Identifikacija zgodnjih označevalcev oksidacije polisorbata 20 s tekočinsko kromatografijo, sklopljeno z masno spektrometrijo

Dražil, Lucija (author), Pajk, Stane (mentor), Lekić, Tinkara (comentor)

Polisorbati so površinsko aktivne snovi, ki se pogosto uporabljajo v formulacijah terapevtskih proteinov za preprečevanje nastajanja agregatov, denaturacije in adsorpcije proteinov na različne površine. Kemično so polisorbati estri višjih maščobnih kislin in polioksietilen sorbitana ali izosorbida. V bioloških zdravilih se najpogosteje uporabljata polisorbata 20 (PS20) in 80 (PS80), ki se razlikujeta po sestavi maščobnih kislin. PS20 vsebuje več nasičenih maščobnih kislin, predvsem lavrinsko kislino, medtem ko PS80 vsebuje več nenasičenih maščobnih kislin, predvsem oleinsko kislino. Polisorbati so občutljivi na hidrolizo in oksidacijo, kar lahko vpliva na kakovost bioloških zdravil. Namen naloge je bil preverjanje stopnje oksidativnega razpada polisorbata 20 v različnih atmosferah in določitev potencialnih označevalcev oksidativnega razpada. V ta namen smo pripravili vodne raztopine, ki smo jih prepihovali s kisikom, zrakom ali argonom. Raztopine smo pri sobni temperaturi spremljali pet tednov, pri čemer smo tedensko odvzeli vzorce. Pričakovali smo največji upad koncentracije PS20 v prisotnosti kisika, nekoliko manjši v prisotnosti zraka, ter zanemarljiv upad v prisotnosti argona. Rezultati so potrdili pričakovanja: največji upad koncentracije PS20 (55,8 % začetne koncentracije) smo zabeležili v raztopini, prepihani s kisikom, medtem ko je bil upad v raztopinah z zrakom in argonom manj kot 1 %. Vzorci so bili nadalje analizirani s tekočinsko kromatografijo sklopljeno z masno spektrometrijo (LC-MS). Prav tako smo poskušali oksidirati PS20 z AAPH, vendar je bil obseg oksidacije, v nasprotju s pričakovanji, zelo majhen, zato vzorcev nismo analizirali z LC-MS. Analiza je pokazala povečanje nezaestrenih polioksietilen sorbitanov, polioksietilen izosorbidov in verig polioksietilena, medtem ko se je koncentracija zaestrenih spojin znižala, kar je posledica cepitve estrskih vezi in/ali polietilenglikolne verige. Zanimivo je, da so se spremembe v vzorcu, prepihanem s kisikom, pojavile z zamikom, saj so bile izrazite šele po tretjem tednu. Za najboljši kazalnik razpada so se izkazale nezaestrene spojine, saj je njihov relativni delež bolj narastel v primerjavi z znižanjem zaestrenih spojin. Verjetno je razlog v tem, da lahko več različnih komponent razpada do istih produktov.

• Language: Slovenian
• Keywords: polisorbat 20, razgradnja, tekočinska kromatografija, masna spektrometrija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-164438
• Publication date in RUL: 25.10.2024

Secondary language

• Language: English
• Title: Identifying early polysorbate 20 oxidation markers by liquid chromatography coupled with mass spectrometry

Abstract

Polysorbates are surfactants that are widely used in therapeutic protein formulations to prevent the formation of aggregates, denaturation and adsorption of proteins to various surfaces. Chemically, polysorbates are esters of higher fatty acids and polyoxyethylene sorbitan or isosorbide. Polysorbate 20 (PS20) and 80 (PS80), which differ in the composition of fatty acids, are most often used in biological medicines. PS20 contains more saturated fatty acids, mainly lauric acid, while PS80 contains more unsaturated fatty acids, mainly oleic acid. Polysorbates are sensitive to hydrolysis and oxidation, which can affect the quality of biologics. In our master's thesis, we focused on the oxidative degradation of PS20 and identify potential markers of oxidative degradation. For this purpose, we prepared aqueous solutions of polysorbate 20, which were blown with oxygen, air or argon. The solutions were monitored at room temperature for five weeks, with weekly samples taken. We expected the greatest decrease in PS20 concentration in the presence of oxygen, a slightly smaller decrease in the presence of air, and a negligible decrease in the presence of argon. The results confirmed expectations: the largest decrease in PS20 concentration (55.8% of the initial concentration) was recorded in the oxygen-purged solution, while the decrease in air and argon solutions was less than 1%. The samples were further analyzed by liquid chromatography coupled to mass spectrometry (LC-MS). We also attempted to oxidize PS20 with AAPH, but the extent of oxidation was, contrary to expectations, very small, so the samples were not analyzed by LC-MS. The analysis showed an increase in non-esterified polyoxyethylene sorbitans, polyoxyethylene isosorbides and polyoxyethylene chains, while the concentration of esterified compounds decreased, which is the result of cleavage of ester bonds and/or polyethylene glycol chain. It is interesting that the changes in the oxygen-purged sample appeared with a delay, being pronounced only after the third week. Non-esterified compounds proved to be the best degradation indicator, as their relative share increased more compared to the decrease of esterified compounds. The reason is probably that several different components can break down to the same products.

• Keywords: polysorbate 20, degradation, liquid chromatography, mass spectrometry