Specific polysorbate fingerprints of CHO hydrolases and implications for indirect assays.

Polysorbate degradation in biopharmaceutical formulations can impact the stability and efficacy of therapeutic proteins. This degradation is predominantly caused by specific residual host cell hydrolases present at sub-ppm concentrations. Their low abundance, combined with the lack of sensitive, enzyme specific detection methods, is a major analytical challenge. This study presents a novel approach using reverse-phase ultra-performance liquid chromatography coupled with mass spectrometry (RP-UPLC-MS) to systematically analyse the specific polysorbate degradation patterns of hydrolases expressed by Chinese hamster ovary (CHO) cells. Our findings reveal distinct degradation fingerprints of five CHO-derived hydrolases, highlighting their unique preferences for different polysorbate species based on ester linkages (e.g. monoester or multiester), hydrophilic head groups and FA chain length. This study is the first of its kind to provide such detailed insights into the enzymatic cleavage patterns of polysorbates using enzymes directly derived from CHO cells. Our results underscore the limitations of indirect polysorbate quantification assays, such as the fluorescence micelle assay (FMA) and 4-methylumbelliferone (MU4)-based hydrolytic activity assays, in accurately reflecting the activity of specific hydrolases. For instance, the FMA tends to overestimate the contribution of certain polysorbate species due to its reliance on micelle formation, while the MU4 assay's predictive reliability is limited by its use of surrogate substrates. In contrast, RP-UPLC-MS enables the precise identification of individual polysorbate species and their degradation products, providing a direct measure of hydrolase activity. The detailed enzymatic fingerprints obtained in this study pave the way for future advancements in enzyme classification and the potential application of computational tools for automated hydrolase identification.