Polysorbate is widely used to maintain stability of biotherapeutic protein formulations. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual enzymes such as lipases and esterases can cause polysorbate degradation. Their high activity, even at low concentration, constitutes a major analytical challenge. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using chemical probes to enrich active serine hydrolases, more than 80 proteins were identified in harvested cell culture fluid (HCCF) from monoclonal antibodies (mAb) production. A total of 8 known lipases were identified by ABPP, while only 5 lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study, phospholipase A2 group VII (PLA2G7) and sialic acid acetylesterase (SIAE) were identified by ABPP as possible root causes of polysorbate-80 degradation. The established ABBP approach can fill the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

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通过基于活动的蛋白质分析对生物治疗中的聚山梨酯降解的活性酶进行分析

聚山梨酯被广泛用于维持生物治疗性蛋白质制剂的稳定性。聚山梨酯的降解会导致药物产品中形成颗粒，这是主要的质量问题和潜在的患者风险因素。来自残留酶（例如脂肪酶和酯酶）的酶促活性可能导致聚山梨酯降解。即使在低浓度下，它们的高活性也构成了重大的分析挑战。在这项研究中，我们评估和优化了基于活性的蛋白质谱分析（ABPP）方法，以鉴定引起聚山梨酯降解的活性酶。使用化学探针富集活性丝氨酸水解酶，从单克隆抗体（mAb）生产中收获的细胞培养液（HCCF）中鉴定出80多种蛋白。ABPP共鉴定了8种已知的脂肪酶，而传统的基于丰度的蛋白质组学（TABP）方法仅鉴定出5种脂肪酶。有趣的是，未发现磷脂酶B样2（PLBL2）（一种众所周知的有问题的HCP）在来自两种不同mAb的过程中间体中具有活性。在概念验证研究中，ABPP将磷脂酶A2 VII组（PLA2G7）和唾液酸乙酰酯酶（SIAE）鉴定为聚山梨酯80降解的可能根源。已建立的ABBP方法可以填补脂肪酶丰度与活性之间的空白，从而为生物治疗的发展提供更有意义的聚山梨酯降解研究。在概念验证研究中，ABPP将磷脂酶A2 VII组（PLA2G7）和唾液酸乙酰酯酶（SIAE）鉴定为聚山梨酯80降解的可能根源。已建立的ABBP方法可以填补脂肪酶丰度与活性之间的空白，从而为生物治疗的发展提供更有意义的聚山梨酯降解研究。在概念验证研究中，ABPP将磷脂酶A2 VII组（PLA2G7）和唾液酸乙酰酯酶（SIAE）鉴定为聚山梨酯80降解的可能根源。既定的ABBP方法可以填补脂肪酶丰度与活性之间的空白，从而为生物治疗发展提供更有意义的聚山梨酯降解研究。