EC-MALS for absolute biophysical characterization

Significance of the topic

Accurate biophysical characterization of biomacromolecules underpins research and development in life sciences and biopharmaceuticals. Determining absolute molecular weight, size, conformation, aggregation state and interactions in native solution is critical for protein engineering, structural biology, quality control of therapeutics and formulation of vaccines. Traditional methods often lack native-state information or rely on calibration standards, leading to uncertainty for non-globular or modified proteins.

Goals and overview

This work presents the combination of size-exclusion chromatography (SEC) with multi-angle light scattering (MALS), dynamic light scattering (DLS), UV absorbance and differential refractive index (dRI) detection—commonly termed SEC-MALS—for absolute, first-principles characterization of proteins, peptides, nucleic acids, polysaccharides and complexes. Applications range from monomer/aggregate quantification to stoichiometry of protein–protein and protein–nucleic acid complexes, and conjugate analysis of glycoproteins, PEGylated proteins and antibody–drug conjugates.

Methodology

SEC separates species by hydrodynamic size but cannot yield absolute molecular weight without assumptions about shape and column interactions. MALS measures scattered light at multiple angles to derive true molar mass independent of elution volume. DLS extends size measurement to small hydrodynamic radii. UV and dRI detectors provide concentration data; dRI is universal, while UV aids conjugate analysis when both detectors are combined.

Used instrumentation

• Wyatt DAWN 18-angle MALS, miniDAWN (3-angle) and microDAWN for UHPLC-SEC
• Optilab and microOptilab dRI detectors
• WyattQELS embedded DLS or external DynaPro modules
• Standard HPLC, UHPLC or FPLC systems and SEC columns
• ASTRA software for acquisition, analysis and reporting

Results and discussion

Monomer, oligomer and aggregate analysis: SEC-MALS distinguishes monomers from soluble aggregates and fragments, even when shoulders or tailing occur. Monoclonal antibody stress-test samples reveal well-resolved dimeric and fragment species and allow quantification of low-abundance aggregates.

Complex stoichiometry: Titration series of p53 wild-type and mutants with S100B demonstrate absolute binding stoichiometry (e.g., one S100B dimer per p53 monomer, or tetrameric complexes), revealing affinity and dynamic equilibrium.

Protein–nucleic acid interactions: Protein Conjugate Analysis in ASTRA determines mass of each component in PFV integrase–DNA complexes, confirming binding of two DNA strands per tetramer.

Conjugate analysis: Three-detector SEC-MALS-UV-dRI quantifies glycoprotein heterogeneity from insect versus mammalian expression, PEGylation reactions and antibody–drug conjugate (ADC) drug-antibody ratios with high sensitivity.

Membrane proteins: Detergent–protein conjugate analysis yields protein and detergent mass separately, guiding detergent choice to preserve functional oligomeric states.

Chromatography optimization: Online DLS coupled with MALS distinguishes non-ideal column interactions from true conformational differences, aiding selection of SEC conditions.

Extension to peptides and polysaccharides: SEC-MALS detects small peptides (down to 500 Da) and measures molar mass distributions of maltodextrin and vaccine polysaccharides for process control.

Benefits and practical applications

• Absolute molar mass and size without standards or shape assumptions
• Characterization of native oligomeric states, aggregates and fragments
• Quantitative conjugate analysis for glycoproteins, PEGylates and ADCs
• Determination of complex stoichiometry and binding affinities in solution
• Support for formulation, vaccine process development, QC and regulatory filings

Future trends and opportunities

Advances in micro-flow instrumentation will enhance sensitivity for UHPLC-SEC peaks. Integration with automated platforms and high-throughput sampling will accelerate process development. Coupling with orthogonal separations (ion exchange, field-flow fractionation) and extension to larger nanoparticles and viruses will broaden applications. Composition-gradient MALS (CG-MALS) will provide deeper insight into transient interactions and affinities.

Conclusion

SEC-MALS, augmented by DLS, UV and dRI detection, provides an absolute and versatile platform for biophysical characterization of biomolecules in native solution. Its broad applicability across proteins, peptides, polysaccharides and complexes makes it indispensable for research, development and quality control in biotechnology and biopharmaceutical industries.

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