Implementation of an experimental and computational tool set to study protein-mAb interactions

Area of Science:

• Biochemistry
• Computational Biology
• Chemical Engineering

Background:

• Understanding protein-monoclonal antibody (mAb) interactions is crucial for biologics manufacturing.
• Host cell proteins (HCPs) can co-purify with mAbs, complicating downstream processing.
• Developing robust methods to characterize these interactions is essential.

Purpose of the Study:

• To develop and validate a combined experimental and computational toolset for studying protein-mAb interactions.
• To elucidate the thermodynamic and mechanistic basis of binding for specific model proteins with a mAb.
• To assess the utility of this approach for analyzing mAb-HCP interactions.

Main Methods:

• Cross-interaction chromatography for initial screening of model proteins.
• Fluorescence polarization assays to determine binding affinities and thermodynamics.
• Protein-protein docking simulations to identify binding interfaces and interaction types.
• Salt sensitivity and temperature dependence studies to probe interaction mechanisms.

Main Results:

• Identified lactoferrin and pyruvate kinase binding to the mAb as entropy-driven and salt-insensitive, suggesting hydrophobic interactions.
• Characterized ribonuclease B binding as enthalpy-driven and salt-sensitive, indicating electrostatic interactions.
• Computational docking revealed the CDR region of the mAb as a key binding site for all tested proteins.
• Binding interfaces showed complementary hydrophobic/electrostatic clusters for lactoferrin/pyruvate kinase, and predominantly electrostatic interactions for ribonuclease B.
• Experimental and computational results were consistent.

Conclusions:

• The combined experimental and computational approach effectively characterizes protein-mAb interactions.
• Different proteins interact with the same mAb via distinct thermodynamic and mechanistic pathways.
• This methodology holds promise for improving the analysis of mAb-HCP interactions in biopharmaceutical development.