Comparison of two different HPLC hydroxylapatite matrices for resolution of IgG idiotypes

High-performance liquid chromatography hydroxylapatite (HPHT) is a well-known matrix utilized to separate macromolecules by differential surface binding to phosphate or calcium sites on the matrix. HPHT columns are commercially available packed with a crystalline structure formed of angular particles conferring a relatively poor elastic resistance to the matrix. In the present paper, we compare a conventional HPHT column to a new ceramic-type hydroxylapatite, which consists of globular particles providing a more mechanically stable matrix. The two hydroxylapatite matrices were compared in the purification of monoclonal antibodies produced by a hybridoma secreting three different types of antibodies. The two HPHT columns were able to separate the three antibodies molecules with equivalent peak resolution, and immunoreactivity analysis identified the active idiotype. HPLC columns packed with the ceramic hydroxylapatite resin exhibited a higher mechanical resistance than the crystalline material allowing significantly higher flow rates.