Affinity chromatography with collapsibly tethered ligands.

We introduce a novel affinity chromatography mode in which affinity ligands are secured to the media surface via collapsible tethers. In traditional affinity chromatography, the immobilized ligands act passively, and their local concentration is static. In collapsibly tethered affinity chromatography, the ligand can move dynamically in response to external stimuli, a design that enables marked changes in both the local concentration of the ligand and its surrounding environment without exchange of solvent. Using the thermoresponsive polymer poly(N-isopropylacrylamide) (PIPAAm) as a scaffold for ligand and hapten attachment, we were able to achieve controlled mobility and microenvironment alteration of the affinity ligand Ricinus communis agglutinin (RCA120). The glycoprotein target, asialotransferrin, was loaded onto a column in which PIPAAm was partially substituted with both RCA120 and lactose. At 5 degrees C, the column retained the glycoprotein, but released most (95%) of the asialotransferrin upon warming to 30 degrees C. This temperature-induced elution was much greater than can be explained by temperature dependency of sugar recognition by RCA120. The simplest explanation is that upon thermally induced dehydration and collapse of the PIPAAm chains, coimmobilized RCA120 ligand and lactose hapten are brought into closer proximity to each other, enabling immobilized lactose to displace affinity-bound asislotransferrin from the immobilized RCA120 lectin.

Regulation of protein binding toward a ligand on chromatographic matrixes by masking and forced-releasing effects using thermoresponsive polymer.

A novel concept of affinity regulation based on masking and forced-releasing effects using a thermoresponsive polymer was elucidated. Affinity chromatographic matrixes were prepared using either poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) or poly(glycidyl methacrylate-co-triethyleneglycol dimethacrylate) beads immobilized with ligand molecule, Cibacron Blue F3G-A (CB), together with poly(N-isopropylacrylamide) (PIPAAm), a polymer with a cloud point of 32 degrees C. Two different lengths of spacer molecules were used for the immobilization of CB while maintaining the PIPAAm size constant. Chromatographic analyses using bovine serum albumin as a model protein showed a clear correlation between spacer length and binding capacity at temperatures lower than the lower critical solution temperature (LCST) of PIPAAm. The binding capacity under the LCST was significantly reduced only when the calculated spacer length was shorter than the mean size of the extended PIPAAm. Furthermore, the adsorbed protein could be desorbed (released) from the matrix surface by lowering the temperature to below the LCST while maintaining other factors such as pH and ion strength. Selective recovery of human albumin from human sera was demonstrated using this newly developed thermoresponsive affinity column.

Selective surface modification technique for improvement of chromatographic separation selectivity for sugar derivatives.

Highly cross-linked macroporous polymers were prepared utilizing ethylene dimethacrylate as a cross-linking agent, in the presence or absence of methyl-alpha-D-glucoside as a kind of template molecule with methacrylic acid as a functional monomer. After the preparation of the polymers, we applied a high temperature to the cross-linked polymers to study the changes of adsorption properties of the polymers for sugar derivatives including the template molecule utilized. Interestingly, the heat treatment up to 250 degrees C afforded improvement of relative adsorption affinity for several sugar derivatives including the template molecule, while heat treatment up to 150 degrees C did not afford those improvements. The detailed studies including polymers prepared using acrylic acid as a functional monomer instead of methacrylic acid prove that temperatures higher than the Tg temperature of the polymer derived from a functional monomer such as methacrylic acid and higher than the melting point (mp) of the sugar template are necessary to afford the observed improvement of relative affinity based on the surface modification effects through the heat treatment to cross-linked polymers.