Rapid purification of monoclonal antibodies by high-performance liquid chromatography.
Three different high-performance liquid chromatographic (HPLC) techniques, i.e., ion-exchange, hydrophobic interaction and hydroxyapatite chromatography, have been used to purify monoclonal antibodies from ascites fluid. The monoclonal antibodies were raised against coagulation factor VIII. Precipitation of the antibodies by ammonium sulphate prior to HPLC made it possible to purify the antibody in one chromatographic step. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis this highly pure preparation revealed only one extra polypeptide besides the light- and heavy-chain immunoglobulin polypeptides. Attempts were also made to purify the antibody without prior ammonium sulphate precipitation. A combination of ion-exchange and hydrophobic interaction chromatography resulted in an antibody preparation comparable in purity to the one obtained from ammonium sulphate-precipitated immunoglobulin. The rapid HPLC techniques were found to be very useful for purification of monoclonal antibodies on a preparative scale, where sample loadings of up to 25 mg of ascites protein were fully resolved in satisfactory yields.
D000645:Ammonium Sulfate; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001202:Ascitic Fluid / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007074:Immunoglobulin G / Q000302:isolation & purification; D051379:Mice; D013056:Spectrophotometry, Ultraviolet
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D000645:Ammonium Sulfate