pH-gradient cation exchange purification of IgG2 disulfide isoforms

Abstract

Immunoglobulin (IgG) based therapies are used to treat a wide range of diseases. The IgG2 subclass can have variable disulfide bond connectivity in the hinge region, leading to different isoforms. Interchain disulfide bonding isoforms that constrain the Fab arm structure may impact potency. Therefore, it is important to understand the abundance of IgG2 isoforms and the impact of function for IgG2s under development. In this work, a pH-gradient cation exchange separation was developed to purify IgG2 disulfide isoforms in their native state. The IgG2 mAb used for this study was not amenable to previously reported purification methods using salt-gradient cation exchange. Collected fractions were analyzed by high-resolution denaturing reversed phase chromatography and isoform content was determined with fluorescence detection. Fractions were then combined to generate solutions with varying amounts of IgG2-B isoform, ranging from 20.3 % to 80.8 % IgG2-B isoform. Across the range of IgG2-B isoform content abundances, all samples had similar levels of product related impurities and were amenable to potency testing. The work herein demonstrates a novel approach to natively fractionate disulfide isoforms for an IgG2 mAb that was not amenable to previous reported methods.

Keywords: Antibody; Cation exchange; Chromatography; Immunoglobulin; Isoform; Purification.