2D-LC/MS Characterization of Charge Variants Using Ion Exchange and Reversed-Phase Chromatography

Significance of the Topic

Monoclonal antibodies (mAbs) are advancing therapeutics with strict quality requirements. Charge variant profiling ensures product consistency, efficacy, and safety by detecting post-translational modifications such as deamidation, oxidation, glycosylation, and C-terminal lysine truncation. Rapid and reliable identification of these variants is critical for comparability studies between innovator and biosimilar products.

Objectives and Study Overview

This application note describes a robust two-dimensional liquid chromatography-mass spectrometry (2D-LC/MS) workflow for charge variant analysis of Rituximab innovator and biosimilar. The study compares three first-dimension separation strategies—phosphate/NaCl salt gradient, phosphate pH gradient, and volatile ammonium formate salt gradient—evaluating retention time precision, resolution, and MS compatibility. The selected fractions are transferred by multiple heart cutting into a reversed-phase (RP) second dimension for desalting, denaturation, and intact mass analysis by Q-TOF MS.

Methodology and Instrumentation

• First dimension: Weak cation exchange chromatography (WCX) using Agilent 1290 Infinity Bio-Inert Quaternary Pump or Binary Pump, testing three gradient types.
• Multiple heart cutting: Two 6-position/14-port valves with 12×40 µL loops to isolate selected peaks.
• Second dimension: RP chromatography on AdvanceBio RP-mAb C4 column for desalting/denaturation (water + 5% formic acid and acetonitrile + 5% formic acid).
• Detection: Agilent 6530 Accurate Mass Q-TOF LC/MS with 1:4 split, and diode array detection at 280 nm.

Main Results and Discussion

The phosphate/NaCl salt gradient provided the best retention time reproducibility, while the phosphate pH gradient and ammonium formate buffer offered higher resolution of biosimilar charge variants. The ammonium formate gradient balanced resolution, precision, and MS compatibility. Innovator Rituximab exhibited a single dominant charge variant peak; the biosimilar displayed three major peaks. Q-TOF MS deconvolution identified these as C-terminal lysine variants (mass shifts ~128 Da). Use of a divert valve time table prevented MS source contamination when non-volatile buffers were used; without it, extensive salt deposition occurred.

Benefits and Practical Applications

• Automated online desalting and denaturation facilitate intact mass analysis without manual buffer exchange.
• Multiple heart cutting allows targeted MS characterization of selected charge variant peaks, improving sensitivity and throughput.
• Quantitative comparison of innovator and biosimilar mAbs supports regulatory comparability and quality control.

Future Trends and Applications

Advances may include integration of native MS for higher-order structure analysis, expanded use of volatile buffers to simplify workflows, and automation of buffer exchange modules. Emerging microfluidic 2D-LC platforms and real-time data analysis with AI-driven peak recognition will further accelerate biotherapeutic characterization.

Conclusion

The Agilent 1290 Infinity 2D-LC solution combined with Agilent 6530 Q-TOF MS provides a powerful platform for detailed charge variant profiling of mAbs. The workflow offers high precision, resolution, and MS compatibility, enabling comprehensive comparability studies of innovator versus biosimilar products.

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