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Mixed-mode resins are specialized materials used in chromatography that are designed to capture a wide range of compounds by utilizing two or more functional groups within the same resin bead. This unique combination of functional groups enables the resin to participate in multiple interaction modes, such as ionic, hydrophobic, and hydrogen bonding.
These interactions make mixed-mode resins very effective in separating and purifying complex mixtures, which is particularly valuable in industries such as pharmaceuticals, biotechnology, and environmental sciences.
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Mixed-mode chromatography (MMC), also known as multimodal chromatography, encompasses chromatographic techniques employing multiple types of interactions between the stationary phase and analytes to achieve efficient separation. What sets it apart from traditional single-mode chromatography is the notable characteristic that the supplementary interactions within MMC must possess a degree of strength that prevents them from being excessively feeble. Consequently, these additional interactions play a significant role in retaining the solutes during the separation process.
The term "mixed mode" refers to the resin’s capability to simultaneously interact with different types of molecules through various binding mechanisms. Here’s how the process typically works:
Ion Exchange: Mixed mode resins include ionic functional groups, such as anionic (negatively charged) or cationic (positively charged) sites. These sites attract and bind molecules based on their charges. This ion exchange helps in capturing charged impurities or molecules.
Hydrophobic Interactions: Many mixed mode resins also have non-polar (hydrophobic) regions. Hydrophobic interactions target non-polar parts of molecules, which is particularly useful for capturing hydrophobic compounds in aqueous solutions.
Hydrogen Bonding and Other Interactions: Some mixed mode resins have groups capable of forming hydrogen bonds, which are particularly useful for binding specific polar compounds.
By using these modes of interaction together, mixed mode resins can selectively attract, capture, and retain a wide array of compounds, especially those that might not be effectively separated by single-mode resins.
Purification of Biomolecules: Mixed mode resins are widely used in the purification of proteins, antibodies, and enzymes. They can selectively capture impurities or target molecules in a mixture based on the unique combination of their binding interactions, resulting in high-purity products.
Separation of Complex Mixtures: In cases where mixtures contain a variety of substances with different properties (like pharmaceuticals or environmental samples), mixed mode resins can separate them more efficiently than traditional single-mode resins, which only target specific types of interactions.
Removal of Impurities: Mixed mode resins are highly effective at removing unwanted compounds, such as salts, heavy metals, and organic contaminants. Their multifunctional binding capabilities allow them to retain impurities that vary in charge, polarity, and molecular size, which makes them popular in water treatment and waste management.
Enhanced Selectivity in Chromatography: Mixed mode resins are used in chromatography, a technique often employed for separating and analyzing compounds in mixtures. The mixed binding modes allow for greater selectivity, which is particularly useful in pharmaceutical research and quality control.
Facilitating Process Simplification: By combining multiple interaction modes in one resin, mixed mode resins allow for fewer purification steps in processes. This can simplify workflows in industrial and research settings, reducing time and costs.
For beginners, mixed mode resins can be a valuable choice because they offer flexibility in handling diverse applications. Unlike single-mode resins that might require sequential purification steps, mixed mode resins can handle complex mixtures in a single step or fewer steps. This makes them ideal for applications with strict purity requirements or where sample complexity is high.
Higher mass transfer velocity
Higher resolution
Simultaneous removal of multiple impurities including aggregates, host cell proteins(HCP), charge isomers, and so on.
MMC can be classified into physical MMC and chemical MMC. In the former method, the stationary phase is constructed of two or more types of packing materials. In the chemical method, just one type of packing material containing two or more functionalities is used.
Based on ion exchange(IEX) interaction, Diamond MMC resin simultaneously possesses hydrophobic, hydrogen bonding, and sulfurophilic interaction, providing the resin with excellent selectivity and alkaline tolerance. Therefore, sample loading can be performed without the exchange of buffer solutions, which significantly simplifies the chromatography process and boosts productivity.
Diamond MIX-A possesses hydrophobic and hydrogen bonding interactions in addition to anion exchange function. The resin can effectively remove various impurities including aggregates, DNA, host cell protein(HCP), endotoxin, and protein A, making it an ideal choice for the polishing step in the two-step antibody chromatographic purification.
Diamond MMC and Diamond MIX-A belong to the mixed-mode resin enjoying hydrophobic interaction. Both resins can tolerate a high volume of sample loading at high salinity conditions, as well as elution method via modification of pH and conductivity or pH-salt gradient elution. Compared with the Diamond IEX, the Diamond IEX Mustang enjoys a smaller bead size, lower ligand concentration, and therefore higher resolution.
