Primerjava klasičnih in visokopretočnih načinov transfekcije in selekcije stabilnih produkcijskih kultur CHO

Petrushevikj, Tamara (author), Narat, Mojca (mentor), Kramer, Lovro (comentor)

Biofarmacevtska industrija teži k večji učinkovitosti pri proizvodnji bioloških zdravil z uporabo večjega števila klonov v krajših časovnicah razvoja, kar omogoča boljše produkcijske celične linije in večji izkoristek proizvodnje. Do sedaj uporabljane rastne plastenke (SF) nadomeščajo plošče s 24 globokimi vdolbinicami (24DW) ter plošče s 96 globokimi vdolbinicami (96DW). Namen magistrske naloge je ovrednotiti vpliv različnih platform transfekcij (klasična - LT ter visoko zmogljiva 96W platforma - HT) na rastne razmere celic CHO in na titer rekombinantnih protiteles, ter ovrednotiti tehnologijo HT za selekcijo stabilno transficiranih CHO celic v gojitvenih platformah 24DW in 96DW. Uspešnost izražanja vstavljenih genov, ki vplivajo na glikozilacijo protitelesa smo ovrednotili z analizo mRNA. Pri primerjavi viabilnosti v točki selekcijske krize v 24DW in 96DW, smo dobili naslednje vrednosti p: < 0,0001 (celična linija 1), 0,1365 (celična linija 2) in 0,0002 (celična linija 3). Selekcija je bila bolj stresna za celice v 96DW v primerjavi z 24DW. Z HT transfekcijsko metodo smo pri celični liniji 1 in 2 dosegli večjo variabilnost viabilnosti. Pri celični liniji 3 smo dobili primerljivi povprečji viabilnosti (p = 0,6916) med LT in HT. V šarži z dohranjevanjem smo dobili primerljive titre na dan 7 in delno primerljive titre na dan 14 med 24DW in 96DW ter LT in HT. Pri vsaki platformi smo dosegli zelo primerljive maksimalne titre. Najvišjo specifično produktivnost smo dobili pri 96DW HT. Dokazali smo, da so dodatni geni za uravnavanje glikozilacije (GE geni) lahko razlog za nižje titre in celično gostoto (VCD). Različni načini transfekcije ne vplivajo na izražanje teh genov (gen A, p = 0,1425; gen B, p = 0,0777), medtem ko smo pri 96DW dobili nižje izražanje GE genov v primerjavi s 24DW (gen A, p < 0,0001; gen B, p < 0,0001).

• Language: Slovenian
• Keywords: Celične linije, sesalske celice, CHO, transfekcije, visokopretočno, selekcija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2024
• PID: 20.500.12556/RUL-162144
• COBISS.SI-ID: 208156675
• Publication date in RUL: 19.09.2024

PETRUSHEVIKJ, Tamara, 2024, Primerjava klasičnih in visokopretočnih načinov transfekcije in selekcije stabilnih produkcijskih kultur CHO [online]. Master’s thesis. [Accessed 14 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=162144

Secondary language

• Language: English
• Title: Comparison of classical and high-throughput methods of transfection and selection of stable CHO production cultures

Abstract

The biopharmaceutical industry strives for greater efficiency in the production of biological drugs by using a larger number of clones in shorter development timelines, which allows for better production cell lines and higher production yields. The previously used shake flasks (SF) are being replaced by 24-deep well plates (24DW) and 96-deep well plates (96DW). The aim of this master's thesis is to evaluate the impact of different transfection platforms (classical - LT and high-throughput 96W platform - HT) on the growth conditions of CHO cells and the titer of recombinant antibodies, as well as to assess the HT technology for the selection of stably transfected CHO cells in 24DW and 96DW cultivation platforms. The expression efficiency of inserted genes affecting antibody glycosylation was evaluated by mRNA analysis. When comparing viability at the selection crisis point in 24DW and 96DW, the following p-values were obtained: < 0.0001 (cell line 1), 0.1365 (cell line 2), and 0.0002 (cell line 3). Selection was more stressful for cells in 96DW compared to 24DW. The HT transfection method resulted in greater variability of viability in cell lines 1 and 2. In cell line 3, comparable average viabilities (p = 0.6916) were obtained between LT and HT. In fed-batch culture, comparable titers were obtained on day 7 and partially comparable titers on day 14 between 24DW and 96DW, as well as between LT and HT. Very similar maximum titers were achieved with each platform. The highest specific productivity was obtained with 96DW HT. It was demonstrated that additional genes for glycosylation regulation (GE genes) could be the reason for lower titers and viable cell density (VCD). Different transfection methods did not affect the expression of those genes (gene A, p = 0.1425; gene B, p = 0.0777), while lower expression was observed in 96DW compared to 24DW (gene A, p < 0.0001; gene B, p < 0.0001).

• Keywords: cell lines, mammalian cells, CHO, transfections, high-throughput, selection