Advancing Host Cell Protein Analyses Through the Combined Use of Microscale 2D RP/RP with CSH C18 and Ion Mobility Enabled MS Detection

Significance of the Topic

The identification and quantitation of host cell protein (HCP) impurities in biotherapeutics is critical to product safety and regulatory compliance. High peak capacity separations and sensitive MS detection are essential to resolve trace-level protein contaminants within complex peptide mixtures.

Objectives and Study Overview

This study evaluates a microscale two-dimensional reversed-phase/reversed-phase (2D-RP/RP) LC-MS workflow combining charged surface hybrid C18 (CSH C18) columns and ion mobility-enabled HDMSE detection. The goal is to improve loadability, resolution, and detection limits for HCP analysis at single-digit ppm levels.

Methodology and Instrumentation

A monoclonal antibody (IgG1K) digest spiked with a calibrant tryptic digest was separated using a 10-step 2D-RP/RP scheme:

• First dimension: high-pH RP on XBridge Peptide BEH C18 trap and analytical column.
• Second dimension: low-pH RP on CSH C18 analytical column or conventional silica C18 for comparison.
• Ion mobility-enabled MS detection (HDMSE) on a SYNAPT G2-S system.

Key instrumentation:

• ACQUITY UPLC M-Class System
• ACQUITY UPLC M-Class CSH™ C18 Column (300 µm × 150 mm, 1.7 µm)
• ACQUITY UPLC M-Class Symmetry® C18 2D HCP Trap Column (300 µm × 25 mm, 5 µm)
• XBridge Peptide BEH C18 Column (1.0 × 50 mm, 5 µm)
• SYNAPT G2-S Mass Spectrometer with HDMSE capability

Main Results and Discussion

The CSH C18 2nd dimension column maintained peak capacity at sample loads five times higher than conventional silica C18, yielding sharper peaks and reduced co-elution. Compared to a 0.2 mg digest with silica C18 and MSE detection (5 HCPs identified), the 1.0 mg digest with CSH C18 and HDMSE enabled detection of 14 HCPs. Hi3 quantitation estimated seven HCPs below 20 ppm, with the lowest at 2 ppm and 6 ppm, demonstrating enhanced dynamic range and sensitivity.

Benefits and Practical Applications

• Improved chromatographic loadability and resolution at high sample loads.
• Reduced spectral crowding and improved peptide fragment assignment via ion mobility.
• Lower detection limits for HCP impurities, enabling more comprehensive safety profiling.
• Robust, user-friendly high-pressure microscale LC-MS workflow applicable to QA/QC and proteomics.

Future Trends and Potential Applications

Advances may include integration of ultra-high-pressure systems (>15K psi), novel stationary phases for enhanced selectivity, and AI-driven data analysis pipelines. Expansion to other low-abundance biomarker studies and streamlined automated workflows is anticipated.

Conclusion

The combined use of microscale 2D-RP/RP with CSH C18 and HDMSE detection significantly advances HCP analysis by improving peak capacity at high loads and achieving single-digit ppm detection. This approach offers a robust platform for comprehensive impurity profiling in biotherapeutics.

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