High-Throughput, Fluorescence-Based Esterase Activity Assay for Assessing Polysorbate Degradation Risk during...

Area of Science:

• Biopharmaceutical development
• Enzyme kinetics
• Protein characterization

Background:

• Polysorbate degradation during cold storage of monoclonal antibody (mAb) products is a concern.
• Residual esterases from bioprocessing are implicated in this hydrolytic degradation.
• Assessing esterase activity offers a potential method to predict polysorbate degradation risk.

Purpose of the Study:

• Develop a robust esterase activity assay for monitoring during mAb bioprocess development.
• Establish a correlation between esterase activity and polysorbate degradation.
• Provide an alternative method to assess polysorbate degradation risk.

Main Methods:

• Developed a fluorescence plate-based assay using 4-methylumbelliferyl caprylate (MU-C8) as a surrogate substrate.
• Assessed assay specificity for substrates, inhibitors, and pH using model enzymes and purified proteins.
• Investigated sample matrix effects on esterase activity rates.

Main Results:

• The assay demonstrated specificity and was tested for matrix effects.
• Esterase activity was quantified using the developed high-throughput method.
• The assay provides rapid protein sample characterization in under three hours.

Conclusions:

• Esterase activity directly correlated with polysorbate degradation.
• The assay provides valuable information for assessing polysorbate degradation risk.
• This high-throughput method aids in drug development and product stability assessment.