An Efficient Workflow for Identification and Monitoring of Host Cell Proteins During Monoclonal Antibody Bioprocessing

Importance of the Topic

Monoclonal antibody (mAb) bioprocessing requires careful monitoring of host cell proteins (HCPs) to ensure product safety and regulatory compliance. Traditional ELISA methods offer limited coverage, whereas liquid chromatography–mass spectrometry (LC–MS) provides broader proteome‐wide analysis. An efficient LC–MS workflow enhances sensitivity and throughput for HCP identification and quantification during mAb purification.

Objectives and Overview

This study presents a two‐step LC–MS workflow combining a discovery assay for comprehensive HCP profiling and a targeted monitoring assay for rapid routine analysis. The aims are to detect low‐abundance HCPs, build a spectral library for streamlined monitoring, and compare chromatographic purification protocols across multiple mAb preparations.

Methodology and Instrumentation

Sample Preparation:

• A well‐characterized NIST mAb standard was diluted to 25 mg/mL, digested overnight with Lys‐C and trypsin, treated with surfactant and reducing/alkylating agents to deplete intact mAb peptides, and spiked post‐digestion with three MassPREP protein standards (yeast ADH, rabbit PHO, bovine BSA).
• Five additional mAb preparations from CHO cell culture were processed identically and spiked with the same standards.

Instrumentation:

• Chromatography: Waters ACQUITY UPLC I‐Class Plus system with CSH C18 column (2.1 x 150 mm, 1.7 µm) at 60 °C; Discovery assay gradient 0–45% acetonitrile over 90 min (0.2 mL/min); Monitoring assay 30 min gradient.
• Mass Spectrometry: Waters Xevo G2‐XS QTof operated in MSE mode via UNIFI 1.9.4; low‐energy scans at 4 eV, high‐energy ramp 15–45 V; scan times 0.5 s (discovery) and 0.3 s (monitoring), mass range 100–2000 Da.
• Data Processing: Progenesis QI for Proteomics 4.0 for database searching against mouse, CHO or UniProt proteomes; construction of a spectral library from discovery data for targeted monitoring.

Main Results and Discussion

The Discovery assay identified six endogenous HCPs alongside three spiked standards down to a detection limit of 5 ppm. Spectral library construction enabled rapid targeted analysis in the Monitoring assay. Five mAb preparations purified by Protein A and four SCX protocols (A–D) were evaluated:

• Protocol D yielded the lowest residual HCP levels and the most consistent peptide signals (RSD <20%).
• Two representative HCPs (low‐affinity IgG Fc receptor peptides) were tracked across all samples, demonstrating robust quantification at ~10–50 ppm.

These results confirm the workflow’s capacity for both broad discovery and high‐throughput monitoring of trace HCPs.

Benefits and Practical Applications

• Comprehensive HCP profiling supports method development and impurity risk assessment in biopharmaceutical manufacturing.
• Targeted monitoring assays enable QC labs to routinely track critical HCPs with minimal analysis time.
• The workflow is adaptable to different mAb constructs and purification schemes.

Future Trends and Opportunities

• Integration of additional orthogonal separation modes (e.g., ion mobility) to further enhance proteome coverage.
• Application of machine learning for improved HCP peptide selection and quantitation.
• Expansion of spectral libraries to include multiple cell lines and post‐translational modifications.
• Automation of sample preparation to increase throughput and reproducibility.

Conclusion

This two‐step LC–MS workflow delivers sensitive discovery of host cell proteins and streamlined monitoring assays for mAb bioprocessing. By coupling long gradient separations for initial profiling with shorter runs for routine QC, laboratories can achieve both depth and efficiency in HCP analysis.

References

• Doneanu C., et al. Anal Chem, 2015, 87, 10283.
• Huang Y.Q., et al. Anal Chem, 2017, 89, 5436.