A Comprehensive Analysis of Novel Disulfide Bond Introduction Site into the Constant Domain of Human Fab

Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bonds can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the H: F130C-L: Q124C, H: F174C-L: S176C, H: V177C-L: Q160C, H: F174C-L: S162C, H: F130C-L: S121C, and H: A145C-L: F116C mutants mostly formed intermolecular disulfide bonds. All these mutants showed increased thermal stabilities compared to those without intermolecular disulfide bonds. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C-F118C mutant showed only a slight decrease in binding activity and β-helix content, owing to adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals the introduction of intermolecular disulfide bonds in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.