Affinity Chromatography (AC) Technology

Affinity chromatography (AC), a major separation technique is used to separate and purify enzymes and other proteins. The technique is based on the reversible interaction between a protein (or a group of proteins) and a specific ligand bound to a chromatographic matrix to separate proteins. CD Formulation integrates cutting-edge AC 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 Affinity Chromatography (AC) Technology?

AC is a liquid chromatography method used to separate, purify, or specifically analyze sample components. The method can be used to separate active biomolecules from denatured or functionally different biomolecules, as well as to separate pure compounds with low concentrations in large amounts of crude samples and eliminate specific impurities. Compared with other chromatographic methods, AC has excellent selectivity, high resolution, and high target protein capacity, which can achieve purification projects that are time-consuming, difficult, or even impossible to complete with other technologies.

Fig. 1 Affintiy chromatography process. (Du M, et al., 2022)

Affinity Chromatography (AC) Principle

AC is based on the affinity of the target molecule for the groups attached to the stationary phase. When a mixture of proteins is passed through the column, those proteins that contain binding sites for the fixed substrate will bind to the stationary phase, while all other proteins will elute in the void volume of the column. Once all other bound proteins have been eluted, the material retained in the column can be eluted in the following several different ways:

• Chang the pH, salt, or organic solvent concentration of the eluent.
• Increase the ionic strength of the buffer, for example, using a sodium chloride gradient.
• Change the pH of the buffer.
• Add a high concentration of substrate (or substrate analog) to the elution buffer.

Our Services Related to Affinity Chromatography (AC) Technology

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

Our experienced team of experts has completed hundreds of AC 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.

Utilizing cutting-edge AC technology, we support the following protein/peptide development and characterization plans, including but not limited to:

Protein Purification

Our scientists use AC for the isolation of specific proteins from crude extracts. By attaching a ligand with a high affinity for the target protein to the stationary phase, this protein is effectively separated from other cellular components. After affinity purification, we will also monitor the purity of the isolated protein using techniques such as SDS-PAGE.

Post-Translational Modification (PTM) Characterization

Certain affinity tags, such as GST or His tags, facilitate protein isolation to characterize modifications such as glycosylation, phosphorylation, or ubiquitination. By analyzing the eluted protein, our scientists can develop assays to examine the presence and extent of these modifications.

Quality Control

We use AC as a quality control method to assess the purity and functionality of protein products at various stages of production.

Protein-Protein Interaction Studies

AC can facilitate protein-protein interaction studies by capturing one binding partner (bait) while allowing unbound protein to wash away. The bound protein can then be eluted and analyzed.

Available Types for Affinity Chromatography (AC)

Protein A/G Affinity Chromatography

This technique utilizes Protein A or Protein G, which can specifically bind to the Fc region of immunoglobulins (antibodies) from different species. It is widely used to purify antibodies from serum, culture supernatants, or other biological fluids. The target protein is eluted by changing the pH or using an excess of Fc-specific peptide.

Antibody Affinity Chromatography

This is a common chromatography method that involves the use of specific antibodies bound to a solid support to selectively bind to a target antigen. It is often used to purify proteins, peptides, or small molecules that specifically interact with the antibody used.

Immobilized Metal Ion Affinity Chromatography (IMAC)

IMAC, a common method for purifying His-tagged recombinant proteins, involves the use of metal ions, such as Ni²⁺, Co²⁺, or Zn²⁺, covalently attached to a solid matrix. His tag binds to the immobilized metal ion and is coordinated with a chelator, such as iminodiacetic acid or nitrilotriacetic acid. The target protein is then eluted by adding excess imidazole or histidine to compete with the His tag for binding to the metal ion.

Lectin Affinity Chromatography

This technique selectively binds to glycoproteins or glycoconjugates present in a sample using lectins. It is primarily used to separate glycoproteins or study glycosylation patterns in proteins.

Dye-ligand Affinity Chromatography

This method uses dye compounds as ligands to interact with biomolecules and can purify proteins with exposed hydrophobic regions. Dye molecules can usually capture various proteins based on their surface properties, and the target protein is subsequently eluted using high salt or competing hydrophobic ligands.

Advantages of Affinity Chromatography (AC) Technology

• AC relies on a specific interaction between the target molecule and a ligand (a specific biomolecule attached to a stationary phase). This allows for high selectivity in the purification process, often resulting in high purity levels of the target.
• AC can often recover a significant proportion of the target biomolecule, with a higher overall yield compared to other purification methods.
• AC can be easily scaled up from the laboratory to industrial applications. It can be used for both small-scale research purposes and large-scale manufacturing processes.
• AC uses different ligands depending on the target and can be used for various types of biomolecules, including proteins, antibodies, enzymes, and nucleic acids.
• The procedure for AC is very simple and often involves fewer steps than other purification techniques.
• Automated, allowing for increased throughput and reduced purification time.

Custom Affinity Chromatography (AC) Services

Why Choose Our Affinity Chromatography (AC) Technology?

• We have a team of experts with rich experience in AC analytical method development and validation.
• We have accumulated decades of expertise and successful project experience using AC technology to support protein/peptide biopharmaceutical development.
• We utilize state-of-the-art equipment and methodologies in our AC processes, ensuring high efficiency and reproducibility.
• Our AC technology is highly customizable, allowing us to tailor the purification process to meet the specific needs of different proteins and peptides.
• We offer end-to-end support throughout the biopharmaceutical development process. From initial method development to scale-up and validation, our team collaborates closely with clients to ensure a seamless workflow and successful outcomes.
• We adhere to strict quality control and regulatory standards to ensure the integrity and compliance of your data.

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 AC 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.