20.500.12556/RUL-174019 Določanje vpliva bakterijskih endotoksinov na proizvodni proces biofarmacevtskih izdelkov magistrsko delo Determination of the impact of bacterial endotoxins on the biopharmaceutical production process master thesis Uvod: Endotoksini so lahko visoko pirogene in imunogene molekule, ki izhajajo iz celične stene po Gramu-negativnih bakterij. Njihova detekcija, preprečevanje vnosa ter odstranjevanje je ključnega pomena v proizvodnji biofarmacevtskih izdelkov pri zagotavljanju varnosti in učinkovitosti zdravil. Dovoljene mejne vrednosti so strogo določene glede na način uporabe zdravila, glavni viri kontaminacije pa vključujejo ostanke bakterij, vodo, materiale, opremo in biofilme. Namen: V magistrskem delu smo preučili vpliv modelnega endotoksina na proliferacijo in viabilnost celic ovarijev kitajskega hrčka ter učinkovitost njegovega odstranjevanja z različnimi kromatografskimi stopnjami tekom biofarmacevtske proizvodnje terapevtskih proteinov. Cilj je potrditi negativen vpliv višjih koncentracij endotoksina na proliferacijo celic in dokazati, da večstopenjsko kromatografsko čiščenje učinkovito zagotavlja vsebnost modelnega endotoksina pod mejo specifikacij. Metode dela: Eksperiment smo razdelili na dva dela: (1) spremljanje vpliva različnih koncentracij endotoksina na proliferacijo in viabilnost celic ovarijev kitajskega hrčka ter vsebnost proteina, (2) ocena učinkovitosti odstranjevanja endotoksina v treh kromatografskih stopnjah (afinitetna, multimodalna, ionsko-izmenjevalna). Vsebnost endotoksina v pridobljenih vzorcih smo določali s testom Limulus amebocitni lizat. Z UV spektrofotometrično metodo smo določali vsebnost proteinov. Rezultate vpliva endotoksina na proliferacijo in viabilnost celic smo statistično opredelili z ANOVA metodo v programu JMP. Rezultati: Različne koncentracije endotoksina niso statistično značilno vplivale na proliferacijo in viabilnost celic ovarijev kitajskega hrčka (ANOVA, p < 0,005). Afinitetna kromatografija (Protein A) je vsebnost endotoksina znižala pod mejo detekcije (< 0,005 EU/mL) in dosegla logaritemski faktor odstranjevanja ? 6,2. Multimodalna in ionsko-izmenjevalna kromatografija sta dodatno prispevali k skupnemu odstranjevanju endotoksina, s kumulativnim logaritemskim faktorjem odstranjevanja 9,4. Izkoristki vseh treh kromatografij so bili skladni s specifikacijami za proizvodni proces, kar kaže, da visoke koncentracije dodanega endotoksina niso imele vpliva na ovrednotene procesne parametre. Razprava in zaključek: Modelni endotoksin statistično značilno ni vplival na proliferacijo in viabilnost celic ovarijev kitajskega hrčka. Afinitetna, multimodalna in ionsko-izmenjevalna kromatografija so skupaj dosegale visok kumulativni logaritemski faktor odstranjevanja, kar potrjuje, da je večstopenjsko čiščenje učinkovito za odstranjevanje modelnega endotoksina tekom biofarmacevtskega postopka. Introduction: Endotoxins are highly pyrogenic and immunogenic molecules derived from the cell wall of Gram-negative bacteria. Their detection, prevention of entry, and removal are crucial in the production of biopharmaceutical products to ensure drug safety and efficacy. Acceptable limits are strictly defined based on the drug's route of administration, with major sources of contamination including bacterial residues, water, materials, equipment, and biofilms. Purpose: In this master's thesis, we examined the impact of model endotoxin on the growth and viability of Chinese hamster ovary cells and the effectiveness of their removal through various chromatographic steps during the biopharmaceutical production of therapeutic proteins. The goal is to confirm the negative impact of higher endotoxin concentrations on cell proliferation and to demonstrate that multi-step chromatographic purification effectively ensures endotoxin levels remain below specification limits. Methods: The experiment was divided into two parts: (1) Monitoring the effect of different endotoxin concentrations on the proliferation and viability of Chinese hamster ovary cells and protein content. (2) Evaluating the efficiency of endotoxin removal across three chromatographic steps (affinity, multimodal, ion-exchange). Endotoxin levels in the obtained samples were determined using the Limulus Amebocyte Lysate test. Protein content was measured using UV spectrophotometry. The results on the impact of added endotoxin on cell growth and viability were statistically analyzed using ANOVA in JMP software. Results: Different endotoxin concentrations did not statistically significantly affect the growth and viability of Chinese hamster ovary cells (ANOVA, p < 0.005). Affinity chromatography (Protein A) reduced endotoxin below the detection limit (< 0.005 EU/mL) and achieved a logarithmic removal factor ≥ 6.2. Multimodal and ion-exchange chromatography further contributed to overall endotoxin removal, with a cumulative logarithmic removal factor of 9.4. The yields of all three chromatographic steps were consistent with process specifications, indicating that high endotoxin concentrations did not affect the evaluated process parameters. Discussion and conclusion: The model endotoxin did not statistically significantly affect the growth and viability of Chinese hamster ovary cells. Affinity, multimodal, and ion-exchange chromatography together achieved a high cumulative logarithmic removal factor, confirming that multi-step purification is effective for endotoxin removal during the biopharmaceutical process. magistrska dela sanitarno inženirstvo endotoksini terapevtski protein celice ovarijev kitajskega hrčka kromatografske stopnje odstranjevanje endotoksinov master's theses sanitary engineering endotoxins therapeutic protein Chinese hamster ovary cells chromatographic steps endotoxin removal true false false [U. Bobnar] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-09-26 07:46:09 2025-09-26 07:46:16 2025-11-16 03:58:34 0000-00-00 00:00:00 2025 0 Ljubljana 0 50 str., [11] str. pril. 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 152768 250774019 54539.pdf 54539.pdf 1 A3D491DA014F0E2F7AD52737C3B41C48 739271a6361c99177f0dfdbb0fc734a005871e64aa3aa3e5677085431ae5a8e4 ee58179a-9a9b-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=218757 Zdravstvena fakulteta 0 0 0