Spremljanje razpada polisorbatov 20 in 80 v različnih placebo formulacijah s tekočinsko kromatografijo ter jedrsko magnetno resonanco

Košir, Pija (avtor), Pajk, Stane (mentor), Lebar, Blaž (komentor)

Polisorbati so najpogosteje uporabljene neionske površinsko aktivne snovi v farmacevtski industriji, saj pripomorejo k ohranjanju ustrezne kemijske in fizikalne stabilnosti terapevtskega proteina med dolgotrajnim shranjevanjem. Kljub temu so nagnjeni k lastni razgradnji preko hidrolize ali avtooksidacije. V okviru magistrske naloge smo želeli preko celovitega vrednotenja vpliva različnih dejavnikov natančneje okarakterizirati oba procesa ter njune razpadne produkte. S pomočjo tekočinske kromatografije visoke ločljivosti, sklopljene z detektorjem nabitih aerosolov, smo uspešno določili vsebnost in profil sestave polisorbatov 20 in 80 v različnih placebo formulacijah tekom študije pospešene stabilnosti. Razpad je bil najobsežnejši v pufru s histidinijevim kloridom, kjer je polisorbat 80 razpadel hitreje in v večjem obsegu kot polisorbat 20. Razpad, ki je potekel preko hidrolize estrske vezi, je bil odvisen od pH; višji kot je bil pH, krajši je bil razpolovni čas obeh polisorbatov, v polisorbatu 80 pa je bila krajša tudi faza zakasnitve razpada. Slednjemu smo nato dodali malatni protiion, ki je pri višjih koncentracijah uspešno stabiliziral polisorbat 80. Pri testiranju različnih primarnih ovojnin smo ugotovili, da do razpada polisorbata 80 v vialah SiO2 ni prišlo. Preučili smo tudi pufer z natrijevim acetatom, kjer je polisorbat 20 razpadel hitreje pri nižjem pH. Primerjava razpada v obeh uporabljenih pufrih je pokazala, da je ta potekel počasneje v pufru z natrijevim acetatom. Intaktna polisorbata sta bila v večji meri v obliki poliestrov. Ob izpostavitvi stresu so najprej razpadli poliestri, medtem ko se je relativni delež monoestrov spremenil šele, ko so poliestri skoraj povsem razpadli. Pri analizi sestavin in razpadnih produktov polisorbata 80 z jedrsko magnetno resonanco smo ugotovili, da je dvojna vez oleinske kisline razpadla hitreje in v večjem obsegu kot estrska. Argonova atmosfera je upočasnila razpad obeh vezi do neke mere, medtem ko je kisik izrazito pospešil razpad obeh vezi. Dobljeni rezultati predstavljajo obetavne rešitve za racionalno načrtovanje bodočih formulacij s terapevtskimi proteini. Predvsem je potrebno izpostaviti problematičnost razpada obeh polisorbatov v pufru s histidinijevim kloridom. Kljub temu pa lahko z določenimi dejavniki kot so nižji pH raztopine, dodatek malatnega protiiona v ustrezni koncentraciji in uporaba SiO2 vial za shranjevanje, stabiliziramo formulacije s polisorbati do določene mere.

• Jezik: Slovenski jezik
• Ključne besede: polisorbat 20, polisorbat 80, razgradnja, tekočinska kromatografija visoke ločljivosti, jedrska magnetna resonanca
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-142859
• Datum objave v RUL: 30.11.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Monitoring of polysorbate 20 and 80 degradation in placebo formulations by liquid chromatography and nuclear magnetic resonance

Izvleček

Polysorbates are the most commonly used nonionic surfactants in pharmaceutical industry, as they help to maintain the adequate chemical and physical stability of the therapeutic protein during long-term storage. However, they are prone to degradation through hydrolysis or autooxidation. In this master's thesis we wanted to more accurately characterize both processes and their degradation products through a comprehensive evaluation of the influence of various factors. Using high-resolution liquid chromatography coupled with a charged aerosol detector we determined the content and composition profile of polysorbates 20 and 80 in different placebo formulations that were subjected to accelerated stability testing. The degradation was the most extensive in histidinium chloride buffer, where polysorbate 80 degraded faster and to a greater extent than polysorbate 20. Degradation via ester bond hydrolysis was pH dependent; the higher the pH, the shorter the half-life of both polysorbates, and in polysorbate 80, the lag phase was also shorter. The malate counterion was then added to the latter, which at higher concentrations successfully stabilized polysorbate 80. When testing different primary packagings, we found that in SiO2 vials the degradation of polysorbate 80 did not occur. We also studied sodium acetate buffer where polysorbate 20 degraded faster at lower pH. When comparing both buffers we found that polysorbate 20 degraded slower in sodium acetate buffer. Intact polysorbates were mostly in the form of polyesters. Upon exposure to stress, these degraded first, while the relative proportion of monoesters changed only when the polyesters were almost completely degraded. Analysis of the components and degradation products of polysorbate 80 by nuclear magnetic resonance indicated that the double bond of oleic acid degraded faster and to a greater extent than the ester bond. The argon atmosphere slowed down the degradation of both bonds to some extent, while the oxygen distinctly accelerated it. The results show promising solutions for the rational design of the future biopharmaceuticals. It is necessary to point out the problematic nature of the degradation of polysorbates in the histidinium chloride buffer. However, with certain factors such as a lower pH of the solution, the addition of malate counterion in an adequate concentration and the use of SiO2 vials, formulations with polysorbates can be stabilized to a certain extent.

• Ključne besede: polysorbate 20, polysorbate 80, degradation, high resolution liquid chromatography, nuclear magnetic resonance