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Activity-Based Protein Profiling Probe for the Detection of Enzymes Catalyzing Polysorbate Degradation
Gao-Yuan Liu1, Song Nie1, Xiaojing Zheng1
1Analytical Chemistry, Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591-6707, United States.
Analytical Chemistry
|June 9, 2022
Summary
A new activity-based protein profiling (ABPP) probe, the REGN probe, effectively identifies lipases that degrade polysorbates in biotherapeutics. This novel probe offers superior enrichment and broader coverage for detecting trace lipases, improving drug product stability.
Area of Science:
- Biochemistry and Biotechnology
- Proteomics and Enzyme Activity Profiling
Background:
- Polysorbates are crucial nonionic surfactants in biotherapeutics, preventing protein aggregation.
- Polysorbate degradation by lipases compromises biotherapeutic shelf-life, leading to aggregates and particles.
- Identifying lipases responsible for polysorbate hydrolysis is vital for biopharmaceutical development.
Purpose of the Study:
- To develop a novel activity-based protein profiling (ABPP) probe for identifying lipases that degrade polysorbates.
- To assess the enrichment efficiency and coverage of the novel probe compared to existing methods.
- To demonstrate the probe's capability in detecting trace levels of lipases in biotherapeutic products.
Main Methods:
- Development of a novel fluorophosphonate activity-based protein profiling (ABPP) probe (REGN probe) mimicking polysorbate structure.
- Enrichment of lipases from Chinese hamster ovary (CHO) cell lysate using the REGN probe.
- Comparison of enrichment efficiency with commercial ABPP probes (FP-biotin, FP-desthiobiotin) and direct tryptic digestion.
- Detection of enriched lipases using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Main Results:
- The REGN probe achieved over 100-fold lipase enrichment from CHO cell lysate.
- It demonstrated higher enrichment efficiency and broader lipase coverage than commercial ABPP probes.
- The REGN probe successfully labeled lipases like lysosomal acid lipase and cytosolic phospholipase A2, missed by commercial probes.
- Trace lipases at 0.08 ppm in drug substances were detected using the REGN probe and LC-MS/MS.
Conclusions:
- A novel ABPP probe (REGN probe) with enhanced lipase enrichment efficiency and broader coverage has been developed.
- This probe is effective for detecting trace levels of lipases in biotherapeutic products.
- The REGN probe facilitates the development and manufacturing of stable biotherapeutics by identifying degradation catalysts.
