20.500.12556/DKUM-90648 Določevanje vpliva multivalentnih fosfatnih in sulfonatnih pomožnih snovi na agregacijo proteinov za uporabo v biofarmacevtskih izdelkih diplomsko delo univerzitetnega študijskega programa I. stopnje The effects of multivalent phosphate and sulphonate ions on protein aggregation for use in biopharmaceuticals V zadnjih desetletjih se je biofarmacevtska industrija osredotočila na razvoj bioloških zdravil. Večinoma gre za kompleksne beljakovinske molekule, ki so zahtevne in drage za izdelavo ter imajo omejen rok uporabe, ki je posledica nestabilnosti samih beljakovin. Na nestabilnost proteinov in agregacijo lahko vplivamo z dodatkom malih molekul in pomožnih snovi kot so površinsko aktivne snovi, pufri, sladkorji, soli itd., ki se na protein neposredno vežejo in ga tako stabilizirajo. Dosedanje raziskave so že preučile vpliv adenozin tripolifosfata in tripoli fosfata na agregacijo biofarmacevtskih proteinov, zato smo v diplomskem delu preučili še vpliv petih drugih že priznanih fosfatnih ionov in za primerjavo izbrali še deset ATP- ju podobnih sulfonatnih spojin, saj so polifosfati nagnjeni k hidrolizi in posledično niso primerni za uporabo kot pomožne snovi. Uporabili smo modelni protein lizocim kokošjega beljaka ter preverjali vpliv pomožnih snovi pri različnih koncentracijah na kinetiko agregacije proteina, ki smo jo spremljali z UV- Vis spektrometrijo. S pomočjo UV-Vis spektrometrije smo opravili tudi presejalni test pomožnih snovi na agregacijo proteina in izračunali delež monomernega proteina. Ugotovili smo, da fosfatne pomožne snovi in večina sulfonatnih učinkovito zaščitijo in stabilizirajo protein in ga tako zaščitijo pred agregacijo kot je ta podvržen temperaturnemu stresu ter ga zaščitijo pred agregacijo v daljšem časovnem obdobju, prav tako tudi zmanjšajo delež monomernega proteina v vzorcu. Preverili smo tudi vpliv pomožnih snovi na agregacijo delno razvitih beljakovin, kar smo dosegli z dodatkom ditiotreitola ali DTT, ki cepi disulfidne mostičke v beljakovini. V povprečju se je izkazalo, da dajejo boljše rezultate fosfatne pomožne snovi in ne sulfonatne. Najboljše rezultate sta med pomožnimi snovmi dala ATP in TPP, preostale pomožne snovi pa dale primerljive rezultate. In recent decades, the biopharmaceutical industry has focused on developing biological drugs. These are complex protein , nucleic acids or carbohydrate molecules. They are difficult and expensive to manufacture and have a limited shelf life, which is a result of the instability of the proteins themselves. Protein instability and aggregation can be influenced by adding small molecules and auxiliary substances, also called excipients,such as surfactants, buffers, sugars, salts, etc., which can directly interact with the protein and thus stabilize it. Previous research has already examined the influence of adenosine tripolyphosphate and tripolyphosphate on the aggregation of biopharmaceutical proteins. In the thesis, we also studied the influence of five other phosphate ions and selected ten ATP-like sulfonate compounds for comparison, since polyphosphates are prone to hydrolysis and consequently, they are not suitable for use as excipients. We used the model protein lysozyme of chicken egg white and checked the influence of excipients at different concentrations on the kinetics of protein aggregation, which was monitored by UV-Vis spectrometry. Utilizing UV-Vis spectrometry, we also performed a screening test of excipients against protein aggregation and calculated the fraction of monomeric protein. We have found that phosphate excipients and most sulfonate excipients effectively protect and stabilize the protein, shielding it from aggregation when subjected to temperature stress, and also prevent aggregation over a longer period of time. We also checked the influence of excipients on the aggregation of partially unfolded proteins, which was achieved by adding dithiothreitol or DTT, which cleaves disulfide bridges in the protein. On average, phosphate excipients have been shown to yield better results compared to sulfonate excipients. Among the excipients tested, ATP and TPP have shown the best results, while the other excipients yielded comparable outcomes. protein agregacija pomožne snovi fosfati sulfonati fazni diagram protein aggregation excipients phosphates sulfonates phase diagram true true false [T. Krepek] Slovenski jezik Angleški jezik Diplomsko delo/naloga 2024-09-12 14:43:05 2024-10-01 16:31:03 2025-01-24 03:12:41 0000-00-00 00:00:00 2024 0 Maribor 0 Maribor 1 spletni vir (1 datoteka PDF (VIII, 20 str.)) 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 544.77.052.2:577.112(043.2) 223561219 UN_Krepek_Tjasa_2024.pdf UN_Krepek_Tjasa_2024.pdf 1 6F8B56E93F2C77B2985DC6338F1A9E6C 9291d0401410d640bca93c093301c731da57f21b57ded14866c367218a996a4d 8908fdd4-7d0b-11ef-998b-00155d000105 https://dk.um.si/Dokument.php?lang=slv&id=185981 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0