Chromatography of polypeptides and proteins on hydroxyapatite columns: Some new developments

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

“…Under physiological, near neutral, conditions, proteins with an isoelectric point (IEP) in the acidic range ("acidic" proteins) showed different elution behaviour than that of "basic" proteins with an IEP in the alkaline range, whereas the elution behaviour of some neutral proteins could be switched from one type to the other by the addition of calcium ions to the mobile phase. Small molecules, such as amino acids, bound only poorly to HA [10,12,13], whereas polypeptides bound to HA, provided that they had a considerable total charge [12,32]. Negatively charged polypeptides could be eluted with 0.35 M phosphate, but not with 3 M KCl or CaCl 2 , and thus, according to Bernardi [12] resembled polynucleotides.…”

“…Small molecules, such as amino acids, bound only poorly to HA [10,12,13], whereas polypeptides bound to HA, provided that they had a considerable total charge [12,32]. Negatively charged polypeptides could be eluted with 0.35 M phosphate, but not with 3 M KCl or CaCl 2 , and thus, according to Bernardi [12] resembled polynucleotides. Basic polypeptides eluted late in the phosphate gradient, but readily in NaCl/KCl or in the presence of low amounts of CaCl 2 .…”

“…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. New production procedures developed in the 1980s resulted in HPLC compatible HA beads [14][15][16].…”

“…However, even in the case of proteins with a net positive charged, retention on HA can rarely be explained by a simple ion-exchange mechanism. The investigation of the retention mechanisms of proteins and polynucleotides on HA was pioneered by Bernardi's group in the 1950s (see [12] and literature cited therein). In 1978, Spencer and Grynpas [30,31] published the chromatographic behaviour of HA as a function of the surface structure (scanning electron microscopy) and the size of the crystallites in the HA particle (X-ray diffraction).…”

“…In the last case, the SVSV fragment was mainly responsible for the interaction, whereas the MKPSP fragment apparently provided a conformational-dependent component that enhanced the peptide's binding, but by itself did not show any specific affinity. In 2009 our group presented a study of HA-interacting peptides at the 5 th Conference on Hydroxyapatite and Related Products: CHT for Bioprocessing (Oct. [11][12][13][14]2009, Rottach-Egern, Germany). The HA interaction (binding/elution) of several amino acids (glycine, serine, phenylalanine, lysine, aspartic acid), two proteins (BSA (IEP 4.7) and lysozyme (IEP 10.7)), was reported together with that of several peptides.…”