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Roles of Acid Ceramidase and Its Cofactor in Biotherapeutics
Sisi Zhang1, Christopher Sevinsky1, Jared Watson1
1Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591-6706, United States.
Molecular Pharmaceutics
|December 9, 2025
Summary
Activated acid ceramidase degrades polysorbates (PSs), causing instability in protein drugs. Effective purification methods, like column regeneration and UF/DF filtration, prevent this degradation.
Area of Science:
- Biochemistry
- Pharmaceutical Sciences
- Protein Therapeutics
Background:
- Polysorbates (PSs) are critical excipients in biopharmaceutical formulations.
- Lipase and esterase activity can degrade PSs, leading to protein drug instability and particle formation.
- Acid ceramidase was not previously recognized as a PS-degrading enzyme.
Purpose of the Study:
- To investigate the potential of acid ceramidase to degrade polysorbates.
- To identify factors that activate acid ceramidase and influence its PS-degrading activity.
- To determine methods for preventing PS degradation in protein drug purification.
Main Methods:
- Analysis of acid ceramidase activity on aged hydrophobic interaction chromatography (HIC) columns.
- Assessment of saposin D's effect on acid ceramidase activity.
- Evaluation of Ultrafiltration/Diafiltration (UF/DF) for saposin D removal.
Main Results:
- Acid ceramidase can be activated on aged HIC columns, enabling polysorbate degradation.
- Saposin D enhances the lipase activity of activated acid ceramidase, accelerating PS degradation.
- Effective HIC column regeneration prevents acid ceramidase activation; UF/DF removes saposin D.
Conclusions:
- Activated acid ceramidase, particularly with saposin D, poses a risk to polysorbate stability during purification.
- Standard purification protocols involving effective column regeneration and UF/DF filtration mitigate the risk of PS degradation.
- The findings suggest that polysorbate degradation by activated acid ceramidase is unlikely in products purified under current guidelines.
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