwamba R, Haydon H, Paul M, Ferrer MP, Ramalingam S, Rech E, Rybicki E, Wigdorovitz A, Yang DC, Thangaraj H. Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies. Plant Biotechnol J. 2013 Dec;11(9):1029-33. doi: 10.1111/pbi.12127. Epub 2013 Oct 14. Erratum in: Plant Biotechnol J. 2017 Jan;15(1):144. PMID: 24119183.

14. Spok A, Karner S. Plant Molecular Farming: Opportunities and Challenges. Brussels: European Communities. 2008.

15. Paul M, van Dolleweerd C, Drake PM, Reljic R, Thangaraj H, Barbi T, Stylianou E, Pepponi I, Both L, Hehle V, Madeira L, Inchakalody V, Ho S, Guerra T, Ma JK. Molecular Pharming: future targets and aspirations. Hum Vaccin. 2011 Mar;7(3):375-82. doi: 10.4161/hv.7.3.14456. Epub 2011 Mar 1. PMID: 21368584; PMCID: PMC3230538.

16. Drake PM, Thangaraj H. Molecular farming, patents and access to medicines. Expert Rev Vaccines. 2010 Aug;9(8):811-9. doi: 10.1586/erv.10.72. PMID: 20673006.

17. Spok A, Karner S. Plant Molecular Farming: Opportunities and Challenges. Brussels: European Communities. 2008.

18. Sparrow PA, Irwin JA, Dale PJ, Twyman RM, Ma JK. Pharma-Planta: road testing the developing regulatory guidelines for plant-made pharmaceuticals. Transgenic Res. 2007 Apr;16(2):147-61. doi: 10.1007/s11248-007-9074-2. Epub 2007 Feb 7. PMID: 17285266.

19. Spok A, Karner S. Plant Molecular Farming: Opportunities and Challenges. Brussels: European Communities. 2008.

20. Wilson KR, Kohler JC, Ovtcharenko N. The make or buy debate: considering the limitations of domestic production in Tanzania. Global Health. 2012 Jun 29;8:20. doi: 10.1186/1744-8603-8-20. PMID: 22747578; PMCID: PMC3413540.

21. Spok A, Karner S. Plant Molecular Farming: Opportunities and Challenges. Brussels: European Communities. 2008.

163 22. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

23. Spok A, Karner S. Plant Molecular Farming: Opportunities and Challenges. Brussels: European Communities. 2008.

24. Foster S, Laing R, Melgaard B, et al. Ensuring Supplies of Appropriate Drugs and Vaccines. In: Jamison DT, Breman JG, Measham AR, et al., editors. Disease Control Priorities in Developing Countries. 2nd edition. Washington (DC): The International Bank for Reconstruction and Development / The World Bank; 2006. Chapter 72. Available from: https://www.ncbi.nlm.nih.gov/books/NBK11723/ Co-published by Oxford University Press, New York.

25. WHO [Internet]. Geneva:WHO. c.2021. The WHO Prequalification Project;2004 [cited 2021 April 28]. Available from:http://mednet3.who.int/prequal/

26. Ofori-Adjei D, Lartey P. Challenges of Local Production of Pharmaceuticals in Improving Access to Medicines. In: Parker, R. and Sommer, M. editors. Routledge Handbook of Global Public Health. Abingdon: Routledge; 2010 17 Dec. Routledge Handbooks Online.

27. Anyakora C, Ekwunife O, Alozie F, Esuga M, Ukwuru J, Onya S, Nwokike J. Cost benefit of investment on quality in pharmaceutical manufacturing: WHO GMP pre- and post-certification of a Nigerian pharmaceutical manufacturer. BMC Health Serv Res. 2017 Sep 18;17(1):665. doi: 10.1186/s12913-017-2610-8. PMID: 28923044; PMCID: PMC5604295.

28. Wilson KR, Kohler JC, Ovtcharenko N. The make or buy debate: considering the limitations of domestic production in Tanzania. Global Health. 2012 Jun 29;8:20. doi: 10.1186/1744-8603-8-20. PMID: 22747578; PMCID: PMC3413540.

29. Murad S, Fuller S, Menary J, Moore C, Pinneh E, Szeto T, Hitzeroth I, Freire M, Taychakhoonavudh S, Phoolcharoen W, Ma JK. Molecular Pharming for low and middle income countries. Curr Opin Biotechnol. 2020 Feb;61:53-59. doi: 10.1016/j.copbio.2019.10.005. Epub 2019 Nov 18. PMID: 31751895.

30. Wilson KR, Kohler JC, Ovtcharenko N. The make or buy debate: considering the limitations of domestic production in Tanzania. Global Health. 2012 Jun 29;8:20. doi: 10.1186/1744-8603-8-20. PMID: 22747578; PMCID: PMC3413540.

