One-step cleavage and purification: a bifunctional IdeS-Protein A fusion protein for on-column generation of antibody fragments

Conventional production of antibody fragments (e.g. Fab/F(ab′)2, VHH/(VHH)2) involves sequential enzymatic cleavage and multi-step purification to remove Fc domains and residual enzymes, a process that is laborious and inefficient for small-scale workflows. To address this, we engineered a bifunctional IdeS-Protein A (IPA) fusion protein, which combines the specific IgG-cleaving activity of IdeS with the Fc-binding capability of Protein A. In solution, this construct enabled simultaneous antibody cleavage and affinity-mediated removal of both the enzyme and Fc fragments in a single step. Furthermore, we immobilized the fusion protein on a chromatographic resin to create an integrated “on-column cleavage-purification” platform. This system allows antibody capture, in-situ cleavage, and direct collection of high-purity F(ab′)2 fragments from the flow-through, while Fc fragments, uncleaved antibodies, and the enzyme are retained on the column. This work establishes a streamlined, one-step method for generating antibody fragments, offering a robust and automatable platform for research and preclinical applications.