IgM Purification with Hydroxyapatite

level: intermediAte H ydroxyapatite (HA) has a long and successful history in the field of antibody purification, and it has worked well for immunoglobulin M (IgM) monoclonal antibodies (MAbs) (1–8). Applications range from initial capture to intermediate purification to final polishing. HA is best known for its superior ability to reduce antibody aggregates, but it also supports excellent reduction of DNA, viruses, and endotoxins. As IgM MAbs exhibit increasing potential in the fields of cancer and infectious disease and in stem-cell therapies, HA’s unique fractionation abilities take on greater importance. Meeting the needs of those new opportunities requires an understanding of how HA interacts with various classes of biomolecules and how such interactions can be coordinated to create selectivities that particularly support the unique requirements of IgM purification. hydroxyaPatite interactions With Biomolecules HA serves as a multimodal chromatography medium. Its principal surface reactivities include cation exchange through negatively charged, surface-exposed HA-phosphate groups and calcium metal affinity through positively charged, surface-exposed HA-calcium atoms (9–12). The positive charge on the calcium atoms may be capable of mediating anion-exchange interactions, but no experimental evidence supports this hypothesis, and it seems to be overwhelmed by the stronger influence of metal-affinity interactions (13). Hydrogen bonding also has been suggested as a possible contributor to HA interactions with biomolecules (10–12), but it has not been investigated systematically, and no experimental evidence has been presented indicating the magnitude of its potential contribution. Phosphoryl cation exchange with biomolecules is intuitively straightforward. It follows the same rationale as cation exchange on familiar chromatography media such as carboxyand sulfo-based cation exchangers. Positively charged amino protein residues participate in electrostatic interactions with negatively charged HA-phosphates. These interactions can be controlled by altering salt concentration or pH. This explains the strong interaction of HA with alkaline proteins, but a given protein need not have an alkaline isoelectric point (pI) to bind strongly to HA, and strong binding is not necessarily an indication of strong cation-exchange binding. HA-calcium binding modulates the net effects of binding through HA-phosphates (13). The mechanism of calcium metal affinity is less intuitive but known to be mediated partly through carboxyl Figure 1: EDTA binding to calcium provides a simplified model of how proteins interact with HA-calcium. Solid lines represent covalent bonds within EDTA; broken lines represent coordination bonds. Note that calcium forms coordination bonds with the two uncharged amide nitrogen atoms, corresponding to peptide backbone nitrogen atoms in proteins; and to coordination bonds with the negatively charged oxygen atoms associated with the four carboxyl groups.