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

Ion exchange chromatography coupled with mass spectrometry (IEX-MS) has become a cornerstone for detailed charge variant analysis of monoclonal antibodies (mAbs). Charge heterogeneity can impact drug potency, stability, and immunogenicity, making sensitive monitoring of deamidation, glycosylation, and other modifications essential during biotherapeutic development and quality control.

Study Objectives and Overview

This work presents the development and validation of an online IEX-MS method that combines a volatile ammonium-based dual salt/pH gradient with a compact time-of-flight (TOF) mass spectrometer to characterize charge variants in mAbs. A case study on pH-stressed trastuzumab demonstrates the method’s ability to resolve and identify acidic and basic species both in intact and subunit (IdeS-digested) formats.

Methodology and Instrumentation

• Chromatography: ACQUITY BioResolve SCX column (3 µm, 2.1×50 mm) at 30 °C, flow rate 0.1 mL/min.
• Mobile phases: Ammonium acetate/formate dual-salt gradient with pH ramp (2–98% B over 20 min), total run time 30 min.
• Mass spectrometry: BioAccord compact TOF MS with RDa detector, capillary voltage 1.5 kV, cone 150 V, desolvation temp 350 °C, intelligent data capture enabled.
• Sample preparation: Trastuzumab buffer exchanged into 100 mM phosphate pH 8.0, half stored at –80 °C, half incubated at 25 °C for 1 week; IdeS digestion for subunit analysis.

Key Results and Discussion

Online IEX-MS delivered well-resolved acidic, main, and basic peaks for both intact and IdeS-digested trastuzumab. Notable findings include:

• Upon 1 week pH stress, (Fab)₂ acidic species increased by 14.1%, and (Fc/2)₂ acidic variants rose by 4.5% relative to unstressed material.
• Detected modifications include deamidation (+1 to +2 Da), sialylation (+292 Da), and potential disulfide or conformational variants.
• Nonreduced analysis revealed an 18.7% increase in total acidic species for intact trastuzumab.
• The method separated C-terminal lysine variants in infliximab and NIST mAb standards, confirming assignments by accurate mass deconvolution.

Benefits and Practical Applications

This streamlined workflow eliminates offline fraction collection and buffer exchange, reducing sample handling artifacts. Direct MS detection of resolved peaks accelerates variant identification and supports fraction collection for downstream peptide mapping or potency assays. High throughput capability suits stability studies and batch release testing in biopharmaceutical environments.

Future Trends and Opportunities

Advances in IEX resin chemistries and miniaturized high-resolution MS promise further improvements in sensitivity and separation power. Integration with automation platforms and machine-learning–driven data analysis may enable real-time monitoring of bioprocess charge variants. Expansion to other biotherapeutic formats (e.g., antibody–drug conjugates) represents an emerging application area.

Conclusion

The optimized online IEX-MS method using volatile salt gradients and a compact TOF instrument provides robust, high-resolution characterization of mAb charge heterogeneity. It offers significant advantages for rapid variant profiling, stress testing, and quality control in antibody development.

References

• Yan Y et al. Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry. Analytical Chemistry 2018;90(21):13013–13020.
• Leblanc Y et al. Charge Variants Characterization of a Monoclonal Antibody by Ion Exchange Chromatography Coupled On-Line to Native Mass Spectrometry: Case Study After a Long-Term Storage at +5 °C. J Chromatogr B Analyt Technol Biomed Life Sci 2017;1048:130–139.
• Fussl F et al. Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to High-Resolution Native Mass Spectrometry. Analytical Chemistry 2018;90(7):4669–4676.
• Diepold K et al. Simultaneous Assessment of Asp Isomerization and Asn Deamidation in Recombinant Antibodies by LC-MS Following Incubation at Elevated Temperatures. PLoS ONE 2012;7(1):e30295.