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A study of selected environmental issues related to the production of plasmid DNA for application in gene therapy

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dc.contributor.advisor Walsh, Gary
dc.contributor.advisor O'Dwyer, Tom
dc.contributor.creator Cliffe, Finola 2010-07-22T11:27:43Z 2010-07-22T11:27:43Z 2010
dc.description non-peer-reviewed en_US
dc.description.abstract The concept of gene therapy involves the transfer of genetic material into a human cell, tissue or organ with the objective of either curing a disease or decreasing the progression of the disease. With the increasing use of plasmid DNA (pDNA) vector vehicles for the delivery of this genetic material, it becomes appropriate to investigate the potential environmental effects of the waste streams discharged from the possible industrial scale manufacture of pDNA. These waste streams can contain biological impurities such as proteins, genomic DNA and endotoxin as well as chemical components such as buffer constituents and unutilised nutrients. Specifically, phosphorus and nitrogen emissions from upstream processing were assessed in order to determine the extent of their use by Escherichia coli (E. coli) during fermentation. The results indicated the spent media waste exhibited only a minor reduction of these nutrient concentrations, indicating minimal uptake of phosphorus and nitrogen by the microorganisms in the fermentation process. The unutilised excess level of phosphorus and nitrogen within the waste streams generated suggested a strong potential for environmental impact in the form of eutrophication. In this regard, waste minimisation studies were undertaken on terrific broth with the aim of reducing unnecessary phosphorus and nitrogen inputs without adversely affecting bacterial growth or plasmid production levels. The results indicated that added phosphorus in the media can be reduced by 98% without significantly affecting the quantity or quality of pDNA produced. However, it was found that the nitrogen content of the media could not be significantly reduced without adversely affecting pDNA yields. The compositions of a number of waste streams generated from the downstream processing of pDNA were established. Mass balances for each of the pollutants investigated (total phosphorus, total kjeldahl nitrogen, ammonia and nitrate) were constructed using SuperPro Designer® 6.0. Process improvements were established by employing the optimised media at bench and projected large scale. The findings from this research were subsequently modelled to evaluate the possible environmental impacts from the pDNA process. The modelling focus was based upon the equivalency potential approach which was used to classify environmental impacts. The model derives a single environmental score which permits identification of the most environmentally adverse emission streams and allows comparison of the efficacy of different waste minimisation strategies. A user-friendly and relatively flexible computer simulation model was also developed to maximise clarity, ease of use and functionality for application of the model. Due to the recombinant nature of pDNA vectors for application in gene therapy, consideration was given to the possible accidental release of such recombinant plasmids from production facilities into the environment. Thermal and enzymatic treatments of pDNA containing waste product streams were evaluated in terms of their suitability for pDNA degradation. Both methods were successful in degrading pDNA in all four waste streams to fragments less than 200 bp in size thus preventing the threat of horizontal gene transfer. Optimisation studies were subsequently undertaken to determine the minimum concentration of nuclease required for pDNA degradation in each of the waste streams. en_US
dc.language.iso eng en_US
dc.publisher University of Limerick, Department of Chemical and Environmental Sciences en_US
dc.subject environmental waste en_US
dc.subject pDNA en_US
dc.subject gene therapy
dc.title A study of selected environmental issues related to the production of plasmid DNA for application in gene therapy en_US
dc.type Doctoral thesis en_US
dc.type.supercollection all_ul_research en_US
dc.type.supercollection ul_theses_dissertations en_US
dc.type.restriction none en

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