Purification of humanized murine and murine monoclonal antibodies using immobilized metal-affinity chromatography.
An affinity purification technique has been developed using mild elution conditions for the isolation of humanized murine and murine IgG1. This technique is based on the innate affinity of IgG1 for metal and utilizes immobilized metal-affinity chromatography. Antibody bound to the metal-affinity column is eluted with a descending pH gradient and IgG1 elutes around pH 6.0. Humanized murine IgG1 isolated from cell culture media using this procedure is approximately 90% pure and does not contain any free light chain, light-chain dimer, or contaminating serum albumin. In addition, murine IgG1 has been isolated from ascites fluid by direct application to the metal-affinity column. Murine IgG1 is recovered essentially free from albumin in approximately 60% purity, the principal contaminant being transferrin. This method facilitates further purification which is easily achieved by cation-exchange chromatography. The near complete removal of albumin makes metal-affinity chromatography an alternative to salt precipitation of antibody from ascites fluid. Recoveries of antibody from the column are high, typically greater than 90%. We have identified the location of the metal affinity in the IgG1 as being near the carboxy terminus of the heavy chain. A histidine-rich sequence is present in this region which offers the possibility of genetically engineering this sequence to form an even higher affinity metal binding site for potential application in antibody imaging and therapeutics.
Hale
J E
JE
Protein Engineering Department, Hybritech Incorporated, San Diego, California 92196.
Beidler
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001201:Ascites / Q000276:immunology; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D003470:Culture Media; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification* / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007074:Immunoglobulin G; D008670:Metals