Ranking the Susceptibility of Disulfide Bonds in Human IgG1 Antibodies by Reduction, Differential Alkylation, and LC—MS Analysis.

One of the basic structural features of human IgGi is the arrangement of the disulflde bond structure, 4 inter chain disulfide bonds in the hinge region and 12 intra chain disulfide bonds associated with twelve individual domains. Disuffide bond structure is critical for the structure, stability, and biological functions of IgG molecules. It has been known that inter chain disulfide bonds are more susceptible to reduction than intra chain disulfide bonds. However, a complete ranking of the susceptibility of disulfide bonds in IgGi molecules is lacking. A method including reduction, differential alkylation, and liquid chromatography-mass spectrometty (LC-MS) analysis was developed and employed to investigate the complete ranking order of the susceptibility of disulfide bonds in two recombinant monoclonal antibodies. The results confirmed that inter chain disulfide bonds were more susceptible than intra chain disulfide bonds. In addition, it was observed that the disuffide bonds between the light chain and heavy chain were more susceptible than disulfide bonds between the two heavy chains. The upper disuffide bond of the two inter heavy chain disuffide bonds was more susceptible than the lower one. Furthermore, disullide bonds in the CR2 domain were the most susceptible to reduction. Disulfide bonds in VI, CL, Vii, and CH1 domains had shuilar and moderate susceptibility, while disulfide bonds in the CH3 domain were the least susceptible to reduction. Interestingly, a difference between IgGit and IgGl.% was also observed. The difference in the susceptibility of inter light heavy chain disulfide bonds and inter heavy chain disuffide bonds was smaller in lgGlic than in IgG1L The intra chain disuffide bonds in the Fab region of IgGlic were also less susceptible than disulfide bonds in the Fab region of lgGl2. [ABSTRACT FROM AUTHOR]