Protein purification by multidimensional liquid chromatography

Recently Giddings discussed the prospect of combining two separation mechanisms in such a way that when "a sample is subjected to two displacement processes oriented at right angles to one another" a two-dimensional separation is carried out. In this review I have focused attention on the various ramifications of this concept in terms of combining two or more chromatographic techniques on-line to conduct MDLC for the purpose of purifying proteins. In general, the MDLC approaches discussed here were classified into two major categories. The first category involves the placement of several separation mechanisms in the same chromatographic work space (the chromatographic column). In this case the displacement processes are collinear. It is hoped that these new chromatographic packings and columns will display surface characteristics capable of a wide range of highly discriminating selectivities that can be modulated by mobile-phase changes to a greater extent than the nonspecific chromatographic techniques such as IEC and HIC. The ability to modulate the mobile phase to generate new selectivities is important in expanding the usefulness of these packings in comparison to the very high selectivity of affinity chromatography, which usually has little use outside its initial intended purpose to purify a particular protein. The second category involves the on-line physical coupling of two or more chromatography columns, each packed with a different chromatography material. Again the idea is to create or design a simple self-contained system that is capable of generating a wide range of high selectivities. Indeed, the on-line coupling of two different chromatographic packings for the purification of a single protein represents a line trace through "a discrete independent 2-dimensional system". These systems are highly attractive in large-scale purification, especially when using "on-off" chromatography, which eliminates the need for sophisticated gradient elution hardware. The purification of a single protein from its biological matrix is usually a multidimensional process utilizing several different separation technologies. By nature this leads to lengthy purifications that are frequently labor-intensive and expensive to scale up. It is my belief that future developments in the concept of on-line MDLC techniques involving complex chromatographic materials and column coupling will merge to create significant improvements in the protein purification process.