Protein adsorption and transport in polymer-functionalized ion-exchangers

Abstract

A wide variety of stationary phases is available for use in preparative chromatography of proteins, covering different base matrices, pore structures and modes of chromatography. There has recently been significant growth in the number of such materials in which the base matrix is derivatized to add a covalently attached or grafted polymer layer or, in some cases, a hydrogel that fills the pore space. This review summarizes the main structural and functional features of ion exchangers of this kind, which represent the largest class of such materials. Although the adsorption and transport properties may generally be used operationally and modeled phenomenologically using the same methods as are used for proteins in conventional media, there are noteworthy mechanistic differences in protein behavior in these adsorbents. A fundamental difference in protein retention is that it may be portrayed as partitioning into a three-dimensional polymer phase rather than adsorption at an extended two-dimensional surface, as applies in more conventional media. Beyond this partitioning behavior, however, the polymer-functionalized media often display rapid intraparticle transport that, while qualitatively comparable to that in conventional media, is sufficiently rapid quantitatively under certain conditions that it can lead to clear benefits in key measures of performance such as the dynamic binding capacity. Although possible mechanistic bases for the retention and transport properties are discussed, appreciable areas of uncertainty make detailed mechanistic modeling very challenging, and more detailed experimental characterization is likely to be more productive.

Figure 1

Transmission electron micrographs of protein loaded on Q Sepharose FF (top) and on Q Sepharose XL at low (middle) and high (bottom) protein loadings. Dark regions show distribution of protein, which is localized to agarose base matrix on FF but partitioned into polymer volumes of increasing size as loading increases. Reprinted from ref. [23] with permission from Elsevier.

Figure 2

Schematic of different structures of polymer derivatives in pores of base matrix. (a) Covalently attached polymer, with multi-point attachment per chain; (b) grafted polymer, with single point of attachment per chain; (c) polymer brush, where grafted polymer chains are too short to form random coil; (d) “gel-in-a-shell” hydrogel.

Figure 3

Schematic of transitions in attached polymer layer. (a) Poor to good solvent, showing swelling of polymer; (b) low to high surface coverage, showing entropic expansion; (c) increase in salt concentration (charged polymer), showing deswelling.