Surfactants are excipients used in biological formulations. Their main tasks are to stabilize and protect the protein from aggregation and denaturation due to interfacial stresses that occur during the production process, but also prevent protein adsorption and subsequent protein loss on surfaces such as filters, tubes, primary containers and sets for intravenous administration. The most frequently represented surfactants in biological formulations are polysorbate 20 and polysorbate 80. In recent years, the pharmaceutical industry has faced major problems due to their heterogeneity and instability in liquid protein formulations, which leads to a decrease in their concentration and, consequently, protective functions on the protein and the direct impact of degradation products on the stability and quality of the drug. The main degradation pathways of polysorbates are auto-oxidation and hydrolysis, whereby hydrolysis can be of chemical or enzymatic origin. It was the instability of polysorbates that led to the research of possible alternative surfactants that could potentially be used in protein formulations. With this aim, we selected 7 commercially available alternative surfactants (Brij-58, TPGS-1000, Poloxamer 188, Kolliphor ELP, SPGS-550-M, polyoxy-32 stearate and poly (ethylene glycol) monooleate) within the master's thesis, which had not yet been used as an excipient in marketed biological medicines. In the first part of the task, we exposed alternative surfactants to 4 different buffers, with two different pH values, which are most often used in parenteral products, and exposed them to elevated temperature for one and two months. In the second part, we exposed alternative surfactants to hydrogen peroxide and iron (III) chloride, with which we wanted to find out whether the oxidation of surfactants occurs and what their breakdown products are. We also wanted to stimulate acid and basic hydrolysis with sodium hydroxide, hydrochloric acid and buffer with pH 11, and analyse the degradation products. Surfactants were analysed using HPLC methods developed for polysorbates, while we also evaluated the appropriate individual methods for determining the concentration and purity of each one. They were also analysed by LC-MS. PS80 was used for comparison in all experiments. In the end, we compared each surfactant with PS80 and found out which of the 7 selected shows the greatest stability in terms of oxidation and hydrolysis.
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