Integrated Effects of Digestion Strategy and LC Gradient Architecture Define Host Cell Protein Detectability in CHO Null Harvests

Abstract Accurate characterization of host cell proteins (HCPs) is critical for biologics development, yet analytical outcomes are strongly influenced by the digestion strategy and chromatographic design. Here, we systematically evaluated digestion parameters, including digestion mode, enzyme ratio, and incubation time, together with LC-MS gradient architecture using a CHO null harvest as a controlled model system to isolate workflow-dependent analytical effects from biological variability. Specifically, the study was designed to determine how these variables jointly influence HCP detection and the physicochemical and biological characteristics of the detected protein populations. By comparing reducing and nonreducing digestion approaches across multiple trypsin ratios and incubation times, we found that under low-enzyme conditions, the primary constraint on HCP recovery is insufficient proteolysis rather than chromatographic undersampling. Reconfiguration of the gradient architecture altered peptide sampling efficiency and MS/MS utilization, enabling controlled comparison of digestion workflows under equivalent acquisition constraints. While higher enzyme ratios increased overall HCP identifications, subcellular localization analysis revealed that low-enzyme native digestion preferentially detected secreted and extracellular proteins with limited access to intracellular species. Extension of native digestion partially restored intracellular protein detection but did not fully recapitulate the coverage achieved by higher enzyme workflows. These findings demonstrate that digestion accessibility and chromatographic spacing impose workflow-dependent effects that shape both the depth and biological origin of detected HCPs, underscoring the need to consider the digestion strategy and chromatographic design together in HCP characterization workflows.