Cooperative multimodal retention of IgG, fragments, and aggregates on hydroxyapatite

Retention mapping of chimeric monoclonal IgG(1), Fc, Fab, F(ab')(2), and aggregated antibody was conducted on hydroxyapatite (HA) by systematically varying phosphate and chloride concentrations during gradient elution in order to characterize the interactions of each solute with calcium and phosphate residues on the solid phase. Lysozyme was used as a control to model cation exchange-dominant interactions. Bovine serum albumin was used as a control for calcium affinity-dominant interactions. Calcium affinity and phosphoryl cation exchange were positively cooperative for IgG-related species. Fc retention was dominated by calcium affinity, while retention of Fab was dominated by cation exchange. F(ab')(2) exhibited a curve shape similar to Fab, but stronger retention. The retention curve for intact IgG incorporated the distinctive elements of its fragments but stronger retention than that predicted by their addition to one another. Aggregate retention paralleled the curve for non-aggregated antibody, with stronger retention by both binding mechanisms. Experimental data revealed evidence of charge repulsion between IgG carboxyls and HA phosphate at low conductivity values. Electrostatic repulsion of amino residues and attraction of carboxyls by HA calcium appeared to be blocked by strong complexation of calcium with mobile phase phosphate.