Whole process of Fab antibody aggregation in intestinal environment and their aggregation regulation: An insight from...

This work revealed the aggregation and aggregation inhibition mechanisms of Fab antibody in a simulated intestinal fluid via static and concentration perturbation aggregations to meet the challenges of oral antibody therapy. Results showed that Fab aggregation was highly concentration dependent, mainly determined by the β-sheet's stacking and amyloid fibers' extension at low (1 mg/mL) and the twine of β-strands' turn at high (20 mg/mL) concentrations. During the incubation of 0-240 min, Fab was continuously aggregating, but with some rearrangements on its spatial conformation: α-helix and β-sheet formation with β-turn and random coil unfolding, which expanded the aggregates' hydrophobic core and extended β-sheet structure through the π-π stacking of aromatic amino acids. The aggregation kinetics indicated that high Fab concentrations promoted high aggregates but the growth of amyloid fibers took a long time, while low to high concentration fluctuation promoted the formation of Fab aggregates. Molecular docking and molecular dynamics simulations suggested that Arg, PEG 10000, and Poloxamer 188 reduced the potential energy; PEG 10000 and Tween 20 enhanced steric hindrance by spontaneously binding through competitive hydrogen bonding without disturbing Fab's conformation. This work can provide promising approaches for our daily health management by facilitating the materialization of Fab-based oral antibody therapy.