Separation of different molecular forms of mouse IgA and IgM monoclonal antibodies by high-performance liquid chromatography on spherical hydroxyapatite beads

Abstract

High-performance liquid chromatography (HPLC) on newly developed spherical beads of hydroxyapatite (HAP) was applied to the separation of different molecular forms of mouse IgA and IgM monoclonal antibodies (mAbs). Monomeric, dimeric, trimeric and tetrameric forms of an IgA mAb were eluted from the column separately with appreciable differences in retention volume by a 20 ml (40 min) gradient of phosphate buffer (pH 6.8) of concentration from 10 to 400 mM. The volumes of respective forms were highly reproducible. In addition, a monomeric form was resolved successfully from a pentameric form of IgM mAbs by HPLC on HAP beads under the same conditions. All forms of IgA and IgM mAbs were eluted almost quantitatively from the column. The results indicate that HPLC on HAP beads is useful for isolation and characterization of different molecular forms of IgA and IgM mAbs.