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Title: Characterisation and monitoring of plasmid DNA vectors of increasing size during processing for gene therapy and DNA vaccination applications
Author: Rock, Cassandra Fernanda
ISNI:       0000 0001 3529 6882
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2005
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The cell lysis step is one of the most critical operations in large-scale plasmid DNA processing as fragments of chromosomal DNA and degraded plasmids remaining in solution have a detrimental effect on downstream operations. In the present thesis the effect of the engineering environment on the bacterial cell suspension used in the lysis step and on solutions containing gene vectors of up to 242 kb in size was investigated. An integral aspect of the thesis was to evaluate current analytic techniques relating to the characterisation of process streams containing plasmid vectors and to identify and develop characterisation techniques suitable for process development and manufacturing. Cell pastes derived from laboratory- and 450 litre-scale fermentations of plasmid-containing bacterial cells were characterised using a variety of analytical techniques. The impact of pilot scale continuous centrifugation equipment used for cell harvesting was then investigated. It was observed that equipment design and operating conditions had an impact on product yield and the molecular weight of contaminant DNA in the process stream. It was found that direct measurement of released nucleic acids at the cell resuspension stage can provide a simple analytical technology to characterise cells before the lysis stage. After cell lysis, the downstream options depend on the susceptibility of the product to chemical and mechanical damage in solution. In the case of circular DNA molecules, it is of particular importance to characterise the backbone integrity. A microwell-based high throughput method to evaluate this for pure double stranded DNA solutions was developed. The method was then validated for reproducibility, accuracy and sensitivity and was successfully adapted for use on a robotic liquid handling system. The method was utilised to investigate the response to hydrodynamic stresses of bacterial artificial chromosomes. It was observed that for a 116 kb vector, average shear rates 104 s-1 (comparable with standard process equipment), caused a 5 fold decrease in the integrity of the molecules in the sample. Finally, the thesis ends with a look towards future process analytical technologies and assay development within the context of DNA-based pharmaceuticals.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available