High Performance Liquid Chromatography of Mouse Monoclonal Antibodies on Spherical Hydroxyapatite Beads

Abstract

High performance liquid chromatography (HPLC) on newly developed spherical beads of hydroxyapatite was applied for the simple purification of monoclonal antibodies (mAbs) secreted into mouse ascitic fluid. Sixteen mAbs including all four subclasses of IgG and IgM were separated successfully from serum albumin a major contaminant in the crude mAb preparation by a 30 min-linear gradient of phosphate ion concentration from 0.01M-0.3M at pH 7.2. Not only IgG mAbs but also IgM mAbs were quantitatively eluted from the column. Each antibody had a different retention time (apparent capacity factor of 2.24–4.14) in the chromatography and no relation was found between the retention time and the type of immunoglobulin (class or subclass). A monomeric form of IgM was also resolved successfully from IgM (pentamer) after its reduction with dithiothreitol; the monomer form of IgM was eluted from the column by a lower concentration of phosphate ion than was the pentamer. These results indicate that HPLC on the hydroxylapatite beads will be useful for the purification and characterization of mouse mAbs.