Hydroxyapatite Chromatography (HAC)

Hydroxyapatite (HA) is a mixed-mode media that has been used extensively for the purification of proteins and DNA since the 1950s. Hydroxyapatite possesses a distinctive selectivity that may be applied in the purification of a wide range of biomolecules: immunoglobulins, alkaline proteins, acidic proteins, and DNA. The functional groups of HA can both attract and repel the carboxyl and amino groups on target molecules. This unique selectivity is due to the modalities that can be employed, which are not possible with traditional anion-exchange and cation-exchange chromatography. HA is a powerful chromatography step for reducing host cell-derived impurities and aggregated product, where a 2-4 log reduction in host cell proteins, aggregates, endotoxin, and viruses are routinely achieved. This chapter describes the procedures for: efficiently packing and evaluating a HA column, purifying IgG and acidic proteins respectively using HA chromatography.