(175aj) Enhancing Separation of Monomer and Aggregate with Mobile Phase Modifiers in Mixed Mode Chromatography

In biopharmaceutical production, the separation of aggregates, specifically dimers, from monomer often employs bind-and-elute chromatographic techniques which require tight operational ranges, gradient elution, or low protein loading. Flow through chromatography can overcome the capacity and gradient challenges, but still often requires narrow operating ranges to balance the trade-off between aggregate removal and monomer yield loss. In this study, we examine the use of mobile phase modifiers in a mixed mode chromatography system to modulate the binding of monomer and aggregates of antibodies and fusion proteins. Small scale column screening studies were performed to understand the impact of mobile phase modifier type and concentration on aggregate clearance, step yield, and host cell protein concentration. Arginine and propylene glycol, had the greatest impact on step yield and aggregate clearance while sodium chloride had a minimal impact on process performance but a significant impact on host cell protein clearance. The combination of mobile phase modifiers, specifically sodium chloride and arginine, was able to inhibit monomer-resin interactions without disrupting the strong aggregate-resin interactions over a robust range of modifier concentrations, increasing step yield greatly while retaining the desired aggregate clearance.