Optimiranje reakcije derivatizacije terapeutskih proteina polietilen glikolom

master's thesis

• Title (english): Optimization of derivatization reaction of therapeutic proteins with polyethylene glycol
• Author: Ana Lukešić
• Mentor: Slobodan Barbarić (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2007-07-17, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Kovalentno vezanje polietilen glikola (PEG) na proteine, poznato kao pegilacija, široko je primjenjivana metoda derivatizacije terapeutskih proteina s ciljem poboljšanja njihovih svojstava. Vezanje PEG reagensa na protein postiže se reakcijom reduktivne alkilacije. Za pegilaciju proteina korišten je linearni, mono-funkcionalni PEG reagens, metoksi polietilen glikol (mPEG) propionaldehid molarne mase 20 kDa. Provođenjem reakcije pod određenim uvjetima, očekivani produkt sadrži uglavnom jednu molekulu PEG-a na N-terminalnoj amino skupini. Različiti reakcijski parametri mogu utjecati na selektivnost i prinos reakcije: pH, sastav pufera, koncentracija proteina, molarni odnos proteina i PEG reagensa te vrijeme trajanja reakcije. U ovom radu ispitivani su spomenuti parametri s ciljem optimiranja reakcijskih uvjeta da se dobije što veći prinos monopegiliranog produkta, uz istodobno što manji udio neželjenih visokomolekularnih struktura. Rezultati su ukazali na kombinaciju reakcijskih uvjeta koji tako primijenjeni omogućuju visoko iskorištenje reakcije. Monopegilirani protein izoliran je iz reakcijske smjese ionskom kromatografijom. Potvrđeno je da se radi o monopegiliranom proteinu.

Abstract (english)

Covalent attachment of polyethylene glycol (PEG) to proteins, known as pegylation, is widely used therapeutic protein derivatization method with the aim of enhancing their properties. The coupling of a PEG moiety to protein was attained using reaction of reductive alkylation. Linear, monofunctional PEG reagent, methoxypolyethylene glycol (mPEG) propionaldehyde of 20 kDa was used for protein pegylation. Conducting the reaction under controlled conditions, the anticipated product essentially contains a single polyethylene glycol molecule conjugated to the N-terminal amino group. Various reaction paramethers can influence the reaction selectivity and yield: pH, composition of the buffer, protein concentration, protein:PEG ratio and reaction duration. In this work mentioned paramethers have been analyzed in order to optimize reaction conditions to achieve as highest content of monopegylated product and at the same time as lowest content of unwanted highmolecular weight structures as possible. The results of this assay have pointed out the reaction conditions, which provide high reaction utilization. Monopegylated protein was isolated from reaction mixture by ion-exchange chromatography. It was confirmed that the product is a monopegylated protein.

• Keywords: pegilacija; polietilen glikol (PEG); reduktivna alkilacija; terapeutski proteini
• Keywords (english): pegylation; polyethylene glycol (PEG); reductive alkylation; therapeutic proteins
• Language: croatian
• URN:NBN: urn:nbn:hr:159:352742
• Study programme: Title: Biotechnology; specializations in: Biochemical-Microbiology; Course: Biochemical-Microbiology; Study programme type: university; Study level: graduate; Academic / professional title: diplomirani inženjer biotehnologije; smjer biokemijsko-mikrobiološki (diplomirani inženjer biotehnologije; smjer biokemijsko-mikrobiološki)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-09-07 07:51:36