Abstract Fab has been extensively studied to elucidate the structural and physicochemical principles underlying antigen recognition. While most investigations have focused on IgG-derived Fabs, comparatively little is known about those from other isotypes. To investigate the role of the IgM constant domain Cμ1, we engineered a chimeric Fab (Cμ1Fab) in which the CH1 domain of adalimumab Fab (IgG1) was replaced by the human IgM Cμ1 domain. Cμ1Fab was expressed in CHO cells and its physicochemical properties were evaluated. SDS-PAGE analysis with or without PNGase F treatment demonstrated that the N166–N167–S168 motif was N-glycosylated. SPR analysis demonstrated that Cμ1Fab retained antigen-binding activity comparable to that of the wild-type Fab, and elimination of the glycosylation motif (N166A) did not alter binding affinity. Notably, N-linked glycosylation within the Cμ1 domain suppressed aggregation, whereas the N166A variant showed marked aggregation. DSC further revealed that the glycosylation affected thermal stability. The melting temperature (Tm) of the N166A variant was 60.5 °C, substantially lower than that of the Cμ1Fab (Tm = 66.0 °C), although Tm of the wild-type Fab was 75.2 °C. Collectively, these findings establish the critical role of native Cμ1 N-linked glycosylation in Fab stability, offering new insights for antibody engineering.
Sugimoto et al. (Mon,) studied this question.