Evaluating the Ruggedness of the Alliance™ iS Bio HPLC System for SEC Separations

Importance of the Topic

Size-exclusion chromatography is a cornerstone technique in the characterization of therapeutic proteins and monoclonal antibodies. In quality control environments, robust and reproducible SEC methods ensure reliable measurements of monomer and aggregate content, guiding critical decisions in biopharmaceutical development and manufacturing.

Objectives and Study Overview

This study compares the ruggedness of the new Alliance iS Bio HPLC System with MaxPeak High Performance Surfaces (HPS) against a legacy stainless-steel HPLC system. Both configurations were evaluated using a compendial SEC method from USP General Chapter <129>. Method parameters including injection volume, flow rate, sample concentration, mobile phase pH, salt concentration, and salt type were systematically varied to assess system performance.

Methodology and Instrumentation

Sample and Reagents:

• NISTmAb RM 8671 monoclonal antibody
• Buffers: 0.20 M potassium phosphate with 0.25 M KCl; phosphate buffered saline (PBS); ammonium acetate

Chromatographic Systems:

• Stainless-steel HPLC System with BioSuite Diol SEC column (250 Å, 5 µm, 7.8 × 300 mm)
• Alliance iS Bio HPLC System with XBridge Premier Protein SEC column (250 Å, 2.5 µm, 7.8 × 300 mm)
• QuanRecovery vials with MaxPeak HPS surfaces
• Detection: UV at 280 nm; software: Empower 3

Main Results and Discussion

Parameter Robustness

• Injection volume, flow rate, sample concentration, and pH shifts produced comparable monomer, HMWS, and LMWS peak areas on both systems.
• The Alliance iS Bio HPLC System achieved higher half-height resolution (2.53 vs 1.88) and narrower peak width at 4.4 % height (0.79 vs 1.52) due to smaller particle size and lower system dispersion.

Salt Concentration and Type

• With potassium phosphate/KCl buffer, the legacy system failed at 0.25× salt strength, losing aggregate detection and showing severe tailing. The Alliance system maintained acceptable performance down to 0.5×.
• Using PBS, the stainless-steel configuration exhibited monomer loss and tailing below 2× concentration, while the Alliance system tolerated 0.5×–2× with stable peak shape.
• Ammonium acetate mobile phases (200–400 mM) were incompatible with the legacy system. In contrast, the Alliance configuration delivered consistent recovery and peak quality across this range.

Benefits and Practical Applications

The Alliance iS Bio HPLC System with XBridge Premier Protein SEC column offers:

• Enhanced tolerance to buffer composition variations
• Improved resolution and peak shape for accurate size-variant quantification
• Greater flexibility for method development and high-throughput QC operations

Future Trends and Potential Applications

Advances in biocompatible surface chemistries and low-dispersion HPLC architectures will support emerging modalities such as antibody-drug conjugates and multispecific proteins. Integration with light scattering and mass spectrometry detectors will further expand SEC capabilities in structural characterization and high-sensitivity aggregate profiling.

Conclusion

The Alliance iS Bio HPLC System paired with an XBridge Premier Protein SEC column demonstrated superior ruggedness and reliability for monoclonal antibody SEC separations. Its broader salt tolerance and enhanced chromatographic performance make it an ideal platform for biopharmaceutical QC laboratories.

Reference

1. USP General Chapter <129> Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies, 2022.
2. Bigos P, Birdsall RE, Nyholm K. Modernizing Compendial SEC Methods for Biotherapeutics Using the Alliance iS Bio HPLC System. Waters Application Note. April 2024.
3. Hong P, Koza SM, Bouvier ESP. Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates. Journal of Liquid Chromatography and Related Technologies. 2012;35(20):2923–2950.