Rapid and refined separation of human IgG2 disulfide isomers using superficially porous particles.

A rapid reversed-phase HPLC separation of recombinant human immunoglobulin gamma 2 (IgG2) disulfide isomers using columns packed with superficially porous particles is reported. Under optimal conditions, a separation of monoclonal IgG2 disulfide isomers was achieved in 10 min using a Poroshell™ 300SB-C8 column via a combination of high column temperature (85°C), mobile phases with high eluotropic strength (e.g. isopropanol) and high flow rate (1.5 mL/min). Thermodynamic stability analyses of chromatographically enriched IgG2 disulfide isomers revealed differences in their individual denaturation temperatures, which correlate with the observed temperature-dependent refinement of peak profiles by reversed-phase HPLC. This reversed-phase HPLC method in conjunction with other orthogonal analytical techniques (e.g. capillary gel electrophoresis, peptide mapping, ion exchange chromatography, etc.) is being used to characterize disulfide isomers in the development of therapeutic IgG2 antibodies.