An Online Two-Dimensional Approach to Characterizing the Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using a 2D-CEX–AEX–MS Workflow

Significance of the Topic

Charge heterogeneity in monoclonal antibodies influences stability pharmacokinetics and therapeutic efficacy Characterizing these variants is essential for quality control process optimization and ensuring consistent product performance

Goals and Study Overview

This work presents an online two dimensional CEX–AEX–MS workflow designed to resolve overlapping charge variants that are challenging to separate in one dimensional analyses Three recombinant mAb samples were investigated to compare standalone cation exchange standalone anion exchange and the combined 2D approach

Methodology and Instrumentation

Volatile ammonium acetate buffers were employed to enable direct coupling with mass spectrometry In the first dimension cation exchange chromatography (CEX) was performed on an Agilent Bio SCX NP5 column followed by a second dimension anion exchange chromatography (AEX) on an Agilent Bio SAX NP5 column Both dimensions were hyphenated with native mass spectrometry Instrumentation

• Agilent 1290 Infinity II two dimensional LC system with integrated 2D valve configuration
• Agilent Bio SCX NP5 and Bio SAX NP5 columns (4.6 × 250 mm 5 μm)
• Agilent 6545XT AdvanceBio LC Q-TOF MS/MS

Main Results and Discussion

The 2D method identified more charge variants than standalone approaches For mAb I thirteen variants were resolved in 2D compared to ten by CEX and eight by AEX Similar improvements were observed for mAb II and mAb III Detected modifications included multiple degrees of deamidation glycosylation heterogeneity aspartic acid isomerization and glycan truncation The volatile salt system eliminated desalting artifacts and enabled in-depth native MS characterization

Benefits and Practical Applications

• Enhanced resolution of closely related charge variants improves product quality assessment
• Reduced sample preparation by avoiding nonvolatile buffer exchange
• Comprehensive variant profiling supports process development regulatory compliance and batch release decisions

Future Trends and Potential Applications

Incorporation of additional orthogonal separations such as capillary electrophoresis or hydrophobic interaction chromatography could further enrich variant detection Advances in high resolution MS and automated 2D-LC platforms may enable high throughput workflows for routine biopharmaceutical analysis

Conclusion

The online 2D CEX–AEX–MS workflow offers a robust strategy for in-depth characterization of monoclonal antibody charge heterogeneity By combining complementary ion exchange modes with native MS this approach reveals subtle variant differences that inform development and ensure product consistency

Reference

• Grunert I Heinrich K Hingar M et al Comprehensive multidimensional liquid chromatography mass spectrometry for bispecific antibody charge variant characterization J Am Soc Mass Spectrom 33 12 2319 2327 2022
• Yan Y Liu AP Wang S et al Ultrasensitive characterization of therapeutic antibody charge heterogeneity using strong cation exchange with native mass spectrometry Anal Chem 90 21 13013 13020 2018
• Kumar S Savane TS Rathore AS Multiattribute monitoring of aggregates and charge variants through native two dimensional SEC MS WCX MS J Am Soc Mass Spectrom 34 1263 1271 2023