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, allowing the desorption of antibodies. The mechanism of adsorption, involving hydrophobic associations and ionic related interaction, is here qualified as dual-mode. Studies on the determination of the apparent dissociation constant for immunoglobulins G are presented. Adsorption of antibodies from crude feedstocks typically occurs without adjustment of pH or ionic strength. The sorbent is then washed with a buffer to eliminate protein impurities and, when lowering the environmental pH, antibodies are desorbed. The solid-phase material is used for the separation of antibodies from an ascites fluid and from a cell culture supernatant, followed by a polishing step on an hydroxyapatite column. Preliminary studies, related to the ability of the solid phase to separate antibody fragments, are also reported. In these studies, it has been demonstrated that both Fab and Fc fragments from polyclonal IgG are adsorbed to the solid phase under typical binding conditions. Under other defined physico-chemical conditions (ionic strength and pH), separation of both fragments in a single step has been achieved.
Guerrier
L
L
Biosepra S.A., Life Technologies, Cergy-Pontoise, France. lguerrie@lifetech.com
Flayeux
I
I
Boschetti
E
E
D000327:Adsorption; D000906:Antibodies / Q000302:isolation & purification*; D001202:Ascitic Fluid / Q000737:chemistry / Q000276:immunology; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification; D007141:Immunoglobulin Fc Fragments / Q000302:isolation & purification; D007128:Immunoglobulin Fragments / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification; D008024:Ligands; D009994:Osmolar Concentration
D000906:Antibodies; D003470:Culture Media; D006882:Hydroxyapatites; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D008024:Ligands