Separation and Characterization of IgG2 Disulfide Isoforms by pH Gradient Cation Exchange Chromatography and Non-Reduced LC/MS - Peptide Mapping

摘要

pH gradient CEX effectively separates charge and disulfide isoforms of a biotherapeutic IgG2 mAb. Acidic, main, and basic species are charge separated by pH gradients 7.3-7.7, 7.7-7.9, and 7.9-8.2 respectively. Structural disulfide isoforms of a biotherapeutic IgG2 mAb are separated into five main fractions by pH gradient CEX, identified by non-reduced LysC peptide mapping - LC/MS. Disulfide isoforms A, A/B, and B are indistinguishable by organic SEC/MS at the intact mAb level. IgG2 isoform A/B was identified in main fractions 1 & 2, IgG2 isoform A was identified in main fraction 3, and IgG2 isoform B was identified in main fractions 4 & 5. Acidic species (deamidation) and basic species (amidated proline, C-terminal lysine, methionine oxidation, and N-terminal glutamine) are determined to be enriched for (> 5X) in the acidic and basic fractions respectively.