Abstract: Polysorbate 20 is a non-ionic surfactant commonly used in the formulation of therapeutic monoclonal antibodies to prevent proteins denaturation and aggregation. It is critical to understand the molecular heterogeneity and stability of polysorbate 20 in protein formulations as polysorbate can gradually degrade in aqueous solution over time by multiple pathways losing surfactant functions 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 protein formulation sample matrix is first reported using 2DLC coupled with charged aerosol detection (CAD) and MS detection. A mixed-mode column that has both anion exchange and reversed-phase properties was used in the 1st dimension to separate protein and polysorbate in the formulation sample, while polysorbate 20 esters were trapped on-line and then analyzed using an RP-UHPLC column in the 2nd dimension to further separate the ester species. The MS served as the 3rd dimension to further resovle as well as to identify the polysorbate ester sub-species. Another 2DLC method using a cation exchange column in the 1st dimension and the same RP-UHPLC method in the 2nd 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 sub-species in real time stability samples of a protein formulation.Summary: We characterized polysorbate 20 molecular heterogeneity and stability in the presence of protein formulation sample matrix using two 2D-UHPLC methods coupled with CAD and MS detector. One method to analyze the change of esters subspecies in polysorbate 20, and another method to monitor the change of degradation products. References:
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