Just a decade ago, experimental work by Guerrier, Flayeux, and Boschetti demonstrated the ability of sodium chloride (NaCl) gradients on hydroxyapatite (HA) to remove aggregates from IgG preparations. This approach has since proven effective with the majority of IgG monoclonal antibodies and become the foundation of HA’s growing popularity.
The “International Conference on Hydroxyapatite,” held late last year in Rottach-Egern, Germany, underlined HA’s emergence as a mature industrial technology, its expansion into new application areas, and its continuing fascination to investigators worldwide. Participants presented new findings on purification of therapeutic proteins, viral vaccines, characterization of biomolecule interactions with HA, and development of new HA materials.
HA is a multimodal chromatography support. In contrast to ion exchangers, for example, which principally exploit biomolecule interactions with a single type of chemical surface, HA exploits two primary binding mechanisms: metal affinity interactions through HA calcium and cation exchange interactions through HA phosphate.
Protein carboxyl residues bind by calcium affinity, amino residues bind by cation exchange. Calcium interactions can be eluted only by ions with high calcium affinity, like phosphate. Cation exchange interactions can be eluted with any salt.
Steve Cramer, professor at Rensselaer Polytechnic Institute, presented results from high-throughput screening experiments and sophisticated modeling programs. Data from a broad panel of model proteins over a wide range of conditions emphasized that positively and negatively charged sites on a given protein are able to bind their complementary chemical surfaces on HA simultaneously.
This cannot occur on ion exchangers and accounts for the unique selectivity of HA. Dr. Cramer also said that, although HA calcium carries a positive charge, NMR studies indicate that anion exchange interactions do not contribute to retention on HA.
Ruth Freitag, professor at the University of Bayreuth, Germany, is working to obtain a more refined understanding of how IgG and its fragments interact with the surface of HA. Experimental data and 3-D computer models revealed how a small region of a protein can dominate its overall interaction with the solid phase. Protein isoelectric point was not a useful predictor of retention behavior, she reported.
Dr. Freitag also talked about HA’s well-known historical ability to achieve separations impossible for other methods and provided several new examples.
Shuichi Yamamoto, professor at Yamaguchi University, addressed a different model system for characterizing HA interactions: DNA.
Polynucleotide phosphates form NaCl-resistant coordination bonds with HA calcium but are electrostatically repelled from HA phosphate. NaCl increases retention by suppressing charge repulsion, allowing the DNA to bind with more HA calcium sites. HA easily separates single- and double-stranded DNA and discriminates among DNA molecules according to size.
This is a substantial benefit over anion exchange chromatography, which supports virtually no discrimination regardless of DNA size or the number of strands. Previous work has shown that HA binds RNA less strongly than DNA. These results highlight the unique utility of HA for DNA plasmid purification.
Alois Jungbauer, professor at the University of Natural Resources and Applied Life Sciences, presented confirmational data. The results illustrated a striking case of preferential orientation, with plasmid DNA binding by the terminus of a roughly linear section and extending out from the HA surface like the bristles of a brush.
Giorgio Carta, professor at the University of Virginia, addressed another aspect of HA surface chemistry. Like all chromatography media possessing a cation exchange functionality, pH descends on HA columns when NaCl is introduced. This results from displacement of hydronium ions from the media surface by sodium ions due to their higher affinity for the cation exchange groups. With HA, the phenomenon also involves the calcium-phosphate equilibrium of HA itself, which has important ramifications for column lifetime.