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

Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.

Investigating pH Effects on Enzymes Catalyzing Polysorbate Degradation by Activity-Based Protein Profiling.

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.