Improving process robustness of cation exchange chromatography with cationic buffers for the reduction of aggregates.

Abstract

Cation exchange chromatography (CEX) is commonly used to separate aggregates from monomers during the industrial manufacturing of recombinant proteins. However, the similar isoelectric point of aggregates and monomers makes the stepwise elution CEX an unstable process. In this study, the performance robustness of sodium chloride stepwise elution and cationic buffers (histidine and Bis-Tris) stepwise elution were compared through Monte Carlo simulation. While all trials achieved acceptable levels of monomer purity, sodium chloride stepwise elution exhibited significant fluctuations in yield and elution volume due to variations in elution pH and eluent concentration. In contrast, histidine or Bis-Tris stepwise elution resulted in more consistent yield and elution volume across a broad operating range. The findings indicate that the dissociation behavior of cationic buffers mitigates the impact of pH variation on ion-exchange equilibrium and thus enables a more robust CEX process.