| Chromatography is a widely used technique in chemistry to separate a mixture by passing it in solution, suspension, or as a vapor through a medium that interacts with the mixture components, moving them at varying rates. This movement is based on the characteristics of the individual molecules, making it possible to separate or purify a unique compound from a complex sample. In liquid chromatography – a resin media is used to capture and polish antibody fragments, vaccines, and other biomolecules using a stationary phase. This process separates a sample into its individual components, allowing for the isolation and purification of molecules. This involves two substances – a stationary phase and a mobile phase. In bioprocessing, a sample is applied to a stationary phase and then moves through it by applying the mobile phase. Both of these phases require their unique media. Chromatography resin is the cornerstone of these processes, as it enables chemical reactions for separation and purification. When considering the right chromatography resin for your procedure, it is critical to first evaluate the following:
- What are the known characteristics of your target molecule?
- What are the characteristics of your sample and its impurities?
- What processing results do you wish to achieve (purification or analysis)?
- Which kind of liquid chromatography meets the demands of your experiment?
Chromatography resin selection can be refined based on your specialized needs for processing. Based on the makeup of your sample material, you can take advantage of resins with diverse properties of nucleic acids, proteins, and small or large molecules to exclude or capture targets of interest from a sample mixture. Depending on your sample’s physical and chemical properties, there is an ideal purification scheme. This can be easily set up with a basic understanding of the different categories of chromatography resins and their respective techniques. What are chromatography resins? The resin used in chromatography, also known as media, is the material used to capture and polish mAbs, antibody fragments, vaccines, and other biomolecules when performing chromatography separations. When using chromatography resin, the media is packed and held in a column during the stationary phase. These particles can be physically or chemically modified to provide specificity to bind or repel particular molecules in a sample. This can be particularly useful, as this process can separate out a target compound, even from a highly complex mixture. These columns typically operate using gravity to move the solution sample, but it has become more common for columns to run using varying levels of pressure via mechanical pumps. Different media resins are available to address a multitude of needs when researchers are purifying a diverse set of target molecules. When performing chromatography, there are two distinct phases: the stationary and mobile phases. The key difference between the two is that the stationary phase does not move with the sample, however the mobile phase solution moves with the sample. In the stationary phase, there is often a resin media used, verses in the mobile phase, it is often a liquid or gas solution that aids in separating the sample materials. Both phases have different interactions with the sample, with the stationary phase typically not moving but still interacting with the sample. A mobile phase solution may dissolve the sample and will migrate through the stationary phase along with the sample. These solutions must be compatible for a successful separation. For more information about chromatography techniques and reagents, explore Avantor® ’s extensive resources on chromatography solutions. Here we will focus on chromatography resins used within the stationary phase of chromatography and the process of selecting the best media for your application. Purification vs. analytical chromatography resin Liquid chromatography can be performed at both analytical or preparative scales. When taking an analytical approach, the objective is often the discovery, identification, and quantification of a target molecule in the sample. Whereas in preparative liquid chromatography, the focus is to isolate and purify compounds using a downstream bioprocessing technique. Protein purification can pose many challenges, requiring protocol optimization for each process phase. Whether your process is a more general selection of molecules, or a highly specific separation, there are many chromatography methods to choose from. Some targets may require a multi-step separation process with multiple column chromatography setups, while others may require a mixed-media resin solution to address specific research needs. Ideally, you will land on a method that requires the fewest steps possible to obtain a purified version of your target molecule. Below we will cover some of the most common forms of liquid chromatography, highlighting the resins used and their specific benefits. Chromatography techniques based on chromatography resin selection Separating materials from a complex sample can prove to be both challenging and laborious when first evaluating your methodologies. Nevertheless, proper purification technique is fundamental in the processes to further characterize and understand the function of a target molecule. Despite the many variables involved, there are a few key areas to focus on when deciding the right technique for your sample material. It is best to start with the primary amino acid sequence of your target. Understanding this will provide information on the molecular weight, isoelectric point (pl), and the solubility characteristics of the target molecule. This information will guide you in selecting the proper chromatography media, as well as the ideal wash and elution buffer conditions that will be used in the purification process. When your target compound is not well understood, there are multimodal or mix-mode resins available for preliminary discovery and purification. Other methods like hydrophobicity plots and secondary-structure predictions can provide guidance when considering the use of hydrophobic interaction chromatography or mixed-mode resins. Additionally, there are techniques like ion exchange, affinity, and size exclusion chromatography that may provide you with the best results for your separation and purification processes. Each of these processes can be customized and implemented to meet your unique needs. To understand the advantages to each technique we have detailed