Chapter 24 Protein Chromatography on Hydroxyapatite Columns

Abstract

The introduction of spherical forms of hydroxyapatite has enabled protein scientists to separate and purify proteins multiple times with the same packed column. Biopharmaceutical companies have driven single column applications of complex samples to simpler samples obtained from upstream column purification steps on affinity, ion exchange or hydrophobic interaction columns. Multiple column purification permits higher protein loads to spherical forms of hydroxyapatite and improved reduction in host cell protein, aggregates, endotoxin, and DNA from recombinant proteins. Adsorption and desorption mechanisms covering the multimodal properties of hydroxyapatite are discussed. The chemical interactions of hydroxyapatite surface with common ions, metals, and phosphate species affect column lifetimes. Adsorbed hydroxonium ions from low ionic strength buffers are noted by a shift in effluent pH during column equilibration. Hydroxonium ion desorption is observed by acidic shifts in the column effluent with the magnitude and duration surprisingly extreme. Buffering reagents with high buffering capacity reduce both the magnitude and duration of the acidic shift. Column packing methods for the robust spherical particles as well as the microcrystalline hydroxyapatite particles are reviewed. Applications covering extracted proteins and recombinant protein purification, especially monoclonal antibodies, with multiple chromatography media in concert with hydroxyapatite are reviewed.