Agilent Biocolumns - Aggregate/Fragment Analysis - Application Compendium

Significance of the topic

Protein aggregation poses significant challenges in biopharmaceutical development, affecting efficacy, stability, and safety of therapeutic proteins. Size exclusion chromatography (SEC) is the industry standard for quantifying monomers, aggregates, and fragments. Agilent AdvanceBio SEC columns, with hydrophilic polymer coatings and sub-2 µm particles, are designed to minimize secondary interactions and deliver high resolution and reproducibility for aggregate analysis.

Objectives and study overview

This compendium of application notes demonstrates a complete workflow for protein aggregate characterization using Agilent SEC solutions. Key goals include:

• Optimizing mobile phase conditions to eliminate nonspecific interactions
• Choosing appropriate pore sizes and column dimensions for various biomolecule sizes
• Calibrating columns with relevant standards for accurate molecular weight estimation
• Incorporating advanced detectors (UV, refractive index, light scattering, MS) to extend quantitative and qualitative capabilities

Methodology and instrumentation

Analyses employ Agilent AdvanceBio SEC columns (pore sizes 100 to 2000 Å, particle sizes 1.9 µm and 2.7 µm) coupled to the Agilent 1260 Infinity II Bio-inert LC or 1290 Infinity II Bio LC systems. Mobile phases typically comprise aqueous buffers (e.g., 150 mM phosphate, pH 7.0) with optional salt, pH, or organic modifiers. Sample preparation emphasizes minimizing nonspecific aggregation. Advanced detection is achieved using the Agilent GPC/SEC multidetector suite with UV, differential refractive index, static and dynamic light scattering, and optional native MS interfaces.

Main results and discussion

– High resolution separation of monomers, dimers, and higher-order aggregates in mAbs, insulin, interferons, and nanobodies using sub-2 µm columns.
– Automated buffer optimization with Agilent Buffer Advisor software and quaternary pumps simplifies screening of salt and pH variations.
– Calibration curves generated with peptide, protein, polysaccharide, and synthetic polymer standards enable accurate molecular weight estimation.
– Light scattering detection distinguishes covalent oligomers from reversible aggregates and provides absolute molecular weight and hydrodynamic radius measurements.
– Native SEC-MS workflows demonstrate direct mass analysis of intact proteins and conjugates under nondenaturing conditions.

Practical benefits and applications

– Robust, reproducible methods suitable for QA/QC in bioprocessing and drug development.
– Improved throughput with shorter columns and higher flow rates, maintaining critical resolution.
– Scalability between analytical and semi-prep formats via column dimension and pore size selection.
– Compliance with pharmacopeia requirements for SEC aggregate testing.

Future trends and possibilities

– Integration of machine-learning guided method optimization for buffer and condition scouting.
– High-throughput, parallel column arrays for screening large sample sets.
– Advanced SEC-MS coupling with real-time deconvolution and proteoform identification.
– Development of novel stationary phases for challenging analytes and surfactant-compatible separations.

Conclusion

The Agilent AdvanceBio SEC line combined with Bio-inert LC systems and multidetector modules delivers a comprehensive solution for protein aggregate analysis. Optimized columns, automated buffer mixing, and advanced detection capabilities provide unmatched resolution, sensitivity, and throughput for modern biopharmaceutical workflows.

References

• Agilent AdvanceBio SEC Application Compendium
• Application Note 5991-3651EN: How-to Guide for SEC of Biomolecules
• Application Note 5994-2709EN: High-Resolution mAb Aggregate Analysis
• Application Note 5994-1739EN: Sensitive Native MS of Macromolecules