Sulfone-aromatic ligands for thiophilic adsorption chromatography: purification of human and mouse immunoglobulins.
New thiophilic matrices and new procedures were used for the purification of immunoglobulins both from human serum and from hybridoma cell cultures containing fetal calf serum. A range of aromatic and heteroaromatic ligands containing hydroxyl or amino groups have been coupled to divinyl sulfone-activated agarose. The resulting affinity matrices have the general formula M-O-CH2-CH2-SO2-CH2-CH2-X-Y, where M is the agarose matrix, X is oxygen or nitrogen, and Y is an aromatic or heteroaromatic compound. Contrary to earlier expectations these matrices showed pronounced thiophilic binding patterns when tested for the selective binding of immunoglobulins from human serum. The binding is influenced by the structure of the aromatic part of the ligand, the ligand concentration, and the concentration and type of lyotropic salt. 2-Hydroxypyridine coupled to divinyl sulfone-activated agarose was used to purify murine monoclonal antibodies (IgG1 and IgM) from hybridoma cell cultures containing fetal calf serum. Compared to previous methods, significantly increased binding capacity (300-1500%) was obtained by using 1.0-1.2 M ammonium sulfate. Purity of the monoclonal antibody may be optimized for each individual clone by washing the column with either a low concentration of ammonium sulfate or polyethylene glycol before elution.
Knudsen
K L
KL
Research Center for Medical Biotechnology, University of Copenhagen, Denmark.
Hansen
M B
MB
Henriksen
L R
LR
Andersen
B K
BK
Lihme
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007075:Immunoglobulin M / Q000302:isolation & purification*; D008024:Ligands; D051379:Mice; D011728:Pyridones; D012492:Salts; D013450:Sulfones
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D008024:Ligands; D011728:Pyridones; D012492:Salts; D013450:Sulfones; C017679:2-hydroxypyridine