General Principles (Complex Interactions):

Hydroxyapatite (HA) chromatography of proteins involves the non-specific interaction of the charged amino or carboxylate group of a protein with oppositely charged groups on the hydroxyapatite, where the net charge of the hydroxyapatite and protein are controlled by the pH of the buffer. HA is used for purification of a wide variety of biomolecules, including proteins, phosphoroteins, carbohydrates, polynucleotides and viral particles. The column is usually equilibrated and the sample applied to a buffer that contains a low concentraiton of phosphate and eluted in an increasing gradient of phosphate salts (see operating conditions). In the alternative, some biomolecules may be eluted in an increasing gradient of chloride salts, but both elution formats impose disadvantages. The high phosphate concentration in which antibodies elute has strong buffer capacity that may interfer with subsequent purificaton steps. The high conductivity at which some biomolecules elute in chloride gradients may also interfer with downstream steps. Both situations require either that the eluted biomolecule be diluted or buffer exhanged which has a negative impact on process economics. As a result, HA steps are often placed at the end of a purification process. This tends to eliminate them from consideration as capture steps.  (Gagnon, US2009/0186396). 

Hydroxyapatite chromatography utilizes a calcium and phosphate based inorganic material with the structural formula of Ca10(PO4)6(OH)2, which forms both the matrix and ligand. Chemically reactive sites include pairs of positively charged calcium atoms and triplets of negatively charged phosphate groups. The interactions between hydroxyapatite and proteins are multi-modal, hence its classification as a mixed mode support. One mode of interaction involves metal affinity of protein carboxyl clusters for crystal calcium atoms. Another mode of interaction involves cation exchange of positively charged protein amino residues with negatively charged crystal phosphates. The individual contributions of the 2 mechanisms to the binding and elution of a particular protein can be controlled in part by the choice of salts used for elution. Due to its biosimilar composition, possible “leachables” present no problem. The material is also known to distinguish clearly between native and denatured variants of the same portein as well as between monomers, dimers and aggregates.

Studies have demonstrated that IgG is retained by HA by a combination of calcium metal affinity and phosphoryl cation exchange, regardless of subclass, light chain type or pI. (Gagnon, J Sep Sci, 2009, 32(22): 3857-3865).

Due to the dual functionality of calcium and phosphate groups comprising the matrix, the specific nature of protein interaction is complex. The amino groups on a protein are attracted to the phosphate sites, but are repelled by calcium sites. The situation is reversed for carboxylic groups as they are attracted to the calcium sites, but repelled by the phosphate sites (Mazzola, WO2009017491). Since proteins usually carry both positive and negative charges that vary in number as a function of the pH, the interaction of such molecules with hydroxyapatite becomes complex. On the other hand, this complexity can be exploited to design highly specific conditions for binding and elution in apatite chromatography. To date hydroxyapatite chromatography is often considered as a “last resort” to be used when everything else fails. This is partically due to the complexity of the interaction, which requires more sophisticated method devlopement than, e.g., Protein A affinity chromatography, but also to some of the physico-chemical properties of the hydroxyapatite itself  (Schubert, J. Chromatography A, 1142 (2007) 106-113). 

Although HA has been widely and successfully used, particularly for the separation of nucleic acids, the mechanism of their operation is little understood. The principles of ion-exchange chromatography cannot be applied to HA since the relation between protein affinity and their electrochemical behavior is not very prounouced in the case of HA. It is known that low MW substances such as amino acids, are poorly or not at all bound to HA. Synthetic polypeptides of various MW bind to HA, provided that they have a considerable total charge. A charge, however, is not a sufficient condition. For a given ionic strenght, the adsorption-desorption process is dependent on pH and for a given pH on ionice strenght (Gorbunoff, Analytical Biochemistry 136, 425-432 (1984). 

Unfortunately, the interaction of proteins with hydroxyapatite columns is not clearly understood. Tehrefore, determining conditions for the desired separation cannot be done theoretically. (Makino, J. Chromatographic Science, 27, 1989). 

Cleaning of HA surfaces

Cummings (US2012/0192901) teaches a method for cleaning an apatite solid surface by neutralizing the apatite solid surface with an alkaline hydroxide such as NaOHand then cleaning the apaptite solid surface such as with a phosphate solution. The invention is based on the discovery that hydrogen (or hydronim) ions can accumulate on an apatite surface following flow through purificaiton of a tareget molecule. If one performs a cleaning step without first neutralizing the column, degradation of the column can occur by displacement of calcium ions in the apatite support.