Ion Exchange Chromatography (IEX) is a protein purification technique that separates proteins based on their different electrostatic interactions with charged ligands at a specific pH. The latter pH is determined based on the pH-Isoelectric Point (pI) at which the net electrical charge of the protein is equal to zero. Therefore, the pH buffer should either be higher or inferior to the pI of the protein of interest.

The following section introduces the basic principles of Ion exchange chromatography and helps you understand when to choose this technique for your protein expression strategy.

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How Does Ion Exchange Chromatography Work?

Ion exchange chromatography is an adsorption chromatography that relies on the electrostatic
interaction between the molecule or biomolecule to be purified and groups of opposite charge
covalently bound to the matrix.

Proteins are composed of amino acid sequences by which they acquire their charges. However, at a certain pH-Isoelectric Point, the net electrical charge of the protein is equal to zero. This parameter is of great importance as it allows the determination of the pH of the buffer used in the IEX protein separation. Therefore, to allow a protein separation based on their charge, the pH buffer should either be higher or inferior to the pI of the protein of interest.

Like all liquid chromatography techniques, IEX is composed of a mobile phase and a stationary phase:

  • Mobile phase: This phase consists of a low ionic strength buffer along with the protein
  • Stationary phase: This phase consists of an inert resin with an immobilized ion exchanger.The latter are spherical beads with attached functional groups granting them the charged property.

Immobilized particles are always oppositely charged to the protein of interest. To this extent, two IEX types can be distinguished; Cation Exchange Chromatography (CEX), and Anion Exchange
Chromatography (AEX).

  • Cation Exchange Chromatography (CEX): It allows the separation of positively charged proteins via the negatively charged ligands.
  • Anion Exchange Chromatography (AEX): It allows the separation of negatively charged proteins via the positively charged ligands.
IEX Cation Exchange Chromatography Anion Exchange Chromatography
Protein Adsorption Positively charged protein Negatively charged protein
pH Buffer pH Buffer < pI pH Buffer > pI
  1. Protein Desorption: To weaken the electrostatic interactions and elute the target protein,
    two methods are possible:
  • Gradient Elution: Increasing the concentration of the competitor, i.e., molecules similarly
    charged to the protein of interest. For example, a gradual increase in salt concentration allows the breaking of the bonds in AEX.
  • pH Change: Changing the buffer pH in the mobile phase can influence the net charge of proteins, same as their ability to bind to the matrix.

What Are The Advantages Of Ion Exchange Chromatography?

  • Resolution: Ion exchange chromatography grants efficient separation of proteins with different charges to allow high purity.
  • Capacity: IEX allows the purification of large volumes with the possibility of getting a
    concentrated product with a considerably decreased volume.
  • Mildness: Ion exchange chromatography is performed under non-denaturing conditions. It
    maintains native protein forms suited for further applications.
  • Diversity: IEX is one of the most versatile liquid chromatography techniques.


What are the Applications of Ion Exchange Chromatography?

  • Characterization: IEX allows analysis and characterization of biotherapeutics, like ADCs and mAbs. It allows the identification of the heterogeneity of the product in terms of size and charge.
  • Product concentration: At the end of the downstream process, IEX is a useful tool to obtain a concentrated protein in a smaller volume.
  • Purification: IEX is an effective technique to isolate the target protein from other protein charge variants and isoforms. IEX is also a successful tool to separate nucleic acids, antibiotics, vitamins, and many other biomolecules.
  • Analysis: IEX is well-known for routine amino acid analysis from mixtures such as blood serum samples. Also, it is suitable to determine the nucleic acid composition via nucleotide analysis.

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