31. Murad S, Fuller S, Menary J, Moore C, Pinneh E, Szeto T, Hitzeroth I, Freire M, Taychakhoonavudh S, Phoolcharoen W, Ma JK. Molecular Pharming for low and middle income countries. Curr Opin Biotechnol. 2020 Feb;61:53-59. doi: 10.1016/j.copbio.2019.10.005. Epub 2019 Nov 18. PMID: 31751895.

32. Murad S, Fuller S, Menary J, Moore C, Pinneh E, Szeto T, Hitzeroth I, Freire M, Taychakhoonavudh S, Phoolcharoen W, Ma JK. Molecular Pharming for low and middle income countries. Curr Opin Biotechnol. 2020 Feb;61:53-59. doi: 10.1016/j.copbio.2019.10.005. Epub 2019 Nov 18. PMID: 31751895.

164 33. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

34. Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer A. Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Curr Pharm Des. 2013;19(31):5531-42. doi: 10.2174/1381612811319310008. PMID: 23394569.

35. Buyel JF, Twyman RM, Fischer R. Very-large-scale production of antibodies in plants: The biologization of manufacturing. Biotechnol Adv. 2017 Jul;35(4):458-465. doi: 10.1016/j.biotechadv.2017.03.011. Epub 2017 Mar 25. PMID: 28347720

36. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

37. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

38. Sparrow PA, Irwin JA, Dale PJ, Twyman RM, Ma JK. Pharma-Planta: road testing the developing regulatory guidelines for plant-made pharmaceuticals. Transgenic Res. 2007 Apr;16(2):147-61. doi: 10.1007/s11248-007-9074-2. Epub 2007 Feb 7. PMID: 17285266.

39. Geigert J. The Challenge of CMC Regulatory Compliance for Biopharmaceuticals. 1st.ed. Boston: Springer; c2004.Chapter 5, Recombinant Source Material: Master/Working Banks; p 83-114

40. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

41. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

42. EMEA,2009. Committee for Proprietary Medicinal Products (CPMP). In: Guideline on the Quality of Biological Active Substances Produced by Stable Transgene Expression in Higher Plants (EMEA/CHMP/BWP/48316/2006). EMA,London,UK.

43. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

44. Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J. GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnol Adv. 2012 Mar-Apr;30(2):434-9. doi: 10.1016/j.biotechadv.2011.08.007. Epub 2011 Aug 12. PMID: 21856403.

165 45. Schillberg S, Finnern R. Plant molecular farming for the production of valuable proteins - Critical evaluation of achievements and future challenges. J Plant Physiol. 2021 Mar-Apr;258-259:153359. doi: 10.1016/j.jplph.2020.153359. Epub 2021 Jan 5. PMID: 33460995.

46. Fujiuchi N, Matoba N, Matsuda R. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression. Front Bioeng Biotechnol. 2016 Mar 8;4:23. doi: 10.3389/fbioe.2016.00023. PMID: 27014686; PMCID: PMC4781840.

47. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

48. Huebbers JW, Buyel JF. On the verge of the market - Plant factories for the automated and standardized production of biopharmaceuticals. Biotechnol Adv. 2021 Jan-Feb;46:107681. doi: 10.1016/j.biotechadv.2020.107681. Epub 2020 Dec 14. PMID: 33326816.

49. Breyer D, Goossens M, Herman P, Sneyers M. Biosafety considerations associated with molecular farming in genetically modified plants. Journal of Medicinal Plants Research. 2009; 3: 825-838..

50. IEA [Internet]. c2021. SDG7:Data and Projections. 2020 October [cited 2021 April 28]. Available from: https://www.iea.org/reports/sdg7-data-and-projections/access-to-electricity.

51. Rosa L, Chiarelli DD, Rulli MC, Dell'Angelo J, D'Odorico P. Global agricultural economic water scarcity. Sci Adv. 2020 Apr 29;6(18):eaaz6031. doi: 10.1126/sciadv.aaz6031. PMID: 32494678; PMCID: PMC7190309.

52. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

53. Tschofen M, Knopp D, Hood E, Stöger E. Plant Molecular Farming: Much More than Medicines. Annu Rev Anal Chem (Palo Alto Calif). 2016 Jun 12;9(1):271-94. doi: 10.1146/annurev-anchem-071015-041706. Epub 2016 Mar 30. PMID: 27049632.

54. Breyer D, Goossens M, Herman P, Sneyers M. Biosafety considerations associated with molecular farming in genetically modified plants. Journal of Medicinal Plants Research. 2009; 3: 825-838.

55. Daniell H. Molecular strategies for gene containment in transgenic crops. Nat Biotechnol. 2002 Jun;20(6):581-6. doi: 10.1038/nbt0602-581. Erratum in: Nat Biotechnol. 2002 Aug;20(8):843. PMID: 12042861; PMCID: PMC3471138.

