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Hydrophobic Interaction Chromatography (HIC)

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Separation using HIC is based on the reversible interaction between a protein and the hydrophobic ligand bound to the chromatography matrix. 

Hydrophobic amino acids of proteins and peptides are usually located away from molecular surfaces. However, many biomolecules have some hydrophobic groups that are sufficiently exposed to allow interaction with hydrophobic ligands on media. Compared to reversed phase chromatography, the density of the ligand on the matrix is much lower and allows mild elution conditions, helping to preserve biological activity. HIC is particularly suitable for samples precipitated with ammonium sulfate or eluted in high salt concentrations since high ionic strength buffers enhance the hydrophobic interaction.

The interaction is enhanced by buffers with high ionic strength, which makes HIC an excellent purification step after ammonium sulfate precipitation or after elution in high salt during ion exchange chromatography (IEX). HIC is well-suited for the capture or intermediate steps in a purification scheme.

During HIC, sample components bind to the column in high ionic strength buffer, typically 1 to 2 M ammonium sulfate or 3 M NaCl. High concentrations of salt, especially ammonium sulfate, may precipitate proteins. Therefore, check the solubility of the target protein under the binding conditions to be used. Elution is usually performed by decreasing the salt concentration, stepwise or using a gradient.

Principles of HIC Media Selection Products Selection Guide

Proteins in aqueous solution have various surface residues exposed to solvent to different degrees, dependent on protein structure. These residues can be hydrophilic, for example they may carry charges, or they can be hydrophobic, such as those in the amino acids phenyl alanine, tyrosine and tryptophan. Hydrophobic groups will prefer to bury themselves internally in the protein 3D structure but some will be exposed. Salts can be used to precipitate or crystallize proteins out of solution – to cause the proteins to self-associate. Scientists have been aware, since Hofmeister, that different salts play a significant role in self-association or the association with hydrophobic surfaces.

These phenomena form the basis for hydrophobic interaction chromatography (HIC). A chromatographic matrix containing hydrophobic groups, binds proteins from aqueous solutions to different extents depending on the protein structures and a range of controllable factors including concentrations of salts, pH, temperature and organic solvents as illustrated in the figure below.

HIC suits all stages of a purification process. Application examples include high-yield capture, polishing monoclonal antibodies, removing truncated species from full-length forms, separating active from inactive forms, and clearing of viruses.

An illustration of the elution of 4 model proteins with their different 3D structures from a HIC column, using a descending salt concentration gradient. Yellow colors indicate hydrophobic residues.

The preferred workflow depends on the application; primarily, whether or not the purification will be scaled up in an industrial process.
For many applications in research, media selection is dictated simply by the purity required. The successful choice of medium depends on finding the right binding selectivity with good product recovery, while the need for method optimization and characterization is generally less than for a process that will be scaled up and used for regular industrial production. The workflow begins in the same way for both types of applications, as illustrated in the figure below.

Workflow for selection of HIC medium and further study of binding and elution conditions.

GE Healthcare introduced the first commercial HIC media in 1977 and now offers one of the broadest ranges of HIC media on the market, covering most common laboratory and industrial applications. In addition to the range of HIC products, hydrophobic interaction frequently plays a role in the use of multimodal chromatography media. These media are often described as salt-tolerant ion exchangers, or ion exchangers offering unique selectivity. GE Healthcare offers Capto MMC and Capto adhere in this class.

Our BioProcess media family is developed and supported for the large-scale manufacture of biopharmaceuticals. This support includes validated manufacturing methods, secure long-term media supply, safe and easy handling, and Regulatory Support Files (RSF) to assist process validation and submissions to regulatory authorities. In addition, Fast Trak Training & Education provides high-level, hands-on training for all key aspects of bioprocess development and manufacturing.

Capture to Polishing
To achieve the full separation potential of HIC in a range of applications demands a wide range of media with different hydrophobicities and operational properties. This is achieved by varying the base matrices and the chemical nature of the ligands. Especially for industrial downstream processes, different bead sizes may be demanded by the application, in particular for capture or polishing.

Bead-size choice considers both the resolving power needed and the pressure drop over the bed – impacted by sample viscosity and often limited by equipment specifications in large-scale applications. Finally, the range covers media with more open bead structures suitable for very large proteins, such as Octyl Sepharose 4 Fast Flow and Butyl Sepharose 4 Fast Flow. The figure shows results from a study based on 100 experiments where six model proteins of varying size, hydrophobicity and charge were eluted in the same ammonium sulfate gradient.

Hydrophobicity map of HIC media. Based on elution studies of the six model proteins: ovalbumin, a-chymotrypsinogen, ribonuclease, lactoferrin, lysozyme, a-amylase.

Select the Appropriate Media
Protein purification can be divided into capture, intermediate purification and polishing, depending upon the goals and nature of the challenges. HIC can be used at any of these stages and Capto media offer a wide range of operating conditions. Choosing the right medium depends very much on getting the binding selectivity right.

  1. In general, use Capto media for optimal productivity in capture or intermediate steps. Average bead size 75 μm
  2. Use Sepharose Fast Flow media for capture at flows up to 300 cm/h and when Capto media do not offer required binding selectivity. Average bead size 90 μm.
  3. Finally, the challenges associated with polishing might require small beads to achieve high resolution, as offered by Sepharose High Performance media. Average bead size 34 μm.


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