eprintid: 1395998 rev_number: 35 eprint_status: archive userid: 608 dir: disk0/01/39/59/98 datestamp: 2013-12-06 12:01:18 lastmod: 2019-01-14 00:59:27 status_changed: 2013-12-06 12:01:18 type: thesis metadata_visibility: show item_issues_count: 0 creators_name: Ordidge, GC title: Rapid microscale evaluation of the impact of fermentation conditions on inclusion body formation, solubilisation and protein refolding yields ispublished: unpub divisions: A01 divisions: B04 divisions: C05 divisions: F47 note: Third party copyright images have been redacted form the e-thesis 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. date: 2013-04-28 vfaculties: VENG oa_status: green full_text_type: other thesis_class: doctoral_open language: eng thesis_view: UCL_Thesis dart: DART-Europe primo: open primo_central: open_green verified: verified_manual elements_source: Manually entered elements_id: 876848 lyricists_name: Ordidge, Gemma lyricists_id: GCORD96 full_text_status: public pagerange: ? - ? pages: 282 institution: UCL (University College London) department: Biochemical Engineering thesis_type: Doctoral citation: 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). Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/1395998/1/1395998_Ordidge_thesis_final_Redacted.pdf