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Mixed mode chromatography

Mixed mode chromatography (MMC) or multimodal chromatography is any chromatographic separation method that exploits more than a single form of interaction between the stationary phase and analytes. These techniques leverage media supports with functionalized ligands that are capable of different modes of interactions such as size exclusion, affinity, ion exchange, or hydrophobic interactions. Combining separation methods enhances selectivity during the protein purification process. Use the Multimodal Chromatography Handbook from Cytiva for practical tips and in-depth information.

In affinity chromatography, a specific site on the protein is the target for ligands. However, mixed mode ligands have no known specificity. MMC interactions depend on one another. When using a mixed mode ligand with ionic and hydrophobic elements, increased salt concentration promotes hydrophobic interactions. An increased ionic strength disrupts ionic bonds. MMC can be physical or chemical in nature. Physical MMC has a stationary phase that comprises two or more types of packing materials, while chemical MMC uses just a single type of packing material with two or more functionalities.

Mixed mode media

Mixed mode media combine various chromatography separation methods, reducing the number of column steps required for purification. Parameters relevant to each separation mode control elution and binding. Commercially available mixed mode media include:

Hydrophobic ion exchange resins

These resins are based on ligands that have ionic and hydrophobic elements. Parameters that affect ion exchange and elution are ionic strength, pH, and buffers. Parameters that affect hydrophobic interactions are salt type, concentration, and additives. Capto™ adhere multimodal resin from Cytiva uses a multimodal ligand with hydrophobic ion exchange, or electrostatic, activities.

Size-exclusion chromatography, ion exchange, and hydrophobic resins

These multimodal resins use a core bead structure in which pore sizes permit only small biomolecules to pass through the bead shell to a core containing a ligand that binds target molecules. Capto™ Core 700 beads are an example. Resins based on these beads are intended to purify target biomolecules in flow-through mode, where impurities are captured on the ligand-activated core while the molecule of interest flows over the resin and is captured in the flowthrough.

Mixed mode resins

Based on media functionalized with ligands that are inherently capable of various chromatographic interactions, mixed mode resins are versatile. Their ability to merge different modes of protein separation enhances selectivity. These resins enable you to remove process impurities in a single-column step, saving time. Mixed mode chromatography resins allow salt-tolerant adsorption of target proteins. They facilitate the binding of desired proteins without dilution or addition of salts, making them a good choice for direct capture steps. You can also use these resins for polishing chromatography in downstream processes.

Mixed mode resins FAQs

Here are answers to some frequently asked questions about protein mixed mode chromatography resins.

What are mixed mode chromatography resins?
Mixed mode resins are resins that contain ligands that can exploit different types of chromatographic separation techniques, such as ion exchange, size exclusion, and hydrophobic interaction.

What is a mixed mode column?
Mixed mode columns are chromatography columns that are prepacked with mixed mode resins.

What are the benefits of mixed mode chromatography?
Mixed mode chromatography can be beneficial for intermediate purification and polishing in a purification protocol. It offers you new options such as high conductivity binding and an alternative selectivity when conditions in the purification workflow are challenging.

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. You might consider mixed mode chromatography:

  • When the selectivity of a conventional 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.

How should I choose a mixed mode resin for my needs?
We offer multimodal resins in a wide range of formats to meet users’ needs at all stages of protein purification process development and manufacturing. Our mixed mode chromatography resins are part of our line of Capto™ resins, which have been developed and supported for production-scale chromatography.

Use our online resin selection tool for further selection guidance.