Auto•Blend Plus Technology for Ion-Exchange Chromatography Method Development and Robustness Testing

Significance of the Topic

Ion-exchange chromatography (IEX) is a cornerstone technique in the biopharmaceutical industry for resolving protein charge variants. Precise separation of isoforms differing in surface charge, even with identical isoelectric points, underpins quality control, biosimilarity assessments, and in-depth characterization of monoclonal antibodies and other therapeutic proteins.

Objectives and Study Overview

This study evaluates Waters Auto•Blend Plus Technology to streamline IEX method development and robustness testing. Through case studies on trastuzumab and a mAb Charge Variant Standard, the work demonstrates how automated blending of concentrated acid, base, salt, and water simplifies gradient creation, pH screening, salt optimization, and method transfer.

Methodology

Samples: Trastuzumab (5 mg/mL in water) and mAb Charge Variant Standard diluted in water.

Mobile phases: 100 mM MES monohydrate (acid), 100 mM MES sodium salt (base), 1 M NaCl (salt), and water.

Gradient design: pH gradients (5.4–6.9) at fixed salt levels, salt gradients (60–500 mM) at fixed pH, and dual pH/salt gradients, all programmed via Auto•Blend Plus tables.

Instrumentation

System: Waters ACQUITY UPLC H-Class Bio with four-solvent quaternary pump.

Column: BioResolve SCX mAb, 3 μm, 4.6×50 mm.

Detection: UPLC TUV at 280 nm.

Software: Empower 3 with embedded Auto•Blend Plus functionality.

Main Results and Discussion

Method programming was simplified by defining buffer pKa and desired pH/salt values, allowing the software to compute solvent blends via Henderson–Hasselbalch relations or empirical tables. pH screening required only changing a single pH value in the method, eliminating preparation of multiple buffers. Robustness testing over pH 6.1–6.3 and salt 55–115 mM demonstrated consistent peak areas and resolution, confirming method stability. Finally, an Auto•Blend Plus method was transferred to a conventional two-solvent method with comparable chromatographic performance.

Benefits and Practical Applications

• Reduces mobile phase preparation time and errors by automating solvent blending.
• Accelerates pH and salt screening without manual titrations or multiple buffer stocks.
• Facilitates rapid robustness assessment by simple parameter adjustments in software.
• Enables straightforward method transfer to instruments lacking quaternary blending.

Future Trends and Opportunities

Integration of intelligent blending technologies with predictive algorithms may enable real-time gradient optimization. Expansion to multi-attribute workflows and automated fraction collection could further enhance biotherapeutic characterization. Adapting Auto•Blend workflows to other chromatographic modes (e.g., HIC, SEC) may broaden applicability.

Conclusion

Waters Auto•Blend Plus Technology significantly streamlines IEX method development and robustness testing for protein charge variant analysis. By automating mobile phase formulation and offering flexible gradient design, it reduces labor, minimizes errors, and delivers reproducible separations, supporting efficient analytical workflows in biopharmaceutical laboratories.

References

1. Du Y., et al. Chromatographic Analysis of the Acidic and Basic Species of Recombinant Monoclonal Antibodies. mAbs 2012, 4(5), 578–585.
2. Harris R.J., et al. Identification of Multiple Sources of Charge Heterogeneity in a Recombinant Antibody. J. Chromatogr. B 2001, 752, 233–245.
3. Yang H., et al. Development of Monoclonal Antibody Charge Variant Analysis Methods Using Waters BioResolve SCX mAb Column. Waters Application Note 720006477EN (2019).