Monoclonal antibody purification with hydroxyapatite

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography

“…The original Tiselius apatite was a soft, powdery substance created by a somewhat irreproducible precipitation process and columns packed from it were characterised by poor flow properties and highpressure drops. In addition, porous agarose beads that incorporated HA crystals became available around that time [11]. Although they gave interesting results [12,13], these materials were clearly not ideal chromatographic supports.…”

“…As a result, interactive points on the HA surface are not organised randomly, but in a pattern. The structured surface of HA has, for example, been used to explain why HA is often able to resolve protein molecules with only subtle structural differences or why denatured proteins tend to bind to HA less strongly than the corresponding native ones [6,8,11]. Contrary to an ion exchanger, with its randomly placed interactive groups, HA will be sensitive to the arrangement of such groups on a protein [22][23][24].…”

“…The Mg ion causes a strong distortion of the environment of the anions, due to the shorter Mg-O distance. Metal ions such as Fe also bind strongly to HA and cause discolouration [11]. …”

“…According to Gagnon [11], C-site interaction is based on calcium chelation by protein carboxyl clusters and calcium coordination by phosphate groups on DNA and other phosphorylated molecules. Anion exchange with negatively charged residues seems unlikely in view of Gorbunoff's [36] experiments exchanging negatively carboxyl groups for other negatively charged ones.…”

“…In this protocol, the basic proteins are eluted first in a gradient of increasing NaCl concentration, then the acidic ones are eluted in the phosphate gradient. More recently [11,25], borate gradients have been proposed as an alternative to phosphate ones, in particular, for antibody purification, for which according to Gagnon [11] they allow better removal of aggregates. In addition, the antibody is eluted at a lower conductivity, which improves the compatibility of the HA step, for example, with a subsequent ion-exchanger step.…”