Easy, fast and reproducible analysis of host cell protein (HCP) in monoclonal antibody preparations

Importance of the Topic

Host cell proteins (HCPs) are residual impurities derived from the cells used to produce monoclonal antibodies (mAbs). Even at low concentrations, these contaminants can impact drug safety, efficacy, and long-term stability. Sensitive, reproducible methods for monitoring HCP profiles are therefore critical in biopharmaceutical quality control and regulatory compliance.

Objectives and Study Overview

This study demonstrates an integrated, automated workflow for HCP analysis in a commercial mAb product. It combines automated magnetic bead digestion with high-resolution peptide mapping and charge variant analysis. The goals were:

• To develop a rapid, reproducible protocol for both HCP characterization and routine peptide mapping
• To identify low-abundance protease contaminants that may contribute to mAb degradation
• To evaluate chromatographic and mass spectrometric performance at high sample loads

Instrumentation

• KingFisher Duo Prime purification system with SMART Digest magnetic beads for automated tryptic digestion
• Vanquish Flex Binary UHPLC with Acclaim VANQUISH C18, 2.1 × 250 mm column for peptide separation and HCP analysis
• Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer for high-resolution accurate mass detection
• Chromeleon CDS and Biopharma Finder for data processing, and PEAKS Studio for HCP database searches

Key Results and Discussion

An automated digestion protocol using the SMART Digest kit on the KingFisher Duo Prime achieved complete mAb sequence coverage and identified 51 HCPs. Six proteins were confirmed by two or more unique peptides. Among these, cathepsin L1, a protease that cleaves the His-Thr motif, was detected at ~4 ppm (ng HCP per mg mAb).
Charge variant analysis coupled to Orbitrap MS revealed low molecular weight mAb fragments consistent with cathepsin L1 activity. Peptide mapping at a high digest load (160 µg) maintained >100% sequence coverage and reproducible chromatographic performance, with only a minor reduction in resolution and asymmetry compared to standard 5 µg loads.

Benefits and Practical Applications

• Automated magnetic bead digestion reduces hands-on time and improves reproducibility across replicate preparations
• The same UHPLC-MS workflow supports both routine peptide mapping and deep HCP profiling without hardware changes
• Detection of low-abundance proteases like cathepsin L1 enables identification of degradation pathways and supports formulation stability studies

Future Trends and Opportunities

Further integration of data-independent acquisition and parallel reaction monitoring could enhance quantitation of trace HCPs. Machine learning models trained on high-resolution MS data may predict HCP risk profiles and guide process optimization. Expanded HCP libraries from diverse host cell lines will improve coverage of unexpected contaminants.

Conclusion

The described automated UHPLC-Orbitrap platform delivers robust peptide mapping and deep HCP analysis in a single workflow. Identification of cathepsin L1 at low ppm levels demonstrates the method’s sensitivity and underscores the importance of comprehensive HCP monitoring for mAb product quality.

References

1. Kim JY, Kim YG, Lee GM. Appl Microbiol Biotechnol. 2012;93:917–930.
2. Tscheliessnig AL, Konrath J, Bates R, Jungbauer A. Biotechnol J. 2013;8:655–670.
3. Shukla AA et al. Biotechnol Prog. 2008;24:615–622.
4. Doneanu C et al. MAbs. 2012;4:24–44.
5. Zhu-Shimoni J et al. Biotechnol Bioeng. 2014;111:2367–2379.
6. Kreimer S et al. Anal Chem. 2017;89:5294–5302.
7. McGivney JB et al. Anal Chem. 2018;90:3262–3269.
8. Kumar A et al. J Proteome Res. 2015;14:4687–4703.
9. Füssl F et al. MAbs. 2019;11(1):116–128.