Heshmatifar, Shahin;
(2023)
Towards Extracellular Release of Recombinant Protein from Escherichia coli During Fermentation: Utilisation of Antisense Technology in Bioprocesses.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Biopharmaceutical and biotechnology businesses use Escherichia coli to make recombinant proteins for medicinal, diagnostic, and analytical uses. Early downstream processing and recovery follow upstream production and processing. Cell lysis procedures damage cellular integrity and release specific periplasmic products. These releases include host cell proteins (HCP) and DNA that will challenge late downstream processing and purification, leading to time-consuming and complex unit operating steps, and an expensive bioprocess and output. Antisense technology can be used to target and suppress the synthesis of indigenous proteins, in this example an outer membrane protein, to promote the release of the recombinant product out of the cell during fermentation with the inner membrane intact, removing the need for a cell lysis phase. This study will investigate the effect of antisense-RNAs that suppress the synthesis of an outer membrane protein (Omp), murein lipoprotein (Lpp), on Escherichia coli fermentation. The bacterium strain was transformed with a model product and antisense-DNA plasmids. This experiment studied recombinant alpha-amylase and fragmented antibodies. Using 0.7 and 4-litre working volumes, reproducible and robust fermentations were achieved. Influential factors affecting product release percentages were identified and studied. Product levels reached 60%, up from 10% in initial studies. After high release levels, the business can alter the approach to eliminate cell lysis processes and minimize the overall number of recovery and purifying procedures. This project's success will attract the industry and lead to a new bioprocess strategy.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Towards Extracellular Release of Recombinant Protein from Escherichia coli During Fermentation: Utilisation of Antisense Technology in Bioprocesses |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2023. 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. |
| Keywords: | Upstream Bioprocess Development, Antisense Technology, Fermentation |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS 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 |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10176515 |
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