Investigating pH Effects on Enzymes Catalyzing Polysorbate Degradation by Activity-Based Protein Profiling.

Area of Science:

• Biopharmaceutical manufacturing
• Protein chemistry
• Analytical chemistry

Background:

• Host cell proteins (HCPs) are critical impurities in biotherapeutics.
• Enzymatic activity of HCPs can degrade polysorbates (PS), impacting drug stability and shelf-life.
• Understanding HCP activity is vital for biopharmaceutical quality control.

Purpose of the Study:

• To investigate the impact of pH on the activity of HCPs capable of degrading polysorbates.
• To identify specific HCPs responsible for polysorbate degradation using activity-based protein profiling (ABPP).
• To evaluate the utility of different chemical probes for HCP activity profiling.

Main Methods:

• Activity-Based Protein Profiling (ABPP) was employed to profile HCP activity.
• Two distinct probes were used: a fluorophosphonate-Desthiobiotin probe and an Orlistat-based probe (OBP).
• Experiments were conducted across varying pH conditions to assess enzyme activity dependence.

Main Results:

• Over 50 HCPs were identified with significant pH-dependent enzymatic activity.
• The Orlistat-based probe (OBP) demonstrated broader coverage for identifying active HCPs compared to the FP probe.
• Three previously unreported active carboxylesterase family HCPs were discovered.

Conclusions:

• pH significantly influences HCP activity relevant to polysorbate degradation.
• Utilizing multiple ABPP probes, like OBP, enhances the comprehensive identification of active HCPs.
• This research provides crucial insights for mitigating HCP-related degradation in biotherapeutics.