Reversed-phase chromatography with large pore superficially porous particles for high throughput immunoglobulin G2 disulfide isoform separation

Reversed-phase liquid chromatography (RPLC) has been commonly used in IgG2 disulfide isoforms analysis. Recently, the columns packed with large pore superficially porous particles (SPP) have become available commercially. This work explores the application of this SPP technology in IgG2 disulfide isoforms separation. A high throughput and improved resolution RPLC method is developed with the optimization of column selection, gradient, temperature and flow rate. Compared with the small particles RP-UHPLC columns, large pore SPP columns provide unique selectivity and several new peaks were resolved and identified to be the free thiol variants of the IgG2 disulfide isoforms. The optimized method enables the detailed characterization of cysteines related variants in a single and fast method.