Combination of Zetaprep mass ion-exchange media and high-performance cation-exchange chromatography for the purification of high-purity monoclonal antibodies.
A procedure involving diafiltration, mass ion exchange on a QAE Zetaprep disk, gel chromatography and cation-exchange chromatography was used for the purification of mouse monoclonal antibodies from hybridoma culture supernatant. Prior to the separation steps, the starting solution was adjusted to the desired pH and conductivity. Diafiltration was used for this purpose in order to keep the volume constant or even to reduce the volume of sample. A QAE Zetaprep disk was used to remove the main protein contaminants from the culture supernatant. After washing unbound proteins out of the Zetaprep disk, slightly bound protein was eluted with a buffer solution containing 50 mM sodium chloride. The monoclonal antibody was eluted with a solution containing 150 mM sodium chloride. The purity of the eluted antibody was 50%, and was increased to 99% by subsequent high-performance cat-ion-exchange chromatography. The purity was determined by means of sodium-dodecyl sulphate polyacrylamide gel electrophoresis and silver staining. The advantage of the two high-performance techniques, mass ion exchange and high-performance cation-exchange chromatography, are the high-flow-rates and the high resolution that can be obtained. These techniques are suitable for the production of injectable therapeutic preparations.
Jungbauer
A
A
Institute of Applied Microbiology, University of Agriculture, Vienna, Austria.
Unterluggauer
F
F
Steindl
F
F
Rüker
F
F
Katinger
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D006825:Hybridomas / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000032:analysis; D007525:Isoelectric Focusing; D051379:Mice; D011506:Proteins / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet; D014568:Urokinase-Type Plasminogen Activator / Q000276:immunology
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011506:Proteins; D014568:Urokinase-Type Plasminogen Activator