Waters ACQUITY Premier Protein SEC 250 Å, 1.7 μm Columns - CARE AND USE MANUAL

Importance of the Topic

Size-exclusion chromatography (SEC) is essential for assessing non-covalent protein aggregation and fragmentation in biopharmaceutical analysis.
Reliable detection of aggregates, monomers, and fragments supports product quality control and stability studies.

Objectives and Overview

This manual presents best practices for Waters ACQUITY Premier Protein SEC 250 Å 1.7 µm Columns and recommended XBridge guards, aiming to:

• Enable generic, platform-type separations for proteins (10 kDa–650 kDa).
• Minimize secondary hydrophobic and ionic interactions via MaxPeak HPS and BEH-PEO particle technology.
• Provide guidelines on system configuration, method setup, and performance verification.

Methodology and Instrumentation

Methodology covers:

• Determination of extra-column dispersion using the 5-Sigma method with caffeine injections.
• Buffer preparation and selection: use high-purity water, 0.2 µm filtration, PBS or low-ionic strength volatile buffers; avoid microbial contamination.
• Column installation, equilibration (≥10 column volumes), temperature control (4–60 °C) and performance benchmarks with Waters mAb Size Variant Standard.
• MS compatibility tests with BioAccord and Xevo G2-XS QTof systems using low concentration ammonium acetate mobile phases.
• Use and benefits of MaxPeak Premier Protein SEC 250 Å 2.5 µm guard columns.
• Cleaning and regeneration protocols (acidic salts or organic modifiers) and recommended storage solvents.

Used Instrumentation

• ACQUITY UPLC H-Class Bio System with low dispersion flow paths.
• ACQUITY Premier Protein SEC 250 Å, 1.7 µm Columns (4.6×150 mm and 4.6×300 mm).
• XBridge Premier Protein SEC 250 Å, 2.5 µm Guard Columns.
• Waters mAb Size Variant Standard (p/n: 186009429).
• Waters BioAccord LC-MS system; Waters Xevo G2-XS QTof.

Main Results and Discussion

Key findings include:

• High resolution separation of mAb monomers, aggregates (e.g. HMWS ~ 2.2 %), and fragments (LMWS ~ 1.1 %) in PBS without organic modifiers.
• Optimized performance at ambient to moderate temperatures (25–35 °C); lower temperatures enhanced ADC peak shapes.
• Recovery of proteins using low-strength ammonium acetate (50–100 mM) in SEC-MS, with minimal background ions above 600 m/z.
• Guard columns effectively trap particulates and preserve analytical column lifetime without compromising separation.

Benefits and Practical Applications

• Robust, generic SEC platform reduces method development time.
• High performance surfaces and particle design minimize secondary interactions.
• Compatibility with standard biological buffers and volatile MS-friendly mobile phases.
• Integrated QC via Waters eCord chip supports real-time tracking of column history and performance.

Future Trends and Possibilities

Potential developments include:

• Further integration of native SEC-MS for in-depth protein characterization.
• Advanced low-dispersion system designs and sub-2 µm column technologies.
• Automation of buffer preparation and column maintenance.
• Data-driven analytics for real-time method optimization and column health monitoring.

Conclusion

The ACQUITY Premier Protein SEC 250 Å 1.7 µm Columns, combined with MaxPeak High Performance Surfaces and BEH-PEO particle technology, deliver high-resolution, reproducible separations for protein therapeutics. Comprehensive guidelines on system setup, method optimization, instrumentation, and QC ensure reliable performance and extended column lifetimes across diverse applications.

References

• Waters Corp. Impact of LC System Dispersion on the Size-Exclusion Chromatography Analysis of Monoclonal IgG Antibody Aggregates and Fragments; Selecting the Optimal Column for Your Method, p/n: 720006336EN.
• Waters Corp. Size-Exclusion Chromatography (SEC) Optimization Guide, p/n: 720006067EN.
• Waters Corp. Waters mAb Size Variant Standard, p/n: 720006811EN.