An Efficient LC/MS Workflow for Identification and Monitoring of Host Cell Proteins for Assisting Monoclonal Antibody Process Development

Significance of the Topic

Host cell proteins (HCPs) are low-level impurities in monoclonal antibody (mAb) production that can impact safety, efficacy and immunogenicity. Regulatory agencies demand robust identification and control of HCPs to ensure product quality and patient safety. Mass spectrometry-based workflows complement conventional immunoassays by offering proteome-wide detection and individual HCP quantification.

Objectives and Overview of the Study

This study presents two LC-MS workflows to support mAb process development:

• Discovery HCP Assay: Data-independent acquisition for broad HCP identification in NIST mAb Reference Material down to 5 ppm.
• Monitoring HCP Assay: Targeted high-throughput analysis for routine quantification of known HCPs using compliance-ready software.

Methodology

Sample preparation employed mAb depletion via precipitation and native enzymatic digestion with recombinant RapiZyme trypsin. Four or five protein digest standards were spiked into digested mAb to benchmark sensitivity. Analytical-scale UPLC separations used ACQUITY Premier CSH C18 columns with 90-min gradients for discovery and 30-min gradients for monitoring. MS data were acquired in positive ESI mode with MSE (DIA) on Xevo G3 QTof and BioAccord systems.

Instrumentation Used

• Xevo G3 QTof Mass Spectrometer
• BioAccord UPLC-MS System
• ACQUITY Premier UPLC with Premier CSH C18 column (2.1×150 mm, 1.7 µm)
• Waters RapiZyme Trypsin
• Data processing: UNIFI, Progenesis QI for Proteomics, Byonic, waters_connect

Main Results and Discussion

• Discovery workflow identified seven endogenous HCPs in NIST mAb RM and two spiked standards, achieving a limit of detection near 5 ppm.
• Chromatographic robustness was demonstrated under high protein loads, yielding sharp peaks and reproducible retention times.
• Cross-validation between Progenesis QI and Byonic confirmed consistent protein identifications.
• Monitoring workflow tracked 43 targeted peptides from five spiked proteins across multiple concentration levels, matching the 5 ppm detection threshold.
• Automated data processing in waters_connect enabled compliant-ready, high-throughput quantification across sample sets (10–50 replicates).

Benefits and Practical Applications

• Enables early-stage HCP discovery to inform process optimization and impurity clearance.
• Facilitates routine QC monitoring with simplified workflows accessible to non-MS experts.
• Supports regulatory compliance through detailed proteomic coverage and audit-ready informatics.
• Applies to release testing and comparability assessments during biopharmaceutical development.

Future Trends and Potential Applications

• Integration of ion mobility and higher-resolution mass analyzers to further lower detection limits.
• Automation and machine learning for predictive HCP profiling and data interpretation.
• Extension of workflows to other biotherapeutic platforms such as fusion proteins and vaccines.
• Development of standardized spectral libraries for cross-laboratory comparability.
• Advances in column chemistry and micro-flow LC for increased throughput and sensitivity.

Conclusion

The dual LC-MS workflows described offer comprehensive HCP discovery and streamlined monitoring capabilities. By coupling robust UPLC separations with DIA acquisition and compliant informatics, both low-level HCP identification and routine quantification can be achieved at the 5 ppm level. These methods enhance process understanding and support quality control throughout mAb development.

References

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