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Optimizacija produkcije in izolacije plazmidne DNA za gensko terapijo
Volk, Manca (Author), Petrovič, Uroš (Mentor) More about this mentor... This link opens in a new window, Peterka, Matjaž (Co-mentor)

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Abstract
Pridobivanje plazmidnih vektorjev za uporabo v genski terapiji je odvisno od donosnosti pridobivanja, ki mora zagotoviti zadostne količine plazmidne DNA v obliki kovalentno zaprtega kroga. Na količino in kakovost plazmidne DNA v zaključnih procesih vpliva več dejavnikov, ki jih je potrebno optimizirati v začetnih, pripravljalnih fazah bioprocesa. Z namenom povišanja donosnosti bioprocesa smo v sklopu magistrske naloge optimizirali pripravljalni proces. Z gojenjem smo pričeli v 3-litrskem bioreaktorskem sistemu, kjer smo lahko tekom gojenja spremljali rast, parcialni tlak kisika ter pH v kontroliranem okolju. Gojenje smo najprej izvedli v šaržu brez dohranjevanja in kasneje v šaržu z dohranjevanjem. Testirali smo vpliv parcialnega tlaka kisika ter temperature na količino plazmidne DNA. Z uporabo šaržnega gojenja z dohranjevanjem smo dosegli 79,5 % višji donos plazmidne DNA v primerjavi s šaržnim gojenjem brez dohranjevanja.

Language:Slovenian
Keywords:plazmidna DNA, genska terapija, pridobivanje plazmidne DNA, optimizacija bioprocesa
Work type:Master's thesis/paper (mb22)
Organization:BF - Biotechnical Faculty
Year:2020
COBISS.SI-ID:19055363 This link opens in a new window
Views:398
Downloads:176
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Secondary language

Language:English
Title:Optimization of production and isolation of plazmid DNA for gene therapy
Abstract:
The manufacturing of plasmid vectors for gene therapy applications depends on the yield of plasmid DNA production, which has to be high enough to provide sufficient amount of super coiled plasmid DNA. In the downstream process the amount and quality of plasmid DNA are affected by many factors, which have to be optimised in the upstream process of bioprocess development. The scope of the thesis was to increase the yield of the upstream process. Bacterial cultivation began in a 3 L bioreactor vessel where the growth of bacteria, pH and oxygen partial pressure were monitored in a controlled environment. Bacterial cultivation was carried out a batch mode and also in a fed-batch mode. The effect of oxygen partial pressure and temperature on the production of plasmid DNA was tested. The use of fed-batch mode increased the yield of plasmid DNA by 79.5% compared to batch mode.

Keywords:Plasmid DNA, gene therapy, plasmid DNA production, bioprocess optimisation

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