below the most common forms of chromatography and the respective resins needed to run each procedure successfully. The four most common resins for chromatography When considering the type of chromatography resin that best suits your needs, there are four categories of resin to focus on. Factors like purity, the surface charge of your target protein, molecule size, or even how water interacts with your sample are all critical in determining the ideal resin. Let’s explore in more detail the specific types of resin based on different chromatography techniques. Affinity chromatography (AC) This highly accurate form of chromatography can achieve high purity in a single step. Affinity chromatography (AC) separates target molecules by using a strong, but reversible interaction between the sample protein and a specific ligand. This binding interaction immobilizes the ligand to a resin, along with the targeted compound. The binding and purification of this technique is highly selective, taking advantage of the target protein’s biological structure or function. Both native and recombinantly generated molecules can be purified using affinity chromatography – making this a versatile and precise method of choice. Some examples of applications include antibody/antigen, enzyme/substrate, and enzyme/inhibitor interactions. Appropriate selection of ligands for affinity chromatography is key for a successful process. There are now novel affinity ligands available on the market, making for less costly and more stable structures chemically. Affinity chromatography provides greater selectivity and often produces faster results with such a specialized interaction – making affinity chromatography the most common first step (and sometimes the only step) in a purification workflow process. 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 performed by matching a chromatographic resin that has the opposite charge to the target compound in your sample. Ion exchange resins are created by covalently linking either positively or negatively charged functional groups to a solid matrix. Some of the more common medias used include cellulose, agarose, polymethacrylate, polystyrene, and polyacrylamide. A protein sample is loaded into an IEX column at low ionic strength and then washed with buffers of increasing ionic strength to remove undesired particulate and impurities. The target protein is then eluted using either defined salt gradients or a shift in pH. When performing elution by salt, it's possible that further processing will be required in preparation before loading the column, whereas elution by pH can work without this added step. This is because exposure to the pH change will cause the target protein to no longer carry a net charge, releasing it from the resin (exploiting the isoelectric point of the target protein). This chromatography technique is ideal for both targeting monoclonal antibodies and as a second purification step after affinity chromatography. In addition, large-bead IEX resin is a great starting point for first column purification. Hydrophobic interaction chromatography (HIC) This technique separates and purifies proteins and other biomolecules based on their surface hydrophobicity. This methodology is useful for separating and purifying proteins while maintaining their biological activity. HIC utilizes buffers, matrices, and parameters that are less denaturing to the sample than other methods – making it ideal for experiments that require your samples to stay intact and be monitored for other biological characteristics. Salt concentrations, pH, and temperature can all affect binding interactions with the media, or even the ligand chemistry that is immobilized to the resin. HIC is commonly used in conjunction with upstream high-salt IEX elution and with downstream sized-exclusion purification procedures. Size exclusion chromatography (SEC) This slightly different methodology utilizes a gel medium to partition proteins based on their size. In this technique molecules will not bind to the chromatography resin but are instead passed through gel filtration. SEC gel comprises spherical beads containing specific-sized pores to either include or exclude molecules within the media. The separation occurs as the sample passes through the column and is eluted in order of decreasing molecular weight. Faction and desalt/buffer exchange are the two most common SEC procedures. These techniques are utilized when methods like IEX or HIC will not separate the proteins to a necessary degree of purification. SEC can often be used as a final step in protein purification. Multimodal chromatography (MM) Mixed-mode chromatography is commonly used as a polishing step in the purification of biomolecules. MM uses resins functionalized with ligands capable of multiple interactions, making this method useful for purifying target molecules without a known specificity. This technique can be used to screen, purify, and potentially identify sites on a target protein that can provide useful affinity and selectivity information. The one limitation to this method is that the target interaction cannot be predicted from simple amino acid sequence analysis, as there are multiple binding and elution properties. This requires additional steps of upfront experimentation on the binding and elution conditions. The key advantage of MM chromatography is to combine complementary chromatography methods while using a single medium. This can save purification steps and minimize the use of precious sample materials. In some cases, this can also provide faster results – especially for product impurities that are similar in makeup to the target molecule. Conclusion Chromatographic resins are an essential tool in research and development, providing scientists with fast and dependable results based on their unique interactions with a sample. Finding a compatible resin media is essential for successful analysis and purification. Selecting the right chromatographic resin does not have to be a challenge, despite the many variables to consider when exploring the options. With a fundamental understanding of your sample and the goals you wish to achieve, there are limitless customizations available to you for perfecting your laboratory protocols. Avantor® supports you in designing a successful chromatography process – whether your research material is well understood or still yet to be discovered. Expand your research capabilities today by building out a chromatography protocol tailored to your goals with Avantor®. |
