Chromatography is a commonly used strategy in chemistry to segregate a mixture. The segregation occurs by passing the mixture in solution, suspension, or vapor through a medium. This medium has interactions with the components of the mixture, moving them at different speeds. The movement is dependent on the individual molecule’s characteristics, resulting in the segregation or purification of a particular compound from a complex sample.
Chromatography resin is the backbone of these purification processes because it allows chemical reactions for separation and purification.
Selection of the chromatography resin is dependent on the specific processing needs. In this article, let us have a look at what chromatography resins are and the four most common resins used for chromatography.
What are Chromatography Resins?
Chromatography resin, also referred to as a media, is the material utilized in chromatography for separating chromatography to capture & polish monoclonal antibodies (mAbs), antibody fragments, vaccines, and various biomolecules.
The media is packed and held in a column during its stationary phase of using these resins. If you want to offer specificity to bind or repel specific molecules in the samples, it is possible to make physical or chemical modifications to the particles. It is an effective method because this technique can segregate a target compound from a highly complex mixture. There are various media resins available, which help researchers meet multiple needs while purifying diverse target molecules.
Stationary and mobile phases are two different phases while performing chromatography. One of the essential differences between these two phases is that the sample does not move in the stationary phase and the sample moves in the mobile phase. A resin media is used in the stationary phase. In the mobile phase, usually a liquid or gas solution is used; which helps in separating the sample materials.
What are The Five Most Commonly Used Resins for Chromatography?
When selecting the right chromatography resin type that suits your needs, there are four categories of resins to consider. It is important to consider the factors such as the purity, the target protein’s surface charge, and the size of the molecule. Additionally, it is also crucial to understand the interaction of water with the sample to determine the best suitable resin. Following are the four types of resin based on various chromatography techniques:
1. Affinity Chromatography (AC)
Affinity chromatography is a highly accurate form of chromatography type that helps get the highest purity in a single step. AC segregates the target molecules by utilizing a powerful but reversible interaction between a particular ligand and sample protein. The binding interaction results in immobilizing the ligand to a resin with the targeted compound.
AC takes benefits of the specific protein’s biological structure or function, making the binding and purification process highly selective. This purification technique can be utilized for both native and recombinantly generated molecules. Affinity chromatography is one of the most preferred versatile and precise methods of purification.
2. Ion Exchange Chromatography (IEX)
IEX separates the molecules depending on their overall surface charge. It is also a reversible interaction that can be executed by matching a
chromatographic resin, which has the opposite charge to the sample’s target compound.
Resin in ion exchange chromatography is generated by linking positive or negatively charged functional groups to a solid matrix covalently. Agarose, polystyrene, polymethacrylate, cellulose, and polyacrylamide are a few of the media used.
The IEX column gets added with a protein sample at low ionic strength. Then, it is washed with increasing ionic strength buffers to extract the unnecessary particles and impurities.
Ion Exchange Chromatography is a perfect solution for targeting monoclonal antibodies. Additionally, this is the second purification step post-affinity chromatography.
3. Hydrophobic Interaction Chromatography (HIC)
HIC segregates and purifies proteins and biomolecules depending on the sample’s surface hydrophobicity. It is an ideal technique for segregating and purification of proteins without impacting their biological activity. Hydrophobic interaction chromatography leverages buffers, matrices, and parameters, which have minimum interaction with the sample compared to the other methods. Hence, HIC is a perfect solution for experiments that need the samples to be intact and tracked for other biological characteristics.
The binding interaction with the media can be impacted by salt concentrations, pH, and temperature. Along with downstream size-exclusion purification techniques and upstream high-salt IEX elution, HIC is generally used.
4. Size Exclusion Chromatography (SEC)
SEC is a different chromatography technique that uses a gel medium to separate proteins depending on their size. This strategy does not bind the molecules to the chromatography resin, rather they are passed through gel filtration. Gel filtration chromatography resin consists of spherical beads with particular-sized pores to either incorporate or eliminate molecules inside the media. The segregation happens once the sample goes through the column and is
The separation occurs as the sample goes through the column and is extracted in order of descending molecular weight.
The two most common Size exclusion chromatography techniques are faction and desalt/buffer exchange. These two strategies are leveraged when techniques such as IEX or HIC do not segregate the proteins to the required degree of purification. It is the last step in protein purification.
5. Multimodal Chromatography (MM)
In order to purify biomolecules, mixed-mode chromatography is usually leveraged. It is an ideal technique for the purification of target molecules without a known specificity, as MM leverages resins functionalized with ligands that have the capability for multiple interactions.
MM can screen, purify, and potentially determine sites on the target protein, which can offer more information about useful affinity and selectivity.
This technique has one limitation: the target interaction cannot be forecasted from simply analyzing the amino acid sequence. The main reason for this limitation is that there are various binding and elution properties.
This technique needs further process of upfront experimentation on the binding and elution conditions. One of the significant benefits of MM chromatography is to mix complementary chromatography methods while using one medium. It helps reduce the purification steps and the use of costly sample materials. In a few cases, MM chromatography can offer quicker results.
Conclusion:
Chromatographic resins play a crucial part in research and development. It offers scientists quick and reliable results depending on their unique sample interactions.
It is essential to find a suitable resin media for successful sample analysis and purification. Selecting the right resin for chromatography does not have to be challenging. There are many variables that scientists and researchers need to consider while exploring the available options. It is important to have a clear understanding of the sample’s characteristics and the results you aim to achieve. There are multiple customization options available that can help enhance the laboratory protocols and help in the purification process.