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

Abstract

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.

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

“…Finally, when proteins have been identified to contain alternative disulfide bonds (disulfide-mediated isoforms), quantifying the amount of the protein that contains the native and alternative disulfide bonds remains a challenge because, many times, the disulfide-mediated isoforms would not be easily separated by standard separation techniques. State-of-the-art separation methods are a possible route forward for solving this problem, though separations of disulfide bond-mediated isoforms have yet to achieve baseline resolution [116, 117].…”