A dual-mode approach to the selective separation of antibodies and their fragments

A novel chromatography method for the separation of antibodies is described . The adsorption of antibodies on the solid phase involves interaction with a ligand that combines mild hydrophobic characteristics and some degree of molecular recognition with a derivative of pyridine. This combined effect results in the adsorption of antibodies in the absence of lyotropic salts. When environmental pH is changed, the ligand becomes ionically charged, all owing the desorption of antibodies. The mechanism of adsorption, involving hydrophobic associations and ionic related interaction, is here qualified a s dual-mode. Studies on the determination of the apparent dissociation cons tant for immunoglobulins G are presented. Adsorption of antibodies from cru de feedstocks typically occurs without adjustment of pH or ionic strength. The sorbent is then washed with a buffer to eliminate protein impurities an d, when lowering the environmental pH, antibodies are desorbed. The solid-p hase material is used for the separation of antibodies from an ascites flui d and from a cell culture supernatant, followed by a polishing step on an h ydroxyapatite column, Preliminary studies, related to the ability of the so lid phase to separate antibody fragments, are also reported. In these studi es, it has been demonstrated that both Fab and Fc fragments from polyclonal IgG are adsorbed to the solid phase under typical binding conditions. Unde r other defined physico-chemical conditions (ionic strength and pH), separa tion of both fragments in a single step has been achieved, (C) 2001 Elsevie r Science B.V. All rights reserved.