Izboljševanje ločljivosti pri analizi Fc-fuzijskega proteina z metodo kapilarnega izoelektričnega fokusiranja

Plahutnik, Jakob (author), Jamnik, Polona (mentor), Kocjan, Boštjan (comentor)

Kapilarno izoelektrično fokusiranje je v zadnjem desetletju postala ena glavnih analitskih metod za določanje naboja in izoelektrične točke analita ter njegovih nabitih različic, še posebej za potrebe proizvodnje bioloških zdravil. Pri analizi fuzijskega proteina s to metodo lahko zaradi kompleksnih lastnosti molekul prihaja do nespecifičnih vrhov na elektroferogramih zaradi agregacije oz. precipitacije proteina. Da bi dosegli čim bolj natančno, zanesljivo in ponovljivo analizo proteina, je potrebno zagotoviti ustrezno topnost in stabilnost molekul v analitski kapilari. Pri izvedbi magistrskega dela smo pripravili nosilne mešanice z različnimi koncentracijami snovi za povečevanje topnosti (urea, formamid, saharoza, zwitterionski detergent CHAPS, zwitterionski NDSB v kombinaciji s taurinom), v katerih smo raztopili preučevano molekulo Fc-fuzijskega proteina. Pomagali smo si tudi z zmanjšanjem koncentracije proteina v nosilni mešanici, kar je izboljšalo ločljivost vrhov in ponovljivost elektroferogramov v primerih, ko je bilo ločevanje dokaj uspešno že pri višjih koncentracijah proteina (npr. pri dodatku formamida in saharoze). Nazadnje smo se poslužili encimske odstranitve negativno nabitih sialičnih kislin, ki zakrivajo proteinski del molekule. Pridobljeni elektroferogrami so bili za razliko od nativne različice pomaknjeni v bazičen del gradienta pH ter imeli manjše število vrhov. Najbolj uspešno ločbo smo dosegli z dodatkom 3 M uree pri koncentraciji proteina 0,4 mg/ml in času fokusiranja 12 minut; vse ostale koncentracije uree in drugih snovi za povečevanje topnosti ter krajši čas fokusiranja niso dosegli zadovoljivih rezultatov.

• Language: Slovenian
• Keywords: analitske metode, separacijske tehnike, izoelektrično fokusiranje, fuzijski proteini, ločljivost, biofarmacevtiki
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [J. Plahutnik]
• Year: 2021
• PID: 20.500.12556/RUL-132946
• UDC: 602.44:543.545:577.112
• COBISS.SI-ID: 83901955
• Publication date in RUL: 07.11.2021

PLAHUTNIK, Jakob, 2021, Izboljševanje ločljivosti pri analizi Fc-fuzijskega proteina z metodo kapilarnega izoelektričnega fokusiranja [online]. Master’s thesis. Ljubljana : J. Plahutnik. [Accessed 18 May 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=132946

Secondary language

• Language: English
• Title: Improving separation of Fc-fusion protein with capillary isoelectric focusing

Abstract

Capillary isoelectric focusing has in recent years increasingly been applied as one of the main analytical methods for determination of protein charge and isoelectric point. It is one of the key tools for monitoring charge heterogeneity, especially in biopharmaceutical industry. However, analysis of fusion proteins can often be challenging due to their structural complexity. As a result, acquired electropherograms may exhibit irreproducible and/or unspecific peaks, which are caused by protein aggregation and precipitation. Therefore, it is crucial to achieve sufficient solubility in the capillary. The aim of the master's thesis was to expose the drug substance to different solubilizers in various concentration (urea, formamide, sucrose, zwitterion detergent CHAPS, and zwitterion NDSB in combination with taurine). In some cases where separation has been partially successful (e.g., formamide and sucrose), results have been improved by decreasing protein concentration. To make separation easier, we enzymatically removed negatively charged sialic acids, which are linked to the protein molecule. Acquired electropherograms are consequently shifted to basic side of pH gradient and have fewer peaks. The most successful separation was achieved by analyzing 0,4 mg/mL of fusion protein in 3 M urea and focusing time of 12 minutes; every other concentration of urea and other tested solubilizers did not produce satisfactory resolution or reproducibility.

• Keywords: isoelectric focusing, separation, fusion proteins, resolution, analytical methods, biopharmaceuticals