Alleviating nonlinear behavior of disulfide isoforms in the reversed-phase liquid chromatography of IgG2

• Reversed-phase liquid chromatography has been used as a tool for characterizing disulfide isoforms of IgG2 monoclonal antibody drugs.
• At high injected concentration, the A1 isoform partially reconverts to the B isoform by the time it elutes.
• Nonlinear chromatography and low resolution impede the analysis.
• Injecting under isocratic conditions and then eluting with a slow gradient alleviates nonlinear chromatography and improves resolution.

Reversed-phase chromatography is an established method for characterizing the disulfide isoforms of IgG2. This work explores the effect of mobile phase gradient profile and sample concentration on the separation of disulfide isoforms. The acidic mobile phase can alter the relative proportions of disulfide isoforms, but only when the level of the reactive A1 isoform is much higher than for typical conditions of separation and typical IgG2 samples. Otherwise, there is minimal disulfide scrambling. A slower gradient and flow rate modestly improve resolution, but the peaks remain heavily overlapped. Resolution is further improved and nonlinear chromatography lessened when injection is performed under non-stacking conditions. Non-stacking conditions also keep the concentration from spiking at the head of the column, reducing noncovalent associations that can promote disulfide scrambling. The higher resolution from non-stacking injection reveals the presence of at least seven species.