New method for the selective capture of antibodies under physiological conditions.
Hydrophobic charge induction chromatography is a recently developed method for protein separation based on the use of dual-mode ligands. They are designed in such a way so as to combine a molecular interaction supported by a mild hydrophobic association effect in the absence of salts. When environmental pH is changed, the ligand becomes ionically charged resulting into the desorption of the protein. This method is applied to the separation of antibodies from ascite fluids and culture supernatants from hybridomas cultured in the presence of fetal bovine serum or in protein free environment. Typically adsorption from cell culture supernatants is accomplished without any pH or ionic strength adjustment; the column is then washed with a typical buffer to eliminate protein impurities. Antibodies are then desorbed using acetate buffer, pH 4. Antibody binding capacity is in the range of 30 mg per ml of resin at 10% breakthrough. Antibody purity varies according to the initial feed stock and can reach values higher than 90% in a single pass. One example of antibody purification process involving hydrophobic charge induction chromatography as a capture step followed by a polishing phase with DEAE Ceramic HyperD is described. Longevity and ligand leakage are compatible with large-scale applications.
Guerrier
L
L
Life Technologies, Cergy Saint Christophe, France.
Girot
P
P
Schwartz
W
W
Boschetti
E
E
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001202:Ascitic Fluid / Q000276:immunology; D002021:Buffers; D002417:Cattle; D002845:Chromatography / Q000379:methods*; D003470:Culture Media; D016895:Culture Media, Serum-Free; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification; D008024:Ligands; D051379:Mice
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002021:Buffers; D003470:Culture Media; D016895:Culture Media, Serum-Free; D007074:Immunoglobulin G; D008024:Ligands