56. Commandeur U, Twyman RM, Fischer R. The biosafety of molecular farming in plants. Ag. Biotech. 2003; Net 5: 1-9.

166 57. Clark M, Maselko M. Transgene Biocontainment Strategies for Molecular Farming. Front Plant Sci. 2020 Mar 3;11:210. doi: 10.3389/fpls.2020.00210. PMID: 32194598; PMCID: PMC7063990.

58. Reichman JR, Watrud LS, Lee EH, Burdick CA, Bollman MA, Storm MJ, King GA, Mallory-Smith C. Establishment of transgenic herbicide-resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats. Mol Ecol. 2006 Nov;15(13):4243-55. doi: 10.1111/j.1365-294X.2006.03072.x. PMID: 17054516.

59. Clark M, Maselko M. Transgene Biocontainment Strategies for Molecular Farming. Front Plant Sci. 2020 Mar 3;11:210. doi: 10.3389/fpls.2020.00210. PMID: 32194598; PMCID: PMC7063990.

60. Gressel J. Dealing with transgene flow of crop protection traits from crops to their relatives. Pest. Manag. Sci.; 2015; 71: 658-667. https://doi.org/10.1002/ps.3850

61. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

62. Brewer HC, Hird DL, Bailey AM, Seal SE, Foster GD. A guide to the contained use of plant virus infectious clones. Plant Biotechnol J. 2018 Apr;16(4):832-843. doi: 10.1111/pbi.12876. Epub 2018 Feb 6. PMID: 29271098; PMCID: PMC5867029.

63. Thresh JM, Hillocks, RJ. Cassava mosaic and Cassava Brown Streak Diseases in Nampula and Zambezia Provinces of Mozambique.Roots. 2003; 8(2), 10–15

64. Ramessar K, Capell T, Twyman RM, Quemada H, Christou P. Calling the tunes on transgenic crops – the case for regulatory harmony.Mol Breeding. 2009; 23, 99–112. 10.1007/s11032-008-9217-z.

65. Spok A, Karner S. Plant Molecular Farming: Opportunities and Challenges. Brussels: European Communities. 2008.

66. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

67. Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J. GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnol Adv. 2012 Mar-Apr;30(2):434-9. doi: 10.1016/j.biotechadv.2011.08.007. Epub 2011 Aug 12. PMID: 21856403.

68. Sparrow P, Broer I, Hood EE, Eversole K, Hartung F, Schiemann J. Risk assessment and regulation of molecular farming - a comparison between Europe and US. Curr Pharm Des. 2013;19(31):5513-30. doi: 10.2174/1381612811319310007. PMID: 23394565.

167 69. Tschofen M, Knopp D, Hood E, Stöger E. Plant Molecular Farming: Much More than Medicines. Annu Rev Anal Chem (Palo Alto Calif). 2016 Jun 12;9(1):271-94. doi: 10.1146/annurev-anchem-071015-041706. Epub 2016 Mar 30. PMID: 27049632.

70. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

71. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

72. Drake PM, Szeto TH, Paul MJ, Teh AY, Ma JK. Recombinant biologic products versus nutraceuticals from plants - a regulatory choice? Br J Clin Pharmacol. 2017 Jan;83(1):82-87. doi: 10.1111/bcp.13041. Epub 2016 Jul 28. PMID: 27297459; PMCID: PMC5338133.

73. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

74. Sparrow PA, Irwin JA, Dale PJ, Twyman RM, Ma JK. Pharma-Planta: road testing the developing regulatory guidelines for plant-made pharmaceuticals. Transgenic Res. 2007 Apr;16(2):147-61. doi: 10.1007/s11248-007-9074-2. Epub 2007 Feb 7. PMID: 17285266.

75. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

76. Gleba Y, Klimyuk V, Marillonnet S. Magnifection--a new platform for expressing recombinant vaccines in plants. Vaccine. 2005 Mar 18;23(17-18):2042-8. doi: 10.1016/j.vaccine.2005.01.006. PMID: 15755568.

77. Paul M, Ma JK. Plant-made pharmaceuticals: leading products and production platforms. Biotechnol Appl Biochem. 2011 Jan-Feb;58(1):58-67. doi: 10.1002/bab.6. PMID: 21446960.

78. Szarka S, Van Rooijen G, Moloney M, inventors; SemBioSys Genetics, Inc., assignee. Method for the Production of Multimeric Immunoglobulins and related compositions. US Patent 7098383B2, 2006 August 29.

79. Wayne C, inventor. Pennsylvania State University, assignee. Growing Cells in a Reservoir Formed of a Flexible Sterile Plastic Liner. US Patent 6709862B2. 2004 March 23.

80. Stomp AM, Dickey L, Gasdaska J, inventors. Byondis BV., assignee. Expression of Biologically Active Polypeptide in Duckweed. US Patent 6815184B2. 2004 November 09.

168 81. Drake PM, Thangaraj H. Molecular farming, patents and access to medicines. Expert Rev Vaccines. 2010 Aug;9(8):811-9. doi: 10.1586/erv.10.72. PMID: 20673006.

