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An engineering study of the alkaline lysis operation for the recovery of supercoiled plasmid DNA for gene therapy

Ciccolini, Laura Anna Stephania; (2000) An engineering study of the alkaline lysis operation for the recovery of supercoiled plasmid DNA for gene therapy. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Advances in recombinant DNA technology are leading to a novel generation of medicines for the treatment of genetic and metabolic disorders, viral infections and cancers. Of practical significance is the advent of gene therapy, in which defective cells are transformed with gene vectors capable of expressing the therapeutic protein. Molecules of plasmid DNA for such treatments are produced by the fermentation of recombinant Escherichia coli host cells. The process involves a series of steps to recover the intracellular plasmid DNA, in its supercoiled form. A key step in the isolation of plasmid DNA is the alkaline lysis operation, which though efficient for small-scale isolation, lacks an engineering basis for its scale-up. This study sets out to characterise the operation and to determine the most efficient way to integrate it with upstream and downstream operations in the plasmid DNA process. A viscometer was used as a scale-down reactor for the alkaline lysis and subsequent neutralisation stages. The viscometer provided both the mixing vessel, capable of producing shear rates from 46 to 581 s-1, and an in-situ measuring device for assessing the viscosity changes. The time of cell lysis reaction was found to range from 30 to 70 seconds, depending on the bacterial strain and the culture medium. Maximal denaturation and entanglement of chromosomal DNA occurred 50 to 90 seconds after release from the cells. The flow behaviour of the cell lysates ranged from shear thinning, to Newtonian and thixotropic. Apparent viscosity rose with the plasmid copy number. The shape of the apparent viscosity-time profiles depended on the culture medium used at fermentation. Upon neutralisation of the cell lysate, the salting-out effect and precipitation of cellular debris and detergent-salt complexes occurred within 10-20 seconds, to produce a two-phase mixture consisting of a crude lysate and a floating gelatinous matrix. For cells cultured using standard medium, homogeneity at neutralisation was not achieved for shear rates up to 116 s-1. Rheological characterisation showed that the crude lysate liquid phase was Newtonian, while the floating gel-matrix was viscoelastic and highly sensitive to shear deformation. Mass balance calculations demonstrated that plasmid DNA yield increased with shear rate and later neutralisation time. Plasmid contamination by chromosomal DNA and suspended solids increased with shear rate and later neutralisation. Contamination by soluble proteins was higher at increased shear rate and earlier neutralisation. Results from the scale-down studies were used to develop a reactor that employs impinging-jets mixing at the lysis stage, followed by jet-mixing in a reaction column and flotation of the gel-matrix at the neutralisation stage. This configuration achieves a plasmid yield above 80% and a plasmid purity of at least 50%. The thesis concludes with recommendations for future work which include: use of the scale-down technique to assess the effect of new strains and plasmids on the operation, pH measurement, optimisation of the reactor configuration and its integration with the upstream and downstream operations in the process for plasmid manufacture.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: An engineering study of the alkaline lysis operation for the recovery of supercoiled plasmid DNA for gene therapy
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10121031
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