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Primerjava in optimizacija metod za gojenje hibridomov v laboratorijskih pogojih.
ID Lovšin, Ema (Author), ID Narat, Mojca (Mentor) More about this mentor... This link opens in a new window

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PID: 20.500.12556/rul/9247ee67-fda5-4f1e-b488-f3c482d19473

Abstract
Monoklonska protitelesa (mAb) so eden glavnih biofarmacevtskih produktov. V industriji jih proizvajajo z rekombinantno tehnologijo, v laboratorijskem merilu pa je pogostejše klasično gojenje hibridomov, ki jih pridobimo s hibridomsko tehnologijo. Pogosto jih gojimo v navadnih gojitvenih posodah, uporabljajo pa se tudi alternativni načini gojenja: v posodah z mešali in manjših bioreaktorjih za enkratno uporabo. Na rast in produktivnost vplivajo tudi gojitveni medij in dodatki, zato se na tržišču pojavljajo vedno nova in bolj izpopolnjena gojišča. V naši raziskavi smo želeli primerjati metode gojenja hibridomov, raziskati vpliv različnih gojišč in načinov gojenja na rast in produkcijo mAb. V prvem delu smo določali dinamiko rasti hibridomov v treh različnih gojiščih (DMEM z 10 % FBS, CCM1 in TurboDoma z 2- in 4-mM glutaminom) ter dinamiko rasti v posodi z mešalom. V drugem delu smo določali njihovo produktivnost v odvisnosti od gojišča in načina gojenja. Hibridomi so dosegli najvišje celične gostote v gojišču DMEM s serumom (FBS), pozitivno na rast in viabilnost je vplivala tudi višja koncentracija glutamina. Boljše rasti v posodi z mešalom nismo uspeli dokazati. Največ mAb so hibridomi proizvedli v gojišču TurboDoma. Vpliv gojišča na dinamiko rasti in produkcijo mAb je bil različen za posamezen klon. Hibridomi so mAb proizvajali tudi po prehodu v stacionarno fazo, visoke koncentracije mAb smo namreč izmerili tudi še v začetni fazi odmiranja celic. Višje celične gostote niso vedno pomenile večje količine proizvedenih mAb. V nekaterih primerih je količina antigena omejitveni dejavnik pri sprotnem testiranju aktivnosti proizvedenih mAb. Za sprotno določanje količine mAb, bi lahko običajno določanje titra mAb z na antigen specifičnim testom ELISA zamenjali s komercialnim testom ELISA, s katerim dobimo podatek o celokupni koncentraciji mAb.

Language:Slovenian
Keywords:Hibridomi, monoklonska protitelesa, optimizacija, produkcija, ELISA
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Publisher:[E. Lovšin]
Year:2017
PID:20.500.12556/RUL-96713 This link opens in a new window
UDC:601.2:576.38:616-097.3(043.2)
COBISS.SI-ID:8833657 This link opens in a new window
Publication date in RUL:12.10.2017
Views:1531
Downloads:655
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Secondary language

Language:English
Title:Comparison and optimization of in vitro hybridoma cultivation
Abstract:
Monoclonal antibodies (mAb) are a common biopharmaceutical product. On industrial scale they are produced using recombinant technology, hybridoma technology is a more common way on laboratory scale. They are often grown in cell culture flasks and alternatively in spinner flasks and smaller disposable bioreactors. Cell culture medium and additives can influence cell growth and productivity, therefore new and more advanced media are emerging on a market. In our study, we wanted to compare different methods of cultivating hybridomas, to study the influence of different growth mediums and cultivation methods on the hybridoma growth and mAb production. Firstly we determined hybridoma growth in three different culture mediums: DMEM with 10 % FBS, CCM1 and TurboDoma with 2 or 4 mM glutamine and growth dynamics in spinner flask. Secondly, we determined their productivity in different culture mediums and cultivation systems. Hybridomas reached the highest cell density in DMEM with 10 % FBS, higher glutamine concentration was also found to be a positive influence on growth and viability. We failed to prove a better growth in spinner flask. Hybridomas produced the most mAb in TurboDoma culture medium. The effect of the culture medium on cell growth and mAb productivity was different for each hybridoma clone. We found that hybridoma production of mAb continued after stationary phase, high mAb concentrations were also measured in the cell death phase. Higher cell densities did not always mean higher amounts of produced mAb. In certain cases, the amount of antigen is a limiting factor in the ongoing testing of mAb production. For the actual determination of the amount of mAb, usual ELISA that determines mAb titre could be replaced with commercial ELISA to obtain mAb concentration.

Keywords:hybridomas, monoclonal antibodies, optimization, production, ELISA

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