Choosing the right protein A affinity chromatography media can remove aggregates efficiently

Area of Science:

• Biochemistry
• Chemical Engineering
• Biotechnology

Background:

• Protein A chromatography (PAC) is a standard capture method for monoclonal antibody (mAb) purification.
• Current selection criteria for PAC often focus on dynamic binding capacity, potentially overlooking aggregate removal efficiency.
• Efficient aggregate removal during initial capture can simplify downstream polishing steps.

Purpose of the Study:

• To develop and validate a method for selecting Protein A chromatography media based on monoclonal antibody (mAb) aggregate removal.
• To evaluate the elution behavior of mAb aggregates and monomers on different PAC columns.
• To compare the efficacy of two stepwise elution protocols for aggregate removal.

Main Methods:

• Linear pH gradient elution experiments were performed using two distinct mAbs on various PAC columns.
• The elution profiles of both mAb aggregates and monomers were quantified.
• Two stepwise elution protocols (A: low pH elution; B: 1.0 M arginine wash followed by elution) were tested for aggregate removal.

Main Results:

• Different mAbs exhibited varied aggregate elution behaviors; one showed stronger retention of aggregates than monomers, while another eluted aggregates in both weakly and strongly retained fractions.
• Protocol A resulted in no detectable aggregate peaks during the wash stage.
• Protocol B, incorporating a 1.0 M arginine wash, effectively monitored aggregate peaks and demonstrated higher aggregate removal and monomer yield compared to Protocol A and other PAC columns.

Conclusions:

• A novel method for selecting PAC media based on aggregate removal efficiency is presented.
• The proposed method, utilizing specific elution conditions, aids in optimizing PAC selection for improved mAb purification.
• Protocol B, with an arginine wash, proved more effective for aggregate removal and maintaining monomer yield, highlighting its utility in PAC media selection.