Novel Technique For Quick And Easy Aggregate Removal Of Monoclonal Antibodies Using Competitive Binding On CEX

By Indraneel Sanyal, Nuresh Manukonda, Ankitha Hirematha and Mukesh Dakua Downstream Process Development, Aragen Biologics Private Limited, Bangalore, India.

In this study, we demonstrated aggregate clearance using competitive binding techniques. Traditionally, cation exchange chromatography (CEX) is a key step for aggregate removal in standard monoclonal antibody (mAb) purification processes. Our research evaluated two distinct approaches: an extended CEX ligand resin and a CEX membrane.

Unlike conventional bind-and-elute CEX, which can capture up to 80 mg/mL, we tested negatively charged CEX resin and membrane in a negative-mode purification strategy for aggregate clearance. Antibody loading was increased to 1000 mg/mL of resin to assess both clearance and recovery. The protein was applied to the resin or membrane after the viral inactivation step at pH 4.5, with a conductivity below 5 mS/cm. The column was equilibrated with a 50 mM sodium acetate buffer at pH 4.5.

Under these conditions, the protein binds to the resin, while aggregates, which exhibit stronger binding, facilitate monomer flow-through over time due to the displacement effect. Initially, both monomers and aggregates bind to the resin until saturation is reached. As loading continues, monomers are displaced first and break through, followed by aggregates once the column is fully occupied.

To optimize recovery and maximize aggregate clearance, we generated breakthrough data for both the CEX resin and CEX membrane, providing valuable insights into their performance in aggregate removal.