Burt, T.H.; (2006) Factors influencing the successful production of large plasmids for use in gene therapy and DNA vaccination. Doctoral thesis , University of London.

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Abstract

A thesis is presented which outlines investigations conducted to determine issues which may be faced should attempts be made to produce large plasmids (>3-10kb) for use in areas such as DNA vaccination and gene therapy. Initial studies attempted to manufacture a series of large Bacterial Artificial Chromosomes (116kb - 242kb) and centered on the fermentation and downstream purification steps required to produce the plasmid material. It was determined that these constructs were not suitable models due to the low yields at which they were propagated. Additionally, many of the analytical protocols employed were found to be inadequate for monitoring the concentration and form of large plasmids. In response to these issues large plasmids were constructed by the insertion of large fragments of S.cerevisiae DNA in to the relaxed, high-copy number vector pGEMl 1. Plasmids ranging from 3kb to 50kb were created and used to probe the upstream and downstream steps of a plasmid production process. Efforts were also made to develop more suitable analytical methods. The production of these plasmids was studied during pilot-scale fermentations at 37 C where it was discovered that the plasmids demonstrated a poor stability profile. In an attempt to improve plasmid stability, reduced temperature (25 C) fermentations were conducted which increased the stability and yields obtained from fermentations of these constructs. Cell paste from the fermentations was subject to a purification process involving alkaline lysis and ultrafiltration steps. Described are balances of plasmid DNA, genomic DNA, RNA and contaminating protein. The topology changes displayed by the plasmids are also described. Finally, studies are outlined describing the binding behaviour of the large plasmids to chromatographic resins used for plasmid purification.

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