magistrsko delo
Eva Vovk (Author), Polona Jamnik (Mentor), Polona Jamnik (Thesis defence commission member), Igor Štern (Thesis defence commission member), Mojca Narat (Thesis defence commission member), Borut Štrukelj (Thesis defence commission member), Igor Štern (Co-mentor)

Abstract

Med razvojem terapevtskih rekombinantnih monoklonskih protiteles je prisilna razgradnja eno od ključnih orodij za pridobivanje informacij o molekuli, analitski metodi, razvoju procesa in proizvodnje. Zato je ne le priporočljivo, temveč zaželeno, da razvoj analitske metode vključuje prisilno razgrajene vzorce, saj to pomembno vpliva na razumevanje o zmožnostih in omejitvah uporabljenih metod. Namen magistrskega dela je bil izpostaviti biološko učinkovino stresnim pogojem, da bi ugotovili, ali in pri kateri izmed izbranih analitskih metod zaznamo spremembe pri določeni (tarčni) biološki učinkovini in v kolikšni meri. Izbrano biološko učinkovino smo izpostavili različnim dejavnikom: povišani temperaturi in različnim vrednostim pH, oksidantu ter reducentu z različnimi koncentracijami, različni jakosti svetlobe, enkratnemu in večkratnemu zamrzovanju in odmrzovanju, deglikozilaciji z encimom v različnih množinah, povišani množini glukoze ter kovinskima ionoma. Z različnimi kromatografskimi metodami (afinitetna kromatografija, kromatografija z obrnjeno fazo, gelska izključitvena kromatografija, kapilarna gelska elektroforeza pod nereducirajočimi in reducirajočimi pogoji) smo analizirali vzorce in ovrednotili povzročene spremembe. Pokazali smo, da so vse metode, razen afinitetne kromatografije, zmožne zaznavanja sprememb za izbrani protein. Hkrati pa smo ugotovili, da je v študiji vključen protein obstojen tudi pri ekstremnih laboratorijskih pogojih: stabilen je v širokem temperaturnem in pH-območju, pri izpostavitvi umetni svetlobi in pri večkratnem zamrzovanju in odmrzovanju.

Keywords

biotehnologija;prisilna razgradnja;analitske metode;monoklonsko protitelo;farmacija;biofarmacevtiki;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [E. Vovk]
UDC: 602:543.54:616-097.3(043.2)
COBISS: 9360249 Link will open in a new window
Views: 721
Downloads: 290
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Other data

Secondary language: English
Secondary title: Forced degradation study of therapeutic monoclonal antibody
Secondary abstract: During the development of a therapeutic recombinant monoclonal antibody, the forced degradation is one of the key tools for obtaining information about the molecule, the analytical method and about the process development and production. Hence, it is recommended that the method development includes foced degradated samples for a better understanding of the method capability and limitations of the method. The aim of the master's thesis was to expose the drug substance to stress conditions in order to determine whether and which of the chosen analytical methods are stability indicating. The chosen drug substance was exposed to various stress conditions: higher temperature, different pH values, addition of an oxidant, addition of a reducing agent, exposure to light, freezing/thawing, (partial) enzymatic deglycosylation, addition of glucose and metal ions. Samples were analyzed with different chromatographic methods (affinity liquid chromatography, reversed-phase chromatography, size exclusion chromatography, capillary gel electrophoresis under non-reducing conditions and under reducing conditions) and the induced differences were evaluated. It has been shown that all the methods except the affinity chromatography are capable to detect changes in the drug substance. Furthermore, the study showed that the studied protein is quite stable under various extreme laboratory conditions: broad temperature and pH ranges, exposure to artificial light, multiple freeze/thaw cycles.
Secondary keywords: biotechnology;forced degradation;analytical methods;monoclonal antibody;pharmacy;biopharmaceuticals;
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: X, 49 f., [4] f. pril.
ID: 11229766