Multi-Attribute Monitoring of Antibody Variants through Hydrophobic and Cation- Exchange Chromatography Coupled with Native Mass Spectrometry

Importance of the Topic

Characterizing multiple structural and charge-related variants of therapeutic monoclonal antibodies is critical for ensuring product quality, safety and efficacy in the biopharmaceutical industry. Integrating orthogonal separation techniques with native mass spectrometry enables comprehensive multi-attribute monitoring in a single automated workflow, reducing analysis time and sample loss.

Objectives and Study Overview

This study aimed to develop and demonstrate a two-dimensional liquid chromatography–mass spectrometry (2D-LC/MS) platform that couples hydrophobic interaction chromatography (HIC) and weak cation exchange chromatography (WCX) under native conditions. The goal was to resolve and characterize both hydrophobic and charge variants of a model monoclonal antibody (mAb A) in an online heart-cut arrangement.

Methodology and Instrumentation

An online 2D-LC arrangement was implemented on an Agilent 1290 Infinity II UHPLC system using volatile buffer salts compatible with native mass spectrometry. The first dimension separated hydrophobic variants on an AdvanceBio HIC column under a decreasing salt gradient. Individual HIC peaks were transferred via a heart-cut method into the second dimension, where charge variants were resolved on a Bio WCX NP5 column. The eluate was then introduced directly into a 6545XT AdvanceBio Q-TOF mass spectrometer operated in native mode.

Used Instrumentation

• Agilent 1290 Infinity II UHPLC with AdvanceBio HIC column (4.6 × 100 mm, 3.5 µm)
• Agilent Bio WCX NP5 column (4.6 × 250 mm, 5 µm) on the same UHPLC platform
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Agilent JetStream ESI source

Main Results and Discussion

• HIC-MS under native conditions resolved mAb A into one major and three minor hydrophobic peaks, exhibiting a charge-state envelope of +21 to +29 and m/z 5000–7000.
• WCX-MS separated charge variants into four acidic peaks, a main peak and two basic peaks, confirming native ionization and high resolution of low-abundance variants.
• In the 2D workflow, heart-cut transfer of HIC fractions into WCX enhanced variant resolution for the main hydrophobic peak, while minor HIC fractions yielded single, low-resolution WCX peaks due to limited sample amount.

Benefits and Practical Applications

The integrated 2D-LC/MS approach streamlines multi-attribute profiling by combining orthogonal separations with direct native mass analysis. It minimizes manual handling, reduces sample consumption and accelerates characterization workflows for monoclonal antibodies in QC laboratories and research settings.

Future Trends and Potential Applications

Advances in 2D-LC automation and MS-compatible chemistries will extend this approach to other classes of biotherapeutics, including fusion proteins and viral vectors. Integration with high-resolution mass analyzers and data-driven analytics will further improve sensitivity, variant identification and real-time process monitoring.

Conclusion

The presented 2D-LC/MS platform effectively separates and characterizes both hydrophobic and charge variants of mAb A under native conditions. This unified workflow offers a robust solution for comprehensive multi-attribute monitoring of biotherapeutic products.

References

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