82. Dunwell JM. Review: intellectual property aspects of plant transformation. Plant Biotechnol J. 2005 Jul;3(4):371-84. doi: 10.1111/j.1467-7652.2005.00142.x. PMID: 17173626.

83. Paul MJ, Thangaraj H, Ma JK. Commercialization of new biotechnology: a systematic review of 16 commercial case studies in a novel manufacturing sector. Plant Biotechnol J. 2015 Oct;13(8):1209-20. doi: 10.1111/pbi.12426. Epub 2015 Jul 3. PMID: 26140440.

84. Emerging Life Sciences Companies Deskbook [Internet].Morgan & Lewis & Bocklus. Freedom to Operate; c2008 [cited 2021 April 28]. Available from: https://www.morganlewis.com/topics/entrepreneurresources/~/media/files/Special-Top ics/ERH_pubs/ERH_FreedomToOperate_ELSCDeskbook.

85. Beachy RN. IP policies and serving the public. Science. 2003 Jan 24;299(5606):473. doi: 10.1126/science.299.5606.473. PMID: 12543937.

86. Drake PM, Thangaraj H. Molecular farming, patents and access to medicines. Expert Rev Vaccines. 2010 Aug;9(8):811-9. doi: 10.1586/erv.10.72. PMID: 20673006.

87. Thangaraj H, van Dolleweerd CJ, McGowan EG, Ma JK. Dynamics of global disclosure through patent and journal publications for biopharmaceutical products. Nat Biotechnol. 2009 Jul;27(7):614-8. doi: 10.1038/nbt0709-614. PMID: 19587663.

88. Shapiro C. Navigating the Patent Thicket: Cross Licenses, Patent Pools, and Standard-Setting. SSRN. 2001 March. Available at SSRN: https://ssrn.com/abstract=273550 or http://dx.doi.org/10.2139/ssrn.273550

89. Using Intellectual Property Rights to Stimulate Pharmaceutical Production in Developing Countries: A Reference Guide. [Internet] United Nations Conference on Trade and Development; c2011 [cited 2021 April 28]. Available from: .https://unctad.org/system/files/official-document/diaepcb2009d19_en.pdf

90. Hall B, Helmers C, von Graevenitz G. Technology Entry in the Presence of Patent Thickets. Oxford Economic Papers. 2021; 73. 10.1093/oep/gpaa034. https://www.ifs.org.uk/uploads/publications/wps/wp201602.pdf

91. Intellectual Property and Developing Countries [Internet]. Rand Corporation; c2010 [cited 2021 April 28]. Available from: https://www.rand.org/content/dam/rand/pubs/technical_reports/2010/RAND_TR804.p df

92. Feldman R. May your drug price be evergreen. J Law Biosci. 2018 Dec 7;5(3):590-647. doi: 10.1093/jlb/lsy022. PMID: 31143456; PMCID: PMC6534750.

169 93. Dunwell JM. Review: intellectual property aspects of plant transformation. Plant Biotechnol J. 2005 Jul;3(4):371-84. doi: 10.1111/j.1467-7652.2005.00142.x. PMID: 17173626.

94. Paul MJ, Thangaraj H, Ma JK. Commercialization of new biotechnology: a systematic review of 16 commercial case studies in a novel manufacturing sector. Plant Biotechnol J. 2015 Oct;13(8):1209-20. doi: 10.1111/pbi.12426. Epub 2015 Jul 3. PMID: 26140440.

95. Grand Challenges [Internet]. Bill & Melinda Gates Foundation;c2003-2021 [cited 2021 April 28]. Available from: https://grandchallenges.org/.

96. Drake PM, Thangaraj H. Molecular farming, patents and access to medicines. Expert Rev Vaccines. 2010 Aug;9(8):811-9. doi: 10.1586/erv.10.72. PMID: 20673006.

97. Paul M, van Dolleweerd C, Drake PM, Reljic R, Thangaraj H, Barbi T, Stylianou E, Pepponi I, Both L, Hehle V, Madeira L, Inchakalody V, Ho S, Guerra T, Ma JK. Molecular Pharming: future targets and aspirations. Hum Vaccin. 2011 Mar;7(3):375-82. doi: 10.4161/hv.7.3.14456. Epub 2011 Mar 1. PMID: 21368584; PMCID: PMC3230538.

98. Stevens AJ, Effort, AE. Using academic license agreements to promote global social responsibility .J.License Exec. Soc. Int.XLIII, 85–101 (2008)

99. Mimura C. Technology licensing for the benefit of the developing world: UC Berkeley’s socially responsible licensing program. AUTM J., 2006; XVIII: 15–28

100. Ma JK, Christou P, Chikwamba R, Haydon H, Paul M, Ferrer MP, Ramalingam S, Rech E, Rybicki E, Wigdorovitz A, Yang DC, Thangaraj H. Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies. Plant Biotechnol J. 2013 Dec;11(9):1029-33. doi: 10.1111/pbi.12127. Epub 2013 Oct 14. Erratum in: Plant Biotechnol J. 2017 Jan;15(1):144. PMID: 24119183.

