Host Cell Protein Analysis Using Agilent AssayMAP Bravo and 6545XT AdvanceBio LC/Q-TOF

Significance of the Topic

The presence of host cell proteins (HCPs) in biopharmaceutical products poses risks to safety and efficacy and must be monitored according to regulatory requirements. Traditional immunoassays lack specificity and coverage for individual HCPs, while LC-MS techniques offer improved identification and quantification. Overcoming challenges in separating low-abundance HCP peptides from the dominant therapeutic protein matrix and achieving broad dynamic range is critical for reliable HCP analysis.

Study Objectives and Overview

This work presents an end-to-end HCP analysis workflow integrating automated sample preparation on the Agilent AssayMAP Bravo, high-throughput LC separation on the Agilent 1290 Infinity II, MS/MS data acquisition with the Agilent 6545XT AdvanceBio Q-TOF, and vendor-neutral data processing via Protein Metrics software. The study evaluates sensitivity, dynamic range, reproducibility, and semiquantitative capability, comparing conventional Auto MS/MS to a novel Iterative MS/MS approach and assessing the benefit of high-pH reversed-phase (HPRP) fractionation.

Methodology and Instrumentation

The workflow includes:

• Sample spiking: Proteomics dynamic range standard (UPS2) spiked into a human IgG1 mAb at 1:1,000 (w/w), with an unspiked control.
• Automated prep: Reduction, alkylation, tryptic digestion, desalting, and optional high-pH fractionation into six fractions using Agilent AssayMAP Bravo with RP-S cartridges.
• LC-MS/MS: Agilent 1290 Infinity II LC with AdvanceBio Peptide Plus column (2.1×150 mm, 2.7 µm) at 60 °C, 0.4 mL/min, 60 min gradient; Agilent 6545XT AdvanceBio Q-TOF with Dual Jet Stream ESI source.
• Acquisition modes: Conventional Auto data-dependent MS/MS and iterative exclusion-based MS/MS, enhancing precursor selection for low-abundance peptides.
• Data processing: Byonic for database search against CHO K1, mAb, and UPS2 sequences (20 ppm precursor tolerance, semispecific trypsin, variable modifications), followed by Byologic for detailed analysis and normalization.

Main Results and Discussion

Comparison of Auto and Iterative MS/MS without fractionation:

• Iterative MS/MS yielded higher unique peptide counts for both mAb heavy and light chains and all UPS2 proteins, including low-abundance species down to 8 ppm.
• The system demonstrated a broad dynamic range (10^3 to 10^8 peak intensities) and excellent LC reproducibility (RSD ≤ 10 % for low-abundance peptides).

Semiquantitative profiling:

• Normalized extracted ion chromatogram (XIC) values correlated with actual UPS2 spike-in levels, enabling semiquantitative estimation of HCP abundance.

Impact of HPRP fractionation:

• On-cartridge high-pH reversed-phase fractionation improved identification sensitivity, detecting all spiked proteins above 2 ppm with high confidence.
• Endogenous CHO HCP identifications increased more than three-fold compared to unfractionated analyses.

Practical Benefits and Applications

This automated workflow delivers high throughput, reproducibility, and scalability, offering improved specificity and coverage for HCP profiling in biotherapeutic development and QC. Iterative MS/MS and HPRP fractionation enhance detection of low-level impurities, supporting regulatory compliance and product safety.

Future Trends and Applications

Advancements may include integration of targeted quantification assays, deeper proteome coverage with additional fractionation strategies, real-time data analytics for process monitoring, and adaptation to novel biologic modalities (e.g., ADCs, cell- and gene-therapy products). Enhancements in software automation and machine learning could further streamline HCP identification and quantification.

Conclusion

The combined Agilent AssayMAP Bravo, 6545XT AdvanceBio LC/Q-TOF, and Protein Metrics software workflow achieves sensitive, reproducible, and semiquantitative HCP analysis. Iterative MS/MS and on-cartridge HPRP fractionation significantly improve detection of low-abundance host cell proteins, providing a robust platform for biopharmaceutical quality control.

Reference

1 ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products
2 Agilent Technologies. Automation for LC/MS Sample Preparation: High Throughput In-Solution Digestion and Peptide Cleanup Enabled by the Agilent AssayMAP Bravo Platform (publication 5991-2957EN)
3 Agilent Technologies. Automation of Sample Preparation for Accurate and Scalable Quantification and Characterization of Biotherapeutic Proteins Using the Agilent AssayMAP Bravo Platform (publication 5991-4872EN)
4 Agilent Technologies. Agilent AssayMAP Bravo Technology Enables Reproducible Automated Phosphopeptide Enrichment from Complex Mixtures Using High Capacity Fe(III)-NTA Cartridges (publication 5991-6073EN)