High-Throughput, Fluorescence-Based Esterase Activity Assay for Assessing Polysorbate Degradation Risk during Biopharmaceutical Development.
Pharm Res
; 38(3): 397-413, 2021 Mar.
Article
en En
| MEDLINE
| ID: mdl-33655394
ABSTRACT
PURPOSE:
Hydrolytic degradation of polysorbate during 2-8°C storage of monoclonal antibody drug products has been attributed to residual enzymes (e.g., esterases) from bioprocessing steps. Robust detection of esterase activity using sensitive, non-polysorbate surrogate substrates can provide an alternate method to assess polysorbate degradation risk, if the correlation between the esterase activity and polysorbate degradation is established.METHODS:
A general esterase activity assay was developed as a monitoring and characterization tool during bioprocess development of monoclonal antibodies.RESULTS:
We report a fluorescence plate-based assay for quantifying esterase activity, utilizing 4-methylumbelliferyl caprylate (MU-C8) as the esterase substrate. The assay was first assessed for substrate, inhibitor and pH specificity using both model enzymes and purified protein samples. The assay was then extensively tested to understand sample matrix effects on activity rates.CONCLUSIONS:
The use of this high-throughput method will allow for rapid characterization of protein samples in under three hours. The esterase activity correlated directly with polysorbate degradation and can provide valuable information on polysorbate degradation risk throughout drug development.Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Polisorbatos
/
Esterasas
Tipo de estudio:
Etiology_studies
/
Risk_factors_studies
Idioma:
En
Revista:
Pharm Res
Año:
2021
Tipo del documento:
Article
País de afiliación:
Estados Unidos