Analytical Chemistry, Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591-6707, United States.
Anal Chem. 2022 Jun 21;94(24):8625-8632. doi: 10.1021/acs.analchem.2c00059. Epub 2022 Jun 9.
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.
聚山梨酯是非离子表面活性剂，已广泛用于生物治疗制剂中，以防止蛋白质聚集和变性。然而，如果生物治疗产品中存在某些脂肪酶，聚山梨酯在长期储存后会降解。由于聚山梨酯的降解会缩短生物治疗产品的保质期，并导致形成不希望的产物，如蛋白质聚集物和亚可见颗粒，因此确定催化聚山梨酯水解的活性酶非常重要。在这项研究中，我们开发了一种新型的氟膦酸酯活性基蛋白谱（ABPP）探针（称为 REGN 探针），该探针模拟聚山梨酯的结构，并针对催化聚山梨酯降解的脂肪酶。我们证明，与直接胰蛋白酶消化相比，REGN 探针可以使 CHO 细胞裂解物中的某些脂肪酶富集超过 100 倍。此外，我们发现 REGN 探针比包括氟膦酸酯-生物素（FP-biotin）和 FP-脱巯基生物素在内的商业 ABPP 探针具有更高的脂肪酶富集效率。值得注意的是，REGN 探针可以富集几种不能被商业探针标记的脂肪酶，如溶酶体酸性脂肪酶和胞质型磷脂酶 A2。此外，我们还表明，通过 REGN 探针富集和液相色谱-串联质谱（LC-MS/MS）分析，可以检测到药物物质中丰度低至 0.08ppm 的脂肪酶。总之，我们开发了一种新型的 ABPP 探针，与商业探针相比，该探针具有更高的富集效率和更广泛的脂肪酶覆盖范围，该探针可用于检测生物治疗产品中痕量的脂肪酶，并有助于其开发和制造。
