Optimizacija graftanja in funkcionalizacije monolitnih kromatografskih nosilcev za učinkovito izolacijo plazmidne DNA

Kodermac, Meta Špela (author), Štefane, Bogdan (mentor)

Pomembna koraka v pridobivanju velikih biomolekul v bioloških procesih, npr. plazmidne DNA (pDNA), sta izolacija in čiščenje. Za ta namen se v farmaciji, medicini in biotehnologiji uporablja mnogo različnih metod čiščenja, ki temeljijo na ionsko-izmenjevalni kromatografiji. Karakteristike kromatografskih nosilcev, npr. dinamična vezavna kapaciteta, so se z razvojem novih materialov in različnih načinov funkcionalizacije bistveno izboljšale. Povečanje učinkovitosti kromatografskih CIM® (angl. convective interaction media) monolitov za izolacijo pDNA sem izvedla z graftanjem – dodajanjem poli-glicidil metakrilatnih (poliGMA) verig na površino. V okviru magistrskega dela sem graftanje optimizirala na CIM® metakrilatnih monolitih s premerom kanalov 2 in 6 μm. Graft polimerizacijo glicidil metakrilata sem izvedla z radikalsko polimerizacijo s prenosom atoma (ATRP). Postopek polimerizacije sem optimizirala tako, da sem dosegla enakomeren graftan sloj po celotni dolžini monolita v smeri toka tekočine. Raziskala sem vpliv sestave in načina vnosa reagenta ter časa reakcije na rast polimernih verig, pretvorbo pa sem opazovala z infrardečo spektroskopijo (IR). Ugotovila sem, da na hitrost rasti polimernih verig vpliva količina monomera, katalizatorja in reducenta v reakcijski mešanici. Za namen kromatografskega testiranja oziroma izolacije pDNA sem na polimerne verige vpeljala anionsko-izmenjevalne terciarne aminske skupine (R–NH+(CH2CH3)2). Na majhnih 0,2-mL CIMmicTM monolitnih enotah sem v primerjavi z negraftanimi monoliti dosegla 5-kratno povečanje kapacitete za pDNA ter 4-kratno povečanje kapacitete za goveji serumski albumin (BSA). Vpliv graftanega sloja na pretakanje tekočine sem določila s prepustnostjo, ki je pokazala, da graftani monoliti z večjim premerom kanalov ne ovirajo pretoka. Prepustnost graftanih monolitov sem izmerila s padcem tlaka. Poleg povečanja kapacitete dietilaminsko modificiranega monolita za pDNA sem se osredotočila tudi na povečanje izkoristka elucije pDNA s kromatografske kolone. S primerno gostoto in dolžino graftanih verig sem dosegla optimalno razmerje med kapaciteto in izkoristkom elucije. V naslednjem koraku sem z optimiziranimi pogoji graftanja funkcionalizirala večjo monolitno kolono. Kromatografske meritve so pokazale 6-kratno povečanje dinamične vezavne kapacitete za pDNA in pa manjše sipanje kapacitet kot na CIMmicTM diskih.

• Language: Slovenian
• Keywords: plazmidna DNA, ionsko-izmenjevalna kromatografija, monoliti, ATRP, graftanje
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-131813
• COBISS.SI-ID: 90925315
• Publication date in RUL: 04.10.2021

Secondary language

• Language: English
• Title: Optimization of grafting and functionalization of monolithic chromatographic supports for efficient plasmid DNA isolation

Abstract

The important steps in the preparation (production) of large biomolecules, e.g. pDNA in biological processes, are isolation and purification. A large number of different approaches (purification methods) based on ion-exchange chromatography are used in pharmaceutical, medical and biotechnology industries. The characteristics of chromatographic supports, such as dynamic binding capacity, have been greatly improved by the development of new materials and various types of functionalization. The efficiency of CIM® (convective interaction media) epoxy methacrylate monoliths for plasmid DNA isolation was increased by grafting polyGMA chains onto the surface. Grafting was optimized using CIM® methacrylate monoliths with channel diameters of 2 and 6 μm. The polymerization of glycidyl methacrylate was carried out with atom transfer radical polymerization (ATRP). The polymerization process was optimized to obtain a uniform grafted layer along the entire length of the monolith (in the direction of the liquid flow). The effects of reagent composition, process, and reaction time on the polymer chain growth were observed using infrared spectroscopy (IR). The growth rate of the polymer chains was affected by the amount of monomer, catalyst and reducing agent in the reaction mixture. To determine the chromatographic and hydrodinamic properties of the grafted monoliths, tertiary amine groups (R—NH+(CH2CH3)) were introduced into the polymer chains as anion exchange groups. The binding capacity of pDNA for 0,2 mL CIMmicTM grafted monoliths was approximately 5-times, whereas for BSA approximately 4-times higher. The effect of the graft layer on fluid flow was determined by permeability, showing that grafted monoliths with larger channel diameters did not impede the flow. The permeability of grafted monoliths was measured by pressure drop. In addition to increasing the capacity of the diethylamine monolith for plasmid DNA, the goal was to increase the efficiency of elution from the chromatography column. With the appropriate chain length and density an optimal ratio between the capacity and elution efficiency was achieved. In the last part of the research, a larger monolithic column with optimized grafting conditions was prepared. Chromatographic measurements showed an approximately 6-times increase in pDNA capacity and less capacity scattering than CIMmicTM disks.

• Keywords: plasmid DNA, ion-exchange chromatography, monoliths, ATRP, grafting