Rapid Polysorbate 80 Degradation by Liver Carboxylesterase in a Monoclonal Antibody Formulated Drug Substance at Early Stage Development

Abstract

Polysorbates (PS) are surfactants commonly added in a therapeutic protein drug product to protect proteins from denaturation and aggregation during storage, transportation, and delivery. Significant degradation of PS in drug products could lead to particulate formation with shortened drug shelf life, and one of the major root causes of PS degradation is the host cell protein (HCP) derived lipase/esterase, which belong to the serine hydrolase family. Typically, PS degradation can only be observed in drug products after a long time of storage if very low levels of host cell protein impurity with PS degradation activities are present. In this study, PS80 degradation was observed in a monoclonal antibody (mAb) within 18 h at 5 °C with a low level of HCP presented (<20 ppm) based on ELISA quantitation. This observation suggested that a trace amount of unknown host cell protein(s) with strong enzymatic activity on polysorbate degradation was present in this drug substance. The activity-based protein profiling (ABPP) method with the ActivX FP serine hydrolase probe was employed to identify host cell proteins that can hydrolyze PS. Two hydrolases, liver carboxylesterase B-1-like protein (CES-B1L, A0A061I7X9) and liver carboxylesterase 1-like protein (CES-1L, A0A061IFE2) were identified with high confidence using the ABPP approach for the first time in a mAb drug substance during early stage development. PS80 became stable in the drug substance sample after the two hydrolases were depleted using the immobilized ActivX FP probe, confirming these two hydrolases were responsible for the rapid PS80 degradation. In addition, the PS80 degradation pattern was found to be equivalent to that generated by their human analog, human liver carboxylesterase-1 (hCES-1) and rabbit liver esterase (rLES). Overall, these results suggest that CES-B1L and CES-1L are the primary cause of PS80 degradation in this mAb drug.