Separation and analysis of charged isomers of monoclonal immunoglobulin G by ceramic hydroxyapatite chromatography

Analysis of monoclonal antibody (MAb) heterogeneity caused by posttranslational modifications is important for pharmaceutical quality assurance of antibody drugs. In this study, by employing small ceramic hydroxyapatite (HAp) particles that were self-manufactured to have a 2.5-mu m diameter, we attempted to separate and analyze MAb isomers. The MAb without N-linked oligosaccharides could be separated by 2.5-mu m HAp chromatography as well as MAb with N-linked oligosaccharides. Hence, a variety of N-linked oligosaccharides do not appear to be involved in the separation mechanism of HAp chromatography. However, there is a difference in retention time between MAb with and without N-linked oligosaccharides, meaning that the presence of N-glycan could influence the retention time of HAp chromatography. Subsequently, the MAb fractions separated by 2.5-mu m HAp chromatography were analyzed by isoelectric focusing, and seven isomers of the MAb having different isoelectric points (pI) were identified. The MAb isomers were eluted in order of lower pI isomers with sodium phosphate buffer, and enzyme-linked immunosorbent assay indicated the immunoreactivity of the fraction including the lowest pI isomers to be remarkably reduced. This study yielded details of the separation behavior of HAp chromatography owing to 2.5-mu m HAp particles.