Discoloration of ceramic hydroxyapatite used for protein chromatography

Hydroxyapatite chromatography was used to purify a recombinant human protein at preparative (400 g) scale. The hydroxyapatite column became progressively discolored as the number of chromatographic cycles increased. Elemental analysis showed that Mn, Fe, Al, Cd, Ba, Cr and Sn were found in used, discolored hydroxyapatite but were below the detection limit (1 ppm) in new hydroxyapatite. Metal ions were not removed from the discolored hydroxyapatite by regeneration with 0.5 M sodium phosphate followed by 0.5 M sodium hydroxide. Chromatographic performance was not affected by the accumulation of metal ions for at least 8 cycles on the preparative column (media volume 56 l) and for at least 12 cycles on the laboratory-scale column (media volume 3.7 ml).