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:
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:
Affinity Chromatography Process
Affinity chromatography can be simplified to a few steps:
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:
Disadvantages:
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:
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:
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.
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