Degradation of Polysorbate 20 by Sialate O-Acetylesterase in Monoclonal Antibody Formulations.

Polysorbates (PS) are surfactants commonly added in biologics formulations that can protect proteins from denaturation and aggregation. However, decreases in polysorbate 20 (PS20) content have been observed in some monoclonal antibody formulations, causing the formation of visible and/or subvisible particles that ultimately compromise the quality and stability of the therapeutic protein products. It was determined that the particles are mainly composed of free fatty acid, suggesting enzymatic hydrolysis of PS is responsible for the degradation of PS. Enrichment of host cell proteins (HCPs) by immunoprecipitation followed by shotgun proteomics have been utilized to identify the HCPs that can hydrolyze PS20. One HCP, sialate O-acetylesterase (SIAE), demonstrated strong enzymatic activity for PS20 degradation even at low concentration (<5 ppm level). Incubation of recombinant SIAE with PS20 resulted in a unique degradation pattern where the hydrolysis of monoester with short fatty acid chain (C12, C14) was observed but not the monoester with long fatty acid chain (C16, C18) or higher-order esters. SIAE was detected and quantitated in several formulated mAbs, and the amount of SIAE was positively correlated to PS20 degradation in these mAbs during incubation. Additional experiments also showed that when SIAE was depleted, PS20 degradation was diminished, suggesting a causality between SIAE and PS20 degradation. The lipase activity of SIAE is specific to PS20, but not to PS 80 (PS80), which contains monoesters with long chain fatty acid (C18) and higher-order esters. The specific esterase activity of SIAE on PS20 suggests a possible solution of using PS80 over PS20 to eliminate surfactant degradation in mAb products.