Charge Variant Analysis of Monoclonal Antibodies and ADCs Using CE/MS

Importance of the Topic

The heterogeneity of protein therapeutics influences key quality attributes including stability, efficacy, and immunogenicity. Accurate characterization of charge variants supports product consistency and safety in biopharmaceutical development.

Objectives and Overview

This work demonstrates a capillary isoelectric focusing–mass spectrometry (CIEF–MS) workflow for detailed charge variant analysis of adalimumab (Humira) and trastuzumab emtansine (Kadcyla). The approach aims to separate species by isoelectric point and directly identify chemical and structural modifications.

Methodology

Sample buffers were prepared using zwitterionic ampholytes spanning pH ranges matched to each therapeutic. A 7100 CE system generated a pH gradient in a coated capillary, driving variants to focus at their isoelectric points. An EMASS-II electrospray interface delivered the separated zones into an Agilent 6545XT AdvanceBio Q-TOF at nanoliter flow rates under low-voltage conditions. MassHunter BioConfirm software enabled deconvolution and assignment of variant masses.

Used Instrumentation

• CMP EMASS-II CE/MS ion source
• Agilent 7100 capillary electrophoresis system
• Agilent 6545XT AdvanceBio quadrupole time-of-flight mass spectrometer
• CIEF/MS reagent kit (CMP Scientific)

Main Results and Discussion

In adalimumab analysis, three charge variants were resolved: two basic forms associated with lysine truncations and one acidic form resulting from deamidation, with mass accuracy within 10 ppm. For trastuzumab emtansine, nine peaks corresponding to drug-antibody ratios from 0 to 8 were separated and identified, confirming glycoform patterns and incremental drug loads of approximately 956 Da.

Benefits and Practical Applications

• Direct coupling of CIEF to MS enables simultaneous separation and identification of charge variants
• Minimal sample and reagent preparation using a dedicated kit streamlines workflows
• Nanoliter flow electrospray enhances sensitivity for low-abundance species
• Flexible method adapts to diverse proteins and antibody-drug conjugates

Future Trends and Opportunities

Integration of automated sample handling and data processing is expected to improve throughput. Advancements in top-down proteomics and microfluidic CE interfaces may further enhance resolution and speed. Real-time monitoring of charge profiles during bioprocessing represents an emerging application.

Conclusion

The presented CIEF–MS platform effectively separates and characterizes charge variants in monoclonal antibodies and ADCs, providing in-depth molecular insights for quality control and development. Its flexibility and sensitivity make it a valuable tool for biopharmaceutical characterization.

References

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3. Wang Y Monoclonal Antibody Charge Variants Identification by Automated CIEF-MS Agilent Technologies Application Note 5994-0672EN 2019
4. Dai J Capillary Isoelectric Focusing-MS for Intact Monoclonal Antibody Charge Variants Anal Chem 2018 90 2246-2254
5. Xia J Identification of NISTmAb Charge Variants by Automated CIEF-MS Agilent Technologies Application Note 5994-1079EN 2019
6. Toraño JS Advances in Capillary Electrophoresis for the Life Sciences J Chromatogr B 2019 1118-1119 116-136