Vpliv ciklov zamrzovanja in odtaljevanja ter filtracije na kakovost vodnih raztopin polisorbatov 20 in 80

Javornik, Tjaša (author), Pajk, Stane (mentor), Lekić, Tinkara (comentor)

Polisorbati so neionske površinsko aktivne snovi, ki se v bioloških zdravilih uporabljajo kot stabilizatorji terapevtskih proteinov. Njihova najpogostejša predstavnika sta polisorbat 20 in polisorbat 80. Kompleksna sestava jim daje edinstvene lastnosti, hkrati pa otežuje njihovo analizo. Uporaba tekočinske kromatografije, sklopljene z detektorjem nabitih aerosolov nam omogoča kvantitativno in kvalitativno analizo. Razgradnja polisorbatov vodi do znižanja njihove koncentracije in nastanka stranskih produktov, oboje pa lahko ogrozi stabilnosti proteina v formulaciji. Dve glavni poti razgradnje sta hidroliza in oksidacija. Med razvojem zdravila je ključnega pomena, da v raziščemo vse dejavnike, ki lahko vplivajo na končni produkt, ki mora biti varno, kakovostno in učinkovito zdravilo. Dva dejavnika, s katerima se srečamo na različnih stopnjah procesa proizvodnje in imata vpliv na kakovost vodnih raztopin polisorbatov 20 in 80, sta ponovna zamrzovanja in odtaljevanja ter filtracija, kar smo tudi preučevali v magistrski nalogi. Ugotovili smo, da število ciklov zamrzovanja in odtaljevanja vodnih raztopin polisorbata 20 in 80 ne vpliva na njuni koncentraciji. Filtre, ki smo jih testirali, v biofarmacevtiki uporabljajo za sterilizacijo manjših serij produktov za potrebe različnih testiranj. Vezava polisorbatov na membrane filtrov je odvisna od materiala membrane in velikosti por. Zaradi proizvajalčevih modifikacij membran, je koristno preveriti vpliv filtracije na kvantitativno in kvalitativno sestavo polisorbata. Za zagotavljanje ustrezne vsebnosti je praksa, da se prvi mililiter filtrata zavrže. Tudi pri naših testiranjih smo ugotovili, da se na začetku filtracije spremeni koncentracija in kvalitativna sestava polisorbata. Po približno pol mililitra filtrata se koncentracija polisorbata ne spreminja več, kvalitativna sestava pa se približa sestavi nefiltrirane raztopine. Zaključimo lahko, da je sedanji protokol, ki zahteva, da se prvi mL filtrata zavrže ustrezen.

• Language: Slovenian
• Keywords: Polisorbat 20, Polisorbat 80, filtracija, zamrzovanje, HPLC-CAD, razpad, detekcija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-175802
• COBISS.SI-ID: 256816899
• Publication date in RUL: 08.11.2025

Secondary language

• Language: English
• Title: The influence of freeze-thaw cycles and filtration on the quality of aqueous solutions of polysorbate 20 and 80

Abstract

Polysorbates are non-ionic surfactants, widely employed in biopharmaceutical formulations as stabilizers of therapeutic proteins. Among them, polysorbate 20 and polysorbate 80 are the most commonly used. Their complex composition not only offers unique functional properties but also poses challenges for their analytical characterization. Liquid chromatography coupled with a charged aerosol detector provides an effective approach for both quantitative and qualitative analysis. However, polysorbate degradation leads to a reduction in their concentration and the formation of by-products, both of which may compromise protein stability within a formulation. The primary degradation pathways are hydrolysis and oxidation. During drug development, it is essential to identify and evaluate all factors that may influence the quality, safety, and efficacy of the final medicinal product. Two such factors, frequently encountered during manufacturing processes, are freeze–thaw cycles and filtration, which were systematically investigated in this master’s thesis. The results demonstrated that the number of freeze–thaw cycles applied to aqueous solutions of polysorbate 20 and 80 did not affect their concentrations. The filters that we tested are used in biopharmaceutical manufacturing for the sterilization of small product batches. The extent of polysorbate binding to filter membranes was shown to depend on both the membrane material and pore size. Given that membranes are often modified by manufacturers, it is advisable to experimentally assess the influence of filtration on the quantitative and qualitative composition of polysorbates. Current practice recommends discarding the first milliliter of filtrate to ensure consistent polysorbate content. Our findings confirmed that both concentration and composition were altered at the beginning of filtration. After approximately 0.5 mL of filtrate, concentrations stabilized and the qualitative profile closely resembled that of the unfiltered solution. These results support the suitability of the current protocol requiring the initial milliliter of filtrate to be discarded.

• Keywords: Polysorbate 20, Polysorbate 80, filtration, freeze-thaw cycles, HPLC-CAD, degradation, detection