Method Development Tools for More Efficient Screening of Biopharmaceutical Method Conditions Using the ACQUITY Arc Bio SystemPart 2 of 2: Screening SEC and IEX Conditions in a Single Experiment

Importance of the Topic

The rapid and systematic screening of chromatographic conditions is critical in biopharmaceutical method development. Efficient evaluation of size exclusion chromatography (SEC) and ion exchange chromatography (IEX) parameters helps ensure product quality, shorten development timelines, and reduce manual labor in buffer preparation.

Objectives and Study Overview

This application note demonstrates how the ACQUITY Arc Bio System, combined with Auto•Blend Plus buffer blending technology, can screen SEC and IEX conditions in a single experiment. In Part 2, the focus is on using a common phosphate buffer system to assess the effects of mobile phase pH and ionic strength on a model monoclonal antibody (trastuzumab).

Methodology and Instrumentation

A single injection series was performed on an ACQUITY Arc Bio System fitted with a CM-A column compartment module. Two columns—a Protein BEH SEC column and a BioResolve SCX mAb IEX column—were placed on separate flow paths. Auto•Blend Plus prepared mobile phases A–D (acid, base, salt, water) to program pH and salt gradients without manual buffer preparation. Empower 3 software controlled valve switching and data acquisition.

Instrumentation Used

• ACQUITY Arc Bio System with CM-A column manager
• 2489 UV/Vis detector at 280 nm
• XBridge Protein BEH SEC column (7.8 × 150 mm, 2.5 µm, 200 Å)
• BioResolve SCX mAb column (4.6 × 50 mm, 3 µm)
• Empower 3 Chromatography Data Software SR2, FR4

Results and Discussion

SEC screening evaluated pH 6.2–7.4 and NaCl 50–350 mM. Higher pH and ionic strength reduced peak tailing, improved monomer peak shape, and increased aggregate recovery. Optimal SEC conditions were identified near pH 7.0–7.4 with 250–350 mM NaCl.

IEX screening held the salt gradient (25–125 mM NaCl) constant and varied pH from 6.0 to 7.4. Peak area analysis of acidic variants, main species, and basic variants indicated the method was most robust between pH 6.2 and 6.6, balancing resolution, tailing, and variant separation.

Benefits and Practical Applications

• High-throughput screening of SEC and IEX in one automated run
• Elimination of manual buffer preparation through Auto•Blend Plus
• Bio-inert flow path minimizes corrosion risk from phosphate and high salt concentrations
• Rapid identification of promising pH and ionic strength ranges for further method optimization

Future Trends and Opportunities

Advances in automated buffer blending and column switching can expand to other chromatographic modes such as hydrophobic interaction and affinity chromatography. Integration with real-time data analytics and machine learning may further accelerate method development. Incorporating larger column arrays or multiplexed detectors could increase throughput and data richness.

Conclusion

The ACQUITY Arc Bio System with Auto•Blend Plus provides a streamlined, high-throughput approach to initial SEC and IEX method screening. By automating buffer blending and column switching, scientists can efficiently map pH and ionic strength effects, reducing manual effort and protecting system integrity. Identified conditions can then be refined with longer columns or specialized stationary phases.

References

1. Waters Corporation. Auto•Blend Plus technology overview. waters.com/autoblendplus