Development of Monoclonal Antibody Charge Variant Analysis Methods Using a BioResolve SCX mAb Column

Significance of the Topic

Charge heterogeneity in therapeutic monoclonal antibodies must be precisely characterized and monitored to ensure consistent biological activity, safety, and product quality. Cation-exchange chromatography (CEX) remains the method of choice for mAb charge variant analysis due to its high selectivity for proteins near physiological pH and its compatibility with routine QC workflows.

Objectives and Study Overview

This work demonstrates a systematic approach to developing fixed-pH salt gradient CEX methods for mAb charge variant profiling. Using Waters BioResolve SCX mAb columns and Auto•Blend Plus Technology within the ACQUITY UPLC H-Class Bio system, the study evaluates key parameters affecting resolution and robustness across multiple antibodies, including trastuzumab, cetuximab, adalimumab, and bevacizumab.

Methodology and Instrumentation

Samples were prepared at 1–5 mg/mL in water and analyzed on an ACQUITY UPLC H-Class Bio system with BioResolve SCX mAb columns (2.1×50 mm, 4.6×50 mm, 4.6×100 mm). Auto•Blend Plus Software generated salt gradients in units of pH and molarity. Mobile phase A/B comprised 100 mM MES at fixed pH, with 1 M NaCl as mobile phase C. Detection employed a TUV detector at 280 nm. Empower 3 Software managed data acquisition and method execution.

Main Results and Discussion

• pH Optimization: Increasing pH reduces mAb retention and shifts resolution between acidic and basic variants. Optimal pH balances acidic peak resolution against basic peak separation.
• Salt Gradient Slope: Shallower gradients (lower Δ mM NaCl per column volume) improve resolution until a plateau is reached, beyond which band broadening limits further gains.
• Gradient Time and Flow Rate: Longer gradient times and higher flow rates can yield shallower effective slopes, enhancing resolution while preserving analysis time. Ultra-high flow rates showed minimal loss in peak capacity due to nonporous particle technology.
• Column Dimensions: Extending column length (from 50 to 100 mm) markedly increased resolution, outperforming equivalent slope changes on shorter columns. Narrow bore (2.1 mm) columns require gradient delay correction and exhibit greater post-column dispersion, but use less solvent.
• Temperature: Within 27–37 °C, retention times varied predictably via van’t Hoff behavior, but selectivity and peak capacity remained stable, supporting temperature control for retention reproducibility.
• Organic Modifiers: Addition of 5 % isopropanol had negligible impact on selectivity or recovery, confirming low hydrophobic interaction on the SCX surface.

Benefits and Practical Applications

The described workflow delivers rapid, reproducible charge variant separations suitable for routine QC and process development. Auto•Blend Plus eliminates manual buffer mixing, streamlining method transfer and robustness testing. The BioResolve SCX mAb column provides high resolution and throughput for biopharmaceutical analysis.

Future Trends and Opportunities

Emerging directions include integration of pH gradient separations, direct coupling of volatile IEX mobile phases to mass spectrometry, miniaturized column formats for limited samples, and application of design of experiments or machine learning to accelerate CEX method development and robustness assessment.

Conclusion

A structured optimization of pH, gradient slope, flow rate, column geometry, temperature, and additives enables robust CEX methods for mAb charge variant profiling. Combining BioResolve SCX mAb columns with Auto•Blend Plus Technology enhances efficiency, reproducibility, and scalability in biopharmaceutical quality control.

References

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