Ločevanje izooblik monoklonskih protiteles z induciranim prehodom pH

Šedivy, Maja (author), Podgornik, Aleš (mentor)

Kromatografske metode zagotavljajo visoko stopnjo čistosti biotehnološko pridobljenih proteinov. Najpogosteje se proteine spira z raztopino soli. Čiščenje s spremembo v pH-vrednosti mobilne faze je teže nadzorovati, vendar je zelo privlačno, saj naknadno odstranjevanje soli ni potrebno. Možnost lažje kontrole prehodov pH pri izolaciji so pokazale mešane kolone, sestavljene iz kombinacije močnih in šibkih ionskih izmenjevalcev. Namen magistrskega dela je bilo ustvariti položen inducirani prehod pH, ki ga dosežemo s preklopom mobilnih faz z različnim pH. Na osnovi induciranega prehoda pH smo testirali ločevanje izooblik monoklonskih protiteles (mAb). Mešano kolono smo naredili z izborom ustreznih ionskih izmenjevalcev: šibki z izjemno majhnimi porami, v katere proteini ne morejo vstopati, namenjen tvorjenju induciranega prehoda pH, in porozen močni, namenjen vezavi tarčnih proteinov. Dvojna funkcija mešane kolone je omogočila ustvarjanje stabilnega prehoda pH z visoko kapaciteto za izbran protein. Kombinacija z različnimi mobilnimi fazami je pokazala, da lahko s koncentracijo in pH-vrednostjo pufra dobro kontroliramo položnost prehoda pH. Šibki (AG® 4-x4) in močni (Fractogel® EMD TMAE Hicap (M)) anionski izmenjevalec sta tako posamezno kot tudi v mešanici delovala dobro, medtem ko z intermediatnim izmenjevalcem (Bio-RexTM 5) nismo dobili pozitivnih rezultatov. Ločevanje izooblik mAb je bilo delno uspešno. V prihodnje bi bilo treba najti optimalno razmerje ionskih izmenjevalcev v mešani koloni in ustrezen pufrski sistem, ki bi omogočal boljše ločevanje različic mAb.

• Language: Slovenian
• Keywords: monoklonska protitelesa, zaključni procesi, ionska izmenjevalna kromatografija, pH-vrednost, induciran prehod pH, ločevanje s spremembo pH
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Šedivy]
• Year: 2017
• PID: 20.500.12556/RUL-99153
• UDC: 602.44:543.544.17:577.112(043.2)
• COBISS.SI-ID: 8894329
• Publication date in RUL: 24.12.2017

ŠEDIVY, Maja, 2017, Ločevanje izooblik monoklonskih protiteles z induciranim prehodom pH [online]. Master’s thesis. M. Šedivy. [Accessed 23 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=99153

Secondary language

• Language: English
• Title: Separation of monoclonal antibody isoforms with induced pH gradients

Abstract

Chromatographic separation techniques ensure purification quality of biotechnically produced proteins. Most of the time, proteins are eluted with salt gradients. Purification with pH gradients is usually difficult to control, however it is appealing because subsequent salt removal steps are unnecessary. Mixed-bed chromatography columns have shown to be an effective way of performing pH gradient-based protein separation. The approach of present work was to create a linear pH gradient, generated by a step change in pH. A separation of protein charge variants with the mixed-bed concept was demonstrated for a monoclonal antibody mixture. A selection of appropriate ion exchange resins has given a mixed-bed column with a dual function: a small-pore weak base resin serves for intracolumn generation of pH gradients, and a large-pore strong base resin serves for interacting with proteins, gaining high binding capacity. A combination of different mobile phases has shown that concentration and pH value of the buffer can regulate the duration of the induced pH gradient. Weak (AG® 4-x4) and strong (Fractogel® EMD TMAE Hicap (M)) anion exchanger have turned to be good candidates in our experiments, however, intermediate anion exchanger (Bio-RexTM 5) has not given us positive results. The separation of monoclonal antibody isoforms has been partially achieved. In future, one would have to define an optimal ratio of weak and strong ion exchange resins, and a suitable buffer system with a higher chromatographic sensitivity for the separation of isoforms.

• Keywords: monoclonal antibodies, downstream processing, ion exchange chromatography, pH value, induced pH gradient, separation with changing of pH