Introduction to Multimodal or Mixed-Mode Chromatography

Unlike affinity chromatography where a specific site on the protein is targeted, with mixed-mode ligands there is no known specificity. Accordingly, screening mixed-mode media becomes a search for sites on the target protein that will provide useful affinity and selectivity.

Mixed-mode chromatography interactions are not independent of one another. For example, when using a mixed-mode ligand containing both hydrophobic and ionic elements, increasing ionic strength will disrupt ionic bonds, however, increasing salt concentration will promote hydrophobic interactions.

Mixed-mode media effectively combine complementary chromatography methods within a single media and can reduce the total number of column steps needed in a purification process. Because these mixed-mode elements are present in a single ligand, it contributes affinity-like binding and selectivity.

Binding and elution are controlled and optimized by the parameters relevant to each mode — salt for hydrophobic interactions and ionic strength for ionic interactions.

There are a number of commercially available mixed-mode media combining different chromatographic elements:

• Hydroxyapatite (CHT/CFT/Bio-Gel HT/HTP) — electrostatic and calcium coordination complexes
• Hydrophobic ion exchange ligands

Hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂, is a form of calcium phosphate used in the chromatographic separation of biomolecules. Sets of five calcium doublets (C-sites) and pairs of –OH containing phosphate triplets (P-sites) are arranged in a repeating geometric pattern. Space-filling models and repeat structure from Raman spectroscopy have also been constructed. Hydroxyapatite has unique separation properties and unparalleled selectivity and resolution. It often separates proteins shown to be homogeneous by electrophoretic and other chromatographic techniques.

Applications of hydroxyapatite chromatography include the purification of:

• Different subclasses of monoclonal and polyclonal antibodies
• Antibodies that differ in light chain composition
• Antibody fragments
• Recombinant proteins
• Viral particles
• Vaccines
• Isozymes
• Supercoiled DNA from linear duplexes
• Single-stranded from double-stranded DNA

CHT™ ceramic hydroxyapatite is a spherical, macroporous form of hydroxyapatite. It has been sintered at high temperatures to modify it from a nanocrystalline to a ceramic form. The ceramic material retains the unique separation properties of crystalline hydroxyapatite, and lot-to-lot control assures reproducibility in large-scale production columns. Unlike most other chromatography adsorbents, CHT is both the ligand and the support matrix. Separation protocols originally developed on crystalline hydroxyapatite can often be transferred directly to the ceramic material with only minor modifications.

Two types of CHT ceramic hydroxyapatite, Type I and Type II, are available in three particle sizes, 20, 40, and 80 μm. Although both types have elution characteristics similar to crystalline hydroxyapatite, there are also some important differences. CHT Type I has a higher protein binding capacity and better capacity for acidic proteins. CHT Type II has a lower protein binding capacity but has better resolution of nucleic acids and certain proteins. The Type II material also has a very low affinity for albumin and is especially suitable for the purification of many species and classes of immunoglobulins.

Protein binding to hydroxyapatite. A is a basic protein. B is an acidic protein. Double parentheses indicate repulsion. Dotted lines indicate ionic bonds. Triangular linkages indicate coordination bonds.

These ligands incorporate hydrophobic and ionic elements. For ion exchange binding and elution, parameters such as salt, pH, ionic strength, and buffer apply. With hydrophobic interactions, salt type, concentration, and additives apply. As mentioned above, it is important to remember that binding and elution factors are not independent and can work counter to one another. For example, increasing the ionic strength for ion-exchange elution will drive hydrophobic binding. Due to the dependency of these interactions, buffer conditions must be optimized for binding, washing, and elution to determine the optimal balance for a highly selective purification scheme.

• Hydrophobic, Anionic Ligand with Hydrogen Bonding — this ligand features hydrogen bonding, a quaternary amine, and a phenyl group
• Mixed-Mode Cationic Ligand with Hydrophobic Binding — this ligand contains a secondary amine and is cationic over a wide pH range, therefore, it behaves as both a hydrophobic interaction resin and an anionic exchange media. At low ionic strength, it can bind acidic proteins as well as proteins with moderately high isoelectric points. However, hydrophobic interactions predominate, as binding capacity increases with temperature and salt
• Mixed-Mode pH-Controllable Sorbents — this ligand contains a 4-mercaptoethylpyridine (MEP) ligand. The pyridine ring is uncharged at neutral and basic pH. As the pH decreases, the pyridine nitrogen becomes positively charged, turning the resin into a mixed-mode media. MEP becomes a pH-controlled mixed-mode ligand, a property that has been termed "hydrophobic charge induction chromatography" (Burton and Harding, 1998)

Example of mixed-mode ligands. A is a hydrophobic, anionic ligand with hydrogen bonding. B is a mixed-mode pH-controllable sorbent. C is a mixed-mode cationic ligand with hydrophobic binding.

* B, bottle; C, cartridge (1 ml or 5 ml); GC, gravity column; SC, spin column; HPLC, high-pressure column; MPC, medium-pressure column.

** +, low suitability; ++, moderate suitability; +++, suitable; ++++, high suitability.

Burton SC and Harding DR (1998). Hydrophobic charge induction chromatography: Salt independent protein adsorption and facile elution with aqueous buffers. J Chromatogr A 814, 71–81.