Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.617910
Title: Rapid microscale evaluation of the impact of fermentation conditions on inclusion body formation, solubilisation and protein refolding yields
Author: Ordidge, G. C.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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Abstract:
Heterologous protein expression in E. coli can lead to the formation of dense insoluble aggregates named inclusion bodies (IB). The refolding of protein derived from IB is often characterised by low yields of active product. Process optimisation is often achieved empirically and requires significant resource and time efforts. Microscale experimentation may provide a valuable alternative by enabling representative process studies to be conducted early on in process development, using minimal quantities of product, parallel experimentation and automated liquid handling procedures. An automated robotic platform has been used to develop a dilution refold microscale process-screening tool with a set of hierarchical assays to rapidly determine optimal refolding conditions. The hierarchical orthogonal assays enable the simplest, cheapest and most generic high-throughput assays to first screen for a smaller subset of potentially high-yielding conditions. Absorbance can be used as an initial filter to measure particulate formation and fluorescence boundaries can then be used to select the conditions with the most native-like tertiary structure. The subset can then be analysed for native protein yield by slower, more expensive or protein specific assays, thus saving resources whilst maximising information output, alleviating the analytical bottleneck. This approach has been demonstrated in this work using lysozyme, with fluorescence boundaries to select 30% of highest yielding samples, and also with DHFR. An automated whole bioprocess sequence comprising fermentation, cell harvest and lysis, inclusion body harvest, denaturation and refolding has been developed at the microscale to study the effect of fermentation conditions on inclusion body yield and quality. The approach has been applied to dihydrofolate reductase (DHFR) and insulin, allowing a more thorough understanding of the effect of fermentation feeding, media and induction strategies on protein refolding yield and purity. This approach allowed yields of active insulin of increased from 10% to 68%. The results obtained from this approach have been compared to larger scales of operation, illustrating the challenges of scale-up. The process sequence, integrated with rapid analytical assays, provides a powerful tool for understanding the interaction between fermentation conditions and downstream processing yields, allowing a whole process approach to optimisation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.617910  DOI: Not available
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