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Title: Escherichia coli-based cell-free protein synthesis of self-assembling particles for vaccine production and gene therapy
Author: Colant, Noelle Angelica
ISNI:       0000 0005 0288 5567
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2020
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The traditional “one-size-fits-all” mass production model commonly used in biologics manufacturing is insufficient to accommodate the advent of personalised medicines and the necessity of on-demand production. The design and validation of novel manufacturing platforms is necessary for on-demand and personalised medicines production. To address this, an E. coli-based cell-free protein synthesis (CFPS) manufacturing platform was developed and applied to self-assembling particles for vaccine and gene therapy production. This in-house CFPS system consistently produces over 400 μg/mL superfolder green fluorescent protein (sfGFP) in 4 hours. A three-step process development strategy that can be completed in under 48 hours was designed and then validated with two products. Using this strategy, sfGFP production was improved by 38% and hepatitis B core antigen (HBcAg) production by 190%. The CFPS system was then used to produce self-assembling products and iterate upon their construct design. Two tandem-core HBcAg virus-like particles (VLPs), called VLP3 and VLP1, that have been modified to display influenza antigens as universal influenza vaccine candidates were produced and assembled. Using a minimal plasmid backbone designed for CFPS improved titres by 1.8 times over the original VLP1 construct and 1.4 times over the original VLP3 construct. Titres were further increased to over 100 μg/mL for VLP3 when the linkers around the influenza inserts were shortened, although improvements in particle quality were not seen. Further, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. Additionally, the three capsid proteins of the adeno-associated virus were produced, which have been shown to form particles in vitro and can be used for the delivery of genetic material, potentially as a gene therapy treatment. Taken together this shows the potential for CFPS systems in the on-demand manufacture of self-assembling vaccine and gene therapy products.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available