magistrsko delo
Kaja Bažec (Author), Mojca Narat (Mentor), Mojca Tajnik (Co-mentor)

Abstract

Zaključni procesi čiščenja rekombinantnih adeno-asociacijskih virusov (rAAV) z uporabo monolitov vsebujejo korake čiščenja, ki so medsebojno odvisni. Za dosego ustreznega rezultata mora biti vsak korak maksimalno optimiziran. Priprava virusne žetve pred kromatografijo zmanjša nečistote in s tem poveča dinamično vezavno kapaciteto na monolitu. Žetev rAAV2/9 iz celic Sf9 smo predhodno pripravili na tri različne načine – acidifikacija, filtracija s tangencialnim tokom (TFF) in TFF v kombinaciji s kriptonazo. Vzorci so bili nato procesirani na kromatografskem koraku zajetja, ki je eden od ključnih korakov v zaključnih procesih čiščenja rAAV. Optimizacija koraka zajetja vodi do povečane produktivnosti zaradi višje dinamične vezavne kapacitete (DVK), večje čistosti produkta in izboljša poznejši korak finega čiščenja rAAV. S CIM® SO3 0,05 mL monolitnimi ploščami s 96 vdolbinicami smo naredili presejalne teste za izbiro optimalne mobilne faze za korak zajetja. Testirali smo pufre z različnimi pH, različnimi koncentracijami natrijevega klorida in poloksamera 188. Z uporabo diska CIMmic® SO3 0,1 mL smo določili pripravo vzorca, ki nam je dala najvišjo eksperimentalno definirano DVK. Pod izbranimi pogoji je ta priprava bila TFF z uporabo kriptonaze (1,44E14 vektorskih kapsid/mL monolita SO3). Rezultati, ki smo jih dobili z eksperimenti na CIM® plošči s 96 vdolbinicami in CIMmic␢ disku, so bili preverjeni tudi na preparativni liniji CIMmultus␢. Elucijska frakcija, ki smo jo dobili na CIMmultus␢ liniji je bila analizirana z metodo BCA in PicoGreen za določitev vsebnosti celokupnih proteinov in dsDNA. Visok vektorski izkoristek in zmanjšanje nečistoč je bilo ugotovljeno za izbrane parametre koraka zajetja, kar potrjuje uspešno optimizacijo koraka zajetja med procesi čiščenja rAAV z monolitnimi nosilci.

Keywords

biotehnologija;genska terapija;monolit;rAAV;ionsko-izmenjevalna kromatografija;biološki procesi;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [K. Bažec]
UDC: 606:616-056.7:602.44:543.544.17:578.822.9(043.2)
COBISS: 125246467 Link will open in a new window
Views: 53
Downloads: 23
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Other data

Secondary language: English
Secondary title: Optimization of recombinant adeno-associated virus purification using monolithic columns
Secondary abstract: rAAV downstream process using monoliths contains a series of purification steps that are mutually dependent, and each step should be optimized to its maximum for sufficient outcome. Lysate preparation prior to chromatography reduces impurities to increase dynamic binding capacity (DBC) on the monolith. We pretreated AAV2/9 clarified harvest from Sf9 cells in three different ways – acidification, tangential flow filtration (TFF) and TFF coupled with KryptonaseTM. Samples were then applied to capture chromatography step, which is one of crucial steps in downstream. Optimization of capture step leads to increased productivity due to high DBC, higher product purity and improves later polishing step. With CIM® SO3 0.05 mL Monolithic 96-well Plate buffers of different pH, sodium chloride concentrations and use of poloxamer 188 were screened to select optimal mobile phase for capture step. Pretreatment of sample that gave us the highest experimentally defined DBC tested using CIMmic␢ SO3 0.1 mL disc under selected conditions was TFF coupled with KryptonaseTM (1.44E14 vector capsids/mL of SO3 monolith). Results obtained with CIM® 96-well plate and CIMmic␢ screening were also verified on preparative CIMmultus␢ line, from which elution was analysed for total protein (BCA) and dsDNA (PicoGreen) content. High vector recovery and great reduction of impurities was determined for selected capture step parameters, confirming successful optimization of rAAV capture step during downstream process using monoliths.
Secondary keywords: biotechnology;gene therapy;monolith;ion exchange chromatography;CIM;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages: 1 spletni vir (1 datoteka PDF (XIV, 93 f., [2] f. pril.))
ID: 16400941