Identification of Acyl-Protein Thioesterase-1 as a Polysorbate-Degrading Host Cell Protein in a Monoclonal Antibody...

Area of Science:

• Biopharmaceutical Science
• Protein Chemistry
• Analytical Chemistry

Background:

• Polysorbate (PS) is crucial for monoclonal antibody (mAb) stability in formulations.
• Low-abundance residual host cell proteins (HCPs), especially hydrolytic enzymes, can degrade PS.
• This degradation impacts the shelf-life and efficacy of mAb therapeutics.

Purpose of the Study:

• To identify the specific HCP responsible for PS degradation in a problematic mAb formulation.
• To validate the identified HCP's role in PS breakdown.
• To explore methods for mitigating PS degradation.

Main Methods:

• Activity-based protein profiling (ABPP) combined with mass spectrometry.
• Recombinant protein expression and activity assays.
• Analysis of PS degradation products and HCP levels.
• Inhibition studies using specific APT1 inhibitors (ML348, ML211).

Main Results:

• Acyl-protein thioesterase-1 (APT-1) was identified as the primary PS-degrading HCP.
• A direct correlation was observed between APT1 levels and the rate of PS degradation.
• APT1 was found to co-purify with a specific mAb.
• PS degradation was successfully inhibited using APT1-specific inhibitors.

Conclusions:

• APT-1 is a critical HCP contributing to PS degradation in mAb formulations.
• ABPP offers a streamlined method for identifying critical HCPs involved in formulation instability.
• These findings support quality-by-design principles for developing stable biopharmaceutical products.