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

Abstract

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.

Illuminating a biologics development challenge: systematic characterization of CHO cell-derived hydrolases identified in monoclonal antibody formulations

“…Figure S1). Strikingly, for LPL, a frequently mentioned hydrolase in the context of PS degradation, 9 , 13 , 14 , 17 the high occurrence in many mAb products correlated with high mRNA and protein expression in all investigated production cell lines. This was also true for SMPD1 and LIPA, although here high expression values correlated only with identification in 5 and 3 of the 7 analyzed mAb products, respectively.…”

“… 17 Nevertheless, a CRISPR/Cas9 mediated LPL KO alone was not able to halt PS degradation completely, explaining the increasing efforts put into identification and characterization of additional hydrolytic HCPs. 9 , 13 , 14 …”

“…Therefore, new innovative analytical methods were developed, including optimized sample preparation prior to mass spectrometric analysis 9–12 and activity-based protein profiling. 13 , 14 , 16 To date, advances in HCP detection have resulted in a comprehensive list of potentially PS-degrading protein contaminants detected at various stages of the bioprocess, but data regarding their enzymatic activity profiles against PS and an understanding of the impact of the downstream purification processes on these enzymes are still lacking. A less explored but promising approach to address this issue is the recombinant overexpression and simultaneous screening of multiple putative polysorbate-degrading HCPs, especially the hydrolase species.…”

Trends in industrialization of biotherapeutics: a survey of product characteristics of 89 antibody-based biotherapeutics

“…Surfactants are used in formulations of antibody-based biotherapeutics to prevent aggregation and denaturation of antibodies at the air–water interface. 173 Figure 5(d) shows use of surfactants in formulations of antibody-based biotherapeutics over time. Of the 79 formulations that utilize surfactants, 53 use polysorbate 80 (67%) at an average concentration of 0.41 ± 0.48 mg/mL (range of 0.04–2.00 mg/mL).…”

Host serine protease ACOT2 assists DENV proliferation by hydrolyzing viral polyproteins

“…Therefore, a direct quantification method is required to study the specific type of protease in complex proteomes. Activity-based protein profiling (ABPP) is a functional proteomic technology that utilizes chemical probes, with the majority active site-directed, to label and monitor the functional status of proteases in vitro and in vivo ( 13 – 15 ). The basic unit of ABPP is the probe, which contains a reactive warhead that selectively binds and covalently labels catalytically active proteases, a reporter tag that is used for detection, enrichment, identification, and a binding group for optimizing the binding affinity or cell permeability ( 16 ).…”