Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444976
Title: Plasmid DNA and bacterial artificial chromosomes processing for gene therapy and vaccination : studies on membrane sterile filtration
Author: Kong, Sim Yee
ISNI:       0000 0001 3601 6766
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2007
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Abstract:
Plasmids currently applied in clinical trials are generally < 20 kb, but the interest in larger circular vectors is rising. However, due to their size and character, filterability and irreversible damage intensified by elongational shear are major concerns during a sterilising filtration procedure. Therefore, key parameters affecting the normal-flow membrane filtration performance of solutions containing purified plasmid DNA and bacterial artificial chromosomes were investigated in this study. Two small scale filtration systems were designed to enable information on material properties to be obtained. Firstly, a pressure driven syringe system was used to conduct constant flux experiments. Data on transmission and degradation could be obtained rapidly and only required small sample volumes (< 1 mL). Secondly, a positive pressure filtration system which permitted operations at constant transmembrane pressure had been applied to determine the filter capacity, this information is useful to facilitate the scale-up of a membrane filtration process. The results showed transmission of DNA vectors decreased linearly with molecular size (6-1 16 kb) and confocal microscopy images confirmed that a fraction of the DNA molecules were being retained by the membrane filters. Degradation increased with molecular weight, flux and number of filtration passes for vectors > 20 kb. The filtration performance was affected by the membrane type used and could be improved by addition of NaCl in the formulation buffer. For filtrations performed at constant pressure, permeate flow decayed with time. As predicted from controlled flux experiments, transmission decreased with increasing molecule size. Initial permeate flux was affected by vector size, DNA concentration and operating pressure. Increase in plasmid size and operating pressure led to reduced membrane capacities. The small scale membrane (filtration area = 1 cm2) capacity was used successfully to predict the performance of a larger scale filtration (filtration area = 4 cm2).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.444976  DOI: Not available
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