Hydrophobic Interaction Chromatography (HIC) Technique

Hydrophobic interaction chromatography (HIC), based on reversible interaction between the hydrophobic surface patch of analyte and the hydrophobic ligand of a chromatographic medium at high salt concentrations, is a widely used technique for separating proteins with different configuration and purifying protein products in their native state. Recent, HIC also has been proved to be suitable for isolating PEGylated proteins and studying protein folding and unfolding.

How Does It Work?

Fig. 1 Schematic illustration of (A) hydrophobic interaction between proteins in an aqueous solution and (B) between proteins and a hydrophobic ligand on an HIC adsorbent. (Methods in enzymology. Academic Press, 2009, 463: 405-414)

Protein that has the hydrophobic chemical structure will self-associate or interact when dissolved in a watery solution. When protein go through the HIC, it is likely to be bonded with the hydrophobic ligand, and separated with other contaminants or small molecules that possess disparate hydrophilia. Thus, HIC is capable to remove assorted impurities in the solution and concentrate protein products.

As regards to the PEGylation, which is the most employed technology to modify the protein and improve its performance in stability, solubility, and activity. Despite the numerous hydroxyl groups on PEG, some types of PEGylated protein that possesses less hydroxyl groups may still maintain hydrophobic properties and can be conjugated with hydrophobic ligand and purified with HIC. The difference of hydrophilia is desired between the chemically modified proteins (i.e., PEGylated protein) and other molecules to isolate and purify the proteins. While most of PEGylated protein have diverse functional groups. Accordingly, the hydrophilia of chromatographic medium need to be tuned to correlate with specific PEGylated protein. HIC has been proved to be a useful polishing step after IEX chromatography and SEC. It is worth noting that in most of the cases, the separation conditions need to be determined empirically due to the apparent increase in molecular weight caused by solubilized conjugates.

General Considerations

• Salts

Various salt ions and ion pairs have the ability to disrupt the orderly arrangement of surrounding water molecules. The composition and concentration of the salt in the mobile phase has the most significant impact on the adsorption capacity of the HIC medium.

• Ionic strength

The ionic strength directly affects the retention value of the sample components in the stationary phase. Generally, it is appropriate to adopt the gradient elution method in HIC experiment to change the ionic strength, which can improve the selectivity of separation.

• pH

In general, the hydrophobicity of the protein can be modulated by changing the pH of the solution. When the pH of the solution is close to the isoelectric point (pI) of the protein, its hydrophobicity increases, which is conducive to interact with the stationary phase ligand.

• Temperature

Generally, when the column temperature rises, the conformation of biological macromolecules will change, the hydrophobic effect will increase, and the adsorption capacity will also increase, which will help to improve the separation ability of the chromatography column.

What Can HIC Be Used For?

• HIC is used in the purification of proteins over a broad range of scales - in both analytic and preliminary scale applications.
• HIC is often employed to remove product aggregate species, which possess different hydrophobic properties than the target monomer species.
• Nowadays, HIC methods have been gradually developed and considered to be suitable for separation of PEGylated proteins, PEGylated antibodies or other conjugates.
• HIC resins can also be modified as an alternative to increase chromatographic resolution. For example, RNase A could be completely separated from PEGylated products and resolution of the mono- and di- PEGylated conjugates was optimized using the 5 kDa PEG modified resin.

Strengths & Weaknesses of HIC

References

1. McCue J T. Theory and use of hydrophobic interaction chromatography in protein purification applications. Methods in enzymology. Academic Press, 2009, 463: 405-414.
2. Ramos-de-la-Peña A M, et al. Progress and challenges in PEGylated proteins downstream processing: a review of the last 8 years. International Journal of Peptide Research and Therapeutics, 2020, 26(1): 333-348.