Developability Assessment of Engineered Monoclonal Antibody Variants with a Complex Self-Association Behavior Using...

Area of Science:

• Biopharmaceutical Development
• Protein Engineering
• Molecular Biophysics

Background:

• Monoclonal antibodies (mAbs) face development challenges at high concentrations, including self-association, high viscosity, and phase separation.
• These issues stem from complex solution properties and can hinder the creation of stable, high-concentration liquid formulations.
• Protein engineering offers a proactive strategy to enhance mAb developability by optimizing molecular properties early in the discovery phase.

Purpose of the Study:

• To identify and reengineer residues responsible for self-association in a model mAb using a combination of analytical and in silico methods.
• To evaluate the effectiveness of protein engineering in mitigating self-association and improving developability for high-concentration formulations.
• To assess correlations between experimental developability assays and computational predictions.

Main Methods:

• Systematic identification and reengineering of problematic residues contributing to mAb self-association.
• Utilized complementary analytical techniques: affinity capture self-interaction nanospectroscopy (AC-SINS), dynamic light scattering (DLS), and PEG-precipitation for solubility assessment.
• Employed predictive in silico tools, including CamSol, to model molecular interactions and guide engineering efforts.

Main Results:

• Identified hydrophobic interactions as a dominant driver of self-association in the model mAb.
• Demonstrated that mutations, including those in complementarity-determining regions (CDRs), can effectively mitigate self-association.
• Established correlations between experimental assays and computational predictions, with CamSol proving effective for screening.
• Engineered mAb variants showed reduced self-association and improved solubility while maintaining binding affinity and thermal stability.

Conclusions:

• Protein engineering is a viable strategy to address mAb self-association and enhance developability for high-concentration formulations.
• Complementary use of analytical and in silico methods enables efficient identification and mitigation of developability liabilities.
• Engineered mAbs exhibit improved biophysical properties suitable for advanced therapeutic applications.