Waters ACQUITY and XBridge Premier Protein SEC 250 Å Columns: A New Benchmark in Inert SEC Column Design

Significance of the Topic

Protein size-exclusion chromatography (SEC) is a cornerstone technique for the characterization and quality control of biotherapeutic proteins, including monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs). Accurate separation of aggregates, monomers and fragments is essential for regulatory compliance, ensuring efficacy and patient safety. However, secondary interactions with column hardware and packing surfaces often compromise resolution, quantitation and method robustness.

Objectives and Study Overview

This study investigates the performance of Waters ACQUITY and XBridge Premier Protein SEC 250 Å columns, which combine a novel ethylene bridged-hybrid particle with hydroxy-terminated polyethylene oxide (BEH-PEO) bonding and hydrophilic MaxPeak High Performance Surfaces (HPS) hardware. The goals are to assess reduction of ionic and hydrophobic secondary interactions, compare to leading commercial SEC columns and demonstrate minimal dependence on salt or organic modifiers.

Methodology and Instrumentation

The evaluation employed an ACQUITY UPLC H-Class Bio system equipped with a Tunable UV detector (280 nm) and Empower 3 data acquisition software. Sample analyses included NISTmAb (RM 8671) and ado-trastuzumab emtansine, using phosphate buffer mobile phases with titrated NaCl (0–200 mM) or organic modifiers (acetonitrile or isopropanol, 0–15%). Comparative columns comprised BioResolve SEC mAb 200 Å (2.5 μm), diol-bonded silica (250 Å, 2 μm) and MeO-PEO silica (300 Å, 2.7 μm). Key parameters:

• Column dimensions: 4.6 × 150 mm, 2.5 μm XBridge Premier Protein SEC 250 Å
• Column temperature: 30 °C; sample temperature: 8 °C
• Flow rate: 0.35 mL/min; injection volume: 1 μL
• Mobile phases: 100 mM sodium phosphate pH 6.8 (A), 1.0 M NaCl (B), 50% MeCN or IPA (C), water (D)

Main Results and Discussion

Ionic interaction testing showed that the Premier Protein SEC 250 Å column displayed virtually constant monomer peak shape and aggregate recovery across 0–200 mM NaCl, whereas traditional stainless steel columns required ≥200 mM NaCl to approach similar performance. Hydrophilic MaxPeak HPS hardware was confirmed as the key contributor by packing identical particles into both conventional and HPS hardware. Hydrophobic interaction assays with a highly hydrophobic ADC demonstrated minimal change in monomer peak efficiency from 0 to 15% acetonitrile or 5% isopropanol, underscoring the high PEO coverage and inertness of the packing material.
Lot-to-lot reproducibility was excellent across seven column batches, with relative standard deviations below 5% for monomer tailing and below 20% for aggregate recovery in ionic tests, and below 10% and 5% respectively in hydrophobic tests.

Benefits and Practical Applications

• Reliable analysis of protein size variants from ~10 kDa to 650 kDa with physiological buffers
• Minimal need for high salt or organic solvents, simplifying method development
• Plug-and-play compatibility with existing SEC protocols for biotherapeutic QC
• Enhanced method robustness, accuracy of aggregate quantitation and peak reproducibility

Future Trends and Potential Applications

Ongoing developments in column surface engineering and hybrid particle chemistries promise further reductions in secondary interactions, enabling higher-throughput SEC and coupling with mass spectrometry for deeper proteoform profiling. Integration of these inert columns into automated workflows and platform methods may accelerate biopharmaceutical development and facilitate compliance with evolving regulatory standards.

Conclusion

The ACQUITY and XBridge Premier Protein SEC 250 Å columns deliver a holistic solution to unwanted secondary interactions in protein SEC by combining BEH-PEO bonded particles with hydrophilic MaxPeak HPS hardware. This innovation achieves excellent resolution, recovery and reproducibility using low concentrations of salts and organic modifiers, simplifying workflows and improving the robustness of biotherapeutic protein analyses.

References

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