Resins:

Chromatography resins:

When examining the type of chromatography resin that best meets your needs, there are four categories of resin that you should focus on. Factors such as purity, surface charge of your target protein, molecular size, and even how water interacts with your sample are all very important in determining the ideal resin. Let’s take a closer look at specific types of resin based on different chromatography techniques.

Affinity chromatography (AC)

Affinity chromatography (AC) is a powerful technique for purifying target molecules based on a strong but reversible interaction between the sample protein and a specific ligand. This binding interaction immobilizes the ligand onto a stationary resin, along with the target compound. The binding and purification in this technique is highly selective and takes advantage of the structure or biological function of the target protein. Both naturally occurring and recombinantly produced molecules can be purified using affinity chromatography, making it a versatile and precise method.

Ion exchange chromatography (IEX)

Ion exchange chromatography works by separating molecules based on their overall surface charge. This interaction is also reversible and can be achieved by matching a chromatography resin that has the opposite charge of the target compound in your sample.

Ion exchange resins are created by covalently attaching positively or negatively charged functional groups to a solid matrix. Some of the more common media used include cellulose, agarose, polymethacrylate, polystyrene, and polyacrylamide.

A protein sample is loaded onto an ion exchange (IEX) column at low ionic strength and then washed with buffers of increasing ionic strength to remove unwanted particles and impurities. The target protein is then eluted from the column using defined salt gradients or pH changes. When performing elution with salt, additional processing may be required before loading the column, while pH elution can be performed without this extra step. This is because exposure to pH changes causes the target protein to lose its net charge, releasing it from the resin (utilizing the isoelectric point of the target protein).

This chromatography technique is ideal for targeting monoclonal antibodies and also serves as a second purification step following affinity chromatography. Additionally, ion exchange resins with large beads provide a suitable starting point for purification in the first column.

Hydrophobic interaction chromatography (HIC)

This technique separates and purifies proteins and other biomolecules based on their surface hydrophobicity. This method is useful for isolating and purifying proteins while preserving their biological activity. Hydrophobic Interaction Chromatography (HIC) utilizes buffers, matrices, and parameters that cause less denaturation of the sample compared to other methods, making it ideal for experiments that require samples to remain intact and be examined for other biological characteristics. Salt concentrations, pH, and temperature can affect the binding interactions with the environment or even the ligand chemistry that is attached to the resin. HIC is typically used in conjunction with high-salt Ion Exchange (IEX) purification methods upstream and size exclusion purification methods downstream.

Size exclusion chromatography (SEC)

This method uses a slightly different approach from a gel environment for the separation of proteins based on their size. In this technique, molecules do not bind to chromatography resin; instead, they pass through gel filtration.

SEC gel is composed of spherical beads that contain pores of specific sizes to accommodate or exclude molecules in its medium. Separation occurs as the sample passes through the column, with molecules eluting in order of decreasing molecular weight.

Separation and salt removal/buffer exchange are the two most common methods of SEC. These techniques are used when methods such as IEX or HIC do not sufficiently purify proteins. SEC is often used as the final step in protein purification.

Multimodal chromatography (MM)

Multimodal chromatography is commonly used as a purification step in the purification of biomolecules. This method utilizes functional resins with ligands capable of multiple interactions, which makes it useful for purifying target molecules without specific properties.

This technique can be employed for screening, purifying, and potentially identifying areas in a target protein that can provide useful information regarding affinity and selectivity.

The only limitation of this method is that the target interaction cannot be predicted through simple analysis of amino acid sequences, as there are multiple characteristics for binding and washing. This necessitates additional preliminary testing on binding and washing conditions.

A key advantage of mixed-mode chromatography is the combination of complementary chromatography methods while using a single medium. This can save purification steps and minimize the use of valuable sample materials. In some cases, it can also provide faster results—especially for product impurities that are structurally similar to the target molecule.