Affinity chromatography on immobilized "biomimetic" ligands synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand

A synthetic bifunctional ligand (22/8) comprising a triazine scaffold subst ituted with 3-aminophenol (22) and 3-amino-1-naphthol (8) has been designed , synthesised, characterised and immobilized on agarose beads to create a r obust, highly selective affinity adsorbent for human immunoglobulin G (IgG) . Scatchard analysis of the binding isotherm for IgG on immobilized 22/8 (9 0 mu mol 22/8/g moist weight gel) indicated an affinity constant (K-a) of 1 .4.10(5) M-1 and a theoretical maximum capacity of 151.9 mg IgG/g moist wei ght gel. The adsorbent shows similar selectivity to immobilized protein A a nd binds IgG from a number of species. An apparent capacity of 51.9 mg huma n IgG/g moist weight gel was observed under the experimental conditions sel ected for adsorption. Human IgG was eluted with glycine-HCl buffer with a r ecovery of 67-69% and a purity of 97.3-99.2%, depending on the pH value of the buffer used for elution. Preparative chromatography of IgG from human p lasma showed that under the specified conditions, 94.4% of plasma IgG was a dsorbed and 60% subsequently eluted with a purity of 92.5%. The immobilized ligand was able to withstand incubation in 1 M NaOH for 7 days without los s of binding capacity for IgG. (C) 2000 Elsevier Science B.V. All rights re served.