Understanding antibody and viral glycoprotein interactions using CG-MALS

Significance of the Topic

Viral surface glycoproteins serve as primary targets for neutralizing antibodies and are central to vaccine design and therapeutic interventions. Accurately quantifying the molecular interactions between antibodies and these glycoproteins in solution is crucial for understanding neutralization mechanisms and guiding the development of post-exposure treatments.

Objectives and Study Overview

This study combined SEC-MALS and CG-MALS to characterize a dimeric viral glycoprotein (vGP) and its binding with a neutralizing monoclonal antibody (IgG). The main objectives were:

• Determine the molar mass, native oligomeric state, and glycan content of the vGP.
• Quantify the stoichiometry and binding affinity of the vGP–IgG interaction.
• Infer epitope location and higher-order complex formation in solution.

Instrumentation Used

• AKTA purifier FPLC with Superdex S200 10/30 SEC column
• Wyatt miniDAWN multi-angle light scattering detector
• Optilab differential refractive index detector
• Wyatt Calypso composition-gradient system
• ASTRA and CALYPSO software for data acquisition and model fitting

Methodology

• SEC-MALS determined the vGP’s native molecular weight, oligomeric state, and glycan fraction via Protein Conjugate Analysis.
• CG-MALS applied automated single- and dual-component concentration gradients to monitor self-association of vGP and hetero-association with IgG.
• Light scattering and concentration signals were fitted to association models to extract stoichiometries and equilibrium constants.

Main Results and Discussion

• SEC-MALS confirmed a glycosylated vGP dimer (~100–118 kDa) with ~17% glycan content and indicated concentration-dependent self-association.
• CG-MALS showed each IgG Fab binds one vGP dimer with Kd ~58 nM, and a minor higher-order complex of two IgGs bound to two vGP dimers was detected.
• Models allowing two IgGs per single vGP dimer failed to fit, suggesting steric hindrance near the dimer interface and pinpointing the epitope location.

Benefits and Practical Applications

• Provides precise binding parameters and stoichiometries vital for antibody engineering and vaccine antigen design.
• Enables accurate glycoprotein characterization for quality control in biopharmaceutical development.

Future Trends and Potential Applications

• Extending CG-MALS to other multivalent viral antigens and antibody formats.
• Combining with high-throughput workflows to accelerate therapeutic antibody screening.
• Advances in detector sensitivity for characterizing weak or transient complexes.

Conclusions

The integrated SEC-MALS and CG-MALS approach provided detailed insights into the molecular weight, glycosylation, stoichiometry, and binding affinity of a viral glycoprotein–antibody system, informing strategies for post-exposure therapies and vaccine design.

References

1. Murin CD, Fusco ML, Bornholdt ZA, Qiu X, Olinger GG, Zeitlin L, Kobinger GP, Ward AB, Saphire EO. Structures of protective antibodies reveal sites of vulnerability on Ebola virus. Proc Natl Acad Sci USA. 2014;111(48):17182–17187.