Enhanced Host Cell Protein Identification and Quantitation with Multi-Reflecting ToF LC-MS

Importance of the Topic

Host cell proteins are low-level impurities in biotherapeutic products that can affect drug safety, efficacy and stability. Comprehensive detection and quantitation of individual HCPs supports process optimization and regulatory compliance. High-resolution mass spectrometry offers an orthogonal approach to traditional immunoassays, enabling detailed protein-level risk assessments and streamlined assay development.

Objectives and Study Overview

This study aimed to implement a multi-reflecting time-of-flight Q-ToF mass spectrometer in a data-independent LC-MSE workflow combined with Hi3 label-free quantitation. The method was applied to the NIST monoclonal antibody reference material (RM 8671) to assess sensitivity, dynamic range and quantitation accuracy for host cell proteins.

Methodology and Instrumentation

Sample preparation employed a non-denaturing trypsin digestion to deplete the monoclonal antibody and enrich HCPs. The peptide digest was analyzed by high-throughput reversed-phase UPLC coupled to the Xevo MRT mass spectrometer in MSE acquisition mode. Data processing used ProteinLynx Global Server with a UniProt Mus musculus database appended with standard and mAb sequences. Hi3 quantitation averaged the three most intense peptides per protein to estimate abundance.

Instrumentation

• Waters ACQUITY Premier UPLC System
• Waters Xevo MRT Q-ToF Mass Spectrometer
• ACQUITY Premier Peptide CSH C18 Column, 2.1×150 mm, 1.7 µm
• PLGS 3.0.3 software for data analysis

Key Results and Discussion

The workflow confidently identified 101 host cell proteins over a dynamic range exceeding four orders of magnitude (128 ppm to 12 ppb). Of these, 58 HCPs were quantified at sub-ppm levels. The method achieved sub-ppm mass errors, high sequence coverage and robust peptide fragmentation patterns. Comparison to previous studies showed strong correlation in relative quantitation and an increased number of unique peptides per protein, highlighting enhanced sensitivity and confidence in low-abundance HCP detection.

Benefits and Practical Applications

• Broad dynamic range and ppb-level sensitivity for early impurity detection
• High mass accuracy enabling unambiguous peptide and protein assignments
• Label-free Hi3 quantitation from a single injection, reducing analysis time
• Orthogonal to ELISA assays, circumventing immunoreagent limitations and accelerating assay development
• Supports process development decisions and risk assessments in biopharmaceutical manufacturing

Future Trends and Applications

Advances in DIA and high-resolution MS will further improve depth of coverage and throughput. Integration with automation and advanced informatics will enable full-scale QC implementation. Emerging regulatory guidelines may increasingly recognize MS-based HCP assays for product release. Continued innovation in sample prep and instrumentation will drive even lower detection limits and streamlined workflows.

Conclusion

The multi-reflecting ToF LC-MSE workflow on the Waters Xevo MRT platform delivers sensitive, high-confidence identification and quantitation of host cell proteins in biotherapeutic samples. This approach outperforms earlier methods in sensitivity, dynamic range and peptide coverage, facilitating safer product development and efficient manufacturing control.

References

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