SIZE EXCLUSION CHROMATOGRAPHY FOR BIOMOLECULE ANALYSIS

Importance of the Topic

Size exclusion chromatography (SEC) is a non-denaturing technique that separates biomolecules on the basis of their hydrodynamic size in solution rather than their chemical interactions. It is widely applied in biotherapeutic development and manufacturing to characterize intact proteins, quantify aggregates and fragments, and remove low-molecular-weight impurities. Regulatory guidelines for therapeutic proteins emphasize the need to resolve and quantify aggregates to ensure product safety, efficacy, and stability.

Objectives and Study Overview

This guide aims to provide a practical foundation for successful SEC separations of peptides, proteins, monoclonal antibodies, and biopolymers. Key topics include:

• Principles of size-based separation and elution order.
• Column selection criteria by pore size, particle size, and dimensions.
• Mobile phase composition, ionic strength, pH, and organic modifiers.
• Method development guidelines and troubleshooting strategies.

Methodology and Instrumentation

SEC separations are typically performed isocratically on columns such as Agilent AdvanceBio SEC (2.7 µm, pores 130–300 Å) or Agilent Bio SEC-5 (5 µm, pores up to 2000 Å). Standard configurations use 7.8 × 300 mm columns at 1 mL/min or 4.6 × 300 mm at 0.35 mL/min. Common mobile phases include 150 mM phosphate buffer (pH 7) with 50–400 mM NaCl, while organic additives (5–10% ethanol, methanol, or acetonitrile) and temperature control (10–30 °C) can improve solubility and resolution. Detectors range from UV (220–280 nm) and DAD to refractive index (RI) and advanced light scattering (static and dynamic) for accurate molecular weight determinations independent of calibration.

Main Results and Discussion

SEC elution profiles demonstrate separation from largest to smallest species: very large molecules elute in the void volume, while smaller species penetrate deeper into pores and elute later. Pore size directly affects resolution of aggregates and monomers, as shown by better peak distinction on 300 Å columns compared to 130 Å columns. Column length and flow rate trade throughput against resolution; a 150 mm column at 2 mL/min can halve run time with acceptable peak separation. Calibration curves differ between proteins, polysaccharides, and synthetic polymers, underscoring the importance of matching standards to analyte type.

Benefits and Practical Applications

SEC offers reproducible, gentle separations for quality control during protein manufacture, formulation, and stability studies. It quantifies aggregates, fragments, and excipients in monoclonal antibodies, vaccines, and conjugated biologics such as PEGylated proteins and antibody–drug conjugates. The technique’s non-interacting nature preserves native conformations, facilitating downstream fraction collection and structural analysis.

Future Trends and Potential Applications

Advances in SEC point toward higher-throughput methods using short columns and sub-2 µm particles, integration with mass spectrometry for sequence and PTM analysis, and multi-detector setups combining UV, RI, and light scattering for comprehensive molecular characterization. Automated buffer preparation and pH scouting software streamline method development, while emerging bio-inert instrumentation designs enable use of harsh solvent conditions for challenging biopolymers.

Conclusion

Size exclusion chromatography remains an indispensable tool in analytical biochemistry and biopharmaceutical development. Careful selection of column chemistry, pore and particle size, mobile phase composition, and detection modes ensures robust, reproducible separations of biomolecules. Continued innovations in instrumentation, column design, and data analysis software will further enhance SEC performance and its role in ensuring the safety and efficacy of therapeutic products.

Used Instrumentation

• Agilent 1260 Infinity Bio-inert Quaternary LC System
• Agilent AdvanceBio SEC columns (2.7 µm, 130–300 Å)
• Agilent Bio SEC-5 columns (5 µm, 100–2000 Å)
• UV/DAD detectors (220–280 nm)
• Refractive index detector
• Multi-angle and dynamic light scattering detectors