Unfolding of IgG domains detected by non-reducing SDS-PAGE

Abstract

Monoclonal antibodies are very important in modern therapeutics and constitute a substantial percentage of newly approved drugs. Every therapeutic monoclonal antibody must be analyzed for structural and functional integrity, and all protein heterogeneities need to be identified and quantified. The conformational stabilities of the monoclonal antibodies are also important for antibody storage and handling stabilities. One of the first and simplest of the structural analysis techniques utilized is SDS-PAGE, which can be performed both with and without prior reduction to break disulfide bonds. This permits sizing of both heavy and light chains in the reduced condition, and sizing of the intact antibody and any disulfide aggregates in the non-reduced condition. Analyzing our human anti-cocaine monoclonal antibody, we noted unexpectedly larger apparent molecular weights and apparent protein size heterogeneities using non-reducing SDS-PAGE. These apparent molecular weight heterogeneities are not consistent with other analysis techniques. Heterogeneities were noted using several heating and pre-electrophoretic sample preparation protocols, and are modified by the inclusion of small concentrations of certain alcohols such as propanol and butanol. All of these unexpected results were also observed for a commercial human IgG1 antibody, suggesting that these observations are applicable to IgGs in general. Thus, careful attention must be paid to the interpretation of non-reducing SDS-PAGE results for IgGs. It is hypothesized that differential thermal unfolding of the Fab, CH2 and CH3 domains of the IgGs in SDS give rise to the stable, discrete bands observed using different heating protocols prior to non-reducing SDS-PAGE.