Assay for Identification and Quantification of Host Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm: An Inter-Laboratory Study

Importance of the Topic

The presence of host cell protein (HCP) impurities in biotherapeutic monoclonal antibodies (mAbs) poses a significant safety risk due to potential immunogenic responses even at concentrations as low as 1 ppm. Regulatory agencies require detailed characterization of these trace contaminants to ensure patient safety and product quality.

Objectives and Study Overview

This interlaboratory study evaluated a generic, bottom-up mass spectrometric assay for simultaneous identification and quantification of HCPs in highly purified mAb samples down to 1 ppm. Three independent laboratories applied the same two-dimensional liquid chromatography and ion mobility mass spectrometry workflow to a standardized NIST reference mAb sample spiked with protein standards.

Methodology and Used Instrumentation

Sample Preparation and Digestion

• The NIST reference mAb was denatured, reduced, alkylated, and digested with trypsin.
• Four non-murine protein standards were spiked post-digestion to monitor assay dynamic range.

Two-Dimensional LC System

• First dimension: high-pH reversed-phase on XBridge Peptide BEH C18 column with 10-step acetonitrile elution.
• On-line dilution and trapping on ACQUITY UPLC Symmetry C18 trap column.
• Second dimension: low-pH peptide separation on ACQUITY UPLC Peptide CSH C18 column with a 40-minute gradient.

Mass Spectrometry and Data Acquisition

• SYNAPT G2-S high-resolution, ion mobility enabled mass spectrometer operating in ESI positive mode.
• HDMS E data independent acquisition with drift time-specific collision energies for fragmentation.
• Data processed with MassLynx and ProteinLynx Global SERVER for peptide and protein identification and quantification.

Main Results and Discussion

Consistency Across Laboratories

• All three sites identified 14 common HCPs in the 1–100 ppm range plus the four spiked standards.
• Quantification used the summed signal of top three peptides per protein against calibration standards.

Role of Ion Mobility Separation

• Precursor-level ion mobility reduced co-eluting interferences, yielding cleaner MS and MS/MS spectra.
• Drift time alignment of precursors and fragments improved confidence in sequence assignment of low-abundance peptides.

Validation of Low-Abundance HCPs

• Targeted MS/MS on quadrupole-isolated, ion mobility-separated precursors confirmed sequence identification for HCPs down to 1–7 ppm.
• Demonstrated potential for extension to sub-ppm monitoring with HD-MRM approaches.

Benefits and Practical Applications

This generic LC/HDMS E assay offers comparable sensitivity to ELISA while providing unambiguous protein identification. It bypasses the need for process-specific immunoassays, reduces development time and cost, and adapts readily to different host cell systems and purification protocols.

Future Trends and Applications

Emerging applications include ultra-sensitive monitoring of known HCPs at sub-ppm levels using targeted mass spectrometry workflows. Integration with high-throughput automation and advanced data analysis algorithms will further enhance HCP coverage and quantification precision, supporting rapid development of biotherapeutics.

Conclusion

The described 2D-LC/ion mobility mass spectrometry method enables reliable identification and quantification of HCP impurities in mAbs down to 1 ppm. Its robustness and sensitivity across multiple laboratories demonstrate its suitability for routine biopharmaceutical quality control and regulatory compliance.

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