Online IEX-MS Characterization and Monitoring of mAb Charge Heterogeneity Using an Optimized Cation Exchange Resin and Compact TOF Mass Spectrometer

Significance of the Topic

The precise analysis of monoclonal antibody (mAb) charge heterogeneity is critical for ensuring drug efficacy, safety, and batch‐to‐batch consistency in biopharmaceutical development. Integrating ion exchange chromatography with mass spectrometry (IEX‐MS) under MS-compatible conditions addresses long‐standing challenges in direct variant characterization.

Objectives and Study Overview

This work demonstrates an optimized volatile‐salt cation exchange method coupled to a compact benchtop time‐of‐flight mass spectrometer. The approach is applied to forced‐degraded trastuzumab and reference mAbs (NIST mAb, infliximab) to monitor and assign acidic, main, and basic charge variants in a single, streamlined workflow.

Methods and Instrumentation

• Chromatography: ACQUITY I-class UPLC with BioResolve SCX column (2.1×50 mm, 3 μm) at 30 °C, 0.1 mL/min.
• Mobile Phases: Proprietary volatile buffers enabling a dual pH/salt gradient for strong cation exchange separation.
• Mass Spectrometry: BioAccord compact TOF with RDa detector; capillary voltage 1.5 kV, cone voltage 150 V, desolvation at 350 °C, Intelligent Data Capture enabled.
• Sample Prep: Trastuzumab buffer‐exchanged into 100 mM phosphate pH 8.0, incubated at 25 °C for 1 week; IdeS digestion for subunit analysis; 10 μL injections of 1 mg/mL samples.

Main Results and Discussion

• Baseline separation of acidic, main, and basic species achieved for NIST mAb, infliximab, and trastuzumab.
• MS deconvolution confirmed deamidation (+1 to +7 Da) and C-terminal lysine variants (+128 Da per lysine addition).
• After 1 week pH 8 stress, trastuzumab acidic variants increased by ~18.7% in nonreduced form; IdeS subunits showed 14.1% increase in Fab acidic species and 4.5% in Fc acidic species.
• Online IEX-MS eliminated offline fractionation and buffer exchange, reducing potential artifacts and enabling native MS detection directly from the chromatographic effluent.

Benefits and Practical Applications

• High‐throughput monitoring of charge heterogeneity essential for stability studies and quality control.
• Rapid hypothesis generation for near‐isobaric variants without specialized MS facilities.
• Ability to collect separated fractions for downstream peptide mapping and potency assays.
• Accessible benchtop platform supports routine biopharmaceutical analytics.

Future Trends and Potential Applications

• Development of next‐generation ion exchange materials for finer resolution of closely spaced variants.
• Automated 2D‐LC-MS workflows combining orthogonal separations for deeper variant profiling.
• Integration of MS/MS experiments for definitive site‐specific modification assignments.
• Adoption as a process analytical technology (PAT) tool for real‐time monitoring in biomanufacturing.

Conclusion

The optimized volatile‐salt IEX-MS workflow with a compact TOF mass spectrometer provides a robust, high‐performance solution for direct characterization and monitoring of mAb charge heterogeneity. This streamlined approach enhances data quality, reduces analysis time, and supports critical decision points in biopharmaceutical development.

References

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