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.

HIC Principles

Hydrophobic interaction chromatography (HIC) is a chromatographic method that uses a filler with moderate hydrophobicity as a stationary phase and a salt-containing aqueous solution as a mobile phase to achieve separation by using the difference in the hydrophobic properties of solute molecules and the strength of the hydrophobic interaction between the stationary phases. Since the separation principle of hydrophobic interaction chromatography is completely different from chromatography techniques such as ion exchange chromatography or gel filtration chromatography, this technique is often used in conjunction with the latter two to separate complex biological samples. At present, the main application field of this technology is in the purification of proteins, and it has become an effective means for the separation of serum proteins, membrane-bound proteins, nuclear proteins, receptors, recombinant proteins, etc., as well as some drug molecules and even cells. The basic principles of HIC separation of proteins are as follows:

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).

1. Hydrophobic interaction chromatography utilizes the different hydrophobic forces of protein molecules on the medium and the different migration rates of each component in the mobile phase to achieve separation.
2. In biological macromolecules, even hydrophilic molecules, there will be local hydrophobic regions, which will hydrophobically interact with the HIC medium. The adsorption of protein molecules on HIC is multi-point coordination. The rate-determining step is the slow conformational change and reorientation of the protein and the surface of the chromatographic medium.
3. The HIC medium is composed of different hydrophobic ligands such as alkyl or aryl groups connected to specific substrates such as agarose. The interaction between HIC medium and hydrophobic biomolecules is considered to be similar to the spontaneous aggregation process of hydrophobic molecules in aqueous solution system, which is driven by the change of entropy increase and free energy.
4. Salt plays a very important role in the hydrophobic interaction, high concentration of salt can have a strong interaction with water molecules, resulting in the formation of holes around the hydrophobic molecules to reduce the water molecules, and promote the combination of hydrophobic molecules and hydrophobic ligands of the medium.
5. Therefore, in the HIC process, a high concentration of salt solution is used in the sample adsorption stage to bind the target molecule to the chromatographic column. In the elution stage, the hydrophobic interaction between the protein and the chromatographic medium was weakened by reducing the salt concentration in the elution machine, so as to elute.

Application of HIC in PEGylated Proteins

PEGylation is a widely used technique in the pharmaceutical industry to improve the stability, solubility, and half-life of therapeutic proteins. However, the presence of polyethylene glycol chains complicates the purification process due to the increased hydrophilicity of PEGylated proteins. Traditional chromatography techniques, such as ion exchange chromatography (IEX) and size exclusion chromatography (SEC), may not effectively separate PEGylated proteins from other impurities.

Fig. 2. General scheme for the purification of commercial PEGylated proteins by chromatography (Front. Bioeng. Biotechnol. 2021, 9: 717326).

HIC has unique advantages in purifying PEGylated proteins due to its ability to separate proteins based on their hydrophobicity. The PEG chains are hydrophilic in nature, while the protein core retains its hydrophobic regions. By using HIC, PEGylated proteins can be separated from other impurities based on differences in their hydrophobic interactions with the stationary phase. This allows efficient purification of PEGylated proteins with high purity and yield. Differences in hydrophilicity between chemically modified proteins (i.e., PEGylated proteins) and other molecules are required to isolate and purify proteins. Most PEGylated proteins have different functional groups. Therefore, the hydrophilicity of the chromatography medium needs to be adjusted to be relevant to the specific PEGylated protein. HIC has proven to be a useful purification step following IEX chromatography and SEC. It is worth noting that in most cases, separation conditions need to be determined empirically since dissolved conjugates result in a significant increase in molecular weight.

