Coupling ion exchange chromatography with mass spectrometry to characterize charge heterogeneity of mAbs under near-native conditions using a ProPac 3R SCX column

Significance of the Topic

Monoclonal antibodies (mAbs) are a rapidly growing class of biotherapeutics whose safety and efficacy depend on detailed characterization of charge variants introduced by post-translational modifications. Coupling ion exchange chromatography (IEX) with high-resolution mass spectrometry (MS) under near-native conditions enables comprehensive analysis of these critical quality attributes directly in a biologically relevant state.

Objectives and Study Overview

This study aimed to develop a robust, MS-compatible cation exchange chromatography method using a new ProPac 3R SCX column and volatile pH gradient buffers to

• Achieve high-resolution separation of mAb charge variants
• Demonstrate compatibility with native MS detection
• Validate method performance across different mAbs and column lots
• Monitor changes in variant profiles following thermal stress

Methodology and Instrumentation

Sample preparation involved diluting commercial durvalumab to 1 mg/mL and subjecting aliquots to heat stress at 50 °C for up to 7 days. Mobile phases were prepared using 25 mM ammonium bicarbonate/30 mM acetic acid at pH 5.3 and 10 mM ammonium hydroxide at pH 10.2, refreshed within 48 h.

The main analytical setup comprised:

• Thermo Scientific Vanquish Flex UHPLC with ProPac 3R SCX column (2×50 mm, 3 µm)
• Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer
• Chromeleon CDS and BioPharma Finder software
• Native MS parameters: HMR mode, 35 000 resolution, 2 500–8 000 Da m/z range, 3.6 kV spray voltage

Results and Discussion

Optimization of the MS-compatible pH gradient (15 to 75 % mobile phase B over 28 min) produced sharp, well-resolved acidic and basic variant peaks. The method was successfully extended to other therapeutic mAbs (rituximab, denosumab) by adjusting the upper pH to accommodate higher pI values.

Lot-to-lot reproducibility was confirmed with two ProPac 3R SCX column batches over three days, showing peak area RSDs below 5 %.

Thermal stress induced progressive increases in acidic variants (deamidation, oxidation) and declines in major basic peaks. Native MS deconvolution mapped:

• Acidic peaks: stepwise deamidation and glycoform shifts
• Main peak: lysine truncation variants
• Basic peaks: C-terminal truncations and oxidation patterns
• New minor peaks after prolonged stress revealed additional modifications

Benefits and Practical Applications

The developed SCX-MS workflow offers:

• High-resolution separation under near-native conditions
• Straightforward buffer preparation without nonvolatile salts
• Applicability to multiple mAb therapeutics
• Robust lot-to-lot and day-to-day reproducibility
• Detailed MS-based mapping of charge variants for CQA assessment

Future Trends and Applications

Further development may include:

• Integration with high-throughput platforms for early-stage screening
• Automation of buffer preparation and gradient optimization
• Expansion to other protein classes and bispecific antibodies
• Coupling with ion mobility or advanced fragmentation for deeper structural insights
• AI-driven data analysis to predict stability profiles

Conclusion

The ProPac 3R SCX column combined with a volatile pH gradient buffer provides a powerful, MS-compatible platform for high-resolution characterization of mAb charge heterogeneity. The method demonstrates excellent reproducibility, broad applicability, and detailed insight into stress-induced modifications, supporting stringent biopharma quality control requirements.

References

1. Wu G et al. High-resolution IEX-MS for mAb charge heterogeneity. Anal Chem 2023, 95, 2548–2560.
2. Liu AP et al. Volatile buffer cation exchange for native MS. Anal Chem 2022, 94, 6355–6362.