Team:Heidelberg/Implementation


Proposed Implementation





Theoretical drug development

Executive Summary

In order to implement our research work on the market, a finished product is required. After hard research we developed a plasmid that can be taken up by natural competent bacteria. The next step is to upscale this process from laboratory scale to industrial size. Therefore, the plasmids can be bred by fermentation. For the applicational use we need to create an appropriate capsule model that dissolves in the intestine.

Figure 1: Final plasmid design. This plasmid contains the phenylalanine ammonia lyase with the suitable L-phenylalanine permease for the in vivo function. Additionally, the β-agarase I and II for the selective advantage. The total size of the plasmid will be about 6 kb. At last a kill switch system for example the cold triggered MazF/MazE with 782 bp length can be considered. Created with BioRender.com

Research and Development - Upscaling

In general, the term fermentation refers to the growth of organisms or cells to produce metabolic products or biomass. On an industrial scale, the goal is to obtain a high yield while keeping costs low. To achieve this, high cell-density culture (HCDC) techniques have been developed for cultivating bacteria, improving productivity, enabling low production costs, producing less wastewater, and allowing smaller culture volumes.

The fermentation takes place in so-called bioreactors, a defined chamber that is sterilised to prevent contamination and thus the growth of undesirable organisms. In order to achieve a maximum production yield, optimal conditions prevail in the chamber for the respective organism. In the bioreactors, parameters such as temperature, oxygen content, pH value or nutrient concentration can be adjusted so that they correspond to the natural habitat of the bacteria [2]. In addition, the conditions are controlled by sensors. Depending on the type of bioreactor, they are equipped with agitators that create a homogeneity of these parameters throughout the reactor chamber. Stirred bioreactors are the most commonly used bioreactors. These cylindrical vessels are equipped with stirrers that achieve homogeneous mixing of all components and at the same time the stirrer breaks down large gas bubbles for optimal oxygen supply. Furthermore, these bioreactors enable the fed-batch strategy. The fed-batch strategy is a process in the industry to achieve HCDC in bioreactors.

In this process, the chamber of the bioreactor is initially half-filled with medium and the cells are inoculated. As soon as the optimal concentration is used up and thus growth is inhibited, new highly concentrated medium and other nutrients are added. Compared to other systems, fed-batch requires more sophisticated equipment as well as more human interaction. However, higher production concentrations are generally achieved due to prolonged cell growth and accumulation of the product [1].
Figure 2: Fermantation process. Created with BioRender.com

Application Model

Requirement

For this purpose, we followed the requirements of the European Pharmacopoeia in order to comply with the guidelines of the European market. Depending on the market of the country you want to introduce your biologics or pharmaceutics to, you need to accomplish the standards of their own Pharmacopoeia, e.g. the U.S. Pharmacopoeia in the United States.

There are two ways to produce the capsules - one type being hard capsules whereas the other type would be soft capsules.

Type of application

Hard capsules

Hard capsules are capsules with a hard gelatin shell. These contain a single dose with two prefabricated cylindrical parts. The active ingredients are usually in solid form. The advantage is to make an individual dosage by manufacturing. To increase the tightness of the seal, it can be increased with the help of a coating.

Figure 3: Hard capsule design. A typical design of a hard capsule with two parts which can be sticked together. Created with BioRender.com

Soft gelatin capsules

From our point of view, this type of encapsulation is the better option. Here, the single doses are packed in only one shell, which is made of gelatin. This can contain one or more active ingredients and is particularly suitable for liquid or semi-solid preparations. Production is generally done in one step, during which the capsule is formed, filled and sealed. The advantage lies in the guaranteed tightness of the capsule in all cases.

Figure 4: Soft capsule design. A typical design of a soft capsule which is tightly sealed. Created with BioRender.com

Both capsule models can be coated with a varnish that enables the delayed release of the active ingredient. These varnishes can be, for example, cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP) or polyvinyl acetate phthalate (PVAP).

Testing

The European Pharmacopoeia describes the following tests: Test of uniformity and Test on time of drug release.

In the test of uniformity, a random sample of capsules gets weighted and must not vary by a certain percentage depending on the capsule weight.

In the test on time of drug release, the capsules dwell for at least two hours in an acidic medium of 0.1 M hydrochloric acid. This should simulate the gastric juice milieu. No capsule should dissolve during this process. In the second step, the capsules are placed in a medium of phosphate buffer solution with pH 6.8 for one hour. Finally, the capsules have to dissolve [3].

Figure 5: Testing sample. A sample of a capsule which releases the active ingredients in the second medium. Created with BioRender.com

User Problems?

