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Protein Glycan Coupling Technology as an alternative method for the production of pneumococcal conjugate vaccines: Investigation of the impact of fermentation and technology-specific factors on recombinant vaccine production

Patel, Neha Naran; (2022) Protein Glycan Coupling Technology as an alternative method for the production of pneumococcal conjugate vaccines: Investigation of the impact of fermentation and technology-specific factors on recombinant vaccine production. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The implementation of glycoconjugate vaccines such as Prevnar-13 against invasive pneumococcal disease has significantly reduced deaths worldwide. However, generally the complex manufacturing process for glycoconjugate vaccines and in particular for Prevnar-13 contributes to the cost per dose of the vaccine to be $200 per dose in the USA. The recombinant production of glycoconjugate vaccines presents a comparatively simplified and cheaper process for vaccine production. The work presented is the first to demonstrate successful recombinant production of a pneumococcal glycoconjugate of serotype 4 in bioreactors and from cells grown in defined medium. Initially cells were grown in a fed-batch protocol with either 30 g/L or 100 g/L glycerol in the batch phase. Despite nearly 3-fold higher cell biomass at harvest in the 100 g/L condition, in normalised densitometry analysis glycoconjugate production was 4.5-fold lower. Strains were engineered and grown in fed-batch mode to investigate the impact of oligosaccharyltransferase (OST) gene location (chromosomal versus plasmidic) and of different acceptor proteins. PiuA a lipopolysaccharide component of an iron transporter in the pneumococcus and ExoA is a ribosyltransferase found in Pseudomonas aeruginosa. The introduction of ExoA was more positively impactful on glycoconjugate production compared to OST location. Densitometry analysis in normalised samples found glycoconjugate production was 4.6-26-fold higher when ExoA was the acceptor protein. ELISA quantification found more glycoconjugate was bound to the same amount of protein for ExoA (between 13-226-fold). A DoE study looking to optimise fermentation using a strain with ExoA as the protein was conducted with an exponential feed pre-induction. Cell biomass at harvest was improved by 3-7.7-fold compared to a starting fed-batch protocol, however, densitometry analysis performed on glycoconjugate bands on normalised samples indicated only three conditions had a higher signal. Post-induction temperature and feed rate had a significant impact on cell biomass and glycoconjugate densitometry signal at harvest as determined by DoE modelling. The work in this thesis lays the foundation for future fermentation and strain optimisation studies.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Protein Glycan Coupling Technology as an alternative method for the production of pneumococcal conjugate vaccines: Investigation of the impact of fermentation and technology-specific factors on recombinant vaccine production
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL
URI: https://discovery.ucl.ac.uk/id/eprint/10144709
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