Our PEGylation Services

BOC Sciences is a leading provider of PEGylation services and PEG products, offering a broad range of solutions to researchers and pharmaceutical companies seeking to improve the properties of drugs and molecules. PEGylation, the process of attaching PEG chains to molecules, has become a popular strategy in drug development due to its ability to improve drug stability, solubility, and half-life in vivo. Our PEGylation services are highly customizable, allowing customers to customize the PEGylation process to meet their specific needs. Our team of experts works closely with customers to design and optimize PEGylation strategies for a variety of molecules, including small molecules, peptides, proteins, antibodies, and more. By leveraging our expertise in chemical and bioconjugation, BOC Sciences can help customers overcome challenges related to drug delivery, immunogenicity and pharmacokinetics.

General Considerations of HIC

Medium

• Matrix

The matrix is divided into soft matrix (polysaccharide microspheres), hard matrix (large pore size silica gel), semi-hard matrix (silica gel coated with a layer of hydrophilic material) and composite matrix (PS-DVB). Ligands are mostly long carbon chain alkyl or aryl groups.

• Ligand

Alkyl ligands simply show hydrophobicity, while aryl ligands have π-π stacking effects. Generally speaking, when other conditions are equal, the binding capacity of HIC media for proteins increases with the increase of alkyl chain length.

• Degree of Ligand Substitution

Increasing the degree of ligand substitution can indeed increase the capacity, but due to steric hindrance, the increase in capacity is not unlimited. Instead, the protein will be more firmly bound to the chromatographic medium and difficult to elute.

• Aperture

Pore size also needs to be considered. 6FF agarose microspheres can be used if the molecular weight is below 200,000, and 4FF agarose microspheres can be used if the molecular weight is above 200,000.

Mobile Phase

• Type and Concentration of Salt

There are two types of salts. One type of salt, such as sodium sulfate and ammonium sulfate, can stabilize the protein conformation in the solution, promote the hydrophobic interaction between the protein itself and precipitate, or promote the hydrophobic interaction between the protein and its ligand to retain it on the column. It is called salting out salt. The other type, such as thiocyanate or guanidine hydrochloride, will increase the water solubility of protein and denature the protein at the same time, which is called salt-soluble salt. Generally, the greater the increase in molar surface tension of a salt, the greater the retention of protein in this salt.

• Mobile Phase pH

Generally choose pH=7. Changes in pH will change the protein surface charge and have an impact on the separation results and retention values, but not significantly. Generally, the farther the eluent is from the isoelectric point of the protein, the more surface charge it has and the weaker its hydrophobicity, which will reduce the adsorption force and make it easier to elute.

• Additives in the Mobile Phase

A small amount of organic matter such as urea, ethylene glycol, sucrose, etc. can be added to reduce the polarity of the mobile phase, thereby weakening the retention of the protein and causing it to be eluted.

• Chromatographic Operating Conditions

Similar to ion chromatography, there are liquid A and liquid B, but the concentration of liquid A in HIC is high and the concentration of liquid B is low. Elution is also divided into equal concentration elution, segmented elution and gradient elution.

• Temperature and Flow Rate

An increase in temperature will stretch the clump-like structure of the protein, exposing more hydrophobic groups, enhancing the hydrophobic effect, and increasing the retention value. Therefore, the temperature can be programmed to elute the protein.

What are the Uses of HIC?

• 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.

What are the Benefits of HIC?

• Using salt solution as mobile phase, the separation conditions are mild, and the activity recovery rate of biological macromolecules is high.
• The samples of high concentration salt solution can be directly separated on the column without treatment.
• Many genetically engineered protein products expressed in prokaryotic cells often exist in the form of inclusion bodies, which need to be dissolved in 7M guanidine hydrochloride or urea. At this time, the protein has been inactivated. Such sample solution can be directly loaded on the HIC column, and the three purposes of removing guanidine hydrochloride, protein refolding and separation can be achieved simultaneously in one chromatographic process, and the refolding results are better than other methods.
• Temperature changes can change the selectivity of protein separation on HIC.

Strengths & Weaknesses of HIC

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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.
3. Sánchez-Trasviña, C. et al. Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies. Front. Bioeng. Biotechnol. 2021, 9: 717326.