101. Fox JL. Puzzling industry response to ProdiGene fiasco. Nat Biotechnol. 2003 Jan;21(1):3-4. doi: 10.1038/nbt0103-3b. PMID: 12511893.

102. Case Studies In Agricultural Biosecurity [Internet]. Federation of American Scientists;c2011. The Prodigene Incident. [Cited 2021 April 28]. Available from: https://biosecurity.fas.org/education/dualuse-agriculture/2.-agricultural-biotechnology/ prodigene-incident.html.

103. Union of Concerned Scientists. Position Paper: Pharmaceutical and Industrial Crops. 2006 October; 1-23.

104. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

170 105. Hundleby PA, Sack M, Twyman RM. Biosafety, Risk Assessment, and Regulation of Molecular Farming. In: Kermode AR, Jiang L, editors. Molecular Pharming. John Wiley & Sons; c2018. p. 327-351. https://doi.org/10.1002/9781118801512.ch13

106. Kaiser J. Is the drought over for pharming? Science. 2008 Apr 25;320(5875):473-5. doi: 10.1126/science.320.5875.473. PMID: 18436771.

107. Allsopp M, Cotter J. Pharm Crops- A Super Disaster in the Making. Greenpeace Research Laboratories. 2005. https://www.greenpeace.to/publications/pharm_crops_2005.pdf

108. Fox JL. Beer giant blindsides Ventria's Pharmacrop. Nat Biotechnol. 2005 Jun;23(6):636. doi: 10.1038/nbt0605-636. PMID: 15940219.

109. Spok A. and Karner S. Plant Molecular Farming: Opportunities and Challenges. Brussels: European Communities. 2008.

110. Ramessar K, Capell T, Twyman RM, Quemada H, Christou P. Calling the tunes on transgenic crops – the case for regulatory harmony.Mol Breeding. 2009; 23, 99–112. 10.1007/s11032-008-9217-z.

111. Levidow, L. “Precautionary Uncertainty: Regulating GM Crops in Europe.” Social Studies of Science, vol. 31, no. 6, 2001, pp. 842–874. JSTOR, www.jstor.org/stable/3182946. Accessed 17 Apr. 2021.

112. Ramessar K, Capell T, Twyman RM, Christou P. Going to ridiculous lengths--European coexistence regulations for GM crops. Nat Biotechnol. 2010 Feb;28(2):133-6. doi: 10.1038/nbt0210-133. PMID: 20139947.

113. Cronica [Internet]. La Cronica Diaria S.A. de C.V. AMLO desoye a cientificos: "No van los transgenicos"; 2019 September 26 [cited 2021 April 28]. Available from: https://www.cronica.com.mx/notas-amlo_desoye_a_cientificos_no_van_los_transgeni cos-1132552-2019.

114. Science Magazine [Internet]. AAAS;c2021. Mexico's New Science Minister is a Plant Biologist who Opposes Transgenic Crops. 2018 October 4 [cited 2021 April 28]. Available from: https://www.sciencemag.org/news/2018/10/mexico-s-new-science-minister-plant-biol ogist-who-opposes-transgenic-crops.

115. Braimah JA, Atuoye KN, Vercillo S, Warring C, Luginaah I. Debated agronomy: public discourse and the future of biotechnology policy in Ghana. Glob Bioeth. 2017 Feb 22;28(1):3-18. doi: 10.1080/11287462.2016.1261604. PMID: 29147107; PMCID: PMC5678443.

116. Tockman J. Global Policy Forum [Internet]. Global Policy Forum; c2005-2021. Venezuela: Chavez Dumps Monsanto. 2004 May 5 [cited 2021 April 28]. Available from: https://archive.globalpolicy.org/socecon/tncs/2004/0505venezuela.htm

171 117. Genetic Literacy Project: Science not Ideology [Internet]. Genetic Literacy Project; c2012-2021. Where are GMO crops and animals approved and banned? [Cited 2021 April 28]. Available from: https://geneticliteracyproject.org/gmo-faq/where-are-gmo-crops-and-animals-approve d-and-banned/

118. Library of Congress Law [Internet]. Library of Congress. Restrictions on Genetically Modified Organisms: Argentina; 2020 December 30 [cited 2021 April 28]. Available from: https://www.loc.gov/law/help/restrictions-on-gmos/argentina.php.

119. Library of Congress Law [Internet]. Library of Congress. Restrictions on Genetically Modified Organisms:Brazil; 2020 December 30 [cited 2021 April 28]. Available from: https://www.loc.gov/law/help/restrictions-on-gmos/brazil.php.

