Helpful ‘Hints’ for Protein SEC Success

Significance of the Topic

The accurate characterization of protein aggregates and fragments is essential in biopharmaceutical development and quality control. Protein aggregation can compromise product efficacy, trigger immunogenic responses, and arise under common stress conditions such as pH shifts, temperature changes, and shear forces. Size exclusion chromatography (SEC) offers a non-interactive, isocratic approach to separating biomolecules by hydrodynamic size, making it indispensable for assessing monoclonal antibodies, enzyme formulations, and complex protein therapeutics.

Objectives and Study Overview

This application note by Jean Lane presents practical guidelines for optimizing protein SEC methods. Key objectives include defining SEC terminology, outlining criteria for column selection, describing mobile phase and buffer considerations, reviewing detector options, and recommending instrument best practices. The aim is to help analytical scientists achieve high resolution, reproducible results while minimizing secondary interactions and analysis time.

Methodology and Instrumentation

Methodology

• Principle: SEC separates analytes by size; large species elute first, small species penetrate pores and elute later.
• Stationary phase: inert, hydrophilic coatings reduce non-specific adsorption.
• Pore size selection: critical to cover target molecular weight range (e.g., 300 Å for monoclonal antibodies).
• Particle size and column dimensions: smaller particles and longer columns improve resolution; shorter columns increase throughput.

Instrumentation Used

• Agilent 1260 Infinity Bio-Inert LC System and 1260 Infinity II Bio-Inert LC System
• AdvanceBio SEC columns (1.9 µm, 2.7 µm; 120 Å, 200 Å, 300 Å pore sizes)
• Bio SEC-3 and Bio SEC-5 columns
• ProSEC 300S and ZORBAX GF columns
• UV/Diode Array Detectors, Refractive Index Detector, Multi-Angle Light Scattering
• Agilent InfinityLab Quick Change inline filters and solvent inlet filters

Main Results and Discussion

Column Performance

• AdvanceBio SEC columns exhibit large pore volumes and narrow pore distributions, delivering superior resolution for monomer, dimer, and low-molecular-weight fragments.
• Optimized 200 Å, 1.9 µm columns provided the widest separation window and highest efficiency compared to competitor columns with similar pore sizes.
• Fast SEC strategies using 7.8 × 150 mm AdvanceBio SEC columns achieved resolution comparable to 300 mm columns but with more than two-fold reduction in run time.

Mobile Phase Optimization

• Phosphate buffer (50–350 mM) at pH 6.6–7.4 with 0–250 mM NaCl balanced electrostatic shielding and minimized secondary interactions.
• Optimal starting conditions: 150 mM sodium phosphate, pH 7.0, with 0.15 M NaCl for robust peak shape and dimer/monomer resolution.
• Agilent Buffer Advisor simplifies buffer preparation and pH scouting studies, reducing manual effort.

Detector Comparison

• UV detection at 220 nm offers basic quantitation of protein species.
• Light scattering detection enhances sensitivity for higher-order aggregates and supports molecular weight determination.

Benefits and Practical Applications

SEC methods refined with these guidelines enable reliable aggregate and fragment profiling in biopharmaceutical workflows. Users can tailor resolution versus throughput by selecting appropriate column dimensions and flow rates. Hydrophilic, inert column chemistries reduce sample loss, extend column life, and improve reproducibility in QC and R&D laboratories. Inline filters and optimized solvent hygiene further protect instrumentation and columns from clogging and contamination.

Future Trends and Applications

Emerging directions include coupling high-resolution SEC with mass spectrometry for detailed proteoform analysis, implementing multi-angle light scattering for absolute molecular weight measurements, and leveraging higher-pressure UHPLC systems for faster separations. Automated buffer preparation and reagent screening tools will accelerate method development, while novel stationary phases may further reduce non-specific interactions.

Conclusion

Size exclusion chromatography remains a cornerstone technique for protein aggregate and fragment analysis. Careful selection of pore size, particle size, column dimensions, mobile phase composition, and detection technology is key to achieving accurate, reproducible results. Agilent’s AdvanceBio SEC columns and Bio-Inert LC systems offer robust performance, high resolution, and streamlined workflows for biopharmaceutical characterization.

References

• Lane J. Helpful ‘Hints’ for Protein SEC Success. Agilent Technologies, May 27, 2020.