Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.639535
Title: Novel engineering tools to aid drug discovery processes
Author: Islam, R. S.
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2007
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Abstract:
A major bottleneck in drug discovery is the production of soluble human recombinant protein for functional, biochemical and structural analyses. The level of recombinant protein expression is controlled by a complex relationship between both biological and engineering variables. Due to the inter-play between these variables and standard experimental methods, the identification of the key variables which lead to improved protein expression can sometimes be missed. This thesis presents a framework which underpins the generation of large quantities of soluble recombinant protein in E. coli in a rapid and cost-effective manner. To achieve this goal, Design of Experiments (DoE) was first employed in combination with microwell plate (MWP) fermentations to investigate the wide array of protein expression variables. These tools are well suited to high-throughput expression requirements as they afford large savings in time, cost and resource requirements. The information generated from these MWP experiments was then exploited to devise a strategy for reproducing the process within stirred- tank reactors (STRs). The DoE methodology was first used to identify relevant protein expression variables including fermentation variables (media type and fermentation time), protein induction variables (inducer concentration and induction time) and environmental variables such as oxygen transfer rate, temperature and pH. Ten factors were screened overall at the microwell scale and three were investigated further through optimisation designs. The application of DoE led to a robust understanding of the process and resulted in protein yields five-fold greater than those obtained under standard shake-flask conditions. The most significant factors were post-induction period and shaking speed, the latter of which is strongly related to the mass transfer coefficient, faa. In order to translate this stable and optimised small-scale expression system to a production-scale stirred-tank reactor (STR), an understanding of the engineering parameters at both scales of operation was crucial. This need was complicated by significant differences between the MWPs and STRs such as geometry, mode of aeration and agitation, and the effects of surface tension. In this work, the MWP fermentation results led to the hypothesis that operation at a constant kia value would facilitate predictable scale translation. However, there currently exists very little published work on the characterisation of kia within MWPs. Miniature oxygen probes were, therefore, used to characterise MWP kia values directly via the static gassing-out method over a range of square-well MWP formats and shaking speeds. This information was then used to translate the performance of a 3ml MWP E. coli fermentation, on the basis of matched faa, to STR working volumes of 51 and 451. The efficacy of scale-up was confirmed by performing F tests on pairs of profiles for cell growth and expression levels of recombinant firefly luciferase. This rapid, accurate and direct method of kia characterisation within MWPs enabled a 15,000-fold direct scale-up of fermentation performance in terms of cell growth and protein expression from MWP to STR.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.639535  DOI: Not available
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