Obtaining Equivalent IEX Chromatographic Performance Through Automated Method Scaling Using Waters Column Calculator

Importance of the Topic

Monitoring charge variants in monoclonal antibody therapeutics is essential to ensure product stability, efficacy and consistent manufacturing performance. As analytical methods move from early development into routine quality control, maintaining equivalent chromatographic behavior across different liquid chromatography platforms reduces the burden of repeated method qualification and validation.

Objectives and Study Overview

This study demonstrates automated scaling of an ion-exchange chromatography method for mAb charge variant analysis using the Waters Column Calculator. An original UPLC method on an ACQUITY Premier System was migrated to an UHPLC workflow on an Arc Premier System. Both pH- and salt-based gradients were evaluated to confirm that selectivity and peak area integrity are preserved after scaling.

Methodology and Experiment Design

An Infliximab drug product (Remicade) solution was injected directly under two sets of conditions. The original UPLC method used a 2.1×100 mm BioResolve SCX mAb Column at 0.10 mL/min, with linear pH or salt gradients generated by BioResolve CX pH Concentrates. The scaled UHPLC method employed a 4.6×100 mm column at 0.48 mL/min. System dwell volumes for both instruments were measured by tracking a caffeine marker response versus programmed gradient. These values and column geometries were entered into the Column Calculator to automatically adjust flow rate, injection volume and gradient timing, including an isocratic hold to offset dwell volume differences.

Instrumentation Used

• ACQUITY Premier System with TUV detector
• Arc Premier System with UV/Vis detector
• BioResolve SCX mAb Columns (2.1 mm and 4.6 mm ID)
• BioResolve CX pH Concentrate A and B
• Empower 3 chromatography software

Main Results and Discussion

After automated scaling, retention time differences between original and migrated methods were below 0.03 relative units, and relative peak area deviations remained under 0.5% for both gradient types. When the dwell volume offset was omitted, retention profiles shifted by nearly two minutes, underscoring the importance of accurate dwell volume compensation. With the recommended isocratic hold, chromatograms from both systems aligned closely, demonstrating reproducible separation of mAb charge variants.

Benefits and Practical Applications

• Rapid, error-free scaling of IEX methods between UPLC and UHPLC platforms
• Consistent selectivity and quantitation of charge variants
• Reduced time and resources for method transfer and validation
• Flexible application to pH and salt gradient protocols

Future Trends and Potential Applications

Automated method scaling tools will become increasingly important as analytical labs adopt diverse chromatographic systems. Integration of dwell volume databases, advanced gradient optimization and machine learning-based prediction models may further streamline method migration. Applications could extend to other separation modes such as hydrophobic interaction and mixed-mode chromatography.

Conclusion

The Waters Column Calculator effectively translates ion-exchange methods from UPLC to UHPLC platforms, maintaining key performance metrics for mAb charge variant analysis. Accurate dwell volume adjustment and column geometry scaling ensure robust, transferable methods that support both upstream development and downstream quality control workflows.

References

1. Birdsall RE, Koshel BM, Yu YQ. Accelerating Charge Variant Analysis of Biotherapeutics with the BioAccord System. Waters Application Note 720007706, 2022.
2. Hong P, McConville PR. Dwell Volume and Extra-Column Volume: What Are They and How Do They Impact Method Transfer. Waters Application Note 720005723, 2018.