Multi-attribute monitoring of aggregates and charge variants of monoclonal antibody through native 2D- SEC-MS-WCX-MS

Importance of the Topic

Biopharmaceutical products such as monoclonal antibodies (mAbs) require rigorous control of critical quality attributes (CQAs), including size and charge heterogeneities. Aggregates and fragments can impact efficacy and safety, while charge variants reflect post-translational modifications that influence stability and function. A robust analytical workflow that simultaneously monitors both attributes in native form enhances process understanding and supports regulatory compliance.

Objectives and Study Overview

This study introduces a novel two-dimensional liquid chromatography–mass spectrometry (2D-LC-MS) method combining size-exclusion chromatography (SEC) and weak cation exchange (WCX) separations, each directly hyphenated to mass spectrometry. The goal was to implement a native 2D-SEC-MS-WCX-MS workflow for a model mAb (mAb A) to achieve concurrent characterization and quantification of aggregates, monomers, and charge variants in a single run.

Methodology and Instrumentation

The first dimension employed an AdvanceBio SEC column (300 Å, 4.6 × 150 mm, 2.7 µm) to resolve size variants under native conditions. A heart-cut of the main SEC peak was diverted to the second dimension via a 2D-LC Active Solvent Modulation (ASM) valve, reducing salt concentration five-fold before loading onto an Agilent Bio MAb WCX column (NP5, 2.1 × 250 mm, 5 µm). Both chromatographic outlets were directly coupled to an Agilent 6545XT AdvanceBio LC/Q-TOF system with Dual Agilent JetStream electrospray ionization for continuous mass analysis. The workflow completed in 25 minutes, contrasting with 90 minutes for separate SEC and WCX runs.

Results and Discussion

The native 2D-SEC-MS-WCX-MS approach produced clear SECUV and WCX profiles of mAb A, with uninterrupted MS acquisition throughout. Charge-state distributions confirmed native ionization. Deconvolution of mass spectra identified multiple acidic variants (deamidation, glycation), the unmodified main species, and basic variants (oxidation). Stress studies under elevated temperature and photo-irradiation demonstrated reliable quantification of monomer, dimer, and charge variant shifts. The method required no manual fraction collection and provided intact mass data for both dimensions in a single sequence.

Benefits and Practical Applications

• Simultaneous monitoring of size and charge CQAs in one run.
• Native mass analysis without offline processing, preserving higher-order structure.
• Reduced total analysis time and simplified workflow.
• Enhanced throughput for stability testing, formulation screening, and comparability studies.

Future Trends and Opportunities

• Integration of additional separation modes (e.g., hydrophobic interaction chromatography) for multi-attribute profiling.
• Automation with real-time data processing and quality control alerts.
• Application to other complex biologics such as fusion proteins or bispecific antibodies.
• Adoption in regulated environments as part of multi-attribute method (MAM) strategies.

Conclusion

The native 2D-SEC-MS-WCX-MS workflow offers a rapid, comprehensive, and automated solution for concurrent analysis of mAb size and charge variants. By streamlining sample handling and leveraging direct MS coupling, this method enhances analytical efficiency and data richness, facilitating accelerated biopharmaceutical development and quality control.

References

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