Al-Marzouqi, I.; (2006) Recovery and purification of plasmid DNA gene therapy vectors using aqueous two-phase systems in a J-type countercurrent chromatograph. Doctoral thesis , University of London.

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Abstract

The use of plasmid DNA (pDNA) as a vector for gene therapy, or DNA vaccination, has been of considerable interest during the last decade with DNA vaccines for HIV, various cancers, cystic fibrosis and influenza presently under development. The size of pDNA molecules and the limitations this imposes on conventional chromatographic matrices mean that attractive pDNA separation techniques need to be found. This work focuses on the downstream processing of naked DNA using a novel technique utilizing countercurrent chromatography (CCC) for the separation of DNA from contaminant RNA, chromosomal DNA and proteins as a primary purification technique. CCC is a liquid-liquid chromatographic technique, in which solutes are fractionated based on their selective partitioning between two immiscible phases. Initial research addressed the need for identifying suitable operating modes for the use of aqueous two-phase systems (ATPS) in CCC in order to obtain high levels of stationary phase retention. The degree of stationary phase retention once a hydrodynamic equilibrium is achieved was shown to be a function of mobile phase flow rate, coil rotational speed, column volume, choice of mobile phase and mobile phase pumping direction with a maximum value of 73.3% v/v obtained. In addition, it was shown to be possible to predict stationary phase retention as functions of mobile phase flow rate and rotational speed. Experiments studying the batch extraction of pDNA using ATPS, and subsequent CCC operations with ATPS, showed successful pDNA purification was possible. This pre-purification procedure was needed to act as a buffer exchange step in order to reduce the disturbance on the hydrodynamic equilibrium, in addition to reducing the amount of contamination present in the lysate. Initial studies examined the degree of pre-purification required prior to CCC separation by testing plasmid DNA partitioning in batch ATP extraction with factors such as volume ratios, plasmid sizes, pH and PEG molecular weights. The recovery yield of plasmid DNA was typically 60% w/w with a reduction in the amount of RNA and chromosomal DNA, and complete removal of proteins. CCC studies were able to obtain overall plasmid DNA recovery yields of 97 % (w/w) with no detection of RNA and chromosomal DNA. Further studies were aimed at optimising CCC performance with regard to larger scale operating strategies. Experiments showed how DNA fractionation at laboratory scale varied with changes in operating variables such as feed type, mobile phase flow rate, rotational speeds and solute loading. In addition, several unique operating modes were tested in order to allow continuous elution of the plasmid DNA. The success of these experiments varied with the highest overall plasmid yields of 58% (w/w) being attainable. The use of unclarified lysates showed the potential to overcome the initial separation steps (filtration and centrifugation) normally utilised in the downstream processing of plasmid DNA with recovery yields of 49% (w/w) being attainable. Overall the results of this work have established the potential of using CCC for the large scale purification of plasmid DNA.

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