Simple and rapid purification of monoclonal antibodies from cell culture supernatants and ascites fluids by hydroxylapatite chromatography on analytical and preparative scales.
A simple and rapid method for the purification of murine and human monoclonal antibodies from ascites fluids and cell culture supernatants is described. The method, based on the use of hydroxylapatite (HAP) column chromatography, is applicable on both analytical and preparative scales. In our work on purification of monoclonal antibodies, we have found that the combination of a single step elution of impurities followed by linear gradient elution of antibody provides an excellent purification of the antibody from cell culture and ascites fluids. The procedure provides very good resolution at high flow rates. The cell culture supernatant can be pumped on the preparative column at the rate of 2-3 ml/min without any measureable back pressure. The binding is independent of the flow rate. This method has been successfully used to purify several monoclonal antibodies of different subtypes from cell culture supernatants.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001202:Ascitic Fluid / Q000032:analysis; D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods; D003470:Culture Media / Q000032:analysis; D006882:Hydroxyapatites; D007074:Immunoglobulin G / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis; D051379:Mice
D000911:Antibodies, Monoclonal; D003470:Culture Media; D006882:Hydroxyapatites; D007074:Immunoglobulin G; D007075:Immunoglobulin M