Chromatography for Protein Purification

Chromatography Definition

Protein purification employs multiple chromatography techniques that separate products according to differences of their properties. Tagged proteins are convenient to be handled by affinity chromatography, which is designed to capture the target protein based on biorecognition of the protein tag.

Protein Purification Chromatography

Chromatography is certainly the principal and commonly used operation in downstream processing. This can be explained by certain advantages of chromatography over other unit operations. For example, chromatography displays high-resolution efficiencies which allow the resolution of complex crude mixtures with very similar molecular properties. In addition, chromatography is ideal for capturing molecules from the dilute solutions encountered in bioprocessing.

The principle of column chromatography is to separate a large pool of proteins into many smaller pools, some of which are enriched in the protein of interest. While expensive and specialized equipment is available for column chromatography, only basic equipment is required.

Protein Purification Systems

Among all chromatographic techniques, affinity chromatography plays a major role. In fact, affinity chromatography is the most specific and effective protein purification technique, providing a rational basis for the purification of target proteins. It exploits the principle of biomolecular recognition, that is, the ability of biologically active macromolecules to form specific and reversible complexes with affinity ligands. As conventional purification protocols for high-value proteins are replaced by more sophisticated procedures based on affinity chromatography, the focus is shifted toward designing and selecting ligands of high affinity and specificity.

The need to obtain a purified protein, economically and in sufficient quantity, applies to any purification, from preparation of an enriched protein extract for biochemical characterization to large-scale production of a therapeutic recombinant protein. Downstream processing has thus been challenged with demands of high yields, resolving power, and cost efficiency.

Affinity chromatography is applied in separating biochemical mixtures based on highly specific interactions. Target protein with well-defined property can be exploited in the purification process.

Ion exchange chromatography works as a common protein purification method that separates ions and polar molecules based on their affinity to the ion exchanger. Soluble molecules bind to oppositely charged insoluble stationary phase while passing through the column.

Size exclusion chromatography, separates molecules by their sizes and molecular weight.

Hydrophobic interaction chromatography separates Target proteins with hydrophobic amino acid side chains on surfaces interact with the hydrophobic groups and bind to them.

Advantages and Disadvantages of Different Purification Methods

  Affinity chromatography Ion exchange chromatography Size-exclusion chromatography Hydrophobic interaction chromatography
Protein property Biorecognition Charge Size Hydrophobicity
Applications Receptor and ligand, enzyme and substrate, antigen and antibody Charged molecules Large molecules, macromolecular complexes Proteins and peptides with hydrophobic amino acid side chains on surfaces
Advantages • Able to isolate one specific protein at a time
• High recovery yield
• Rapid separation
• High accuracy and precision
• High matrix tolerance
• High selectivity
• High recovery yield
• Well defined separation time
• Narrow bands available
• High selectivity
• Mild, non-denaturing conditions
Disadvantages Demand ligand with high selectivity Inconsistency from column to column Demand differences in MW Too strong interactions


Adamikova J, et al. (2019) Chromatographic purification of recombinant human erythropoietin. Biotechnol Lett 41(4-5): 483-493.
Labrou NE (2014) Protein purification: An overview. Methods in molecular biology (Clifton, N.J.) 1129: 3-10.
Joshi H, et al. (2017) Novel method to rapidly and efficiently lyse escherichia coli for the isolation of recombinant protein. Anal Biochem 528: 1-6.

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