This is a very important point, as overestimating HCP coverage due to missing identifications is a known risk in LC‑MS workflows.

In practice, this is managed through a combination of robust library design and controlled data analysis workflows. The database or spectral library is carefully curated to be specific to the production system, ensuring relevant host‑cell proteins are included while minimising false identifications.

On the data analysis side, conservative identification criteria are applied, such as strict FDR control and consistent peptide‑to‑protein mapping, to avoid overcalling proteins. In addition, quantification approaches focus on summed peptide signals rather than simply counting identified proteins, which reduces the risk of overestimating coverage due to missing IDs.

Finally, the workflow is typically supported by validated and standardised data processing pipelines, ensuring reproducibility, traceability, and consistent interpretation of results within a GMP framework.

Thomas Kofoed, PhD, CEO of Alphalyse in Denmark, discussed key factors in implementing GMP-compatible LC-MS methods for release assays and highlighted successful cases where this approach has replaced traditional ELISA methods. Don’t miss his presentation and expert insights at the panel discussion.