Fundamentals of mixed mode (multimodal) chromatography

Mixed mode chromatography can be advantageous for intermediate purification and polishing in a purification protocol/process. 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. 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

 

To explore examples of applications, see Application notes, posters and articles at the bottom of the page.

We recommend you explore a wide range of chromatography conditions early to increase process understanding and increase the likelihood of developing a robust purification process. This is the case for both traditional chromatography and MM chromatography.

When you compare an MM resin with traditional IEX and HIC resins, several questions may come to mind:

• What will happen on a multimodal resin that contains both interactions?
• Will the protein of interest bind in a high-conductivity environment?
• Under which conditions will it be possible to elute the target protein?

 

With an MM resin, it may be more difficult to predict the answers to these questions. The answers will be determined by the multimodal functionality of the resin, by the operating conditions, and by the target molecule itself. Optimization is therefore critical for success (Fig 3).

Fig 3. A contour map in a design of experiments (DoE) contour chart of static binding capacity (SBC). The contour map is measured under different salt and pH conditions on Capto MMC ImpRes resin. The map shows you that multiple optimal conditions are possible for this mixed mode resin. Unlike a traditional IEX resin, Capto MMC ImpRes can have high SBC even at high salt concentrations.

We provide additional recommendations for you in our MM chromatography handbook.

Bind/elute vs flow-through mode

In MM chromatography, the choice between bind/elute and flow-through mode is more complex than when using a single method such as IEX alone. This is because multiple types of interactions occur in MM chromatography, and the strength of these individual interactions often depends on your overall process conditions. The pH range for binding is generally extended for a multimodal resin compared with a traditional IEX resin, which gives the MM resin novel selectivity and a wider operational window (Fig 4).

Fig 4. Net charge of a protein vs pH. Schematic illustration of the extended pH binding range for multimodal AIEX/CIEX (light green) compared with traditional anion exchange (AIEX)/cation exchange (CIEX) resin (light blue).

Because the isoelectric point (pI) of a protein is generally not a good indicator for choosing the correct pH for binding and elution with multimodal resin, screening of conditions is paramount. This can be performed with high-throughput formats, such as microplates or minicolumns. The experimental setup for screening studies should be performed using DoE, and the factors most typically screened for are pH and conductivity.

To help select the pH range to screen, you can perform a pH gradient elution experiment where an analytical amount of sample is loaded on a small column. The experiment will establish the elution pH of the sample. A salt gradient or a combined salt and pH gradient can also be run if you need better understanding of the mixed-mode behavior. An example is shown in Figure 5.

Fig 5. Establishing suitable conditions for DoE on Capto adhere in bind/elute mode.

Salt types and additives

Different salt types and additives can modulate the interactions of target molecule with MM resin.
Hydrophobic interaction is one of the interactions often involved, and your choice of salt may play an important role. Different salt types will affect the strength of interaction according to the Hofmeister series (Fig 6).

Typical ions used in HIC are found to the left in the series. The chaotropic ions to the right in the series, for example, iodine, reduce the hydrophobic interaction through the salting-out effect.

Anion: SO₄^2- > HPO₄^2- > acetate > Cl^- > NO₃^- > Br^- > ClO₃^- > I^- > ClO₄^- > SCN^-
Cation: NH₄⁺ > K⁺ > Na⁺ > Li⁺ > Mg²⁺ > Ca²⁺ > guanidium

Fig 6. Hofmeister series. Organic solvents such as ethanol and isopropyl alcohol (isopropanol) decrease the strength of hydrophobic interactions. This can affect the binding of biomolecules to your MM resin.

Detergents and antifoaming agents such as Tween™ 80 can have a similar effect. Hydrogen bond disruptors such as urea and guanidium hydrochloride also affect the strength of interaction on your MM resin. Urea and guanidium salts are chaotropic and disrupt hydrogen bonds. Studies on several other modifiers, for example, amino acids or polyethylene glycol, have also been published.