120. Library of Congress Law [Internet]. Library of Congress. Restrictions on Genetically Modified Organisms:South Africa; 2020 December 30 [cited 2021 April 28]. Available from: "https://www.loc.gov/law/help/restrictions-on-gmos/south-africa.php. Accessed April 28th 2021.

121. Murad S, Fuller S, Menary J, Moore C, Pinneh E, Szeto T, Hitzeroth I, Freire M, Taychakhoonavudh S, Phoolcharoen W, Ma JK. Molecular Pharming for low and middle income countries. Curr Opin Biotechnol. 2020 Feb;61:53-59. doi: 10.1016/j.copbio.2019.10.005. Epub 2019 Nov 18. PMID: 31751895.

122. Milne, R. Public Attitudes Toward Molecular Farming in the UK. AgBioForum. 2008; 11(2): 106-113.

123. Zerbe N. Feeding the Famine? American Food Aid and the GMO Debate in Southern Africa. Food Policy. 2004; 29: 593-608. 10.1016/j.foodpol.2004.09.002.

124. Spok A, Karner, S. Plant Molecular Farming: Opportunities and Challenges. Brussels: European Communities. 2008.

125. Fischer R, Buyel JF. Molecular farming - The slope of enlightenment. Biotechnol Adv. 2020 May-Jun;40:107519. doi: 10.1016/j.biotechadv.2020.107519. Epub 2020 Jan 16. PMID: 31954848.

126. Kuntz M. Destruction of public and governmental experiments of GMO in Europe. GM Crops Food. 2012 Oct-Dec;3(4):258-64. doi: 10.4161/gmcr.21231. Epub 2012 Jul 24. PMID: 22825391.

127. Knight, A. Differential Effects of Perceived and Objective Knowledge Measures on Perceptions of Biotechnology. AgBioForum. 2005; 8(4): 221-227

128. Einsiedel EF, Medlock J. A public consultation on plant molecular farming. AgBioForum. 2005; 8: 26-32.

172 129. Pardo R, Engelhard M, Hagen K, Jørgensen RB, Rehbinder E, Schnieke A, Szmulewicz M, Thiele F. The role of means and goals in technology acceptance. A differentiated landscape of public perceptions of pharming. EMBO Rep. 2009 Oct;10(10):1069-75. doi: 10.1038/embor.2009.208. Epub 2009 Sep 18. PMID: 19763146; PMCID: PMC2759743.

130. Milne, R.. Public Attitudes Toward Molecular Farming in the UK. AgBioForum. 2008; 11(2): 106-113.

131. Einsiedel EF, Medlock J. A public consultation on plant molecular farming. AgBioForum. 2005; 8: 26-32.

132. Knight A. Does Application Matter? An Examination of Public Perception of Agricultural Biotechnology Applications. AgBioForum. AgBioForum. 2006; 9(2); 121-128.

133. Pardo R, Engelhard M, Hagen K, Jørgensen RB, Rehbinder E, Schnieke A, Szmulewicz M, Thiele F. The role of means and goals in technology acceptance. A differentiated landscape of public perceptions of pharming. EMBO Rep. 2009 Oct;10(10):1069-75. doi: 10.1038/embor.2009.208. Epub 2009 Sep 18. PMID: 19763146; PMCID: PMC2759743.

134. Kirk D, McIntosh K. Social Acceptance of Plant-Made Vaccines: Indications from a Public Survey. AgBioForum. 2005; 8(4): 228-234.

135. Knight, Andrew. . Does Application Matter? An Examination of Public Perception of Agricultural Biotechnology Applications. AgBioForum. AgBioForum. 2006; 9(2); 121-128.

136. Menary J, Hobbs M, Mesquita de Albuquerque S, Pacho A, Drake PMW, Prendiville A, Ma JK, Fuller SS. Shotguns vs Lasers: Identifying barriers and facilitators to scaling-up plant molecular farming for high-value health products. PLoS One. 2020 Mar 20;15(3):e0229952. doi: 10.1371/journal.pone.0229952. PMID: 32196508; PMCID: PMC7083274.

137. Milne, R.. Public Attitudes Toward Molecular Farming in the UK. AgBioForum. 2008; 11(2): 106-113.

138. Aerni P, Bernauer, T. Stakeholder attitudes toward GMOs in the Philippines, Mexico, and South Africa: The issue of public trust. World Development. 2006; 34: 557-575. 10.1016/j.worlddev.2005.08.007.

139. Einsiedel EF, Medlock J. A public consultation on plant molecular farming. AgBioForum. 2005; 8: 26-32.

140. Joly PB, Lemarié, S. Industry consolidation, public attitude and the future of plant biotechnology in Europe. AgBioForum. 1998;1(2): 85-90.

173 141. Aerni P, Bernauer, T. Stakeholder attitudes toward GMOs in the Philippines, Mexico, and South Africa: The issue of public trust. World Development. 2006; 34: 557-575. 10.1016/j.worlddev.2005.08.007.

