Researchers looking to separate proteins and peptides focus on ion exchange chromatography to meet their needs and IEC developers look to help them.
Ion exchange chromatography (IEC) has been around for decades. And this powerful tool has been used for every application imaginable from removing heavy metals from drinking water to separating proteins in biological fluids.
One researcher currently using ion exchange chromatography as a tool is Robert S. Hodges, PhD, F.R.S.C., professor of biochemistry and molecular genetics, University of Colorado at Denver School of Medicine, Aurora. The objective of the Hodges’ laboratory is to develop novel antimicrobial cyclic and ?-helical peptides (for eventual commercialization) with enhanced antimicrobial activity, low toxicity, and with broad-spectrum activity or activity profiles for selected clinical indications. These compounds are designed to replace the ever-problematic class of drugs called antibiotics due to issues with drug resistance. “The compounds that we are developing do not bind to receptors but work by disrupting the bilayer of pathogens; thus the ability of the pathogen to develop resistance to these compounds is minimal,” says Hodges.
In the course of creating novel peptides with the desired activity profile, Hodges inserts amino acid substitutions into the peptide’s structure in an effort to understand the relationship between structure and function. These antimicrobial peptides are inherently amphipathic—meaning that, structurally, they have a hydrophilic/polar face and a hydrophobic/nonpolar face. Amino acid substitutions are made on either the hydrophobic face or on the hydrophilic face of the peptide. To test the effect of amino acid substitutions on a peptide’s structure, Hodges has developed novel chromatographic methods.
Hodges explains that “when making substitutions on the hydrophobic face or the hydrophilic face, you need the best method of chromatography to analyze the effects of those substitutions.” Essentially, a reversed-phase chromatographic (RPC) method is best suited to monitor changes to hydrophobicity of the nonpolar face. It is the hydrophobic face of these amphipathic helices and amphipathic beta sheets that binds to the nonpolar face of the reversed-phase column. “On the other side of the coin, RPC is poor at monitoring substitutions on the polar or hydrophilic face of a molecule. So we came up with a method for using hydrophilic interaction cation exchange chromatography as a way to monitor the effects of substitutions on the hydrophilic face of the peptide,” says Hodges. The latter is a high performance liquid chromatography method officially called hydrophilic interaction/cation exchange chromatography, or HILIC/CEX, for short. This method was described in detail in a recent review article authored by Hodges (Mant et al. J Sep Sci. 2008 Aug; 31(15):2754-73). In that paper, Hodges described the use of HILIC/CEX for separation and analysis of antimicrobial peptides including, but not limited to, cyclic peptides, alpha-helical peptides, and random coil peptides.
The coupling of these two chromatographic methods has proven useful in drug design. But chromatography is not the only way in which the effects of amino acid substitution on peptides are measured in the Hodges’ lab. The activity of each peptide is also ascertained, using a battery of biological assays and biophysical methods. Moreover, data from chromatographic and other non-chromatographic assays are integrated to provide a complete profile of the biological/biophysical and structural properties of each peptide. For example, another analytical use of chromatographic methods during the drug development process is in testing for peptide dimerization. According to Hodges, peptides tend to dimerize by their nonpolar faces due to their amphipathic nature. “But the question is ‘Is dimerization good for activity?’,” asks Hodges.
A novel reversed-phase chromatography method called temperature profiling in RPC (reversed-phase runs are carried out every 3°C from 5 to 80°C to give a temperature profile) can detect dimerization of these molecules. When data from the dimerization assay are correlated with all of the other assay results for a given peptide molecule, Hodges has demonstrated and published (Chen et al, Antimicrobial Agents and Chemotherapy. 2007;51:1398-1406) that too much dimerization of these peptides correlates with loss of antimicrobial activity but not toxicity to normal cells, which translates into a dramatic decrease in therapeutic index. Thus, the trick for novel peptide antimicrobial therapeutics is to disrupt dimerization in aqueous conditions while maintaining desired hydrophobicity for maximum antimicrobial activity and minimum toxicity in biological membranes, i.e., specificity for the membranes of pathogens. In addition, Hodges’ laboratory is involved in the generation of a peptide vaccine for Pseudomonas aeruginosa and the development of peptide-based therapeutics and antibody therapeutics against the SARS virus and influenza virus.
Developing better exchangers
Dionex Corporation (Sunnyvale, Calif.) is a developer of ion exchange columns. By developing columns using a number of different synthesis strategies, each with a different purpose, Dionex is able to tackle the issues of selectivity and capacity of their columns as well as the speed of the chromatographic method. In terms of definitions, selectivity basically means the ability of the column to separate a certain set of analytes from one another. Capacity means that one can load more sample volume on a column. Jeff Rohrer, PhD, director of applications development at Dionex, leads groups of scientists to develop applications of Dionex columns for a number of different industries including the pharmaceutical and biotech industries. For example, Rohrer points out that the pharmaceutical industry has used their columns to separate and detect drug degradation products, and the biotech industry has used the columns to do carbohydrate analysis.
Dionex has several proprietary synthesis strategies for the ion exchange columns. “The principle that made Dionex famous or got our business going is what we call a pellicular resin,” says Rohrer. To create that resin, they immerse solid polymer beads that are resistant to pH 0 to 14 in hot sulfuric acid to add sulfonate groups, thus producing a cation exchanger. Although they once made cation exchange resins in this manner, this intermediate in the production of a pellicular anion-exhange resin is no longer used as a cation exchange resin. The approach they use to create pellicular-coated beads for anion exchange is to take small beads coated with quaternary amine groups (which make the beads cationic), and then use that positive charge to allow the small beads to form a very tight electrostatic bond with the large sulfonated bead. “What that gives us is very short diffusion path lengths, which means that we get very high resolution for ions.” Another synthesis approach Dionex takes is to graft (like adding branches to a tree) ion exchange groups onto the beads. In this case, they often use porous beads to increase their surface area or capacity. They graft nearly all of their cation exchange columns and a few of their anion exchange columns.
Dionex also produces a family of ion exchange columns for protein analysis marketed under the name ProPac columns. The column is produced by first adding a proprietary hydrophilic surface to the resin and then later grafting an anion exchange surface on top of that surface. “In a protein column, when you are doing ion exchange, you would like to [just] do ion exchange and not hydrophobic interaction, too. By having a hydrophilic surface, we prevent those hydrophobic interactions; then we can do straight ion exchange.” One of the columns in the ProPac family—a weak cation exchanger—”has really become the method of choice for analyzing C-terminal lysine variants in monoclonal antibodies,” says Rohrer.
In summary, regardless of application, IEC is powerful tool applied to solving a plethora of chemical and biological problems. And with continued development, IEC stands to remain a dominant chromatographic method.
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 10, October, 2008, pp. 42-45.
Filed Under: Genomics/Proteomics