Displacement Chromatography Comes of Age
An indispensable tool for the purification of biomacromolecules
by Dr. Charles Little
When Russian botanist Mikhail Tsvet announced in 1901 to a gathering of scientists in St. Petersburg, Russia that he had separated plant pigments on a column of calcium carbonate, using organic solvents flowing through the column under the force of gravity, the modern science of chromatography was born.
Today, we would call the technique that Tsvet announced "elution mode" chromatography. By 1943, Swedish scientist Arne Tiselius had come to understand that the entire field of liquid-solid chromatography could be organized around three distinct modes: elution mode, frontal mode and displacement mode. We can make a further distinction between "methods" of chromatography, such as adsorption and partition, and "modes" of chromatography, such as elution and displacement.
To be more precise, what Tsvet announced in 1901 was the discovery of adsorption chromatography, operating in the elution mode. Tsvet went on to give the first description of displacement mode chromatography in 1906, after he observed that sample displacement occurred under conditions of overloaded elution chromatography.
Since the 1950s, great advances have been made in the practice of elution mode chromatography, to the near complete neglect of other modes. It is fair to say that the chromatographic methods most often used today to purify biomolecules—ion exchange (IEX), hydrophobic interaction (HIC) and reverse-phase HPLC (RP-HPLC)—are usually performed in elution mode. Indeed, the term "chromatography," without further qualification, today usually refers only to elution mode chromatography.
In elution mode, substances ideally emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of peaks can be achieved only with gradient elution and low column loadings. Thus, two significant drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings.
These limitations of elution mode chromatography are virtually unknown in displacement mode chromatography, where isocratic flow of mobile phase and high column loadings are the rule. But beyond these operational advantages, there is the further advantage that displacement mode often is more effective in separating complex mixtures into their components than is elution mode. It is therefore quite surprising that displacement mode chromatography is so rarely taught.
In recent years, though, the benefits of operating chromatographic processes in displacement mode have been brought to a wider audience, thanks to the work of the late Professor Csaba Horvath and his many students, in particular Professor Steven Cramer. Among the significant advances since the early 1990s that allowed this was Cramer’s discovery that certain low-molecular-weight compounds make effective displacers, and that these compounds can easily be removed from product biomolecules by ultrafiltration/diafiltration (UF/DF). Now, with the commercial availability of high-purity compounds specifically designed as displacers for various chromatographic methods, displacement chromatography is set to become a routine purification tool for biomolecules.
Our intent here is to highlight the benefits of displacement chromatography over elution chromatography for the purification of biomolecules. The meaning of the major points enumerated below will become clear as we discuss theory and practice in later sections.
Displacement chromatography advantages can be realized in a variety of product purification stages, including:
• late-stage product polishing,
• early-stage product capture,
• after an affinity purification step, and
• enriching and purifying a product initially present only as a minor component in a complex mixture.
Displacement chromatography benefits can be achieved in small scale (2 mL) and large-scale preparative work (50 L). Typical, small, analytical IEX columns (2 to 6 mL) become remarkably productive preparative tools when used in displacement mode. Scale up from small to large columns is very simple and remarkably straightforward, even with different particle size matrices.
Additional advantages of using displacement chromatography include:
• more useful than elution mode for difficult separations,
• simple operation—no gradients,
• product is isolated in dilute buffer—no salt contamination that has to be removed before the next step,
• product is recovered in a more concentrated form,
• sharper separations for higher yields—less product is recycled,
• operation on standard stationary phases, thus requiring no special chromatography medium, and
• displacer is easily cleared from product by UF/DF.
The principle of displacement chromatography is described as such:
A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.
The three stages of a displacement separation are illustrated in Figure 1.
As shown in Figure 1, a displacement chromatography process can be broken down into three distinct phases: loading, displacement and regeneration. For the sake of simplicity, examples of biomolecule purification given here will be restricted to proteins. In principle, all biomolecules of commercial interest today, including oligonucleotides and antibodies, can be purified in displacement mode with the right choice of column matrix and displacer.
