magistrsko delo
Tina Modrijan (Author), Mojca Narat (Mentor), Ajda Trdin (Co-mentor)

Abstract

Koncentracijo monoklonskih protiteles (mAb) in fuzijskih proteinov lahko določamo z različnimi bioanalitskimi metodami in napravami. Med slednjimi afinitetna tekočinska kromatografija visoke ločljivosti (HPLC) velja za zlati standard. Ker je pridobivanje rezultatov s HPLC zelo zamudno, so se na trgu začele pojavljati številne alternativne visokozmogljive bioanalitske metode in naprave. Mednje lahko uvrstimo naprave družine Cedex, kjer določanje koncentracije produkta poteka z metodo imunske turbidimetrije, in Octet, ki temelji na interferometriji bioloških plasti, ter metodo ValitaTiter, s katero koncentracijo mAb in fuzijskih proteinov določamo na podlagi sprememb v polarizaciji svetlobe. Namen magistrske naloge je bil raziskati odstopanja med meritvami na HPLC in preostalimi visokozmogljivimi metodami oziroma napravami. Poleg tega smo želeli preveriti, ali proces ločevanja produktov od večjih nečistoč in zamrzovanje vzorcev vplivata na meritve naprav Cedex Bio HT. Za proučevanje potencialnega vpliva strukture molekul rekombinantnih proteinov na razlike med meritvami naprav, smo v eksperiment vključili štiri celične linije CHO, ki proizvajajo IgG1, IgG2, IgG4 in Fc-fuzijski protein. Tekom 14-dnevne produkcijske faze je potekalo dnevno vzorčenje bioprocesnih brozg. Ugotovili smo, da zamrzovanje vzorcev in postopek primarnega ločevanja nimata vplivata na meritve koncentracije produktov in da je odstopanje meritev na preučevanih visokozmogljivih napravah od meritev na HPLC predvsem odvisno od koncentracije produkta v vzorcu, umeritvenih krivulj ter od strukture rekombinantnega proteina. Med vsemi preučevanimi napravami oziroma metodami smo največje ujemanje s HPLC zabeležili pri analizatorju Octet QK.

Keywords

CHO;Cedex;monoklonska protitelesa;fuzijski proteini;analitične metode;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [T. Modrijan]
UDC: 602.1:543.544.5:57.083.34:591.111.8(043.2)
COBISS: 131174147 Link will open in a new window
Views: 13
Downloads: 6
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Other data

Secondary language: English
Secondary title: Comparison of analytical methods for determination of monoclonal antibody and fusion protein concentration
Secondary abstract: The concentration of monoclonal antibodies (mAbs) and fusion proteins can be determined by various bioanalytical methods and devices. Among these, affinity high-performance liquid chromatography (HPLC) is considered as the gold standard. As obtaining HPLC results can be very time-consuming, many high-performance alternatives have started to appear on the market. These include the Cedex family of analysers, where product concentration measurements are based on immunoturbidimetry, Octet analysis systems, which are based on bio-layer interferometry, and ValitaTiter IgG quantification assay, which detects changes in light polarization. The purpose of the master’s thesis was to investigate possible discrepancies in measurements between the before-mentioned high-throughput alternatives and HPLC. In addition, we wanted to check whether the separation of the product from larger impurities and whether the freezing of samples affects the measurements of Cedex Bio HT devices. To study the potential influence of recombinant protein molecule structures on device measurements, four CHO cell lines producing IgG1, IgG2, IgG4, and Fc-fusion protein were included in the experiment. During the 14-day production phase, daily sampling of bioprocess slurries took place. We found that the primary separation process and the freezing of the samples prior to analysis does not affect product concentration measurements and that deviations between measurements mainly depend on product concentration, calibration curves, and the structure of the recombinant protein. Among all studied devices or methods, the lowest discrepancies from HPLC were observed on the Octet QK analyser.
Secondary keywords: biotechnology;monoclonal antibodies;fusion proteins;analytical methods;
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: 1 spletni vir (1 datoteka PDF (XII, 65 f., [14] f. pril.))
ID: 17196444