Host cell proteins (HCPs) are process-related impurities that can negatively impact the quality of biotherapeutics. Some HCPs possess enzymatic activity and can affect the active pharmaceutical ingredient (API) or excipients such as polysorbates (PS). PSs are a class of non-ionic surfactants commonly used as excipients in biotherapeutics to enhance the stability of APIs. The enzyme activity of certain HCPs can result in the degradation of PSs, leading to particle formation and decreased shelf life of biotherapeutics. Identifying and characterizing these HCPs is therefore crucial. This study employed the Activity-Based Protein Profiling (ABPP) technique to investigate the effect of pH on the activity of HCPs that have the potential to degrade polysorbates. Two probes were utilized: the commercially available fluorophosphonate (FP)-Desthiobiotin probe and a probe based on the antiobesity drug, Orlistat. Over 50 HCPs were identified, showing a strong dependence on pH-milieu regarding their enzyme activity. These findings underscore the importance of accounting for pH variations in the ABPP method and other investigations of HCP activity. Notably, the Orlistat-based probe (OBP) enabled us to investigate the enzymatic activity of a wider range of HCPs, emphasizing the advantage of using more than one probe for ABPP. Finally, this study led to the discovery of previously unreported active enzymes, including three HCPs from the carboxylesterase enzyme family.
Title: Activity-Based Protein Profiling Probe for the Detection of Enzymes Catalyzing Polysorbate Degradation Liu GY, Nie S, Zheng X, Li N Ref: Analytical Chemistry, 94:8625, 2022 : PubMed
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.
Title: Novel lipid droplet-associated serine hydrolase regulates macrophage cholesterol mobilization Goo YH, Son SH, Kreienberg PB, Paul A Ref: Arterioscler Thromb Vasc Biol, 34:386, 2014 : PubMed
OBJECTIVE: Lipid-laden macrophages or foam cells are characterized by massive cytosolic lipid droplet (LD) deposition containing mostly cholesterol ester (CE) derived from the lipoproteins cleared from the arterial wall. Cholesterol efflux from foam cells is considered to be atheroprotective. Because cholesterol is effluxed as free cholesterol, CE accumulation in LDs may limit free cholesterol efflux. Our objective was to identify proteins that regulate cholesterol trafficking through LDs. APPROACH AND RESULTS: In a proteomic analysis of the LD fraction of RAW 264.7 macrophages, we identified an evolutionarily conserved protein with a canonical GXSXG lipase catalytic motif and a predicted alpha/beta-hydrolase fold, the RIKEN cDNA 1110057K04 gene, which we named LD-associated hydrolase (LDAH). LDAH association with LDs was confirmed by immunoblotting and immunocytochemistry. LDAH was labeled with a probe specific for active serine hydrolases. LDAH showed relatively weak in vitro CE hydrolase activity. However, cholesterol measurements in intact cells supported a significant role of LDAH in CE homeostasis because LDAH upregulation and downregulation decreased and increased, respectively, intracellular cholesterol and CE in human embryonic kidney-293 cells and RAW 264.7 macrophages. Mutation of the putative nucleophilic serine impaired active hydrolase probe binding, in vitro CE hydrolase activity, and cholesterol-lowering effect in cells, whereas this mutant still localized to the LD. LDAH upregulation increased CE hydrolysis and cholesterol efflux from macrophages, and, interestingly, LDAH is highly expressed in macrophage-rich areas within mouse and human atherosclerotic lesions. CONCLUSIONS: The data identify a candidate target to promote reverse cholesterol transport from atherosclerotic lesions.
