Characterization and stability study of polysorbate 20 in therapeutic monoclonal antibody formulation by multidimensional ultrahigh-performance liquid chromatography-charged aerosol detection-mass spectrometry.

ABSTRACT

Polysorbate 20 is a nonionic surfactant commonly used in the formulation of therapeutic monoclonal antibodies (mAb) to prevent protein denaturation and aggregation. It is critical to understand the molecular heterogeneity and stability of polysorbate 20 in mAb formulations as polysorbate can gradually degrade in aqueous solution over time by multiple pathways losing surfactant functions and leading to protein aggregation. The molecular heterogeneity of polysorbate and the interference from proteins and the excipient in the formulation matrix make it a challenge to study polysorbate in protein formulations. In this work, the characterization and stability study of polysorbate 20 in the presence of mAb formulation sample matrix is first reported using two-dimensional liquid chromatography (2DLC) coupled with charged aerosol detection (CAD) and mass spectrometry (MS) detection. A mixed-mode column that has both anion-exchange and reversed-phase properties was used in the first dimension to separate protein and polysorbate in the formulation sample, while polysorbate 20 esters were trapped online and then analyzed using an reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) column in the second dimension to further separate the ester species. The MS served as the third dimension to further resolve as well as to identify the polysorbate ester subspecies. Another 2DLC method using a cation-exchange column in the first dimension and the same RP-UHPLC method in the second dimension was developed to analyze the degradation products of polysorbate 20. Stability samples of a protein drug product were studied using these two 2DLC-CAD-MS methods to separate, identify, and quantify the multiple ester species in polysorbate 20 and also to monitor the change of their corresponding degradants. We found different polysorbate esters degrade at different rates, and importantly, the degradation rates for some esters are different in the protein formulation compared to a placebo that has no protein. The multidimensional UHPLC-CAD-MS approach provides insights into the heterogeneous stability behaviors of polysorbate 20 subspecies in real-time stability samples of a mAb formulation.