Compared with conventional single IEX chromatography, mixed-mode chromatography can function beyond the isoelectric point (PI), expanding the pH range in process development and making the optimized purification process for complex molecules possible.
Diamond Layer 400 and Diamond Layer 700 are innovative mixed-mode chromatography resins with an exclusion limit of 400KD and 700KD in Mol. Wt respectively. The double-layered structure consists of core microspheres with anion exchange and hydrophobic interaction as well as a thin shell functioning as size exclusion. Impurities smaller than the set Mol. Wt can enter the microspheres and bind with ligands while target molecules are bigger than the set Mol. Wt will be blocked outside the microspheres.
Thus, compared with traditional SEC resin, the resins dramatically improve the volume of sample loaded and effectively reduce column volume during scale-up. The resins are specially designed for the intermediate purification and polishing of macro-biomolecules including viruses, making both ideal options in the virus purification process of vaccine production.
You can perform mixed-mode chromatography either in bind/elute or in flow-through mode. Table 1 shows how you use MM chromatography in four main steps in bind/elute mode. In bind/elute, the target molecule binds to the ligand coupled to the resin through mixed-mode interactions. Changes in the buffer composition and pH release the molecule from the resin to allow collection.
| 1. Equilibration |
Prepare the column to the desired start conditions. When equilibration is reached, all charged groups of the stationary phase are associated with exchangeable counter ions such as chloride or sodium ions. |
|---|---|
| 2. Wash |
The target molecule binds to the ligand on the resin via multimodal interactions. The sample buffer needs to have the same pH and ionic strength as the starting buffer. |
| 3. Elution | The target molecule is released from the stationary phase by changing the buffer composition and the buffer pH. |
| 4. Regeneration |
Removes all molecules still bound to the stationary phase. Ensures the full capacity of the resins is available for the next run. 1 M NaOH is often used to clean the resin. |
Mixed-mode resins are especially suited for the purification of target molecules that are difficult to purify using common purification platforms. Mixed-mode media can be used for direct processing of clarified feedstocks at physiological salt concentrations as well as for intermediate and polishing applications, such as mAb aggregate removal.
In contrast to affinity chromatography, which specifically targets particular sites on a protein, mixed-mode ligands lack known specificity. Consequently, exploring mixed-mode media involves searching for sites on the target protein that can offer valuable affinity and selectivity, without the constraints of predetermined specificity.
The interactions in mixed-mode chromatography are interconnected and interdependent. For instance, when utilizing a mixed-mode ligand containing both hydrophobic and ionic elements, an increase in ionic strength disrupts ionic bonds, while an elevation in salt concentration promotes hydrophobic interactions.
Mixed-mode media seamlessly integrate complementary chromatography methods within a singular medium, thereby reducing the total number of column steps required in a purification process. The presence of diverse mixed-mode elements within a single ligand contributes to an affinity-like binding and selectivity.
Control and optimization of binding and elution processes hinge on parameters relevant to each mode—salt concentration for regulating hydrophobic interactions and ionic strength for managing ionic interactions.
Bestchrom is a trusted manufacturer and supplier of high-quality chromatography resins, with a specialty in producing customized mixed resins to meet the unique needs of our international clientele. Our resins are designed with precision to ensure superior performance across a wide range of chromatography applications, offering consistency and reliability in each batch. Whether you’re involved in biotech, pharmaceuticals, or analytical research, Bestchrom’s chromatography resins deliver outstanding separation and purification results.
Our commitment to excellence in quality control and innovative solutions makes us a preferred choice for companies worldwide seeking highly effective and customizable mixed resin products. At Bestchrom, we not only provide top-tier products but also offer comprehensive customer support to ensure the success of your projects. Discover the Bestchrom advantage and enhance your research and production processes with resins designed for maximum efficiency and high performance.
Mixed mode chromatography offers you alternative solutions in purification workflows by widening the window of operation where traditional resins are not as effective as you had hoped. These circumstances may be encountered, for example:
When the selectivity of traditional resin is insufficient to provide the required purity of the target protein
If salt tolerance is required, for example, when the loading conductivity of the sample is too high for a traditional ion exchange resin
When there is a need to reduce the number of purification steps
Bestchrom offers resins in a variety of formats to meet user needs at all stages of protein purification process development and manufacturing. If you don't know how to choose resin, you can contact us directly and we have the best mixing mode resin for your project!
That’s all about mixed-mode resin. If you would like to find out if a mixed mode resin is the best product for your project, please visit our chromatography resins page or contact us directly and we can provide you with the most professional answers and service.
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