Identification and Characterization of Polysorbate-Degrading Enzymes in a Monoclonal Antibody Formulation

Characterization of radicals in polysorbate 80 using electron paramagnetic resonance (EPR) spectroscopy and spin trapping

“…As shown in different studies, the released free fatty acids can cluster into visible particles and thereby compromise the drug product quality ( Dixit et al, 2016 ; Labrenz, 2014 ; Tomlinson et al, 2015 ). Hydrolysis, especially enzymatically driven, is linked to the presence of remaining host cell proteins, such as esterases and lipases ( Labrenz, 2014 ; Graf et al, 2021 ; Zhang et al, 2021 ; Glücklich et al, 2021 ). Additionally, chemical hydrolysis driven by pH is possible but highly unlikely in the considered pharmaceutical relevant pH range from 5 to 7 ( Dwivedi et al, 2020 ).…”

The measurement and control of high-risk host cell proteins for polysorbate degradation in biologics formulation

“…High dynamic range translates to detection of less abundant peptides in a milieu of more abundant ones. Most commercial MS instruments have a dynamic range of about 4 to 5 orders of magnitude; however, high-risk HCPs, such as PSDEs, could have a direct impact on PS degradation at ppb levels [ 26 , 27 ]. This challenge is amplified in high concentration formulations, where the ppm level of HCP may not change, but the absolute concentrations of HCPs (ng/mL) significantly increase [ 28 ].…”

“…Several sample preparation methods to reduce high-abundance proteins or enrich low-abundance proteins have been used in HCP proteomics analysis to expand the dynamic range and increase the identification sensitivity of the TABP approach, such as native digestion [ 30 ], ProteoMiner [ 31 ], DS depletion with Protein A [ 32 ], anti-HCP affinity chromatography [ 27 , 33 ], molecular weight cutoff filtration [ 34 ], and hydrophilic interaction chromatography enrichment [ 35 ]. In addition to advancements in sample preparation, improvements in LC–MS analysis have also been made to improve sample dynamic range and improve the sensitivity of HCP identification.…”

“…Additional gas phase separation with ion mobility, such as high-field asymmetric waveform ion mobility spectrometry[ 32 ], and different mass spectrometry acquisition techniques, such as BoxCar and DIA [ 37 , 38 ], have been employed to improve both HCP identification and quantification. Several PSDEs, including LPLA2 (PLA2G15, phospholipase A2, group XV), LPL (lipoprotein lipase), CES (liver carboxylesterase), LIPA (lysosomal acid lipase), PPT1 (palmitoyl-protein thioesterase 1), and SIAE (sialate o-acetylesterase) have been identified by TABP and have shown to have a direct impact on PS-80 or PS-20 degradation in biotherapeutics [ 15 , 26 , 27 , 33 , 39 ]. Drawbacks of these extensive sample preparation and LC–MS analytical approaches include reduced testing throughput and potential HCP loss, and hence impaired assay robustness.…”

“…Our previous study also indicated that chemical probes against SHs with different mechanisms of action may provide synergistic or complementary information for inhibition or enrichment of various classes of lipases/esterases [ 10 ]. All PSDEs that have been reported to impact PS degradation have been identified by our ABPP approach [ 10 ], such as LPLA2 [ 26 ], LPL [ 39 ], CES [ 15 ], LIPA [ 27 ], PPT1 [ 27 ], and SIAE [ 33 ]. In addition, PLA2G7 was identified and confirmed to directly contribute to PS degradation for the first time in our previous publication [ 10 ].…”