Ordidge, GC; (2013) Rapid microscale evaluation of the impact of fermentation conditions on inclusion body formation, solubilisation and protein refolding yields. Doctoral thesis , UCL (University College London).

Preview

PDF 1395998_Ordidge_thesis_final_Redacted.pdf Download (4MB)

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.

Download activity - last month

Export as CSVXMLJSON

Download activity - last 12 months

Export as XMLCSVJSON

Downloads by country - last 12 months

Export as XMLCSVJSON

Archive Staff Only

View Item