Spremljanje produktov oksidacije polisorbata 80 in 20 v modelnih formulacijah s tekočinsko kromatografijo, sklopljeno z masno spektrometrijo

Zavadlav, Veronika (avtor), Pajk, Stane (mentor), Lekić, Tinkara (komentor)

Polisorbati predstavljajo eno najpogosteje uporabljenih skupin neionskih površinsko aktivnih snovi v formulacijah bioloških zdravilnih učinkovin. Njihova struktura vsebuje sorbitansko jedro s pripetimi polioksietilenskimi enotami in zaestrenimi dolgoverižnimi maščobnimi kislinami – lavrinska kislina v primeru polisorbata 20 in oleinska kislina pri polisorbatu 80. Razgradnja polisorbatov poteka prek več mehanizmov, najpogosteje hidrolize in oksidacije. Razpad polisorbatov v formulaciji pomeni izgubo površinske aktivnosti, ki preprečuje agregacijo in obarjanje proteinov. Dodatne težave pa povzročajo produkti razpada, kot so proste maščobne kisline, aldehidi, ketoni in peroksidi. Ti lahko tvorijo delce ali reagirajo z aminokislinami na površini proteinov, kar lahko vodi do imunskega odziva na učinkovino. Namen magistrske naloge je bilo spremljanje in določitev produktov oksidacije polisorbata 80 in 20 v modelnih formulacijah, ki smo jih izpostavili kisiku, argonu in zraku v časovnem okvirju petih tednov. Koncentracije v vzorcih s polisorbatoma smo določali s tekočinsko kromatografijo, sklopljeno z detektorjem nabitih aerosolov in s pomočjo tekočinske kromatografije, sklopljene z masnim spektrometrom. Skušali smo tudi sprožiti oksidacijo z dodatkom 2,2'-azobis(2-amidinopropan) diklorida (AAPH), vendar nismo dobili pričakovanih rezultatov, saj nismo uspeli v zadostni meri sprožiti oksidacije. Pri polisorbatu 80 smo po pričakovanjih dobili večjo raznolikost razgradnih zvrsti spojin. Največji upad deležev obeh polisorbatov je bil v kisikovi atmosferi, kjer je bil obseg oksidacije najbolj izrazit. Pri polisorbatu 20 je bila razgradnja najmanj izrazita v atmosferi z argonom, medtem ko je pri polisorbatu 80 atmosfera z zrakom pokazala najmanjši obseg razgradnje. Rezultati meritev so nam pokazali širok obseg razgradnje polisorbata 80 v atmosferi argona, kjer gre za kombinacijo procesov oksidacije in hidrolize. Med 63 dobljenimi spojinami smo prepoznali nekaj obetavnih označevalcev razgradnje polisorbata 20 in 80.

• Jezik: Slovenski jezik
• Ključne besede: Polisorbat 20, Polisorbat 80, oksidacija, tekočinska kromatografija, masna spektrometrija
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-174232
• Datum objave v RUL: 30.09.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Tracking oxidation products of polysorbate 80 and 20 in model formulations using liquid chromatography coupled with mass spectrometry

Izvleček

Polysorbates represent one of the most commonly used groups of nonionic surfactants in formulations of biologic drug substances. Their structure consists of a sorbitan core with attached polyoxyethylene units and esterified long-chain fatty acids - lauric acid in the case of polysorbate 20 and oleic acid in polysorbate 80. The degradation of polysorbates occurs through multiple mechanisms, most commonly hydrolysis and oxidation. The breakdown of polysorbates in a formulation results in the loss of surface activity, which is essential for preventing protein aggregation and precipitation. Additional complications arise from degradation products such as free fatty acids, aldehydes, ketones, and peroxides. These can form particles or react with amino acids on the surface of proteins, potentially triggering an immune response to the active substance. The aim of the master’s thesis was to monitor and identify the oxidation products of polysorbate 80 and 20 in model formulations, which were exposed to oxygen, argon, and air over a five-week period. The concentrations in the polysorbate-containing samples were determined using liquid chromatography coupled with a charged aerosol detector and liquid chromatography combined with mass spectrometry. We attempted to induce oxidation by adding 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), but the expected results were not achieved, as the oxidation was not sufficienlty triggered. As expected, polysorbate 80 exhibited a greater diversity of degradation species. The most significant decrease in both polysorbates occurred under the oxygen atmosphere, where the extent of oxidation was most pronounced. For polysorbate 20, degradation was least evident in the argon atmosphere, whereas for polysorbate 80, the air atmosphere showed the lowest degree of degradation. Measurements results revealed a broad range of degradation for polysorbate 80 in the argon atmosphere, indicating a combination of oxidation and hydrolysis processes. Among the 63 identified compounds, we recognized several promising markers of degradation for both polysorbate 20 and 80 degradation.

• Ključne besede: Polysorbate 20, Polysorbate 80, oxidation, liquid chromatography, mass spectrometry