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Type: Thesis
Title: A statistical methodology to optimise soluble production of virus-like particle protein by fermentation of Escherichia coli
Author: Huynh, Nhat Hoang
Issue Date: 2021
School/Discipline: School of Chemical Engineering and Advanced Materials
Abstract: Hepatitis B core protein (HBc) virus-like particle (VLP) is a self-assembled nanoparticle that resembles the native conformation of a virus without viral genome. HBc has been widely applied as a vaccine production platform to present foreign antigens. Escherichia coli is recognised as an industrial microbial factory that offers high yield of products and economics of process. However, poor protein folding attributes to the accumulation of newly synthesised recombinant protein in inclusion bodies (IB). Complicated purification process, low yield and high cost are the consequence of IB expression from fermentation. Therefore, the soluble intracellular protein expression with correct conformation of HBc-VLP is desirable in fermentation process. As biological processes involve many factors and reactions concomitantly, a conventional approach of one-factor-at-a-time is not suitable. However a statistical approach, also known as design-of-experiment, offers an improved means to determine the effects of multiple factors to achieve the most advantageous setting. A research program was therefore undertaken with the aim to apply statistical approach to investigate the fermentation process factors impact soluble expression of chimeric HBc VLP when they carry foreign epitope in E. coli fermentation. Additionally, findings extrapolated from a careful statistical approach are necessary for optimised cultivation at fermenter scale. A logical and stepwise approach was implemented as a research strategy. This is the first time that design of experiment is used to boost the soluble expression of HBc VLP. Two (2) VLP protein models includes HBc carrying Hepatitis C virus and HBc carrying Epstein–Barr virus nuclear antigen which are listed as EBNA1-HBc and HCV-HBc. Fractional factorial design (FFD) was applied to study the effects process factors on the soluble expression of chimeric HCV-HBc in shake-flask fermentation. The greatest yields achieved were 89.7 mg g⁻¹ dry cell weight (DCW) and 84.4 mg from one (1) L of culture media. The important process factors were ranked as 1) cell density at induction, 2) post-induction rotation speed of shaker, and 3) post-induction temperature. Similar to HCV-HBc case, the significant factors for the soluble expression of EBNA1-HBc were determined from FFD. These process factors, particularly cell density at induction, post-induction rotation speed of shaker, and post-induction temperature and were optimised by response surface methodology (RSM). The highest volumetric yield and the cellular yield achieved were 272.0 mg L⁻¹ of culture media and 210.5 mg g⁻¹ DCW, respectively. It was found that using Terrific Broth (TB) as culture media provided optimal conditions for EBNA1-HBc production at an induction time of six (6) h. The production of EBNA1-HBc was scaled up to 5 L fermenter cultivation using the optimal setting achieved from RSM and TB media. Results in fermenter-scale agreed with findings in shake-flask, showing that the soluble yields decreased as the culture was induced at greater cell density. It was hypothesised that the nutrient in the media is depleted when culture reaches to higher cell density, limiting the protein synthesis. Two (2) feeding strategies were applied in fed-batch mode, namely, 1) constant feeding and 2) dissolved-oxygen (DO) stat feeding. Results showed that both feeding strategies improved productivity of EBNA1-HBc in case of induction of OD600 of 20. Constant feeding is significantly improved over DO-stat-based feeding. The greatest yield achieved from constant feeding was 1800 mg L⁻¹, which is greater than from DO-stat-based feeding of 2.57 x times. By using constant feeding strategy, EBNA1-HBc cost per media therefore decreases by a significant 10 %, compared with batch-mode. Additionally, it was found that the thermal stability of ENBA1-HBc is beneficial to couple with SEC to quantify the EBNA1-HBc capsid in clarified crude lysate. Results from this research confirm that the newly applied statistical approach is a practical tool to determine the impact of process factors on fermentation and to provide robust data optimise conditions. Process factors, media composition, induction time and feeding strategy contribute to the soluble expression of chimeric HBc-VLP and need to be optimised to boost productivity. However there are present limitations of this research that need to be determined in future work, namely 1) quantitative variation of complex media between batches, 2) an explanation of why this is the optimal combination of factors, and; 3) the reason why carbon limitation from DO-stat-feeding limits the production. The justification for this is that these findings will aid a detailed understanding of process factors that contribute to soluble expression of chimeric HBc VLP. Not only navigate the design space, but this application of statistical approach is also a general framework for process development and scaling up. This research is original and not incremental work. Findings will be of direct interest and benefit to recombinant protein production in E. coli in general and HBc platform in particular, and the wider biopharmaceutical industries.
Advisor: Bi, Jingxiu
Davey, Kenneth
Jin, Bo
Dissertation Note: Thesis (MPhil.) -- University of Adelaide, School of Chemical Engineering and Advanced Materials, 2021
Keywords: Bioprocessing
microbial fermentation
statistical approach
Hepatitis B core
virus-like particle
E coli
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
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