Improvement in protein separation by pH excursion modulated ion-exchange chromatography

• Suppression of acidic pH excursion during salt-based elution • Hydroxyl ion pulse from anion exchange cartridge • Sharper eluted protein peak • Significant increase in resolution • Suitable for analytical and preparative separation The transient change in pH during the elution step in ion exchange chromatography is referred to as pH excursion. Acidic pH excursion is commonly observed during salt-based elution in cation exchange chromatography. This could have a detrimental effect on protein stability as well as on protein separation. Acidic pH excursion could delay protein elution and thereby decrease resolution of sequentially eluted proteins. In a recent study, a method for suppressing or modulating pH excursion during salt-induced elution in cation exchange chromatography has been discussed. In the current study, the feasibility of using such pH excursion modulation for increasing resolution in protein separation is examined. Modulation of pH excursion during elution resulted in rapid release of a weakly bound protein from cation exchange media while the release of a strongly bound protein remained largely unaffected. This differential effect was then utilized to increase the resolution in binary protein separation. The resolution obtained in pH excursion modulated cation exchange chromatography was significantly greater than that obtained with unmodulated cation exchange chromatography, i.e., where acidic pH excursion was allowed to happen as usual. This approach for increasing resolution in protein separation could potentially be utilized in analytical as well as preparative protein chromatography applications.