Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization

Area of Science:

• Biopharmaceutical manufacturing
• Protein chemistry
• Immunology

Background:

• Monoclonal antibodies (mAbs) are crucial therapeutics but manufacturing faces challenges due to complexity and regulatory demands.
• Protein aggregates, a concern for immunogenicity, can form during viral inactivation steps like acidic treatment.
• Acidic treatment, while necessary for viral inactivation, can induce mAb denaturation and aggregation, leading to product loss.

Purpose of the Study:

• To investigate the mechanisms of monoclonal antibody (mAb) denaturation and aggregation at low pH and during subsequent neutralization.
• To identify strategies for mitigating mAb aggregation during manufacturing processes.
• To enhance product yield by optimizing viral inactivation procedures.

Main Methods:

• Systematic investigation of two mAbs under low pH conditions and after neutralization.
• Analysis of changes in mAb surface hydrophobicity and molecular size.
• Evaluation of the impact of d-sorbitol addition and temperature reduction on mAb stability.

Main Results:

• At low pH and low ionic strength, mAb surface hydrophobicity increased without significant changes in molecular size.
• Neutralization of acidic mAb solutions led to decreased monomer fraction and increased average molecular size, indicating aggregation.
• Electrostatic repulsion at low pH prevents aggregation, which is initiated upon neutralization.
• Limiting denaturation with d-sorbitol or reduced temperature improved monomer recovery post-neutralization.

Conclusions:

• Electrostatic repulsion plays a key role in preventing mAb aggregation under acidic conditions.
• Neutralization triggers aggregation by reducing electrostatic repulsion between denatured mAb molecules.
• Strategies like d-sorbitol addition or temperature control during low pH treatment can significantly improve mAb monomer recovery and manufacturing yields.