Assessing the polysorbate degradation fingerprints and kinetics of lipases - how the activity of polysorbate degrading hydrolases is influenced by the assay and assay conditions

Abstract

Two of the most widely used surfactants to stabilize biologicals against e.g. interfacial stresses are polysorbate 20 (PS20) and polysorbate 80 (PS80). In recent years, numerous cases of hydrolytic polysorbate (PS) degradation in liquid formulations of biopharmaceuticals have been observed. Concomitant with the degradation of PSs, formulated proteins become inherently instable and more susceptible to aggregation. Furthermore, poorly soluble fatty acids (FA) are released from the PSs, which might lead to FA precipitation and the formation of visible and subvisible particles. Therefore, possible particle inducing factors have to be monitored closely. The major root cause of hydrolytic PS degradation in biologicals is the presence of enzymatic active host cell proteins (HCP), like lipases and esterases, which are co-purified with the active pharmaceutical ingredient. Such contaminants can be detected via their hydrolytic activity, either using ester-based substrates or PS itself. However, each approach has its up- and downsides, which makes the comparison of the results from other publications difficult. It was therefore the aim of the present study to investigate the impact of lipase specificities on the assay readouts. This study evaluates three different surrogate (model) lipases with distinctively different degradation kinetics and substrate specificities using specific analytical methods. The analytical panel contains on one hand two lipase activity assays with ester-based substrates, either detecting the release of para-nitrophenol or 4-methylumbelliferone, and on the other hand two PS-based monitoring analyses (fluorescence micelle assay and reverse phase high performance liquid chromatography - charged aerosol detection), which detect hydrolytic "activity" directly in the target substrate. Thereby, strengths and weaknesses of each assay are discussed, and recommendations are made for the respective use cases. Our results show that the determined lipase activities vary not only from assay to assay, but also significantly for the lipase tested, thus showing a different degradation fingerprint in the RP-HPLC-CAD chromatogram. This demonstrates that a comprehensive monitoring approach is essential to assess potential HCP contaminations.

Keywords: CAD; Degradation kinetics; FMA; Free fatty acids; HCPs; Hydrolysis; Lipase; Polysorbate; Protein formulation; Tween®.