142. Einsiedel EF, Medlock J. A public consultation on plant molecular farming. AgBioForum. 2005; 8: 26-32.

143. Menary J, Hobbs M, Mesquita de Albuquerque S, Pacho A, Drake PMW, Prendiville A, Ma JK, Fuller SS. Shotguns vs Lasers: Identifying barriers and facilitators to scaling-up plant molecular farming for high-value health products. PLoS One. 2020 Mar 20;15(3):e0229952. doi: 10.1371/journal.pone.0229952. PMID: 32196508; PMCID: PMC7083274.

144. Aerni P. Stakeholder attitudes towards the risks and benefits of genetically modified crops in South Africa. Environ. Sci. Policy, 2005 Oct; 8(5):464–476

145. Małayska A, Twardowski T. The Influence of Scientists, Agricultural Advisors and Farmers on Innovative Agrobiotechnology. AgBioForum. 2014; 17(1).

146. Vilella-Vila M, Costa-Font J, Mossialos E. Consumer involvement and acceptance of biotechnology in the European Union: a specific focus on Spain and the UK. Int J Consum Stud [Internet]. 2005 Mar 1 [cited 2019 Mar 27];29(2):108–18. Available from: http://doi.wiley.com/10.1111/j.1470-6431.2004.00425.x

147. Menary J, Hobbs M, Mesquita de Albuquerque S, Pacho A, Drake PMW, Prendiville A, Ma JK, Fuller SS. Shotguns vs Lasers: Identifying barriers and facilitators to scaling-up plant molecular farming for high-value health products. PLoS One. 2020 Mar 20;15(3):e0229952. doi: 10.1371/journal.pone.0229952. PMID: 32196508; PMCID: PMC7083274.

148. Nevitt J, Mills B, Reaves D, Norton G. Public Perceptions of Tobacco Biopharming. AgBioForum. 2006; 9(2): 104-110.

149. Appendix

150. Kirk D, McIntosh K. Social Acceptance of Plant-Made Vaccines: Indications from a Public Survey. AgBioForum. 2005; 8(4): 228-234.

151. Nausch H, Sautter C, Broer I, Schmidt K. Public funded field trials with transgenic plants in Europe: a comparison between Germany and Switzerland. Curr Opin. Biotechnol. 2015 Apr;32:171-178. doi: 10.1016/j.copbio.2014.12.023. Epub 2015 Jan 19. PMID: 25597648.

152. Connor M, Siegrist M. Factors Influencing People’s Acceptance of Gene Technology: The Role of Knowledge, Health Expectations, Naturalness, and Social

174 Trust. Sci Commun [Internet]. 2010 Dec 18 [cited 2019 Apr 16];32(4):514–38. Available from: http://journals.sagepub.com/doi/10.1177/1075547009358919

153. Knight A. Differential Effects of Perceived and Objective Knowledge Measures on Perceptions of Biotechnology. AgBioForum. 2005; 8(4); 221-227

154. Wilsdon J, Willis R. See Through Science: Why Public Engagement Needs to Move Upstream [Internet]. Demos; c2004 [cited 2021 April 28]. Available from: http://sro.sussex.ac.uk/id/eprint/47855/1/See_through_science.pdf

155. Arnall AH. Future Technologies: Today's Choices [Internet]. Greenpeace Environmental Trust; c2003 [cited 2021 April 28]. Available from: https://www.yumpu.com/en/document/read/22079593/future-technologies-todays-cho ices-greenpeace-uk

156. Wilsdon J, Willis R. See Through Science: Why Public Engagement Needs to Move Upstream [Internet]. Demos; c2004 [cited 2021 April 28]. Available from: http://sro.sussex.ac.uk/id/eprint/47855/1/See_through_science.pdf

157. Hargreaves I, Lewis J, Spears T. Towards a Better Map: Science, the Public and the Media [Internet]. Swindon: ESRC; c2003 . Available from: http://www.esrcsocietytoday.ac.uk/ESRCInfoCentre/Images/Mapdocfinal_tcm6-5505. pdf

175 Conclusion- Citations

1. Rademacher T, Sack M, Blessing D, Fischer R, Holland T, Buyel J. Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering. Plant Biotechnol J. 2019 Aug;17(8):1560-1566. doi: 10.1111/pbi.13081. Epub 2019 Feb 14. PMID: 30672078; PMCID: PMC6662111.

2. Margolin EA, Strasser R, Chapman R, Williamson AL, Rybicki EP, Meyers AE. Engineering the Plant Secretory Pathway for the Production of Next-Generation Pharmaceuticals. Trends Biotechnol. 2020 Sep;38(9):1034-1044. doi: 10.1016/j.tibtech.2020.03.004. Epub 2020 Apr 1. PMID: 32818443.