In a conversation with our pediatric clinic expert, we were also made aware of the problem of ingestion for children. Of course, we thought about this. One possibility is to produce a syrup or sugar-coated tablets. The problem with this dosage form is that our active ingredient is digested in the gastric juice. Since it is a relatively sensitive active ingredient, we had to rule out this option. We also cannot market our product in the form of a suppository. The problem is that the plasmids are exposed to high temperatures. This can lead to a loss of the chemical and physical properties of the plasmids. However, there are already approaches to solving this problem in industry. For example, very small capsules can be produced, as is the case with Espumisan®. This drug serves as a defoamer and is used in the accidental ingestion of detergents. Therefore, it is essential to offer this drug to children [4].

Figure 6: Comparison of different capsule sizes.There are standard sizes from #000 to #2. In addition, on the right side is the extremely small capsule which is inspired by the Espumisan® Capsule. Created with BioRender.com

Clinical trials

Phase I

In phase 1 of the clinical trials, the active substance is tested and verified in the laboratory. A major part of the research here has to deal with tolerability and efficacy. Questions that arise here above all are:
    Do the plasmids survive the intestinal milieu?
    Do the right bacteria take up our plasmids?
    Is phenylalanine degraded?
    Are there any side effects?

Tolerance and dosage must be checked and tested. If the body recognizes the plasmids as foreign bodies or pathogens, severe immune reactions may occur. To prevent this, further tests and trials need to be done, which require years of research.
Figure 7: Release of active ingredients in the microbiome. To identify possible side effects clinical trials are important. The clinical trials show how the body reacts to the biopharmaceuticals. Created with BioRender.com

Phase II

In the second phase of the trial, small patient groups will be formed once tolerability of the compound has been confirmed. The correct dosage and safety of the product will be investigated here. Therefore, we will have a look whether the treatment works well enough so we can test it in phase III with a larger number of people. Another aim is to find out more about the side effects.

Phase III

After the correct dosage has been determined, larger patient groups can be formed. Due to the larger subject group, further side effects can be determined.

Phase IV

Long-term efficacy and tolerability of the drug in everyday care are found out here. This is the final phase of the study and decides on a possible approval [5].

Market Access

Risk-Benefit Assessment

In Europe there is the European Medicines Agency which can recommend the authorisation of a medical product. Their opinion decides whether a drug is approved or not. This is done by weighing up the risks and benefits, which can only be seen in full after the clinical trials [6]. So far, we mainly see the benefits of our research.
We use the natural competence of our cells. Therefore, similar comparisons can be made as with a stool transplant. The cells are not influenced by novel enzymes or drugs Instead, plasmids are used. These are not foreign to the body and can hopefully be absorbed better. Nevertheless, to banish the risk of degeneracy from our plasmid, a killswitch could be incorporated into the plasmid sequence. In our proof of concept the risks and modes of action are described in more detail.

Monetization

Our product is essentially a plasmid which is produced on a large scale. The advantage is the cheap production and high yield through natural transformation. Our product still requires some research, which is why the product generates high start-up costs. However, this is a market niche that can be a money pit for companies. Apart from the fact that there is no comparable treatment option in the field of phenylketonuria, the implementation of this method on the current market is an advance into new technologies. This means that the monetization of our product can be set high to cover research costs, as it is a non-competitive product, and a market for follow-on products can be developed.

How Covid-19 could promote our project

Many teams have suffered a disadvantage due to the Covid-19 pandemic. This is only partially the case for us. Because of the approval of the mRNA vaccines, the market for new techniques in synthetic biology has been expanded. So hopefully, the technology of Natural Transformation will be better accepted. In order to create a matrix for the plasmids, the success and research of the mRNA vaccines can be used as a guide for this. In relation to this, storage and transport can be used in the same medium. This matrix could consist of liposomes. Since plasmids are about 100 times larger than mRNA and consist of a double strand, we suspect that they will be much more stable.

References

[1] Chmiel, H., Takors, R., & Weuster-Botz, D. (2018). Bioprozesstechik. Springer Spektrum. https://doi.org/10.1007/978-3-662-54042-8.

[2] Lee SY. High cell-density culture of Escherichia coli. Trends Biotechnol. 1996 Mar;14(3):98-105. doi: 10.1016/0167-7799(96)80930-9. PMID: 8867291.

[3] Bundesinstitut für Arzneimittel und Medzinprodukte, Ph. Eur. 10. Ausgabe Grundwerk 2020: Band 1 Allgemeiner Teil Monographiegruppe, Deutscher Apotheker Verlag Leipzig

[4] Schneider, Detlef; Richling, Frank (2013): Checkliste Arzneimittel A - Z, 6. Auflage, Georg Thieme Verlag, Stuttgart

[5] Cancer Research UK (2019): Phases of clinical trials : https://www.cancerresearchuk.org/about-cancer/find-a-clinical-trial/what-clinical-trials-are/phases-of-clinical-trials (Last viewed: 2021-10-18 08:43 am)

[6] European Medicines Agency: Benefit-risk methodology: https://www.ema.europa.eu/en/about-us/support-research/benefit-risk-methodology (Last viewed: 2021-10-18 08:49 am)