Ion exchange chromatography (IEX) is a chromatographic separation method essentially based on the net charge of the protein, and is generally used to follow deamidation and succinimide formation. There are two types of IEX, cation exchange and anion exchange chromatography. At buffer pH values above this IP, the protein is negatively charged (anionic); at pH values below that, the protein is positively charged (cationic).
All proteins exhibit a net charge that depends on the amino acid composition of the protein and any covalently attached modifications. The net charge of a protein is influenced by the pH of the solvent that it is dissolved in, as solvents exchange hydrogen ions with proteins. Typically, binding of a protein to IEX must be determined by trial and error, using solvents with a range of pH values, to determine the optimum pH for protein retention. A solvent pH that is about one pH unit away from the pI is usually sufficient for protein binding.
Ion Exchange Chromatography is the most widely used chromatographic mode in biopurification and applied in most downstream processing platforms. The typical scheme in IEX of binding the target, washing the column and eluting the target is called 'bind/elute mode' and is often applied in intermediate purification steps for example in downstream processing of antibodies. Anion Exchange Chromatography is an integral part of most plasma protein purification platforms, such as Factor VIII purification.
The first step is the equilibration of the stationary phase. When equilibrium is reached, all stationary phase charged groups are bound with exchangeable counterions, such as chloride or sodium. The pH and ionic strength of the start buffer are selected to ensure proteins of interest bind to the medium.
Sample Application and Wash
The goal in the second step is to bind the target molecule(s) and wash out all unbound material.
The proteins with the lowest net charge at the selected pH will be the first ones eluted from the column as ionic strength increases. Similarly, the proteins with the highest charge at a certain pH will be most strongly retained and will be eluted last.
A final wash with high ionic strength buffer regenerates the column and removes any molecules still bound. The column is then re-equilibrated in start buffer before starting the next run.
Kosanovic M, et al. (2017) Ion-exchange chromatography purification of extracellular vesicles. Biotechniques 63(2): 65-71.
Clark DD, et al. (2018) Virtual protein purification: A simple exercise to introduce ph as a parameter that effects ion exchange chromatography. Biochem Mol Biol Educ 46(1): 91-97.