Future Proofing the Biopharmaceutical QC Lab:Benefits of Automating Mobile Phase Delivery to Improve pH Consistency in Size Exclusion Chromatography Methods

Importance of Mobile Phase pH Consistency in SEC Methods

Size exclusion chromatography in biopharmaceutical quality control relies on precise buffer composition. Variations in pH or salt concentration during manual buffer preparation can cause shifts in protein retention times and impact aggregate measurements. Automated mobile phase blending offers a strategy to minimize variability and improve method robustness.

Objectives and Study Overview

This study evaluates the conversion of conventional SEC methods to automated buffer delivery using Auto•Blend Plus Technology on the ACQUITY UPLC H-Class Bio System. Key aims include:

• Comparing retention times and peak areas for a protein standard mix and a monoclonal antibody (infliximab) between manual and automated buffer preparation.
• Demonstrating equivalence for both SEC-HPLC and SEC-UPLC formats.
• Highlighting operational benefits in QC environments.

Methodology

Two buffer preparation workflows were examined:

• Manual preparation of 20 mM phosphate, 200 mM NaCl at pH 6.8.
• Automated blending of four stock solutions (100 mM NaH2PO4, 100 mM Na2HPO4, 1 M NaCl, water) to target the same pH and ionic strength.

Both workflows were applied to:

• SEC-HPLC: 7.5 × 300 mm BioSuite SEC column, 20 µL injection, 0.400 mL/min, 35 min run.
• SEC-UPLC: 4.6 × 150 mm Protein BEH SEC column, 4 µL injection, 0.885 mL/min, 3 min run.

Instrumentation

The ACQUITY UPLC H-Class Bio System included:

• Quaternary Solvent Manager with Auto•Blend Plus.
• Sample Manager and Tunable UV Detector (Ti flow cell).
• BioSuite SEC 10 µm, 250 Å column (7.5 × 300 mm) and Protein BEH SEC 200 Å, 1.7 µm column (4.6 × 150 mm).
• Empower 3 chromatography data software for method setup and data processing.

Main Results and Discussion

Both SEC-HPLC and SEC-UPLC runs produced nearly identical retention times and relative peak areas when comparing manual versus automated buffer delivery. Protein standard components (thyroglobulin, IgG, BSA, myoglobin, uracil) and infliximab monomer/dimer peaks showed deviations below 0.1 min and relative area differences under 1%. Automated blending maintained pH accuracy across salt concentrations by using empirical data tables to correct for ionic effects.

Benefits and Practical Applications

Auto•Blend Plus offers:

• Enhanced reproducibility and precision in pH-dependent separations.
• Reduced operator variability and risk of pH meter calibration errors.
• Faster buffer preparation and seamless transition between method conditions.
• Scalability for high-throughput QC laboratories.

Future Trends and Opportunities

Advances may include integration of automated buffer blending with laboratory information management systems, expansion to multi-component buffer systems (including organic modifiers), and use of predictive models to optimize buffer compositions. Adoption of green solvents and real-time pH feedback loops could further enhance sustainability and method control.

Conclusion

Automated mobile phase delivery via Auto•Blend Plus on the ACQUITY UPLC H-Class Bio System delivers equivalent chromatographic performance to manual buffer preparation for both SEC-HPLC and SEC-UPLC. This approach enhances robustness, reduces variability, and supports efficient QC workflows in biopharmaceutical laboratories.

References

1. Future-proofing the Biopharmaceutical QC Laboratory: Using the ACQUITY UPLC H-Class Bio to Run SEC HPLC and SEC UPLC. Waters Application Note, 2014; 720005057EN.