Activity-Based Protein Profiling Probe for the Detection of Enzymes Catalyzing Polysorbate Degradation

• Published in: Analytical chemistry (Washington) Vol. 94; no. 24; pp. 8625 - 8632
• Main Authors: Liu, Gao-Yuan, Nie, Song, Zheng, Xiaojing, Li, Ning
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.06.2022
• Subjects: analytical chemistry; Biotin; Chemistry; Cricetulus griseus; Degradation; Denaturation; digestion; drugs; Enrichment; Enzymes; hydrolysis; Lipase; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Nonionic surfactants; Phospholipase A2; polysorbates; Probes; Protein denaturation; Protein interaction; Proteins; Shelf life; storage time; tandem mass spectrometry
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.2c00059

Polysorbates are nonionic surfactants that have been widely used in biotherapeutic formulations to prevent protein aggregation and denaturation. However, polysorbates are subject to degradation after prolonged storage if certain lipases are present in the biotherapeutic product. Because the degradation of polysorbates compromises the shelf life of biotherapeutics and leads to the formation of undesirable products such as protein aggregates and subvisible particles, it is important to identify the active enzymes that catalyze polysorbate hydrolysis. In this study, we developed a novel fluorophosphonate activity-based protein profiling (ABPP) probe (termed the REGN probe), which mimics the structure of polysorbate and targets lipases catalyzing polysorbate degradation. We demonstrated that the REGN probe could enrich certain lipases from Chinese hamster ovary (CHO) cell lysate by more than 100-fold compared with direct tryptic digestion. Furthermore, we found that the REGN probe had higher lipase enrichment efficiency than commercially available ABPP probes including fluorophosphonate-biotin (FP-biotin) and FP-desthiobiotin. Remarkably, the REGN probe can enrich several lipases that cannot be labeled by commercial probes, such as lysosomal acid lipase and cytosolic phospholipase A2. Additionally, we showed that lipases with abundances as low as 0.08 ppm in drug substances were detected by the REGN probe enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Collectively, we have developed a novel ABPP probe with higher enrichment efficiency and broader coverage for lipases compared with commercial probes, and this probe can be used to detect the trace level of lipases in biotherapeutic products and to facilitate their development and manufacturing.