Almulla, Fatma Mohamed Ebrahim Alhamer;
(2025)
Optimisation of an alkaline lysis
process to improve plasmid DNA
production at scale – challenges and opportunities.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Renewed interest in plasmid DNA (pDNA) production is the result of its role in the supply chain of novel biopharmaceuticals.” pDNA lies at the heart of viral and mRNA vector production because it provides the coding sequences for gene-based advanced therapy medicinal products (ATMPs). This thesis attempted to upscale the pDNA production process with effective controls laid on the processes and product quality. Chemical treatment such as alkaline lysis methodology has been employed to extract the pDNA. This thesis work aimed at understanding the involvement of process parameters such as physiological conditions, mixing and rheological properties, and its influence in synthesizing high-quality supercoiled pDNA. Biomanufacturing in different process systems: Inversion (laboratory), Batch-mode in 1L stirred tank reactor (STR) and Continuous-flow In-line static mixer were attempted in this study. Key steps in alkaline lysis viz., Lysis and Neutralisation were segregated and independently optimised using the Design of Experimental (DOE) approaches, to address the scale-up issues and facilitate technology transfer to the biopharmaceutical industry. Preliminary investigation showed significant parameters like pH, mixing time and agitation speed to be influential during the lysis step in batch 1L STR. Following optimisation of the lysis step using central composite design, the maximum plasmid yield (13 mg/g cells, R2=95%) and % supercoiling (85%, R2=92%) were observed at 0.2M NaOH (pH of 12.5), incubation of 15 minutes and a tip speed of 2.3 m/s. For the neutralisation step, improved plasmid yield (4.7 mg/g cells, R2=67%) and supercoiling content (92%, R2=98%) were achieved at 20 minutes incubation and a tip speed of 2.3 m/s. In the continuous static mixer, the optimal lysate and neutralised solution flow rates were determined as 165 ml/min and 83 ml/min, respectively, achieving a desirability score of 0.55 for maximum plasmid yield (2.06 mg/g cells, R2=84%) and minimal genomic DNA impurities (1005 w/w%, R2=96%). This report stands first of its kind in comparing large-scale plasmid productivity in different systems, focusing on improved productivity and product quality.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Optimisation of an alkaline lysis process to improve plasmid DNA production at scale – challenges and opportunities |
| Language: | English |
| Additional information: | Copyright © The Author 2025. 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 UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10203919 |
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