Shivakumar, Tejasvi
(2024)
Cell-free protein synthesis for on-site and on-demand biomanufacturing in extreme and low resource environments.
PhD thesis, University of Nottingham.
Abstract
Humans are starting to explore extreme environments such as space due to advancements in Science and Technology. Although current manned missions have involved short-durations with emergency supply or return to earth, future long-duration space travel will not have this luxury. Moreover, 87% of current spaceflight medications have a limited shelf-life of less than 2 years. Therefore, there is a severely limited capacity to supply a pharmacy for human exploration of Mars, which is a combined goal of 15 space agencies in the next decade. This research project, under the theme ‘astropharmacy’, aims to address this challenge through on-demand and on-site synthesis of biopharmaceuticals.
The increasing share of biologics in the pharmaceutical market can be attributed to their therapeutic abilities against challenging targets, but they are conventionally produced in centralized, large-scale facilities using living cells, and typically require cold chain storage and transportation. This severely limits their applicability in low-resource and extreme environments, where a largely untreated human population are present. A platform based on cell-free protein synthesis (CFPS) technology is capable of surpassing many limitations of cell-based expression and is the system of choice for the astropharmacy vision in this thesis.
This thesis begins by describing the development and optimisation of an in-house CFPS system, which was optimised for high-level protein production, using chromogenic reporter superfolder green fluorescent protein (sfGFP) as a model. Next, the system was benchmarked against commercial systems such as the NEB PURExpress and Promega E.coli S30A extract system for circular DNA, with sfGFP yields increasing and surpassing commercial systems and others reported in literature. The sfGFP yield improvements are seen from 0.8 mg/mL in experiments described in the initial chapter to > 5 mg/mL in that of the final chapters.
Experimentation with freeze-drying, microglassification™ and cellulose stacks were carried out in order to explore platforms with ease-of-storage and distribution. Lyophilised pellets and cellulose stacks were two main approaches that met some astropharmacy goals. The latter platform was composed of lyophilised CFPS components on cellulose discs, which were layered and rehydrated to kickstart protein synthesis. Such paper-encompassed reactions were shown to be capable of robust expression after drying, and the system can be modulated by simply changing the DNA element.
To further address the gap in our knowledge of storing and transporting active CFPS compounds for longer periods of time, stability studies were conducted. This was particularly important for VITA (Visualising In-situ Tx-Tl Astropharmaceuticals); a student payload being developed with the European Space Agency. VITA aims to become Nottingham’s first astropharmacy technology demonstration mission on the International Space Station. The thesis finishes with a short, but final note on producing therapeutic proteins-of-interest using the developed platform, along with ideas and initial experiments for in situ purification. As this research progresses, numerous applications in healthcare for space and Earth, such as elimination of transport and provision in extreme environments, are hoped to be realized.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Williams, Phil
Dreveny, Ingrid
Croft, Anna
Conradie, Alex |
| Keywords: |
Cell-free protein synthesis; on-demand biomanufacturing; protein therapeutics; freeze-drying; sfGFP reporter; stability testing; protein purification; quality control |
| Subjects: |
R Medicine > RM Therapeutics. Pharmacology |
| Faculties/Schools: |
UK Campuses > Faculty of Science > School of Pharmacy |
| Item ID: |
76780 |
| Depositing User: |
Shivakumar, Tejasvi
|
| Date Deposited: |
24 Jul 2024 04:40 |
| Last Modified: |
24 Jul 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/76780 |
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