Monoclonal Antibody Charge Variants Identification by Fully Automated Capillary Isoelectric Focusing – Mass Spectrometry

Importance of the Topic

Monoclonal antibodies are key biotherapeutics whose efficacy, stability and safety depend on precise control of charge heterogeneity arising from modifications such as deamidation, glycosylation and C-terminal variants. Direct and reliable identification of these charge isoforms accelerates development, supports quality control and ensures regulatory compliance.

Objectives and Study Overview

This work presents a fully automated capillary isoelectric focusing–mass spectrometry workflow for direct mass determination of mAb charge variants. The approach leverages an Agilent 7100A CE instrument coupled via a CMP Scientific EMASS-II CE/MS ion source to Agilent 6230 accurate-mass TOF or 6545 Q-TOF mass spectrometers. System performance, reproducibility and application to the IgG1 therapeutic daratumumab are demonstrated.

Methodology

• Capillary isoelectric focusing in a 75 cm PS1 coated capillary using 1 percent formic acid anolyte and 0.2 N ammonium hydroxide catholyte, both containing 15 percent glycerol.
• Sample buffer composed of 5 mM ammonium acetate, 1–1.5 percent ampholyte pH 3–10 and 15 percent glycerol.
• Sequential injection under 950 mbar: catholyte plug, then sample plug; focusing at 18.8 kV and chemical mobilization by acidic sheath liquid.
• Nanospray ESI with a 30 µm tip, 2.2–2.4 kV spray voltage, sheath liquid of 20 percent acetic acid/25 percent acetonitrile.
• TOF/Q-TOF acquisition at 0 V capillary voltage, 6 L/min drying gas at 365 °C and 380 V fragmentor voltage.

Instrumentation Used

• Agilent 7100A Capillary Electrophoresis system
• CMP Scientific EMASS-II CE/MS ion source
• 75 cm PS1 coated capillaries (0.75 mm id)
• Agilent 6230 accurate-mass TOF LC/MS
• Agilent 6545 Q-TOF LC/MS

Main Results and Discussion

• System performance verified by CZE-MS of lysozyme and ribonuclease A, showing sharp peaks, minimal tailing and clear separation.
• CIEF-MS validation with cytochrome C and myoglobin yielding focused peaks corresponding to known pI values.
• Five overnight injections of daratumumab showed relative migration time RSD of 5.8 percent, confirming reproducibility.
• Daratumumab charge profile resolved into a basic variant (N-terminal pyroglutamate), main form and two acidic variants (deamidation and lysine glycation) by intact mass deconvolution.

Benefits and Practical Applications

• Automated CIEF separation and MS detection reduces hands-on time and risk of sample loss.
• Direct intact mass measurement of charge isoforms streamlines biopharmaceutical characterization and comparability studies.
• High resolution of charge heterogeneity enhances quality control and process monitoring.

Future Trends and Potential Applications

Ongoing developments may extend this workflow to diverse biomolecules including bispecific antibodies and fusion proteins, introduce negative-ion mode for acidic analytes, improve capillary coatings for higher throughput, and integrate with data analytics and laboratory information systems for real-time release testing.

Conclusion

The fully automated CIEF-MS workflow on Agilent CE and TOF/Q-TOF platforms with the EMASS-II ion source delivers robust, high-throughput separation and direct mass spectrometric identification of monoclonal antibody charge variants, supporting pharmaceutical R&D and quality assurance of biotherapeutics.

References

• Zhu G Sun L Dovichi NJ Simplified Capillary Isoelectric Focusing with Chemical Mobilization for Intact Protein Analysis J Sep Sci 2017 40(4) 948–953
• Dai J et al Capillary Isoelectric Focusing-Mass Spectrometry Method for the Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants Anal Chem 2018 90(3)
• Dai J Zhang Y A Middle-Up Approach with Online Capillary Isoelectric Focusing/Mass Spectrometry for In-Depth Characterization of Cetuximab Charge Heterogeneity Anal Chem 2018 90(24) 14527–14534
• Ouyang Y et al Negative-Ion Mode Capillary Isoelectric Focusing Mass Spectrometry for Charge-Based Separation of Acidic Oligosaccharides Anal Chem 2019 91(1) 846–853