The market for recombinant antibody fragments has rapidly expanded, with small-sized fragments providing alternatives to full-length monoclonal antibodies in antibody-based therapeutics. Single-chain antibody fragments (scFv) are the smallest fragments retaining a complete antigen-binding site, making them ideal for applications requiring effective tissue penetration. However, the high titers of recombinant proteins pose challenges for developing efficient continuous purification processes that simplify isolation compared to traditional multi-step methods. This work explores the theoretical design and experimental validation of a Simulated Moving Bed (SMB) chromatography process using Immobilized Metal Ion Affinity Chromatography (IMAC) for purifying a single-chain antibody fragment. Initially, the cell culture supernatant from Bacillus megaterium was characterized through stepwise pH-gradient batch chromatography. The impact of solvent composition (pH) on the adsorption isotherm parameters for the antibody fragment and impurities was assessed in single-column runs. Based on these parameters, a feasible multicolumn open-loop 3-zone pH-gradient SMB process is proposed to continuously isolate the fragment. The design utilizes a recursive solution of an equilibrium stage model from an equivalent True Moving Bed (TMB) process. Theoretical operating conditions were simulated and experimentally validated in a lab-scale SMB unit, wit
