Biological macromolecules such as proteins and polynucleotides have become of increasing commercial interest in medicine as pharmaceutical products. Productivity of synthetic processes is frequently limited by purification methods available. Products of biosyntheses are frequently contaminated by structurally similar impurities that must be removed before the product can be used. Chromatographic methods are typically the most effective purification methods, but the physical and chemical similarities between the desired product and the impurities frequently require laborious multiple separations.
Elution chromatography is the mode almost exclusively known and used. However, a chromatographic system may also be operated in a displacement mode, and operation in this mode can have important advantages for purification of bioproducts, particularly on a preparative and/or an industrial scale. Displacement chromatography is distinguishable from elution chromatography both in theory and in practice. In elution chromatography, a solution of the sample to be purified is applied to a stationary phase, commonly in a column. As the mobile phase is passed over the stationary phase, equilibrium is established between the mobile phase and the stationary phase. Depending on its affinity for the stationary phase, the sample species pass along the column at speeds which reflect their affinity relative to other components that may occur in the original sample.
A modification and extension of isocratic elution chromatography is found in step gradient chromatography wherein a series of eluants of varying compositions are passed over the stationary phase.
Displacement chromatography is fundamentally different from elution chromatography (e.g., linear gradient, isocratic or step gradient chromatography). The displacer, having an affinity higher than any of the feed components, competes effectively for adsorption sites on the stationary phase. An important distinction between displacement and desorption is that the displacer front always remains behind the adjacent feed zones in the displacement train, while desorbents (e.g., salt, organic modifiers) move through the feed zones. The implications of this are quite significant in that displacement chromatography can potentially concentrate and purify components from mixtures having low separation factors. In the case of desorption chromatography, however, relatively large separation factors are generally required to give satisfactory resolution.
In displacement chromatography the eluant (i.e., the displacer) has a higher affinity for the stationary phase than do any of the components in the feed. This is in contrast to elution chromatography, where the eluant usually has a lower affinity. The essential operational feature which distinguishes displacement from elution or desorption chromatography is the use of a displacer molecule. In displacement chromatography, the column is first equilibriated with a carrier solvent under conditions in which the components to be separated all have relatively high binding. The feed solution is then introduced into the column following which the displacer is passed through the column. If the displacer and the mobile phase are appropriately chosen, the products exit the column as adjacent square waves zones of highly concentrated pure material in the order of increasing affinity of adsorption. Following the zones of purified components, the displacer emerges from the column. Finally, after the breakthrough of the displacer, the column is regenerated by desorbing the displacer from the stationary phase to allow the next cycle of operation.
Displacement chromatography has some particularly advantageous characteristics for process scale chromatography of biological macromolecules such as proteins. Displacement chromatography can achieve product separation and concentration in a single step unlike elution chromatography which results in product dilution during separation. Since displacement operates in the non-linear region of the equilibrium isotherm, high column loadings are possible. This allows better column utilization than elution chromatography. Finally, displacement can concentrate and purify components from mixtures having low separation factors unlike the relatively large separation factors which are required for satisfactory resolution in desorption chromatography. Displacement is thus a powerful preparative technique that can offer high production rates, resolving power and elevated yields and purity of a desired byproduct.
The main disadvantage of displacement chromatography, and what has limited its application in bioseparations, is the need to identify a displacer molecule for use in each separation. An effective displacer has greater affinity for the stationary phase than the bioproduct to be purified. Additionally, it should cause separation of the bioproduct from impurities on the column. Finally, it should be readily separable from the bioproduct, so that it does not become an impurity itself.
Identification of an effective displacer has been a laborious and tedious task. Displacer candidates are typically screened individually in column experiments using trial and error. While column experiments indicate the exact behavior of displacer molecules in the column, the time required for screening a large number of molecules is a major limitation. A technique for the high throughput screening of potential displacers would enable rapid screening of molecules generated, for example, from a combinatorial library. Screening of a large number of molecules would also provide sufficient data for a predictive QSAR model to actually direct the design of a displacer molecule for a particular bioproduct or a particular stationary phase. This would enable the identification of important properties for a particular interaction or for similar interactions on different stationary phases. Therefore, a need exists for a rapid method for screening a large number of displacer candidates.