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Optimizacija metode kapilarnega izoelektričnega fokusiranja za analizo treh novih bioloških molekul
ID Marinček, Eva (Author), ID Kerč, Janez (Mentor) More about this mentor... This link opens in a new window, ID Kocjan, Boštjan (Comentor)

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Abstract
Biološka zdravila so večinoma proteinske molekule, ki imajo v primerjavi z majhnimi sinteznimi učinkovinami mnogo večjo molekulsko maso ter kompleksno zgradbo, zato sta proces proizvodnje in karakterizacija zgradbe v njihovem razvoju ključnega pomena. Terapevtska monoklonska protitelesa postajajo ena izmed najhitreje rastočih skupin bioloških zdravil. Monoklonska protitelesa so heterogene molekule po velikosti in naboju. Nabite različice monoklonskih protiteles so eden izmed kritičnih atributov kakovosti, ki jih je treba karakterizirati in količinsko opredeliti ter določiti njihov vpliv na varnost in učinkovitost zdravila. Kapilarno izoelektrično fokusiranje (cIEF) je ena izmed metod, ki se uporablja za analizo in karakterizacijo nabitih različic. Na podlagi izoelektrične točke se različice ločijo v glavne, kisle in bazične skupine in jih na elektroferogramu vidimo kot ločene vrhove. V magistrski nalogi smo proučili primernost generične metode izoelektričnega fokusiranja pri razvoju treh bioloških molekul. Ker pri eni molekuli nismo dosegli zadovoljive ločbe, smo metodo optimizirali tako, da smo spreminjali sestavo reakcijske mešanice in pogoje fokusiranja. V generični metodi uporabljamo farmalit, ki pokriva široko pH območje (3-10), koncentracijo sečnine 2,0 M in čas fokusiranja 6 minut. V optimizirani metodi smo ločljivost uspeli izboljšati z dodatkom farmalita, ki pokriva ožje pH območje (8-10,5) ter s podaljšanjem časa fokusiranja na 10 minut. Za vse tri molekule smo določili območje, v katerem je metoda linearna, in testirali vpliv razsoljevanja. Ugotovili smo tudi, da je metoda primerna za zaznavanje sprememb v nabitih različicah proteina, ko tega izpostavimo stresnim pogojem.

Language:Slovenian
Keywords:Biološka zdravila, monoklonska protitelesa, kapilarna elektroforeza, izoelektrično fokusiranje, nabite različice
Work type:Master's thesis/paper
Organization:FFA - Faculty of Pharmacy
Year:2022
PID:20.500.12556/RUL-137832 This link opens in a new window
Publication date in RUL:02.07.2022
Views:2104
Downloads:133
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Secondary language

Language:English
Title:Optimization of a capillary isoelectric focusing method for analysis of three new biological molecules
Abstract:
Biopharmaceuticals are mostly protein molecules that have a much higher molecular weight and more complex structure compared to small synthetic active pharmaceutical ingredients. Production process and characterization of the structure is crucial in development of these molecules. Therapeutic monoclonal antibodies are becoming one of the fastest growing groups of biopharmaceuticals. Monoclonal antibodies are heterogeneous molecules in both size and charge. Charge variants of monoclonal antibodies are one of the critical quality attributes. They have to be characterized, quantified, and their impact on drug safety and efficacy has to be determined. Capillary isoelectric focusing (cIEF) is one of the methods used to analyze and characterize charge variants that are separated into main, acidic and basic groups based on their isoelectric point and are presented as seperated peaks on the electropherogram. In the master's thesis we tested the suitability of the generic isoelectric focusing method in the development of three biological molecules. For one of the molecules we did not achieve a good resolution, therefore we optimized the method by changing composition of reaction mixture and focusing conditions. In the generic method we use pharmalyte, which covers a wide pH range (3-10), urea concentration 2.0 M and focusing time 6 minutes. In the optimized method, resolution was improved by adding a different pharmalyte, which covers a narrower pH range (8-10.5) and by extending the focusing time to 10 minutes. For all three molecules we determined the range in which the method is linear and tested desalting of samples. We also confirmed that the method is suitable for detecting changes in charged variants of a protein when exposed to stress contiditons.

Keywords:Biopharmaceuticals, monoclonal antibodies, capillary electrophoresis, isoelectric focusing, charge variants

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