Quantification of Host Cell Protein Impurities using the Agilent 6495C Triple Quadrupole LC/MS

Importance of the Topic

Monoclonal antibody therapeutics produced in host cells can contain trace amounts of host cell protein (HCP) impurities. Regulatory agencies require tight control of HCPs to ensure drug safety and efficacy. Traditional immunoassays, such as ELISA, lack the ability to identify and quantify individual HCPs in complex biologic matrices. High-sensitivity LC/MS workflows offer enhanced specificity and coverage, enabling precise quantification at sub-ppm levels.

Objectives and Overview

The aim of this study was to establish a robust, fully integrated workflow for the quantification of low-abundance HCPs in an IgG1 monoclonal antibody matrix using MRM-based isotope dilution on an Agilent 6495C triple quadrupole LC/MS. Key goals included automated sample preparation, high-resolution chromatographic separation, sensitive MS detection, and streamlined data analysis.

Methodology and Instrumentation

• Equipment Used:
  
  • Agilent AssayMAP Bravo automated sample preparation system for protein denaturation, reduction, alkylation and trypsin digestion.
  • Agilent 1290 Infinity II UHPLC with high-speed pump, multisampler with cooling, and thermostatted column compartment.
  • Agilent 6495C triple quadrupole LC/MS with Jet Stream ESI source operating in positive ion dMRM mode.
• Sample Preparation:
  
  • Monoclonal antibody purified from CHO cells subjected to denaturation, reduction, alkylation and on-cartridge trypsin digestion.
  • Three heavy stable isotope-labeled (SIL) peptides representing two exogenous protein standards (SUMO1 and SYHC) and one endogenous HCP (CHO S100-A11) spiked at eight concentration levels (6.25–125,000 amol/μg).
• LC/MS Conditions:
  
  • Reversed-phase C18 column at 60 °C, flow rate 0.5 mL/min, 13 min gradient from 3 % to 90 % acetonitrile with 0.1 % formic acid.
  • dMRM acquisition with optimized collision energies, cycle time 500 ms, JetStream source settings: 150 °C gas temp, 19 L/min drying gas, 35 psi nebulizer, 250 °C sheath gas.
• Data Processing:
  
  • LC-dMRM method development and retention time scheduling via Agilent Automation tool integrated with Skyline and MassHunter Quantitative Analysis.
  • Quantitative analysis performed in Skyline and MassHunter using standard curves and light/heavy peak area ratios.

Main Results and Discussion

Standard curves for the three SIL peptides (LLLEYLEEK, VFDVIIR, DPGVLDR) displayed excellent linearity (R² ≥ 0.999) across a dynamic range spanning low sub-ppm to over 1,000 ppm (6.25 amol/μg to 125 fmol/μg). The lower limit of quantitation (LLOQ) reached as low as 0.13 ppm for CHO S100-A11, 0.24 ppm for SUMO1, and 0.35 ppm for SYHC, with precision (RSD ≤ 18 % at LLOQ) and accuracy within 15 % at LLOQ and better than 10 % at higher levels. Retention time reproducibility was outstanding (RSD ≤ 0.5 %). Absolute quantification of the endogenous HCP S100-A11, using either external calibration or light/heavy ratio methods, yielded comparable results (1.05 vs. 1.47 ppm), demonstrating the workflow’s reliability for trace-level measurement.

Benefits and Practical Applications

• Sub-ppm sensitivity and broad dynamic range for HCP quantification.
• Automated sample prep increases throughput, reproducibility and reduces manual error.
• Integration of Skyline and MassHunter enables rapid method development, optimization and data review.
• Applicable for process development, quality control and regulatory compliance in biologics manufacturing.

Future Trends and Applications

Advances in high-resolution MS, automation, and data processing software are poised to further lower detection limits and expand HCP coverage. Emerging technologies such as microflow LC, ion mobility and machine-learning–driven data analysis will enhance specificity and throughput. The integration of HCP quantification into continuous manufacturing and real-time release testing represents a promising direction for biopharmaceutical quality assurance.

Conclusion

The presented workflow combining Agilent AssayMAP Bravo, 1290 Infinity II LC, and 6495C triple quadrupole LC/MS with robust software integration offers a powerful solution for accurate, sensitive quantification of host cell protein impurities at sub-ppm levels. This platform supports regulatory requirements and provides a scalable approach for biologics quality control.

References

1. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products.
2. Host Cell Protein Analysis Using Agilent AssayMAP Bravo and 6545XT AdvanceBio LC/Q-TOF. Agilent Technologies Application Note, 5991-9300EN, 2018.
3. Agilent Triple Quadrupole LC/MS Peptide Quantitation with Skyline. Agilent Technologies Application Note, 5990-9887EN, 2017.