Eluting Possibilities with Mixed-Mode Chromatography - Pharmaceutical Technology

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Eluting Possibilities with Mixed-Mode Chromatography
Mixed-mode chromatography sorbents and custom ligands aim to optimize protein purification.

Pharmaceutical Technology

BioSepra (now Pall Life Sciences) introduced "MEP HyperCel" as the first mixed-mode resin (originally developed to replace protein A for antibody capturing). The ligand, MEP (4-mercaptoethylpyridine) contains a heterocyclic ring and a thioether linkageknown to have an affinity for antibodies. MEP HyperCel allows antibody binding at neutral pH directly out of the feedstock and, unlike protein A, binds all species and isotypes, including IgM. Elution can be carried out at pH 4 or higher.

Andrew Lees, scientific director at Fina Biosolutions LLC (Rockville, MD, http://www.finabio.com/) explains that elution from MEP HyperCel is conducted by reducing its pH. As the pH decreases, the pyridine ring picks up a positive charge, so the sorbent goes from being hydrophobic to one that has a positive charge (similar to an anion exchanger).

Figure 1
Decreasing the pH also causes the protein to become more positively charged, so it is repelled. Proteins that are more basic (i.e., have a net positive charge), including most antibodies, are eluted first and, as the pH decreases, more acidic proteins are eluted. In addition, the column also retains its hydrophobic characteristics. Therefore, the first materials that will elute are those that are more basic and more hydrophilic; materials that tend to elute last are more acidic and more hydrophobic (see Figure 1). Because antibody aggregates tend to be more hydrophobic compared with the monomer, they will be more tightly bound to the sorbent and so will elute at a lower pH than the monomer.

MEP HyperCel has also been effective in binding recombinant protein from Escherichia coli. Unlike antibodies, which bind by both affinity and hydrophobic interactions, most recombinant proteins will bind only by hydrophobic interactions and require some lyotropic salt (e.g., ammonium suflate) to effect binding. Because MEP HyperCel is much more hydrophobic than most hydrophobic sorbents used for proteins, lower concentrations of salt can be used.

Major contaminants from E. coli are lipopolysaccharide (LPS), host-cell proteins, and nucleic acid. Those components tend to bind tightly to MEP because they are all hydrophobic and/or negatively charged. LPS is very negatively charged (highly acidic) and very hydrophobic, so it elutes only under very extreme conditions. Thus, as the protein elutes, the LPS stays bound to the column. Nucleic acid is negatively charged, so as the sorbent becomes more positively charged and the protein elutes, the nucleic acid will bind more tightly. Many host-cell proteins also bind tightly to MEP HyperCel. If used as the polishing step following Protein A capture, mixed-mode chromatography with MEP could be used to remove leached protein A, which is acidic.

Two recently introduced sorbents, "HEA" and "PPA" (Pall Life Sciences) are hydrophobic resins that have different ligands (a hexyl group and a phenyl group, respectively) and a positive charge until pH 8 or higher. Adsorption to HEA and PPA is performed at a pH above their isoelectric point (pI) and eluted by decreasing the pH below their pI. As the protein becomes positively charged, they are repelled from the positive charges on the HEA and PPA resins. Proteins can bind to HEA and PPA even below their pI by adding lyotropic salts such as ammonium sulfate. Elution of the protein from the sorbent in this case is effected by lowering the salt concentration. "The selectivity of HEA and PPA differs and is not always predictable, but this adds another tool to the chromatographer's kit," says Lees. "The properties of mixed-mode sorbents not only provide unique selectivities but, by combining features of multiple modes, can reduce the total number of column steps needed."


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