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

The aim of this study was to classify the retention time of recombinant human monoclonal antibodies (rhMAbs) in hydroxyapatite chromatography with sodium-phosphate gradient elution according to their physicochemical properties. We analyzed 37 rhMAbs and found that (i) the structures of both the constant and variable regions affected retention time, (ii) the number of basic amino acid residues in the variable region, particularly in the heavy chain, correlated well with retention time, and (iii) this correlation was more pronounced (e.g. r(2) = 0.87 for 18 K IgG(1) rhMAbs) when the surface accessibility of those residues are taken into account. These findings provide a useful guide for investigators and purification-process developers working with monoclonal antibodies.