Characterizing Thermal Transitions of IgG with Mass Spectrometry

Variable temperature electrospray ionization (ESI) is coupled with mass spectrometry techniques in order to investigate structural transitions of monoclonal antibody immunoglobulin G (IgG) in a 100-mM ammonium acetate (pH 7.0) solution from 26 to 70 degrees C. At 26 degrees C, the mass spectrum for intact IgG shows six charge states + 22 to + 26. Upon increasing the solution temperature, the fraction of low-charge states decreases and new, higher-charge state ions are observed. Upon analysis, it appears that heating the solution aids in desolvation of the intact IgG precursor. Above similar to 50 degrees C, a cleavage event between the light and heavy chains is observed. An analysis of the kinetics for these processes at different temperatures yields transition state thermochemistry of Delta H-double dagger = 95 +/- 10 kJ mol(-1), Delta S-double dagger = 8 +/- 1 J mol(-1) K-1, and Delta G(double dagger) = 92 +/- 11 kJ mol(-1). The mechanism for light chain dissociation appears to involve disulfide bond scrambling that ultimately results in a non-native Cys(199)-Cys(217) disulfide bond in the light chain product. Above similar to 70 degrees C, we are unable to produce a stable ESI signal. The loss of signal is ascribed to aggregation that is primarily associated with the remaining portion of the antibody after having lost the light chain.