Validacija metode mapiranja peptida

master's thesis

• Title (english): Validation of peptide mapping methods
• Author: Mislav Sladić
• Mentor: Vladimir Mrša (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2011-03-18, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Svrha ovog rada je validirati metodu mapiranja peptida proteina Filgrastima (rekombinantni humani faktor stimulacije granulocitnih kolonija, rhG-CSF). Analitička metoda mapiranja peptida Filgrastima je identifikacijska metoda kojom se dokazuje prisutnost peptidnih fragmenata Filgrastima dobivenih enzimatskim cijepanjem i time potvrđuje njegova primarna struktura. Analiza peptidnih fragmenata Filgrastima provedena je primjenom tekućinskog kromatografa visoke djelotvornosti (HPLC) uz separaciju kromatografijom obrnutih faza. U ovom radu ispitano je više validacijskih parametara među kojima su: specifičnost, robusnost, preciznost te stabilnost uzoraka. U svrhu potvrde specifičnosti metode, pojedini peptidni fragmenti identificirani su korištenjem tehnike spektrometrije masa. Kao dodatna potvrda specifičnosti metode, provedena je i analiza mutiranog Filgrastima koji na svojem C-terminalnom kraju ne sadrži aminokiselinu prolin. U sklopu validacije ključnih parametara analitičke metode mapiranja peptida uspješno su razdvojeni fragmenti analiziranog proteina te su potvrđeni prvobitno postavljeni uvjeti. U sklopu rezultata ispitivanja specifičnosti metode određeno je vrijeme zadržavanja na koloni, te relativno vrijeme zadržavanja za radni standard, referentnu otopinu i mutirani Filgrastim-M4 kod kojeg je vidljivo da dolazi do pomaka u vremenu zadržavanja fragmenta A12, čime je dokazana selektivnost metode.

Abstract (english)

The purpose of this study was validation of protein Filgrastim (recombinant human granulocyte colony stimulating factor, rH G-CSF) by the method of peptide mapping. Analytical method of peptide mapping Filgrastim, which will be elaborated in this thesis, is an identification method that proves the presence of Filgrastim peptide fragments obtained by enzymatic cleavage, thus confirming its primary structure. Analysis of peptide fragments Filgrastim was performed using High-Performance Liquid Chromatograph (HPLC) with reversed-phase chromatography separation. In this thesis different validation parameters including the specificity, robustness, precision, and stability samples were tested. In order to confirm the specificity of the method, specific peptide fragments were be identified using mass spectrometry technique. As additional confirmation of the specificity of the method, the analysis of mutant Filgrastim, which its C-terminal end does not contain the amino acid proline, was be carried out. Within validation of the key parameters of the analytical method of peptide mapping, the peptide fragments of analyzed protein were successfully separated and the initially set conditions were confirmed. Within the test results of the specificity of the method, retention time on the column and the relative retention time were determined for the working standard, reference solution, and Filgrastim-M4 mutant. For Filgrastim-M4 mutant is evident that there is a shift in the retention time of the fragment A12, which demonstrated selectivity of the method.

• Keywords: tekućinska kromatografija visoke djelotvornosti; validacija; mapiranje peptida; Filgrastim
• Keywords (english): High Performance Liquid Chromatography; validation; the mapping Peptide; Filgrastim
• Language: croatian
• URN:NBN: urn:nbn:hr:159:968968
• 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: 2017-05-30 12:09:08