Characterization and Monitoring of mAb Charge Variants via Online IEX-MS

Significance of the Topic

Monoclonal antibodies (mAbs) represent a major class of biotherapeutics whose clinical efficacy and safety depend strongly on their charge heterogeneity. Variations such as deamidation, glycosylation and C-terminal lysine processing can affect stability, activity and immunogenicity. Rapid and detailed profiling of these charge variants is essential for quality control, biosimilar comparison and formulation development.

Objectives and Study Overview

The presentation addresses the development and application of a direct online coupling between ion exchange chromatography (IEX) and mass spectrometry (MS) for mAb charge variant characterization. Key goals include:

• Eliminating tedious fraction collection and 2D-LC workflows
• Maintaining native protein structure during analysis
• Achieving robust, reproducible separation across multiple mAbs
• Optimizing MS parameters for signal and resolution

Methodology

Chromatographic separation was performed using a BioResolve SCX column under native conditions. A volatile ammonium-based dual salt/pH gradient enabled effective elution of acidic, main and basic variants while preserving native structure. Salt content and pH were systematically varied by SEC-MS experiments to balance separation efficiency and MS sensitivity. Instrument parameters such as source temperature, desolvation gas flow and cone voltage were optimized on a Vion IMS QTof platform to enhance signal by over three-fold.

Instrumentation

• Waters BioResolve SCX, 3 µm, 2.1 × 50 mm and 2.1 × 100 mm columns
• Ammonium acetate/formate dual salt buffers for volatile IEX gradients
• Waters Vion IMS QTof mass spectrometer
• Optimization: source temp. 120 °C, desolvation temp. 350 °C, cone gas 100 L/h, desolvation gas 600 L/h
• SEC-MS setup for pH and ionic strength studies

Main Results and Discussion

A robust IEX-MS method was demonstrated with triplicate injections of infliximab showing consistent UV and conductivity gradients. A single gradient profile resolved charge variants for NIST mAb, rituximab, infliximab, trastuzumab and cetuximab. MS deconvolution confirmed C-terminal lysine variants (+128 Da, +256 Da) and distinct glycoforms (G0F, G1F, G2F, Man5). Forced degradation of trastuzumab at pH 8 highlighted increases in acidic species mapped predominantly to the Fab region via IdeS digestion. Biosimilar comparison of infliximab revealed subtle shifts in variant abundances, confirmed online by MS.

Benefits and Practical Applications

• Rapid decision-making without offline fractionation
• Native-state analysis preserves functional variants for downstream assays
• High throughput profiling of multiple mAbs with a single gradient
• Accurate mass assignments of deamidation, glycosylation and lysine clipping
• Streamlined workflows reducing dependency on 2D-LC and manual collection

Future Trends and Possibilities

Advances may include integration with automated sample preparation, expansion to multi-attribute monitoring (MAM) workflows, use of higher-resolution MS platforms and AI-driven data analysis. The approach could be extended to other complex biologics, bispecifics and antibody-drug conjugates. Continued optimization of volatile buffer chemistries will further improve sensitivity and robustness.

Conclusion

Online coupling of IEX with MS under native conditions provides a powerful, streamlined platform for comprehensive mAb charge variant characterization. This method delivers robust separations, high-confidence mass assignments and significant reductions in analysis time and complexity compared with traditional fractionation-based workflows.

References

• Protein Cell. 2018 Jan;9(1):86–120
• Birdsall R, et al. Characterization of Biotherapeutics: ACQUITY UPLC H-Class Bio with 2D Part 2 of 3. Waters Corporation.
• Ayoub D, et al. mAbs. 2013;5(5):699–710