The purification starts with a column equilibrated with a loading buffer much like for elution chromatography. The feed mixture containing the impure proteins in buffer is loaded onto the column at a fairly slow rate, under conditions where the materials are well retained. After the feed mixture has been loaded, approximately one column volume of loading buffer is passed through the column. The purpose of these steps is to help the displacement train start to form during the sample loading. This is shown in the graphic as the mixture of proteins begins an early separation into yellow and purple components.
After the sample has been loaded, the column is fed a solution of a displacer at a fairly low concentration (typically 5 mM) in the same buffer used to load the sample. The displacer is designed to bind more tightly to the matrix than any of the biomolecules and thus "pushes" all components of the mixture off the matrix ahead of it. How does this happen? As long as each sample component and the displacer is not irreversibly adsorbed on the column matrix, there is some of each continually adsorbing to, and desorbing from, the matrix.
In elution mode, optimal results are obtained when concentrations are so low that individual components act independently and do not compete for binding sites on the matrix. In displacement mode, sample components are introduced in much more concentrated form, and so it is possible for a stronger binding component—either the displacer or one of the proteins in the sample mixture—to compete for binding sites. The stronger binding components (initially, the displacer itself) then displace by successfully competing for binding sites.
Based on their individual binding strengths, each component in the original sample then becomes a "displacer" for the next less tightly bound component. Thus, a displacement train is established as adjacent, focused bands with little overlap (yellow and purple in the graphic). In a successful run, the train is fully established before the components of interest arrive at the bottom of the column. Fractions can be collected and the desired product cuts made.
When the displacer breaks through, i.e., begins to emerge from the column, the run is complete and column regeneration can begin. Regeneration is accomplished by using a buffer that can remove the displacer from the matrix, followed by an equilibration with loading buffer in preparation for the next run. Since displacers must bind more strongly than any protein being purified, it is to be expected that their removal from the column should require some special conditions. A non-trivial part of the design of a good displacer, then, is the incorporation of some structural feature that allows for complete removal from the matrix under some conditions.
Following the process described above will yield a chromatogram that might appear odd to a scientist who has only practiced elution chromatography. Components are not separated into widely spaced, Gaussian-shaped bands. Instead, they emerge from the column in concentrated, adjacent, nearly square zones. A typical displacement train is shown in Figure 2.
Weakly bound components may elute off the column in advance of the displacement train, which is composed of the more tightly bound components. In the literature, displacement bands are often represented as vertical bars. In practice, the component band shapes are trapezoids, with the steepness of the sides being dependent on such factors as flow rate and specific binding interactions of the components with the matrix and each other.
The sloped sides of the bands give rise to transition zones that will be a mixture of components. In displacement mode, the transitions are self-sharpening so the overlap of components is much less than in overloaded elution mode. It should also be noted that minor impurities show up as small triangle-shaped peaks concentrated in the transition zones between the major components. This is because the concentrations of the minor components are not high enough to saturate the column capacity and thus participate in the displacement phenomenon. They are simply carried along by a major component that is displacing another major component. This does not hinder the purification since these impurities are in the transition zones only and are not mixed in the main component zones.
If displacement chromatography is used in an early stage of product purification, it is possible that the desired product is one of the minor peaks in a transition zone and would be contaminated with the other minor components and some of the major components. In that case, one would capture the transition zone fragment and run that mixture on a much reduced column size such that the loading is high enough to establish a true displacement train. The second run would allow isolation of the desired product in pure form.
It should be noted that no component should be contaminated with displacer except for the very last component, which shares a transition zone with the displacer itself. Even for that component, only the very last fraction is likely to be contaminated with displacer, which is easily removed by UF/DF.
Figure 2: A typical displacement train looks nothing like the Gaussian-shaped peaks resulting from elution chromatography. (Click on graph to view larger)
Displacement mode chromatography is fast becoming an indispensible tool for purification of all biomolecules of commercial interest, including oligonucleotides. It enables a higher degree of purification in fewer steps than most commonly practiced techniques. Best of all, it requires no specialized equipment or materials other than a high-purity, efficient displacer, and can be practiced on the bench or in the plant. n
Dr. Charles Little is a Principal Scientist at SACHEM. To find out more information about displacement chromatography, he may be contacted at ChromatographyTechniques@advantagemedia.com.