Zhang_2025_Mol.Pharm__

Polysorbates (PSs) are frequently used as excipients in protein drug formulations. However, recent findings suggest that lipases and esterases can degrade PSs even at very low concentrations, thereby resulting in biotherapeutic instability and particle formation during stability assessments. Acid ceramidase, a lipase frequently present in drug formulations, was previously believed not to be a PS degrading enzyme. ( J. Pharm. Sci. 2024, 114, 1002-1009. J. Pharm. Sci. 2023, 112, (5), 1351-1363.) However, our study shows that acid ceramidase can be activated during purification process on an aged hydrophobic interaction chromatography column that subjected to multiple use cycles without effective regeneration procedures. After activation, low-abundance acid ceramidase degrades PS. Furthermore, the enzyme saposin D increases the lipase activity of activated acid ceramidase, thus accelerating PS degradation. Our study also demonstrated that effective regeneration of the HIC column can prevent acid ceramidase activation, and the cofactor saposin D can be eliminated by Ultrafiltration/Diafiltration (UF/DF) filtration through a 50 kDa membrane. Consequently, the rapid PS degradation by activated acid ceramidase and its cofactor observed herein is less likely to occur in drug products purified according to standard protocols and guidelines.