What is mix-mode resin? The definitive guide to 2024
Article directory
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What is mixed-mode resin?
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What are the characteristics of mixed-mode resins?
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What are the types of mixed-mode resins?
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How does mixed mode resin work?
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What are the application areas for mixed-mode resins?
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Things to note when working with blend mode resins?
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Who are the major suppliers of mixed-mode resins?
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Frequently asked questions about mixed-mode resins
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Conclusion
What is mixed-mode resin?
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.
What are the characteristics of mixed-mode resins?
Higher mass transfer velocity
Higher resolution
Simultaneous removal of multiple impurities including aggregates, host cell proteins(HCP), charge isomers, and so on.
What are the types of mixed-mode resins?
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.
HIC+IEX Diamond MMC / Diamond MIX-A
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.
SEC+HIC+IEX Diamond Layer
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.
How does mixed mode resin work?
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. |
What are the application areas for mixed-mode resins?
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.
Things to note when working with blend mode resins?
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.
Who are the major suppliers of mixed-mode resins?
Several prominent suppliers offer a variety of mixed-mode resins for chromatography applications. Some key players in the market include but are not limited to:
GE Healthcare Life Sciences: Renowned for its diverse range of chromatography products, GE Healthcare provides mixed-mode resins designed for efficient and selective separations.
Bestchrom: Specializing in chromatography and bioprocessing solutions, Bestchrom provides a range of mixed-mode resins known for their reliability and effectiveness in protein purification.
Merck Millipore: As a leading supplier in the life science industry, Merck Millipore offers a comprehensive selection of mixed-mode resins tailored for various chromatographic needs.
Thermo Fisher Scientific: A major player in the scientific and biotechnology markets, Thermo Fisher Scientific delivers mixed-mode resins designed for versatility and high performance in chromatographic applications.
Frequently asked questions about mixed-mode resins
When should I use mixed mode chromatography?
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
Tips for getting started with mixed mode chromatography
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Screen pH and conductivity for optimal running conditions
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Use Design of Experiments (DoE)
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Consider parallel screening formats to speed up the process
How to select mixed-mode resins and columns?
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!
Conclusion
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.