Therapeutic monoclonal antibodies (mAbs) represent a majority of biopharmaceuticals and are primarily recombinantly expressed in Chinese hamster ovary (CHO) cells. A major class of process-related impurities are the host cell proteins (HCPs) released from the producing cell line. Among these, certain hydrolases can degrade excipients such as polysorbates, causing formulation instability, shortening product’s shelf life, and threatening its quality and safety. This doctoral work addresses gaps in detecting and mitigating low-abundance enzymatically active HCPs through an activity-centric framework that integrates activity-based protein profiling, HCP–mAb interaction screening, targeted proteomics, enzyme assays, and rational downstream process design.
First, we show that the enzyme-driven polysorbate degradation in a mAb formulation can be attributed to specific enzymatically active HCPs enriched by covalent activity-based probes. Using the fluorophosphonate-biotin probe and mass spectrometry, we identified acyl-protein thioesterase-1 (APT1) as a previously unreported polysorbate-degrading HCP. Several lines of evidence confirm that APT1 is indeed responsible for polysorbate hydrolysis: the correlation between APT1 abundance and polysorbate loss, replication of the monoester-selective degradation fingerprint with recombinant APT1, and prevention of polysorbate degradation using selective APT1 inhibitors ML348 and ML211.
Second, we find that persistence of critical HCPs can arise from specific HCP–mAb associations. APT1 exhibits product-dependent hitchhiking, co-eluting with the monomeric mAb fraction during preparative size-exclusion chromatography and binding the mAb’s CH1 region as determined by the biolayer interferometry.
Third, we demonstrate that implementing alternative chromatographic techniques and optimizing chromatographic conditions can reduce critical (product-detrimental) HCPs. In a flow-through anion-exchange case study, membrane adsorbers outperformed bead resin for total HCP clearance. Additionally, factors such as feed pH, feed conductivity, and loading density had a significant impact on esterase clearance. We further show that total HCP abundance does not necessarily correlate with polysorbate-degrading risk, and that function-focused readouts better prioritize conditions that meaningfully reduce hydrolase activity.
The workflows outlined here are broadly applicable to downstream process designs targeting effective enzymatic HCPs removal, ultimately supporting the production of stable, high-quality, and safe biopharmaceutical products.
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