TY  - THES
AB  - Plasmid DNA is currently used in gene therapy and genetic vaccination as a vector system for the delivery of therapeutic genes. Clinical trials as well as future therapeutics demand large amounts of high quality plasmid DNA that fulfils the specifications set by regulatory authorities. This thesis describes the development, analysis, and evaluation of pharmaceutical plasmid DNA production processes comprising cultivation, product isolation, and purification as well as stability assessment during storage and application. Cultivations on defined media have been analyzed and compared to state of the art cultivations on semidefined medium. The influence of amino acid supplementation as well as the effect of the physiological conditions of the inoculation culture on growth and product formation have been determined. In this way, batch cultivation processes utilizing glycerol based defined media could be established having yield coefficients Y_x/s and product selectivities S_p/x comparable to or exceeding the values obtained with a semidefined medium. Additional proteome analysis indicated a stringent response that could influence plasmid replication. A semi continuous alkaline lysis was combined with froth flotation for large scale product isolation. The procedure was able to produce a highly clear lysate that could directly be applied to subsequent purification. This process was compared to other modes of operation with respect to product formation, contamination with chromosomal DNA, and plasmid form distribution. For the purification of plasmids, DNA-binding proteins were analyzed as potential affinity ligands. In addition, a recombinant RNase has been produced and its capability for RNA depletion could successfully be demonstrated. The partitioning of nucleic acids in reverse micellar two-phase systems was examined and used to develop an extraction process for plasmid purification which could be integrated into different purification schemes that allowed the complete depletion of all contaminants. Finally, the stability of purified plasmid DNA during long term storage and its implication on the effectivity after gene transfer has been investigated.
DA  - 2008
KW  - Produktaufarbeitung
KW  - Plasmid
KW  - DNS
KW  - Gentherapie
KW  - Fermentation
KW  - Plasmid DNA
KW  - Gene therapy
KW  - Proteomanalyse
KW  - Bioverfahrenstechnik
KW  - Biochemical engineering
KW  - Downstream processing
LA  - eng
PY  - 2008
TI  - Processes for the production of pharmaceutical grade plasmid DNA
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:361-15115
Y2  - 2024-11-21T23:20:45
ER  -