Factors influencing antibody stability at solid-liquid interfaces in a high shear environment

Area of Science:

• Biochemistry
• Protein Science
• Biopharmaceutical Development

Background:

• Human monoclonal antibodies of the IgG4 isotype are critical therapeutic proteins.
• Understanding protein stability under stress is crucial for bioprocessing and drug formulation.
• Interfacial shear can impact protein structure and function.

Purpose of the Study:

• To investigate the effects of interfacial shear on the structural integrity of IgG4 antibodies.
• To identify key factors influencing IgG4 monomer loss under high shear conditions.
• To elucidate the primary denaturation pathway of IgG4 under interfacial stress.

Main Methods:

• Utilized a rotating disk shear device to apply controlled interfacial shear.
• Analyzed IgG4 structural integrity using SDS-PAGE, IEF, dynamic light scattering, and LC-MS peptide mapping.
• Varied solution conditions (pH, ionic strength, surfactant concentration, temperature) and interface properties (surface roughness).

Main Results:

• The primary denaturation pathway observed was the formation of large, insoluble IgG4 aggregates.
• No evidence of soluble aggregation, primary structure breakdown, or chemical modifications was detected.
• pH and nanometer-scale surface roughness were dominant factors affecting IgG4 monomer loss.
• Surfactant addition showed a stabilizing effect up to 0.02% (w/v).
• Temperature (15-45°C) was not a significant factor in IgG4 monomer loss.

Conclusions:

• Interfacial shear predominantly leads to IgG4 aggregation, not fragmentation or chemical modification.
• Controlling pH and surface roughness at the solid-liquid interface is critical for maintaining IgG4 structural integrity during bioprocessing.
• Surfactants can enhance IgG4 stability under shear stress.