magistrsko delo
Špela Kralj (Author), Iztok Prislan (Reviewer), Aleš Podgornik (Mentor), Urh Černigoj (Co-mentor)

Abstract

V magistrskem delu je bilo opravljeno primerjalno testiranje šibkih ionsko izmenjevalnih kromatografskih nosilcev CIMmultus␢ DEAE za čiščenje plazmidne DNA (pDNA). Namen dela je bilo iskanje optimalnega premera pretočnih kanalov znotraj kromatografskega nosilca, ki bi omogočal čiščenje pDNA. Tekom preparativnega kromatografskega čiščenja je pogosto opažen izrazit padec tlaka na koloni, kar lahko vodi v podaljšanje procesa ali uničenje kromatografske kolone. Povečevanje premera pretočnih kanalov vodi v zmanjšano vezavno kapaciteto kolon. Predvidevali smo, da k padcu tlaka prispevata velikost pDNA ter delež izoforme oc. S preučevanjem delovanja kolon z različnimi premeri pretočnih kanalov (2 µm, 3 µm, 6 µm) smo poiskušali določiti dimenzije, ki bi omogočale kromatografsko čiščenje brez izrazitega padca tlaka in ugoden izkoristek čiščenja. Potrdili smo začetno hipotezo, da je za čiščenje pDNA večjih od 7 kbp potrebno uporabljati pretočne kanale s premerom večjim kot 2 µm. Kromatografska kolona s premerom pretočnih kanalov 3 µm ni bila ustrezna. Za čiščenje pDNA večjih od 7 kbp se priporoča uporaba kromatografskih kolon s premerom pretočnih kanalov 6 µm. Potrdili smo vlogo izoforme oc pDNA – ujetje na površino monolita, ki vodi v povečevanje padca tlaka na koloni in znižanje izkoristka.

Keywords

kromatografske tehnike;anionska kromatografija;CIM;monoliti;monolitne kolone;plazmidna DNA;čiščenje plazmidne DNA;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [Š. Kralj]
UDC: 602.44:543.544.17:577.2
COBISS: 142105603 Link will open in a new window
Views: 24
Downloads: 7
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Other data

Secondary language: English
Secondary title: Comparison of monolith anion-exchange columns for plasmid DNA purification
Secondary abstract: Comparative testing of weak anion exchange CIMmultus␢ DEAE chromatographic media was conducted in following work. During preparative pDNA purification pressure drop on the columns is often observed. It leads to prolonged process time and in extreme cases to irreversible column damage. Search for optimally channel diameter (2 µm, 3 µm, 6 µm) that would allow for efficient pDNA purification was the objective. Pressure drop and mass balance were two crucial parameters when evaluating different chromatographic media. Increased channel diameter leads to reduced dynamic binding capacity for target molecule. We expected plasmid size and oc isoform percentage are the drives of pressure drop. During testing of chromatographic columns with different channel diameters (2 µm, 3 µm, 6 µm) we aimed to determine optimal diameter that would allow for process without significant pressure drop and favourable mass balance. We confirmed starting hypothesis implying use of increased channel diameter when working with pDNA above 7 kbp. Column with 3 µm channel diameter was not optimal, when working with pDNAs above 7 kbp 6 µm channel diameter is advised. We confirmed oc isoform mediated pressure drop and column clogging due to oc isoform entrapment onto chromatographic surface.
Secondary keywords: chromatographic techniques;anion chromatography;CIM;monoliths;monolithic columns;plasmid DNA;purification of plasmid DNA;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za mikrobiologijo
Pages: 1 spletni vir (1 datoteka PDF (XIV, 57 str.))
ID: 18019757