Introduction to Ion Exchange Chromatography

For decades ion exchange chromatography has been used across numerous disciplines - from water purification and softening to food and clinical research to fermentation. In this Purolite How to Series, we'll explore the basics of ion exchange chromatography for the purification of monoclonal antibodies (mAbs) and recombinant proteins.
 
The purification procedure of monoclonal antibodies can be broken down into three stages: capture, intermediate purification, and polishing. Ion exchange chromatography is the separation and purification of molecules based on charge; specifically, the separation of molecules that are similar in character to the target molecule identified for purification and thus difficult to separate using other chromatography techniques.
 
Ion exchange resins are concentrated, highly selective, and have a high capacity. While ion exchange is one of many techniques used to separate and purify proteins and biomolecules, it can be used in all three stages of purification.

 

Ion Exchange Types & Structure

To perform ion exchange chromatography, a resin is coupled with a positively or negatively charged molecule which interacts with the target molecule selected for purification.
 
The resin attached to the ligand should possess three key characteristics. The resin should be:
 

1. Macro porous to flow efficiently through the column.
2. Hydrophilic or neutral to prevent non-specific protein interactions.
3. Chemically and physically stable to withstand multiple rounds of the purification procedure.

 
Due to the unique properties required for chromatography resins, Purolite resins for both affinity and ion exchange chromatography purposes are manufactured from Agarose.

 

Cation vs Anion Exchange Chromatography 

Two types of ion exchange chromatography are applied during the purification procedure: anion exchange chromatography and cation exchange chromatography.
 
Cation exchange resins, such as Purolite’s SP product line, carry a negatively charged ligand and bind to positively charged molecules. In contrast, anion exchange resins, such as Purolite's Q product line, carry a positively charged ligand, thus binding to negatively charged molecules.

 

The Basic Steps of Ion Exchange Chromatography

Five key steps are required during ion exchange, as outlined below:
 
1. Equilibration
 
Begin by equilibrating the column with a starter buffer. Typically, equilibration requires 5-10 column volumes of buffer to reach a baseline. The purpose of this step is to prepare the column to bind to the sample, so the buffer selected should possess all the conditions that will encourage the sample to bind to the column.
 
2. Column Loading
 
Load the sample in the column, ensuring it has the same conditions as the binding buffer selected so it will bind to the column too.
 
3. Washing
 
Wash the column to return to baseline.
 
4. Gradient Elution
 
Gradually increase the salt concentration within the column. As salt concentration increases, proteins with opposing charges will elute. Gradient elution, the process of slowly increasing salt concentration, helps to ensure that proteins with the highest interaction with the column will elute last.
 
5. Wash & Re-Equilibration
 
Complete the process by washing the column once again with a high salt solution to remove any remaining molecules bound to the column and begin re-equilibration to perform another purification.