Vrednotenje hidrolize polisorbatov z lipazami in esterazami

Orehek, Meta (avtor), Mravljak, Janez (mentor), Lekić, Tinkara (komentor)

Zdravila sestavljajo različne pomožne snovi od površinsko aktivnih snovi, antioksidantov, ojačevalcev okusa in tako naprej. Med površinsko aktivnimi snovmi so najpogosteje uporabljeni polisorbati. To so ne-ionske površinsko aktivne snovi, ki pripomorejo k ohranjanju ustrezne kemijske in fizikalne stabilnosti terapevtskega proteina med dolgotrajnim shranjevanjem. Nagnjeni so k lastni razgradnji preko hidrolize ali avtooksidacije. V okviru magistrske naloge smo želeli ovrednotiti razgradnjo polisorbata, na katero imajo vpliv gostujoči celični proteini (HCP), ki jih dobimo medčiščenjem terapevtskega proteina. V ta namen smo polisorbat izpostavili delovanju različnih encimov. 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, v katerih so bile dodane esteraze, lipaze in proteaze in jih izpostavili stresnim pogojem, da so bili encimi v temperaturnem območju, kjer so najbolj aktivni in tako pospešili razgradnjo polisorbata. Encimi so različno vplivali na polisorbata 20 in 80. Polisorbat 20 je najhitreje razgrajevala zajčja jetrna esteraza, polisorbat 80 pa lipaza iz kandide. Razgradnjo smo primerjali z bazično razgradnjo polisorbatov. Primerjali smo tudi profile polisorbata 20, pri razgradnji z encimoma, ki sta ga najbolj razgrajevala (zajčja jetrna esteraza in lipaza kandide) in ugotovili, da pri razgradnji z esterazo nastane več nezaestrenih zvrsti. Najpočasneje sta se polisorbata 20 in 80 razgrajevala s prašičjimi encimi, in sicer polisorbat 20 z jetrno esterazo, polisorbat 80 pa z lipazo iz slinavke. Dobljeni rezultati nakazujejo, da moramo pri formulacijah, ki jih sestavljamo s polisorbati, paziti na prisotnost encimov in poleg ovrednotenja vpliva encima na zdravilno učinkovino upoštevati tudi vpliv encima na polisorbat. Posredno je treba upoštevati tudi, kako razpadli produkti polisorbata vplivajo na zdravilno učinkovino.

• Jezik: Slovenski jezik
• Ključne besede: polisorbat 20, polisorbat 80, razgradnja, tekočinska kromatografija visoke ločljivosti, encimi
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-166721
• Datum objave v RUL: 23.01.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Evaluation of polysorbates hydrolysis by lipases and esterases

Izvleček

Medicines consist of various excipients from surfactants, antioxidants, flavour enhancers and so on. Among surfactants, polysorbates are the most used. These are non-ionic surfactants that help to maintain adequate chemical and physical stability of the therapeutic protein during long-term storage. They are prone to self-degradation through hydrolysis or auto-oxidation. As part of the master’s thesis, we wanted to evaluate the degradation of polysorbate, which is affected by guest cell proteins obtained during protein purification. For this purpose, polysorbate was exposed to the action of various enzymes. Using high-resolution liquid chromatography coupled with a charged aerosol detector, we successfully determined the content and composition profile of polysorbates 20 and 80 in various placebo formulations to which esterases, lipases and proteases were added and subjected to stress conditions that the enzymes were in the temperature range where they were most active and at the same time accelerated the degradation of polysorbate. Enzymes have different effects on polysorbates 20 and 80. Polysorbate 20 was broken down most rapidly by rabbit liver esterase and polysorbate 80 by candide lipase. The degradation was compared to the basic degradation of polysorbates. We also compared the profiles of polysorbate 20 when degraded by the enzymes that most degraded it (rabbit liver esterase and candide lipase) and found that several non-esterified species were formed when degraded by esterase. Polysorbates 20 and 80 were broken down most slowly by porcine enzymes, with polysorbate 20 being liver esterase while polysorbate 80 being salivary lipase. The results obtained indicate that in formulations consisting of polysorbates, attention should be paid to the presence of enzymes and, in addition to evaluating the effect of the enzyme on the active substance, should also be paid to the presence of enzymes and, in addition to evaluating the effect of the enzyme on the active substance, the effect of the enzyme on the polysorbate should also be taken into account. Indirect consideration should also be given to the effect of enzyme-mediated polysorbate products on the active substance.

• Ključne besede: polysorbate 20, polysorbate 80, degradation, high-performance liquid chromatography, enzymes