Quantitative Analysis Reveals Hitchhiking Drives Polysorbate Hydrolase Persistence Via Host Cell Protein-Antibody...

Polysorbate-degrading host cell proteins (HCPs) represent a critical challenge in the manufacturing of monoclonal antibody therapeutics due to their potential to persist during downstream processing. While their enzymatic activity has been characterized, the role of direct HCP-mAb interactions, particularly those involving polysorbate degrading HCPs, remains poorly understood. In this study, we systematically investigated the binding behavior of four representative polysorbate-degrading HCPs (CES1F, LPLA2, PAF-AH, and PPT1) to a panel of mAbs using biolayer interferometry (BLI). All tested HCPs showed specific, transient interactions characterized by fast-on/fast-off kinetics, with apparent equilibrium dissociation constants (K_D) in the low nanomolar range (40-90 nM for strong binders) and rapid dissociation kinetics (k_d > 0.01 s^-1). This indicates a binding mode characterized by relatively high affinity but limited kinetic stability. Due to incomplete saturation and partially not meeting the quality criteria for kinetic fitting, we complemented model-based analysis with equilibrium-derived descriptors. The initial slope of the binding isotherm correlated well with kinetic parameters and enabled robust ranking of interaction strength. To assess hitchhiking relevance during downstream processing, we performed a Protein A chromatography experiment using PLBL2 as a model HCP and two mAbs with different interaction profiles. PLBL2 levels in Protein A elution pools correlated well with interaction propensity confirming that transient interactions can contribute to HCP co-elution. Our results provide the first systematic and quantitative comparison of polysorbate hydrolase-antibody interactions. They also demonstrate that direct mAb-HCP interaction is a relevant mechanism contributing to HCP persistence during downstream processing.