Improved Separation Performance of Monoclonal Antibody and Antibody Drug Conjugates Using Waters XBridge Premier Protein SEC 250 Å, 2.5 μm Column

Significance of the topic

Monoclonal antibodies and antibody drug conjugates have become essential to modern therapeutics, accounting for a significant portion of leading biopharmaceutical revenues. Accurate analysis of size variants such as aggregates and fragments is critical to ensure safety, efficacy, and regulatory compliance. Size exclusion chromatography remains the standard approach for this characterization but can be undermined by undesired secondary interactions between proteins and column hardware or packing materials.

Objectives and overview of the study

This study evaluates the performance improvements offered by the Waters XBridge Premier Protein SEC 250 Å, 2.5 µm column—featuring an ethylene bridged hybrid particle with BEH-PEO bonding and hydrophilic MaxPeak Premier HPS hardware—against a conventional silica-based SEC column. Three protein standards were assessed: a proprietary mAb size variant standard, the NISTmAb reference material RM 8671, and the antibody drug conjugate ado-trastuzumab emtansine (KADCYLA). Performance metrics included resolution of high and low molecular weight species, percent peak areas, peak widths, and column efficiency.

Methodology and instrumentation

• LC system: Agilent 1260 Infinity Bio-Inert with quaternary pump, high-performance autosampler, and DAD with BioInert flow cell (10 mm, 13 µL)
• Column comparison: Waters XBridge Premier Protein SEC 250 Å, 2.5 µm versus silica-based dSEC-2 200 Å, 3 µm (both 7.8×300 mm)
• Mobile phase: 2× phosphate buffered saline (20 mM phosphate, 276 mM NaCl, 5.4 mM KCl, pH 7.4)
• Flow rate: 0.58 mL/min; injection volumes: 10 µL for mAb standards, 7.2 µL for ADC
• Detection: UV at 280 nm, sampling at 5 Hz; system dispersion measured at 37 µL (5σ)
• Sample prep: mAb size variant standard reconstituted in water; NISTmAb prepared in histidine buffer; ADC prepared in water

Main results and discussion

The Waters column delivered superior separation across all three standards. For the ADC, it achieved higher percent area for high molecular weight species (1.72 % vs 1.51 %), narrower peaks, and improved half-height resolution. In the mAb size variant standard, the innovative column resolved low molecular weight fragments (LMWS1&2) as distinct peaks, whereas the silica column only generated a shoulder on the main peak. With the NISTmAb reference material, partial resolution of low abundance fragments and enhanced signal-to-noise for minor species were observed using the XBridge Premier column. Overall plate counts, percent area accuracy for aggregates, and peak shapes demonstrated consistent improvements attributed to reduced secondary interactions.

Benefits and practical applications

• Enhanced resolution of aggregates and fragments supports robust characterization throughout biopharmaceutical development and quality control
• Improved accuracy in percent area measurements ensures reliable monitoring of critical quality attributes
• Higher column efficiency accelerates method throughput and sensitivity
• Simplified method development using a common PBS eluent stream streamlines workflow

Future trends and possibilities

Advancements in surface inertness and novel stationary phase chemistries are expected to further minimize unwanted interactions, extending SEC applicability to more complex biomolecules. Integration with orthogonal detection methods such as multi-angle light scattering or mass spectrometry and adoption of microflow formats may yield greater sensitivity and throughput. The combination of AI-driven method optimization and next-generation hardware promises accelerated development cycles for emerging biotherapeutics.

Conclusion

The Waters XBridge Premier Protein SEC 250 Å, 2.5 µm column with MaxPeak Premier HPS hardware significantly improves separation performance for monoclonal antibodies and antibody drug conjugates. By mitigating ionic and hydrophobic secondary interactions, it delivers superior resolution, efficiency, and quantitation of size variants compared to a conventional silica-based column, enhancing confidence in biopharmaceutical characterization.

References

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6. Jung M Lauber M Demonstrating Improved Sensitivity with MaxPeak HPS Application Note, 2021
7. Kizekai L Lauber M ACQUITY and XBridge Premier Protein SEC Columns Application Note, 2022
8. Waters mAb Size Variant Standard Care and Use Manual, 2022