During biopharmaceuticals development programme the main challeng is to develop safe, effective and high-quality drug product that have as long shelf-life as possible. The vast majority of biopharmaceuticals on the market are therapeutic proteins, and in contrast to conventional pharmaceuticals, these formulations are less stable. They have complex degradation pathways, mainly due to the large sizes and diverse structures of these proteins. To protect them from interfacial stress, and to counter their aggregation and denaturation in aqueous solutions, surfactants are added to these formulations. Polysorbates are the most commonly used surfactants in the pharmaceutical industry, and they are included in more than 80% of commercial monoclonal antibody formulations. As part of my doctoral project, polysorbates were comprehensively evaluated as stabilisers of biopharmaceuticals. Ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry was developed and optimised for quality control of input polysorbates, identification of individual polysorbate species, comparisons between different polysorbate qualities across different sources, identification of polysorbate impurities, and monitoring the formation of polysorbate degradation products. Free fatty acids are important indicators of the quality of intact polysorbates and their degradation, which is one of the main reasons for particle formation in protein formulations. As the only current pharmacopoeia method for evaluation of free fatty acids in polysorbates is a non-specific method that determines the acid value, a rapid and simple nuclear magnetic resonance method for quantitative evaluation of free fatty acids in polysorbates was designed and set up. The effects of formulation composition on the stability of polysorbates were also evaluated. The stabilities of polysorbates in the buffers most commonly used in biopharmaceuticals were determined and the influence of proteins and other excipients on the degradation of polysorbates was investigated. While polysorbates can undergo hydrolysis and oxidation, the mechanisms for polysorbates degradation in a histidine chloride buffer were further investigated. According to the polysorbates degradation products, a new degradation pathway of polysorbates was defined: aminolysis. Histidine buffer systems with different counterions were investigated and counterions which prevented histidine catalyzed hydrolysis of polysorbates were identified. The degradation mechanism of polysorbates in histidine buffer was also confirmed by quantum mechanics calculations. The addition of chelators to formulations with polysorbates and therapeutic proteins was examined. The use of an ascorbate redox system assay and peptide mapping demonstrated that in combination with metal ions, oxygen and ascorbate, some chelators can even accelerate the protein oxidation, instead of preventing it. Therefore, inclusion of chelators in such pharmaceutical formulations should be based on experimental data, and not on »common practice«. Based on the polysorbates stability data and their degradation pathways, and based on the influence of other excipients on polysorbates degradation in such formulations, their design can now follow a more rational approach.
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