Demonstrating a Fit-for-Purpose Solution for Biopharmaceutical Separations with the ACQUITY UPLC H-Class PLUS Bio Binary System

Significance of the Topic

The development and quality control of biopharmaceuticals requires reliable chromatographic methods that can be run on a single instrument platform. Laboratories often lack dedicated LC systems for each assay, making method transfer and system versatility critical for efficient workflows.

Study Objectives and Overview

This study evaluates the ACQUITY UPLC H-Class PLUS Bio Binary System across three common biopharmaceutical separations: HILIC for glycan mapping, HIC for antibody–drug conjugate characterization under high salt conditions, and SEC for size variant analysis. Comparative assessments against a quaternary LC system demonstrate method equivalency without altering established protocols.

Methodology

HILIC analysis employed a RapiFluor-MS labeled glycan standard on an ACQUITY UPLC Glycan BEH Amide column with fluorescence and mass detection. HIC separations of a cysteine-conjugated ADC were performed using a Protein-Pak Hi Res HIC column in a multi-day robustness study. SEC experiments used a BioResolve SEC mAb column to resolve high and low molecular weight variants in a mAb size variant standard.

Instrumentation Used

• ACQUITY UPLC H-Class PLUS Bio Binary System (binary pump configuration)
• ACQUITY UPLC H-Class PLUS Bio System (quaternary pump configuration)
• ACQUITY UPLC Glycan BEH Amide Column, 130 Å, 1.7 µm, 2.1 × 150 mm
• Protein-Pak Hi Res HIC Column, 2.5 µm, 4.6 × 100 mm
• BioResolve SEC mAb Column, 2.5 µm, 4.6 × 150 mm
• ACQUITY FLR Detector (λEx 265 nm, λEm 425 nm)
• ACQUITY QDa Mass Detector (ESI+, 350–1250 m/z)
• Empower 3 Chromatography Data Software

Key Results and Discussion

HILIC separations on the binary system produced glycan profiles and glucose unit (GU) values nearly identical to those from the quaternary system, with an R2 of 0.99996 and negligible differences in peak areas. In HIC, retention time and drug-to-antibody ratio reproducibility were maintained over a 25-hour injection series across three days, with stable system pressure and no evidence of salt precipitation or clogging. SEC on the low-dispersion binary system achieved clear separation of high and low molecular weight species from the monomer, offering improved resolution of fragments compared to legacy methods.

Benefits and Practical Applications

• Method equivalency between binary and quaternary platforms without protocol changes
• Enhanced robustness for high salt, non-denaturing separations
• Improved resolution of size variants using modern column technology
• Operational efficiency by consolidating multiple assays on a single LC system

Future Trends and Potential Applications

Integration of binary pump systems in QC labs will support a wider range of analytical methods with minimal method transfer risk. Combining low-dispersion LC technologies with advanced mass detection offers deeper molecular insights. Future developments may include longer columns for higher resolution, automated sample preparation, and expanded use in protein characterization workflows.

Conclusion

The ACQUITY UPLC H-Class PLUS Bio Binary System is demonstrated as a fit-for-purpose solution for HILIC, HIC, and SEC biopharmaceutical separations. It delivers reproducible, high-quality results across diverse assays without requiring method modifications, enabling labs to future-proof their analytical operations.

Reference

1. Dao D, Koshel BM, Birdsall RE. Demonstrating Method Equivalency of the ACQUITY UPLC H-Class PLUS Bio Binary System and an ACQUITY UPLC I-Class PLUS System Through Method Transfer of a Reversed-Phase Peptide Mapping Method. Waters Application Note (2021).
2. Dao D, Koshel BM, Birdsall RE. Repeatability of the ACQUITY UPLC H-Class PLUS Bio Binary System Under Challenging Separation Conditions. Waters Application Note (2020).
3. Waters Corporation. RapiFluor-MS Glycan Performance Test Standard Care and Use Manual. (2015).