A Comprehensive Approach for HCP Identification, Quantification, and Monitoring Based on a Single Dimension (1D) LC Separation

Significance of the Topic

Residual host cell proteins (HCPs) are low-level impurities in protein biopharmaceuticals that can trigger immunogenic responses, compromise drug stability, or accelerate degradation. Regulatory agencies require comprehensive HCP profiling to ensure product safety and efficacy. While ELISA assays provide total HCP measurements, mass spectrometry (MS) offers proteome-wide coverage and individual HCP quantitation, addressing critical quality attributes in biologics development.

Objectives and Study Overview

This study presents a two-step one-dimensional LC-MS workflow for HCP discovery and monitoring in a monoclonal antibody (mAb) digest. The first step employs a 90 min peptide separation in SONAR data-independent acquisition (DIA) mode to identify low-abundance HCPs and build a spectral library. The second step uses a 30 min MS^E assay for high-throughput quantitation and process monitoring of HCP levels across purification stages. The National Institute of Standards and Technology (NIST) mAb reference material served as the test sample.

Methodology

• Sample Preparation: The NIST mAb was denatured with RapiGest, reduced (DTT), alkylated (IAM), and digested with Lys-C/trypsin. Surfactant degradation by formic acid and centrifugation removed precipitates.
• Discovery Assay: Post-digestion spiking with three protein standards (ADH, PHO, BSA) enabled peptide identification during a 90 min LC gradient in SONAR mode.
• Monitoring Assay: Four digest samples containing variable amounts of four protein standards (ADH, BSA, PHO, CLP-B) were analyzed over a 30 min gradient in MS^E mode for quantitation against an internal standard.
• Data Processing: Progenesis QI for Proteomics 4.0 performed database searches, assembled SONAR spectra into a retention-time aligned library, and quantified HCPs using the Hi3 approach.

Instrumentation

• LC System: Waters ACQUITY UPLC I-Class with CSH C18 column (2.1×150 mm, 1.7 µm) at 60 °C, 200 µL/min flow.
• MS System: Waters Xevo G2-XS QTof with ESI(+) ionization, operating in SONAR DIA and MS^E modes with alternating low/high collision energy.
• Software: MassLynx 4.1 for acquisition and Progenesis QI for Proteomics 4.0 for data analysis.

Main Results and Discussion

• Discovery Phase: SONAR DIA reduced spectral complexity and enabled identification of four HCPs in the NIST mAb, including two low-abundance proteins (beta-2-microglobulin and low-affinity IgG gamma Fc receptor) at 10–30 ppm. SONAR spectra outperformed MS^E and DDA in detecting low-level peptides under high sample loads.
• Spectral Library: MS/MS spectra for 24 unique HCP peptides were compiled into a library with retention times, precursor m/z values, and charge states, facilitating targeted monitoring.
• Monitoring Phase: MS^E assays tracked spiked proteins down to ~20 ppm across twenty LC-MS runs. Quantitation against a constant CLP-B internal standard showed excellent correlation (RSD < 10 %) between known and measured protein levels.

Benefits and Practical Applications

• Enhanced Selectivity: SONAR’s scanning quadrupole isolates co-eluting precursors, yielding cleaner MS/MS spectra and improved detection of low-abundance HCPs compared to conventional DIA or DDA.
• Accelerated Monitoring: The curated spectral library enables rapid, high-throughput HCP profiling during process development and quality control.
• Regulatory Compliance: The workflow satisfies stringent guidelines for HCP identification and quantitation in biotherapeutic products.

Future Trends and Possibilities

• Integration with Ion Mobility: Coupling SONAR with ion mobility separation could further enhance HCP coverage and confidence.
• Automated High-Throughput Workflows: Shorter LC gradients and real-time library matching may support process analytical technology (PAT) in biomanufacturing.
• Expanded Spectral Libraries: Building comprehensive HCP libraries across various host-cell systems will standardize MS-based impurity analysis in the industry.

Conclusion

A streamlined 1D LC-MS workflow combining SONAR DIA for spectral library generation with MS^E for targeted monitoring delivers a sensitive, selective, and high-throughput solution for identifying and quantifying host cell protein impurities in monoclonal antibody preparations. This approach enhances analytical confidence at low ppm levels and supports regulatory compliance in biopharmaceutical development.

References

1. Doneanu CE et al. Enhanced Detection of Low-Abundance Host-Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry. Anal Chem, 2015, 87, 10283–10291.
2. Huang L et al. A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies. Anal Chem, 2017, 89, 5436–5444.
3. Chen W et al. Improved Identification and Quantification of Host Cell Proteins in Biotherapeutics Using LC/MS. ACS Symposium Series, 2015, Vol 3, 357–393.
4. Gethings LA et al. Lipid Profiling of Complex Biological Mixtures by LC/MS Using a Novel Scanning Quadrupole DIA Strategy. Rapid Commun Mass Spectrom, 2017, 31, 1599–1606.
5. Moseley MA et al. Scanning Quadrupole DIA – Part A. Qualitative and Quantitative Characterization. J Proteome Res, 2018, 17, 770–779.
6. Silva JC et al. Absolute Quantification of Proteins by LC-MSE: A Virtue of Parallel MS Acquisition. Mol Cell Proteomics, 2006, 5, 144–156.