Affinity chromatography

Affinity chromatography is a separation and purification method that is based on the specific binding interactions between a ligand immobilized to the chromatography column, and a binding partner/target to that ligand in the mobile phase.

As a rule of thumb, this method is especially fit for the selection of complex, large, and unique molecules like long amino acid chains & proteins, complex and large molecules, and nucleic acid chains with a complex three-dimensional structure (especially mRNA).

The too-small or too-simple compounds might be not a good fit for affinity chromatography. The interaction between the target and ligand also needs to be easily reversible.

The concept of affinity chromatography is an especially pertinent choice for high-specificity ligand-partner interactions that already occur in nature, of which there are many different types possible, for example, the following pairs:

• Enzyme/substrate.
• Antibody/
• Enzyme/enzymatic inhibitors.
• Receptor/activator.

With many such interactions documented and studied in great detail, it is possible to design a customized affinity chromatography resin with extremely high specificity for a unique target, while not retaining almost any other compound.

Other product categories are commonly purified through resin affinity chromatography, including many high-value compounds or biologically active molecules:

• Recombinant proteins (see below “Recombinant Proteins and Tags”).
• Hormones.
• mRNA.
• Other nucleic acids.

Affinity Chromatography Process

Affinity chromatography can be simplified to a few steps:

1. The chromatography column is manufactured with a specific ligand tied to the bead of the column matrix with a spacer (see below for more details on column design). The matrix of the column will also need to be a good fit for the media and target.
2. The liquid mixture of biological products, often a protein extract, is loaded into a chromatography column.
3. Wash buffer, often applied under pressure, is used to push the initial volume of biological product through the column.
4. The desired targets bind to the ligands, getting trapped in the column, while the unwanted molecules (like all the other untargeted proteins for example) are flowing through.
5. An elution buffer flushes through the columns, breaking the ligand-partner/target bound, allowing the collection of the purified biochemical molecule, like a unique protein for example. The elution buffer will need to break the bound without damaging the target molecule.

Pros vs Cons

Affinity chromatography is especially adapted to any application where the target molecule is a highly complex one, with ideally a unique three-dimensional configuration, like many proteins. The more unique the target, the more likely the affinity bound will be specific and strong enough for high extraction yield. Like all chromatography methods, affinity chromatography has several advantages and disadvantages compared to other methods:

Advantages:

• Affinity being a highly unique property, it should deliver much stronger selectivity than almost any other purification method.
• The targeting can be wide enough to aim for a whole class of biological compounds, for example binding all the antibodies in a solution, but no other protein types, using protein A or protein G ligands.
• The high degree of specificity can eventually allow for a one-step purification process. Or at least greatly reduces the need for further steps and their complexity.
• Alternatively, affinity chromatography can be used for the removal of a specific impurity instead of selecting a specific target to purify.
• The high affinity normally results in a high-purity final product.

Disadvantages:

• Aiming for a novel target might require the design and manufacture of a custom affinity chromatography resin, which will likely be more costly than using a pre-established mass-produced template.
• Low-solubility proteins might be difficult to purify with this method as they will not be very soluble in liquid media. This is a problem generally common to all chromatography methods.
• A ligand for the targeted protein or molecule needs to be well studied and be easy & cheap to manufacture and attached to the column matrix. The ligand also needs to be a stable molecule, or the column performance will degrade over time.
• High specificity is not always associated with high affinity, potentially leading to a very pure result, but not a very efficient purification, losing some or most of the targeted molecule. Such low yield can be a problem for low-concentration or expensive molecules where a high extraction yield is required for commercial viability.
• Some impurity types, including proteins like biotin, are known to easily bind to many ligands, potentially interfering with the affinity chromatography process and saturating the column. This becomes especially a problem if the bounding of biotin (or a similar molecule) to the ligand is permanent, potentially rendering the column useless.
• Depending on the media condition (ionic, pH, etc…) and the 3D configuration of the target, the target might not be reachable for the ligand, or the bond might not be strong enough.
• Like for all chromatography techniques, the solvents or media used should not in any way damage the resin’s matrix (both beads and spacers). In the case of affinity chromatography, it also should not damage the ligand or reduce its ability to bind with the target. 

Recombinant Proteins and Tags

Another option where affinity chromatography resin can be used is where instead of using a naturally occurring amino acid sequence as the target for the column ligand, the protein is engineered so it carries a sequence specifically designed to function as a “tag”. It is that tag that is later binding to the ligand. This is a popular method in synthetic biology and the biomanufacturing of high-value compounds in bioreactors, as it makes the desired product easier to detect and purify.

This tag should be designed to be highly accessible to the ligand, especially avoiding the risk of its being folded inside the three-dimensional structure of the protein. It should also not be too large to not cause steric encumbrance: taking too much space, or be too large in order to not interfere with the ligation of the flow between the matrix beads.

The recombinant protein tag should also be designed according to the following set of principles:

• It does not match the sequence of naturally occurring proteins.
• It does not reduce the bioactivity of the proteins, or worse, make it less stable, especially through a change of its tree dimensional configuration.
• It does not unintentionally bind with other proteins or compounds present in the media.
• The tag does not interfere with the later steps of purification, detection, or the intended usage of the protein.
• If possible, the tag should to the contrary be equally useful for later steps, like immobilization or detection, giving it a dual purpose. This can also make the selection through affinity chromatography a “filter” to purify and select proteins with a functional and available tag, making sure the required tag is present and functional for later steps.
• If required, the chosen tag can also help increase the solubility of the protein, its stability, or any other parameters that will be beneficial to the extraction, purification, or bioactivity of the final product.

Such a tag commonly used in biochemistry processes is the polyhistidine tag. The most common version of this tag is a hexahistidine tag, with 6 histidine in a row, with other histidine tag lengths or dual tags also used. The histidine tag is known to bind to a nickel affinity resin, with most if not all other proteins binding to the metal with only a very low affinity or not at all.

Other metals with bioactive properties have also been used in metal-based ligand affinity resin, notably copper, cobalt, and zinc, but nickel is generally considered the most common choice thanks to its highest yield.

Other commonly used tags include:

• Glutathione-S-Transferase (GST) tag.
• Maltose-Binding Protein (MBP).
• Calmodulin-Binding Protein (CBP).
• Streptavidin/Biotin-Based Tag.
• Strep-Tag® Peptide System.

Column Design

Because affinity chromatography’s main advantage is very highly specific binding between the target and the ligand, it is especially important to avoid any non-specific interactions.

Therefore, special attention needs to be paid to the core and spacer component of the column, for its composition itself to not have any binding property, nor interfere with the ability of the ligand or the target to bind with each other.

For protein purification, agarose bead material for the column core is the most commonly used. Other materials can be used as well, including silica gel, aluminum oxide, acrylate, or organic polymers.

Spacers are used to distance the ligand from the column matrix, creating more space to avoid for example the full size of a protein to be to constrained by available space and not managed to interact with the ligand. A balance between spacers’ length and column stability/durability is usually required.

Most column designs will require finding a balance between all the needed parameters, from high selectivity and affinity and high yields, to column stability and costs. Our experts can help you find the best option, either from existing columns or through a custom design.