Polysorbates are among the most commonly used surfactants in biopharmaceuticals, where they prevent protein aggregation and denaturation. The most widely used types are polysorbate 20 and 80. In addition to ensuring protein stability, polysorbate stability is also important, as their degradation can result in loss of functionality and increased immunogenicity. The main degradation pathways are hydrolysis and oxidation.The purpose of this master thesis was to identify markers of oxidative degradation of polysorbate 80. A sample solution of polysorbate 80 was prepared and exposed to an atmosphere of argon, oxygen, and air, and samples were taken weekly over five weeks. Using liquid chromatography coupled with a CAD detector, we found that the greatest decrease in polysorbate content occurred in the argon atmosphere, while the smallest decrease was observed in the air. These results contradicted expectations, as the highest degradation was anticipated in oxygen and the lowest in argon. Mass spectrometry analyses confirmed the same degradation trends across all conditions. We identified 34 compounds from the chromatogram and monitored their relative abundance. With this wide range of compounds, we confirmed the heterogeneity of the polysorbate mixture. We confirmed the presence of mono- and diesters of polyoxyethylene chains and polyoxyethylated isosorbide, as well as mono-, di-, tri-, and tetraesters of polyoxyethylated sorbitan. Due to a broad signal, tetraesters were not included in further monitoring. The mentioned compounds were esterified with oleic, linoleic, palmitic, or stearic acid, and could also be present in unesterified form. As expected, the concentration of unesterified compounds increased, while the concentration of esterified compounds decreased. In the argon atmosphere, in addition to oxidation, hydrolysis likely occurred as well, since there was a complete breakdown of all monoesters. We found that esters with linoleic acid degraded the fastest and to the greatest extent, which we attribute to the presence of two double bonds. Monoesters with stearic acid showed the least degradation, while oleic and palmitic acids behaved similarly. We also showed that monoesters were more prone to degradation than di- and triesters, with no substantial differences between the latter two. Additionally, no major differences were observed among diesters with individual fatty acids, except for those with linoleic acid. Expected AAPH-induced oxidation did not occur, likely due to insufficient AAPH.
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