Hydrophobic Interaction Chromatography (HIC) is a protein purification technique based on the protein’s degree of hydrophobicity and hydrophobic interaction with immobilized ligands. In this section, you will find a detailed description of HIC chromatography. This is not a stand-alone service. All our protein purification techniques are included in our Protein Expression Services

When To Use Hydrophobic Interaction
Chromatography For Protein Purification?

Each protein expression project is different, so it is of great importance to establish an adapted protein expression strategy well suited to your project needs. In this matter, the purification step is a determining factor to achieve your goals. Here are some key characteristics to help you define if this purification technique is suitable for your protein project :

  • To maintain the proper structure and function of the protein: HIC is well-suited for structural studies and protein characterization. The former is performed in non-denaturing conditions. Therefore, proteins maintain their native forms.
  • To get high sensitivity: HIC is well-suited for downstream applications such as Mass Spectrometry (MS), as it ensures increased detection and sensitivity of the targeted protein.
  • To obtain efficient separation: Thanks to its low viscosity, HIC guarantees greater diffusion and faster protein separation in the column compared to other purification methods.
  • To increase purification and concentrate the protein sample: Hydrophobic interaction chromatography applied to protein purification is suited for any downstream phase. It complements other chromatography techniques and can operate after capture, intermediate, or polishing steps:
    • To perform protein purification after ion-exchange separation (IEX): Proteins eluted from ion exchange chromatography are highly concentrated in salt, making them ready for HIC purification which requires high salt concentration for loading.
    • To concentrate the sample directly perform size-exclusion chromatography (SEC): To remove excess salt from IEX-eluted proteins and to eliminate small aggregates, SEC chromatography can be performed during the final purification steps. Finishing up with SEC chromatography will not only allow salt removal but also provides the highest resolutions.

What is Hydrophobic
Interaction Chromatography?

Hydrophobic Interaction Chromatography is a protein separation technique. It is based on interactions between the hydrophobic region on the surface of proteins and hydrophobic ligands immobilized in the resin. The mechanism of action relies on the equilibrium between adsorption and desorption in the presence or not of salt. The higher the salt concentration is, the higher the protein-ligand interactions are.

HIC separation has both a stationary phase and a mobile phase:

  • Stationary phase: Resin immobilized hydrophobic ligands such as alkyl and aryl.
  • Mobile phase: Aqueous buffer with a high salt concentration usually ammonium sulfate.

How to perform Hydrophobic
Interaction Chromatography?

Like other chromatography techniques, HIC is also composed of 3 major steps:

  • Loading: The first step involves column equilibration with a high salt concentration buffer, followed by protein mixture loading.
  • Binding: Under this salt-concentrated environment, hydrophobic proteins interact with ligands to form a hydrophobic bond. This phenomenon is known as “protein adsorption”, i.e., protein binding. Weak interactions and inbound proteins are washed out of the column. Only strong hydrophobic interactions remain.
  • Elution: Finally, the elution of proteins takes place by decreasing the salt concentration. The interaction created between protein-ligand break results in the release of proteins in order of increased hydrophobicity, i.e., from weak hydrophobic reactions to the strongest interactions.

What Are The Applications
Of Hydrophobic Interaction Chromatography?

Hydrophobic interaction chromatography is a versatile separation tool. It can be used in many application areas:

  • Aggregate removal: This technique has a high selectivity to eliminate the misfolded proteins and other protein aggregates. It is mostly employed when the protein is required in a soluble form and free from contaminants. Generally, aggregates exhibit a higher hydrophobicity degree than the targeted monomeric protein. Therefore, under an appropriate HIC environment, aggregates bind to the resin allowing the elution of the targeted proteins only.
  • Protein concentration: HIC is well-known for contaminant removal; however, it is also an efficient tool to obtain a concentrated product at the end of the protein expression process.
  • Protein characterization: Hydrophobic interaction chromatography is ideal for protein characterization studies. Since it holds the non-denaturing feature, proteins eluted with HIC keep their structure and function.
  • Analytical and preparatory applications: HIC can be employed in the biopharmaceutical industry or even at a research scale. One of its famous uses is the separation and analysis of Antibody Drug Conjugates (ADCs).

How To Optimize Hydrophobic Interaction

To accomplish protein purification with HIC and achieve the awaited goals, it is of great importance to select the proper parameters for this purification. Here are some of the factors to consider before applying hydrophobic interaction chromatography:

  • Salt: Salt plays a critical role in hydrophobic interaction chromatography. To understand the effect of salt on proteins, it is important to address the mechanism of salting-out effect The latter describes the decreased solubility of the sample due to the increase of salt concentrations in the sample. Adding salt to the protein sample promotes protein binding to ligands and can result in protein aggregation.

In this context, there are two parameters that can influence protein separation:

    • Salt concentration: Salt concentration is proportional to protein binding. Specifically, increasing the salt concentration results in higher hydrophobic interactions between proteins and ligands and vice versa. However, high salt concentration can sometimes result in an undesirable effect, notably, protein precipitation. To counter this side effect, it is recommended to opt for an intermediate concentration providing an effective separation with low precipitation risks. Moreover, placing the protein in the same buffer used in the column equilibration helps to lower the precipitation effect.
    • Salt type: Salts have different salting-out effects. For this reason, different protein separation profiles appear when using different salt types. Some salts appear to have a more salting-out effect than others. For example, ammonium sulfate or sodium chloride are commonly used to achieve better interactions.
  • pH: The buffer’s pH can influence protein binding. It is recommended to test several pH variations to see the best option that delivers a higher resolution.
  • Temperature: Temperature usually increases the hydrophobic interactions, thus protein binding. Yet, increasing this parameter can lead to negative consequences on the course of the chromatography as well as on protein structure. In other words, proteins can lose their correct structure therefore negatively impacting their solubility and their adsorption to the resin.
  • Ligand: Ligands play an important role in the hydrophobicity property and protein binding. Alkyl and aryl ligands are the most common ligands used in HIC protein separation.
  • Flow rate: Flow rate is a key parameter to optimize the chromatography process. An increased rate and lower adsorption of proteins, lead to yield loss.

To sum up, hydrophobic interaction chromatography is a versatile tool for protein purification. Based on the hydrophobic interactions created between proteins and ligands, HIC allows protein separation. This technique can be combined with other chromatography methods to concentrate the final product and obtain high resolutions.