20.500.12556/RUL-146843 Razvoj analitskih kromatografskih metod za spremljanje procesa proizvodnje lentivirusnega vektorja Development of analytical HPLC fingerprint method for lentiviral vector production monitoring Posledica vzpona genske terapije je povečana potreba po lentivirusnih vektorjih. Nov razred kompleksnih bioloških zdravil je proizvajalce postavil pred številne razvojne in proizvodne izzive. Med najbolj perečimi je vzpostavitev ponovljive proizvodnje, ki bi zagotavljala varnost in učinkovitost končnega izdelka. Kot pomemben del rešitve so bile v okviru FDA pobude procesno analitskih tehnologij prepoznane enostavne in hitre analitske tehnike. Za boljše razumevanje in vzpostavitev celovitejšega nadzora smo za proizvodnjo letnivirusnega vektorja razvili metodo kromatografskih prstnih odtisov. Namen je bil vzpostaviti metodo, ki bi proizvodni proces spremljala od vstopnih surovin za bioproces preko korakov čiščenja vse do kontrole kakovosti končnega produkta. Kromatografske prstne odtise smo ovrednotili z izborom skupnih kromatografskih vrhov, spremljanjem njihove višine, relativne višine v primerjavi s preostalimi skupnimi vrhovi ter spremljanjem celotne površine. Osredotočili smo se na analizo poteka bioprocesa, odstranjevanja DNK, bistrenja bioprocesne brozge, koncentriranja in diafiltracije ter preparativne kromatografije. Z vzpostavljeno metodo smo v vzorcih odvzetih tekom bioprocesa zaznali spremembe v sestavi dovedenega svežega gojišča, med encimskim odstranjevanjem DNK smo spremljali razgradnjo domnevnega vrha DNK in zaznali nove nečistoče, ki jih, kot smo opazili, popolnoma odstrani naslednji korak bistrenja. Metodo smo nato vpeljali v testiranje filtrirnih kaset za koncentriranje in diafiltracijo, kjer smo opazili znatne razlike v dinamiki odstranjevanja komponent vzorca. Analiza preparativnega kromatografskega koraka je pokazala, da se večino nečistoč odstrani prav med kromatografijo. Razvita metoda omogoča spremljanje bioprocesa s primerjavo relativnih sprememb posameznih vzorcev, vendar kvantifikacije celotne koncentracije lentivirusnih vektorjev ne omogoča. Recent advances in the field of gene therapy significantly increased the need for lentivirus vectors. Significant pressure fell on the production where several serious bottlenecks throttling the expanding field were uncovered. With robust and reproducible production being a prerequisite for a safe and efficient final product, quick and simple analytical methods have been recognized as a crucial enabling element in the FDA sponsored Process Analytical Technology initiative. In this regard, the chromatographic fingerprint method was developed for deeper process understanding and better process control. The goal is to have a method that would be able to cover the process from the control of the upstream raw materials, downstream process steps all the way to the final product quality control. Chromatographic fingerprints were evaluated through common peak selection, common peak height, relative height, and whole chromatogram area. The focus was on the bioprocess progression, DNA removal, broth clarification, concentration, buffer exchange, and chromatography. Change in the composition of the inflown fresh growth media was detected during the upstream virus proliferation, presumable DNA peak reduction was observed, while new emerging impurities were detected and subsequently removed during clarification. The developed method was also applied to the crossflow filtration cassette test with results indicating significant differences in their operation. Analysis of the chromatographic step suggested that most of the impurities, the ones dominating chromatograms in all steps upstream, were removed with chromatography. The developed chromatographic analytical method enables process monitoring through a comparison of relative changes between specific process samples. It does, however, not enable quantification of lentiviral particle concentration. biotehnologija procesna analitika lentivirus proces čiščenja biotechnology process analytics lentivirus purification process true false false Slovenski jezik Neznan jezik Magistrsko delo/naloga 2023-06-15 08:43:45 2023-06-15 08:43:51 2023-10-06 13:05:11 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 217792 156774659 32864.pdf 32864.pdf 1 7E83CA821B6C8CE68F91CFC2D8EFC179 f8c19fffc90db7349a1da2cbfc15f4921d03119d2c58dd23094c6d73c923d6b6 a293c700-0b47-11ee-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=170149 Biotehniška fakulteta 0 0 0