Light Scattering and Size Exclusion Chromatography (SEC) in Biopharma

Importance of the topic

Size exclusion chromatography (SEC) combined with light scattering detection has become a cornerstone technique in biopharmaceutical analysis. It enables direct measurement of molecular weight, size distribution and structural conformation of proteins, monoclonal antibodies, polymers and nanoparticles under native conditions. This capability is critical for quality control, characterization of aggregates, and ensuring batch-to-batch consistency in drug development and manufacturing.

Objectives and study overview

This white paper reviews theoretical and practical aspects of integrating static (SLS) and dynamic light scattering (DLS) detectors with SEC. Key goals are:

• To illustrate how multi-angle static light scattering yields weight-average molar mass (Mw) and radius of gyration (Rg).
• To show how DLS provides hydrodynamic radius (RH) and resolves size distributions.
• To demonstrate real-world examples in biopharma, including analysis of monoclonal antibodies (mAbs), serum albumin, and protein aggregates.

Methodology and instrumentation

Light scattering detection was implemented in an Agilent InfinityLab GPC/SEC system equipped with:

• Agilent 1260 Infinity II Multi-Angle Light Scattering Detector (20 angles, 12°–164°).
• Agilent WinGPC Software for automated Zimm and Guinier analyses.
• Agilent 1260 Infinity II Bio-SEC Multidetector System combining DLS (15°/90° angles), VWD and refractive index detectors.
• PROTEEMA 300 Å or mAb SiO2 3 µm analytical columns.

In SLS, scattered intensity vs. angle and concentration was extrapolated to θ→0 and c→0 to extract Mw and Rg. DLS autocorrelation functions were fitted with single‐ or bi-exponential models to determine RH and distribution profiles.

Principal results and discussion

Examples include:

• Bevacizumab on a mAb SiO2 column:
  – Mw = 147 kDa (±1 % by 20-angle Zimm plot)
  – Rg ≈ 11 nm
• Bovine serum albumin (BSA) in PBS:
  – Resolved monomer (RH ~4 nm) and aggregates (RH ~8 nm) by DLS
• IgG aggregates:
  – Primary structure RH ~6 nm, higher associates RH ~11 nm
• Thyroglobulin:
  – Rg = 12 nm and 16 nm for monomer and dimer
  – RH = 9 nm and 14 nm respectively
  – ρ = Rg/RH ratio provided topology insights

Combining SLS and DLS yields z-average mass and size, while the Rg/RH ratio differentiates spherical, coil-like or hollow structures.

Benefits and practical applications

• Direct, calibration-free determination of molar mass and size.
• Fraction-specific analysis under physiological conditions.
• Enhanced detection of aggregates, critical-quality attributes in biotherapeutics.
• Topology assessment through combined Rg and RH measurements.

Future trends and applications

Advances will focus on faster detectors, integration with high-throughput autosamplers and microfluidic columns. Emerging areas include characterization of lipid nanoparticles, extracellular vesicles, adeno-associated viruses and mRNA therapeutics. Machine-learning algorithms may improve deconvolution of overlapping size distributions.

Conclusion

Integration of static and dynamic light scattering with SEC provides a robust platform for comprehensive physicochemical characterization in biopharma. The approach delivers precise, fraction-resolved molecular weight, size and topology data, supporting quality control, formulation development and regulatory compliance.

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