3. Jutras PV, Dodds I, van der Hoorn RA. Proteases of Nicotiana benthamiana: an emerging battle for molecular farming. Curr Opin Biotechnol. 2020 Feb;61:60-65. doi: 10.1016/j.copbio.2019.10.006. Epub 2019 Nov 22. PMID: 31765962.

4. Gengenbach BB, Opdensteinen P, Buyel JF. Robot Cookies - Plant Cell Packs as an Automated High-Throughput Screening Platform Based on Transient Expression. Front Bioeng Biotechnol. 2020 May 5;8:393. doi: 10.3389/fbioe.2020.00393. PMID: 32432097; PMCID: PMC7214789.

5. Huebbers JW, Buyel JF. On the verge of the market - Plant factories for the automated and standardized production of biopharmaceuticals. Biotechnol Adv. 2021 Jan-Feb;46:107681. doi: 10.1016/j.biotechadv.2020.107681. Epub 2020 Dec 14. PMID: 33326816.

6. Buyel JF, Stöger E, Bortesi L. Targeted genome editing of plants and plant cells for biomanufacturing. Transgenic Res. 2021 Mar 1. doi: 10.1007/s11248-021-00236-z. Epub ahead of print. PMID: 33646510.

7. Demir, F., Niedermaier, S., Villamor, J.G. and Huesgen, P.F. (2018), Quantitative proteomics in plant protease substrate identification. New Phytol, 218: 936-943. https://doi.org/10.1111/nph.14587

8. Jutras PV, Dodds I, van der Hoorn RA. Proteases of Nicotiana benthamiana: an emerging battle for molecular farming. Curr Opin Biotechnol. 2020 Feb;61:60-65. doi: 10.1016/j.copbio.2019.10.006. Epub 2019 Nov 22. PMID: 31765962.

9. Baruch Y, Budhwar P, Khatri N Brain drain: Inclination to stay abroad after studies. Journal of World Business. 2007: 42: 99-112. doi.org/10.1016/j.jwb.2006.11.004.

10. Ma JK, Christou P, Chikwamba R, Haydon H, Paul M, Ferrer MP, Ramalingam S, Rech E, Rybicki E, Wigdorovitz A, Yang DC, Thangaraj H. Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies. Plant Biotechnol J. 2013 Dec;11(9):1029-33. doi: 10.1111/pbi.12127. Epub 2013 Oct 14. Erratum in: Plant Biotechnol J. 2017 Jan;15(1):144. PMID: 24119183.

176 11. Ijsselmuiden C, Marais DL, Becerra-Posada F, Ghannem H. Africa's neglected area of human resources for health research - the way forward. S Afr Med J. 2012 Mar 7;102(4):228-33. PMID: 22464504.

12. Wilsdon J, Willis R. See Through Science: Why Public Engagement Needs to Move Upstream [Internet]. Demos; c2004 [cited 2021 April 28]. Available from: http://sro.sussex.ac.uk/id/eprint/47855/1/See_through_science.pdf

177 APPENDIX An Examination of Publicly Available Material on Plant Molecular Pharming

To establish some sense of the material available to the general public on plant molecular pharming, a targeted search was conducted in October 2020 using Google News. The search parameters were as follows:

● Search term: (molecular farming OR biopharming OR pharming) AND (plant) AND (pharmaceutical OR vaccine OR drug OR treatment OR therapy) ● Timespan= January 2015- October 2020 ● Data noted: ○ Is the article retrieved relevant to the plant molecular pharming topic? ■ Determined via reading the article. ○ Which country is the site hosting data from? ■ Determined through examining the site publishing the article. ○ Is it written for a general audience or for a professional, in industry/research audience? ■ Determined through examining the site publishing the article. ○ What type of content is written about? ■ Determined through examining the article and the site publishing it.

In total, around 30 pages of results, and n=305 articles were examined. Of these 305 articles, n=62, or around 20% of total findings, were deemed to be relevant to the plant molecular pharming topic.

Figure 1. Proportion of relevant to irrelevant articles found in search. (n=305)

Of these 62 relevant articles, most originated from US- and Europe-based sites and platforms, and most were professional media written for an in-industry audience, not for general public education. The strong US and European bias of the search terms may have, in part, been due to the use of English language search terms or the use of US-based Google.

178 Figure 2. Article platform site country of origin (n=62)

Figure 3. Article platform site target audience (n=62).

Topics discussed in professional and general public oriented articles also differed. Professionally oriented articles were dominated by either business related articles or pieces related to scientific disciplines, generally those related to medicine or life sciences. Publicly oriented articles either represented daily news stories for a local or academic community, or simple biotechnology or general science related pieces.

Figure 4. Professional article subject matter topics (n=40)

179 Figure 5. Public article subject matter topics. (n=22)

All raw data collected from this search, which was summarized above, can be found at: https://docs.google.com/spreadsheets/d/1DBQN3FCToTUDcS

This includes all data found on the Google News search, as well as all relevant articles with summaries of content, how categories were defined and assigned, and links.

180