Relationship between human IgG structure and retention time in hydroxyapatite chromatography with sodium chloride gradient elution

Accurate prediction of the elution tendency of monoclonal antibodies in column chromatography would be beneficial for the efficient setup of purification procedures. Hydroxyapatite chromatography experiments using 37 recombinant human monoclonal antibodies were performed by sodium chloride gradient elution with 5 mM sodium phosphate to correlate the retention times with antibody structures (subclass and light‐chain isotypes). The contribution of metal affinity interactions in the interaction of antibodies with hydroxyapatite was investigated by (i) eliminating 5 mM sodium phosphate in buffers, (ii) comparing sodium chloride versus sodium phosphate gradient elutions, and (iii) using IgG₄ antibodies with a leucine→glutamate mutation. By using antibodies of different subclasses but with identical Fab regions, the elution behavior in sodium chloride elution could be classified by subclass and type of light chain. It is considered that the retention of monoclonal antibodies to hydroxyapatite is affected by the cooperation of phosphoryl cation exchange and metal affinity interactions. The contribution of the metal affinity interactions is greater in the sodium chloride gradient elution method than in the sodium phosphate gradient elution method.