Day, Andrew Francis;
(2002)
The use of ultra scale-down for the fast process characterisation of plasmid DNA processes for gene therapy.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
In the production of plasmid DNA for gene therapy the product is separated from chromosomal DNA by an alkaline lysis operation. During this key step a typical rheological profile with respect to time is developed and this has previously been examined using a viscometer as an ultra scale down reactor. The aim was to elucidate changes in rheological behaviour and plasmid DNA yield of E. coli alkaline lysates as a function of fermentation time. This data was used to determine operational windows for fermentation harvesting. A comparative study of two rheometers showed that the optimum shear rate for the viscosity vs. time profile was 180 s-1. Any rheometer used must achieve this shear rate without breaching the torque limit. Investigations into E.coli cell resistance to physical breakage showed that cells had the least resistance (disruption rate constant (K) 0.9) during exponential growth phase, compared to early exponential and stationary phase values of 0.6 and 0.4 respectively. However, the same effect was not seen for chemical cell lysis, where total cell lysis due to alkaline lysis was maintained at 40 s throughout batch and fed-batch fermentation. The viscosity vs. time profile for E.coli DH1 was seen to change as a function of fermentation time. During exponential growth phase the secondary peak appeared later every hour (from 120 s to 180 s), and the apparent viscosity was higher. This change was due to an increase in chromosomal DNA during exponential growth. Plasmid DNA yield was seen to peak at stationary growth (69 mg/kg). During early exponential and exponential growth phases the plasmid DNA yield was seen to increase with increasing neutralisation time, except for stationary phase where plasmid yield dropped after 100 s. The reasons for this are undetermined. Yield did not change as a function of fed-batch fermentation time. In conclusion late exponential / early stationary was determined as the ideal point for fermenter harvest, with the alkaline lysis process requiring efficient mixing to achieve the highest plasmid yield.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | The use of ultra scale-down for the fast process characterisation of plasmid DNA processes for gene therapy |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. |
| Keywords: | Applied sciences; Gene therapy |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10099445 |
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