An Enigma Unmasked : How Hydroxyapatite Works , and How to Make It Work For You

products on the market, the number of fundamentally unique selectivities is small. Differences among anion exchangers, for example, represent differences of degree, not of kind. Those differences are certainly significant but not to the extent that it would normally be considered beneficial to develop a purification procedure with two or more anion exchange steps. The same is individually true for cation exchange, hydrophobic interaction, size exclusion, and even affinity chromatography. This limitation restricts opportunities for maximizing orthogonal complementarity in multistep purification schemes. In this context, ceramic Hydroxyapatite (cHA) represents an important tool for process developers: a truly unique selectivity with chromatographic performance features on a par with the best of more widely used chemistries. The objective of this article is to highlight cHA’s features and describe how to exploit them to your best advantage. Composition and mechanisms of adsorpt i o n . H y d r o x yapatite (HA) is a crystalline mine ral of calcium phosphate. It has been ava i l a b l e for purification of proteins and nucleotides since 1956, but mostly in the physical form of easily fractured rectanglular plates, genera l l y unsuitable for industrial column applications. In the late 1980s, new synthesis procedures yielded HA in hexagonal-cross section microrods. These rods could be agglomerated into spheres of similar diameter, then sintered at high temp e rature to bond their structures. This “cera m i c ” HA is the subject of this article. The functional groups of HA consist of positively charged pairs of crystal calcium ions (Csites) and the six negatively charged oxygen atoms associated with triplets of crystal phosphates (P-sites). C-sites, P-sites, and hydroxyl groups are distributed in a fixed topogeographic pattern on the crystal surface. This combination of active groups supports retention by at least three distinct mechanisms: cation exchange with P-sites, calcium coordination with C-sites, and anion exchange with C-sites. Hydrogen bonding has been noted as being theoretically possible, but hasn’t been described. Which mechanism or combination of mechanisms dominates in a given application depends on the operating pH, buffer composition, and the surface properties of the protein (or other solute) applied to the column. Strongly alkaline proteins tend to adsorb mostly by phosphoryl cation exchange of positively charged amino acid residues with P-sites (Figure 1). Adsorption becomes stronger with reduced operating pH due to the increasing charge on these residues. As with classical cation exchange, there is a loose and limited correlation between elution order and isoelectric point (pI). The most alkaline proteins generally elute last in a linear gradient of NaCl or other salts. Although alkaline protein interactions with HA are dominated by cation exchange with P-sites, concurrent amine repellance by C-sites, and the crystal surface distribution of both Pand C-sites impart a unique stereochemical influence that renders the selectivity of HA wholly distinct from traditionalcation exchangers. Binding of strongly acidic proteins at acidic and neutral pH is heavily dominated by formation of metal coordination complexes between C-sites and carboxyl clusters on protein surfaces (Figure 2). That this mechanism is distinct from classical anion exchange has been prove n experimentally by evaluating retention of proteins on wh i ch the carboxyls have been replaced by sulfo groups. Binding is reduced draAn Enigma Unmasked: How Hydroxyapatite Works, and How to Make It Work For You