Hydrophobic Interaction Chromatography (HIC) Technology

Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomers and aggregates in the purification of protein therapeutics. CD Formulation integrates cutting-edge HIC technology into our protein characterization technology platform to provide reliable analytical support for all stages of the development and production of your protein/peptide drugs, including formulation development, process development, drug release testing, and stability testing.

What is Hydrophobic Interaction Chromatography (HIC) Technology?

HIC is a technique used in biochemistry and molecular biology to separate and purify proteins, peptides, and other biomolecules based on their hydrophobicity. This chromatographic method exploits differences in the hydrophobicity of target molecules, allowing for selective binding and elution under specific conditions. The technique is ideal for capture or intermediate steps in a purification protocol and can provide valuable information about the hydrophobicity of a protein, which is important for understanding its structure, stability, and function. It can also be used to determine the number of hydrophobic regions in a protein or to assess the effects of mutations or chemical modifications on its hydrophobicity.

Fig. 1 Schematic diagram of the interaction between a protein and a hydrophobic ligand on a HIC sorbent. (McCue JT, et al., 2009)

Hydrophobic Interaction Chromatography (HIC) Principle

HIC uses the reversible interaction between proteins and the hydrophobic ligands of the HIC resin for separation. The principle of protein adsorption to HIC media is complementary to ion exchange and size exclusion chromatography. In HIC, protein molecules containing hydrophobic and hydrophilic regions are applied to a HIC column in a high salt buffer. In the binding buffer with a high salt concentration, the proteins bind to the hydrophobic ligands on the hydrophobic interaction chromatography resin. When the ionic strength of the buffer is reduced, the interaction is reversed. Therefore, the least hydrophobic proteins elute first and the most hydrophobic proteins elute last. Elution is usually performed to elute the sample from the column using a decreasing salt gradient to increase hydrophobicity. Elution can also be performed by adding organic modifiers or detergents to the elution buffer.

Fig. 2 Schematic chromatogram showing a gradient elution of a protein mixture using hydrophobic interaction chromatography. (McCue JT, et al., 2009)

Our Services Related to Hydrophobic Interaction Chromatography (HIC)

Thanks to decades of experience supporting protein/peptide biopharmaceutical development and manufacturing using HIC technology, our team of highly qualified experts offers a range of HIC-related services to accelerate the implementation and success of your project.

Our experienced team of experts has completed hundreds of HIC separation and purification projects for protein and peptide, supporting all stages of your protein/peptide drug development and manufacturing - from early studies to downstream process monitoring and GMP batch release testing.

Since HIC is sensitive to salt concentration, our scientists focus on the effects of salt conditions on separations when characterizing and purificating protein using HIC. Utilizing cutting-edge HIC technology, we support the following protein/peptide development and characterization plans, including but not limited to:

Protein/Peptide Purification

HIC is an effective method for concentrating proteins. HIC can be used as a polishing step to obtain highly pure proteins after initial purification by other methods such as affinity or ion exchange chromatography.

Protein Conformation Characterization

Evaluate how changes in various conditions (such as salt concentration, temperature, or pH) affect protein binding to infer thermal stability and folding properties to determine the optimal conditions for maintaining active protein conformation.

Protein/Peptide Aggregation Characterization

Aggregated proteins often exhibit altered hydrophobicity, which can be separated from monomeric species using HIC to better understand aggregation mechanisms and conditions that affect protein stability.

Quality Control Testing

We integrate HIC into our quality control processes to assess the purity and identity of protein therapeutics. By establishing retention curves for different batches or formulations, consistency can be monitored and deviations in product quality can be detected.

Therapeutic Protein/Peptide Development

Automated HIC systems enable high-throughput screening of multiple protein samples, facilitating rapid assessment of protein properties and helping to select candidates for further analysis or development. We use HIC for the purification and analysis of drug targets and for screening small molecule libraries for potential drug candidates.

Advantages of Hydrophobic Interaction Chromatography (HIC) Technology

• HIC can perform separations under mild conditions, thereby maintaining the stability and functionality of the purified protein.
• HIC can separate proteins and other biomolecules based on their hydrophobicity, providing an additional separation dimension compared to other chromatographic methods that rely on charge or size differences.
• HIC can achieve high-resolution separations, allowing the separation of closely related protein variants or isomers.
• Compatibility with a variety of buffer systems.
• HIC can be used for a variety of protein types, including antibodies, enzymes, membrane proteins, recombinant proteins, peptides, oligonucleotides, and plasmids.
• HIC can be easily scaled up from the laboratory to industrial processes, making it suitable for both small-scale research and large-scale biomanufacturing.

Custom Hydrophobic Interaction Chromatography (HIC) Services

Why Choose Our Hydrophobic Interaction Chromatography (HIC) Technology?

• We have a team of experts with rich experience in HIC analytical method development and validation.
• We have accumulated decades of expertise and successful project experience using HIC technology to support protein/peptide biopharmaceutical development.
• Our laboratories are equipped with the latest HIC equipment and technology, enabling innovative methods and high-quality data generation.
• From initial consultation and method development to validation and scale-up, we provide comprehensive support to guide you through each phase of your project, ensuring smooth transitions and successful outcomes.
• We adhere to strict quality control and regulatory standards to ensure the integrity and compliance of your data.
• We provide flexible experimental design and customized solutions.

Publication

CD Formulation aims to provide a powerful analytical tool for the separation, purification, and characterization of proteins and peptides. Please feel free to contact us if you are interested in our services. Learn how our HIC technology can support the smooth implementation of your protein/peptide biopharmaceutical program.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.