Improving Protein Separations with Mixed-Mode Chromatography -

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Improving Protein Separations with Mixed-Mode Chromatography

Jan 1, 2009
By: Ronald E. Majors, Andrew Lees, Marty Burkhardt
LCGC North America

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Ronald E. Majors

Because proteins are polyions with both hydrophobic and hydrophilic surfaces in distinct spatial arrangements, each protein presents unique purification challenges. Although protein interactions with chromatography sorbents generally are considered in terms of single modes such as ionic or hydrophobic interactions, in fact, protein chromatography often involves multiple modes of interaction, some of which are unintended "secondary" interactions with the sorbent bead or spacer–linker structure carrying the nominal ligand. In contrast to unintended interactions, mixed-mode protein chromatography uses a sorbent intentionally functionalized with ligands capable of multiple modes of interaction to effect a protein separation process, including binding, washing, and elution. The advantages of mixing modes deserve much wider recognition. Mixed-mode chromatography can enhance selectivity beyond that of chromatography with the same single modes performed separately, can reduce the number of column steps needed for protein purification, and indeed, sometimes solves protein purification problems that are otherwise intractable.

Figure 1

These multiple modes can include ion exchange, hydrophobic, affinity, and size exclusion, as well as hydrogen bonding, π–π and thiophilic interactions, as illustrated by the imaginary multimodal ligand shown in Figure 1. In viewing the process of mixed-mode chromatography, two considerations are especially important. First, the distinct and fixed spatial array of structural elements comprising the mixed-mode ligand create a pseudoaffinity sorbent that will tend to bind to complementary structures on the protein. In contrast to the usual view of an affinity ligand binding to a specific site, a priori, for a mixed-mode ligand, there need be no known specific site. Indeed, screening various mixed-mode sorbents against a particular protein or feedstock constitutes a search for sites on the target protein (or impurities) that will provide useful affinity and selectivity. Second, there is an inherent orthogonality to the mixed-mode chromatography process, as multiple types of chromatography occur simultaneously. However, interactions between modes mean the chromatography processes are not independent. Thus, for example, in a mixed-mode ligand containing hydrophobic and ionic elements, increasing ionic strength will disrupt ionic bonds but the increasing salt concentration will promote hydrophobic adsorption.

Mixed Mode is More Common Than Recognized

Chromatographers should not be surprised to find they already practice mixed-mode chromatography. It is well known that base matrix, linker, and linker chemistry influence the chromatographic process through interactions that are generally different than the primary mode. These interactions constitute a hidden form of mixed-mode chromatography. The hydrophobic properties of the linker used in the weak ion-exchange sorbent DEAE-Sephadex are an instance of a less obvious mixed-mode sorbent. Affinity ligands virtually always involve mixed modes of interactions and bind to a specific site on the protein. For example, Cibachrome Blue, a hydrophobic dye containing sulfonates, amines, and benzyl groups, binds to a nucleotide pocket on proteins. A variety of "custom ligands," such as the dye-based mixed-mode sorbents offered by Prometics Life Sciences (Cambridge, U.K.), interact via multiple modes. Protein-based affinity ligands also interact with their targets through a plethora of interactions. For example, the well-known antibody affinity ligand protein A binds to the Fc region of antibodies through hydrophobic, hydrogen bonding, and ionic interactions. Elution from protein A is effected by lowering the pH so that a highly conserved histidine on the antibody is protonated, setting up a charge repulsion with nearby cationic sites on protein A (1).

Other common examples of mixed-mode sorbents include many of the "aqueous" reversed-phase columns that incorporate ionic groups into their hydrophobic phase to promote wettability. Solid-phase extraction sorbents also frequently operate using mixed-mode interactions, but the pore sizes of these sorbents are usually too restrictive for protein chromatography. Recently, however, analytical high performance liquid chromatography (HPLC) wide-pore columns containing ionic and hydrophobic ligands have been introduced (for example, ProMix from SIELC, Prospect Heights, Illinois) that are suitable for protein analysis.

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