Polysorbates are the most commonly used nonionic surfactants in pharmaceutical industry, as they help to maintain the adequate chemical and physical stability of the therapeutic protein during long-term storage. However, they are prone to degradation through hydrolysis or autooxidation. In this master's thesis we wanted to more accurately characterize both processes and their degradation products through a comprehensive evaluation of the influence of various factors.
Using high-resolution liquid chromatography coupled with a charged aerosol detector we determined the content and composition profile of polysorbates 20 and 80 in different placebo formulations that were subjected to accelerated stability testing. The degradation was the most extensive in histidinium chloride buffer, where polysorbate 80 degraded faster and to a greater extent than polysorbate 20. Degradation via ester bond hydrolysis was pH dependent; the higher the pH, the shorter the half-life of both polysorbates, and in polysorbate 80, the lag phase was also shorter. The malate counterion was then added to the latter, which at higher concentrations successfully stabilized polysorbate 80. When testing different primary packagings, we found that in SiO2 vials the degradation of polysorbate 80 did not occur. We also studied sodium acetate buffer where polysorbate 20 degraded faster at lower pH. When comparing both buffers we found that polysorbate 20 degraded slower in sodium acetate buffer. Intact polysorbates were mostly in the form of polyesters. Upon exposure to stress, these degraded first, while the relative proportion of monoesters changed only when the polyesters were almost completely degraded.
Analysis of the components and degradation products of polysorbate 80 by nuclear magnetic resonance indicated that the double bond of oleic acid degraded faster and to a greater extent than the ester bond. The argon atmosphere slowed down the degradation of both bonds to some extent, while the oxygen distinctly accelerated it.
The results show promising solutions for the rational design of the future biopharmaceuticals. It is necessary to point out the problematic nature of the degradation of polysorbates in the histidinium chloride buffer. However, with certain factors such as a lower pH of the solution, the addition of malate counterion in an adequate concentration and the use of SiO2 vials, formulations with polysorbates can be stabilized to a certain extent.
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