When designing our project, we learnt about the recent surge in use of plants for therapeutics, such as for the Zika and Ebola vaccines, and were interested in plant therapeutics’ future applications. From this we chose to follow the plant pharmaceutical route, to produce a treatment for Graves’ disease. During our project, we began to learn just how diverse the range is for plant therapeutics, and began wondering why, until now, it has been a relatively small field. We then started to ask more questions, such as what has limited the growth of this field, as these were issues that would have to be faced, if our project worked and was to be commercialised. The issues that we discovered included public and media perception, and the issues that companies face with laws regarding genetic modification (GM). Therefore, we created a survey to understand the public’s perception of GM therapeutics, and began to speak to companies that have succeeded in producing plant therapeutics such as Azargen, Leaf Expression Systems, and Medicago. The contact with these companies, along with literature research, was very helpful in providing insight in choosing to use plant expression system rather than bacterial or mammalian cell expression. As well as how companies manage public perception and the laws and restrictions of GM pharmaceuticals. The results from our survey, provided more knowledge on what the public thought of the use of different expression systems to produce medicines, showing more support for plant and bacterial expression, rather than the use of animals for production of medicines. These issues not only shaped our project and human practices, but worked alongside our modelling to try to understand how to commercialise our project, make the product and administer it to patients. A lot of research that was recommended by professionals was used for the modelling page, and references for these are shown there.

We focused our attention on some main questions that we had for plant biotechnology companies, and were looking for during our research.
- Why choose plants, rather than bacteria or CHO cells for expression?
- The issues companies face due to legislations on genetic modification
- The issues companies face with public perception of genetic modification
- Why plant biotechnology is less established than other biotechnology, such as bacterial expression systems?

Why choose plants, rather than other expression systems?
This was our initial question when beginning to think about plant expression to produce therapeutics. When speaking to companies, this was one of the easiest questions for them to answer. Azargen provided a lot of information for this, especially relating to the ease of scalability and efficiency of plant biotechnology. The most common use of plant therapeutics is to produce novel vaccines, due to the quick production and ease of scalability in case of a pandemic. This is replacing the egg-based vaccine production which requires attenuated viruses and a long production time of 6-9 months, limiting production scalability (Wirz 2012). Bacterial and mammalian cells, can also be used for vaccine production, but they have difficulties with processing and the need for expensive equipment. Plants, as they are eukaryotes, can perform post-translational modifications that bacteria are unable to perform, therefore requiring less downstream processing.
Tobacco plants (Nicotiana benthamiana and Nicotiana tabacum) are commonly used due to the high biomass, leaf: plant ratio, and susceptibility to Agrobacterium tumefaciens. The leaves are easily and simultaneously infiltrated using vacuum infiltration, after 4-7 days the plant biomass is harvested and purified (Holtz et al. 2015). Agrobacterium infiltration is the gold standard for gene infiltration in plants (Rybicki 2010). Due to the small scale of our project, we have been using syringes to infiltrate the plants with agrobacterium, but in larger scale production of therapeutics, vacuum infiltration of agrobacterium is used to optimise efficiency, allowing easy scalability for our project.

The issues companies face due to legislations on genetic modification
When speaking to Dr Philip Cater and Dr Nicholas Holton from Leaf Expression Systems, we were informed about the strict compliance issues that biotechnology companies face. The need for cGMP (current Good Manufacturing Practice) in order to maintain clean production and manufacturing of the genetic product. Because the plants are grown in strict laboratory conditions there is a level of control, however, the control is limited because of the growth of plants, so there is no official protocol between all companies. They told us issues would arise if the plants were grown in a farm setting, where there would be problems with consistency and risk of spread of the modified plant. One company (Wirz 2012) has grown their Nicotiana tabacum under soil free, hypotonic medium for further control of their production.
There are no issues with the compliances of purification techniques, as it is the same as the purification of naturally occurring substances in plants. After purification and extraction there is no genetically modified DNA in the final product, reducing risk of spreading the modified DNA.

The issues companies face with public perception of genetic modification
This question is something we discovered after creating our survey, to investigate the public’s perception on the use of genetic modification, of different expression systems, to produce medicine. We had an overall positive response for the use of GM plants for medicines, 77.6% responses for support or strongly support; but, found some responses that believed GM was unethical, so we wanted to continue studying that further. When speaking to companies, they believed that when it came to pharmaceuticals, as there is less advertisement as GM, the public do not tend to associate these pharmaceuticals as genetically modified. Therefore, the perception of GM in pharma is different to the negative perception of GM crops for food, and Medicago believed there is positive reception to their products, despite the stigma of GM, due to the help that these pharmaceuticals may have.

Why plant biotechnology is less established than other biotechnology, such as bacterial expression systems?
During our research, we found many reasons why companies have chosen to use plant expression systems, rather than other more established expression systems, such as the use of bacteria and CHO cells. From this, we wondered why these systems were previously preferred and are still more widely used. During our phone call with Leaf Expression Systems, we learnt that there was an initial push for plant biotechnology 20 years ago, during the time where GM was very controversial. The technology for plants was less advanced than bacterial biotech, which had been established since the 1960s. Therefore, there was more knowledge for the use of bacteria, and so, plant biotech was seen as less efficient and less favourable. But, as the knowledge of plant biotechnology increased, the advantages of plants rather than bacteria, made plants, in some cases, the better choice.

Scalability and Integration of Modelling
After speaking to Dominic Berry from Edinburgh University, who suggested to view our iGEM project not just as a summer project, but also view it as a possible business opportunity. We began investigating our project in a different way and asking more questions, like how would a potential company be doing things differently to us. We realised that we were looking at our project on a very small scale, and began to look at the larger picture. This is when we started investigating modelling as a way to see what our project could be on a larger scale, if it were to be commercialised as a business. Once we started, we realised how well modelling fit into our project, and how what we had been investigating for human practices could prove useful to modelling. We used modelling to allow us to understand what our project would look like on a larger scale; such as, how many plants we would need to produce Xg of our product, and how much of our therapeutic we would need to treat Y patients.

References
H., Wirz (2012). Automated production of plant‐based vaccines and pharmaceuticals. JALA. 17:449-457.

Holtz, B. R., Berquist, B. R., Bennett, L. D., Kommineni, V. J. M. et al. (2015). Commercial‐scale biotherapeutics manufacturing facility for plant‐made pharmaceuticals. Plant Biotechnology Journal 13:1180-1190.

Rybicki, E. P. (2010). Plant‐made vaccines for humans and animals. Plant Biotechnology Journal 8:620-637.