Protein A functionalized hierarchically ordered nanofibrous sponge enabling high-efficiency IgG purification

Immunoglobulin G (IgG) purification remains costly, largely due to limitations of conventional bead-based affinity media. This study presents a novel Protein A-functionalized nanofibrous sponge (NFS-Protein A) with hierarchically ordered lamellar pore channel structure designed for efficient IgG separation. This medium is fabricated through an integrated approach combining in-situ grafting of EVOH nanofibers with a polyethyleneimine (PEI) molecular amplifier and directional freezing technology. A key innovation is the use of PEI to exponentially amplify surface aldehyde groups, overcoming the intrinsically low grafting efficiency of the nanofiber substrate and enabling high-density, stable Protein A immobilization. The resulting sponge features long-range ordered lamellar pores (~20 μm), a high specific surface area (25.318   m²/g), and robust mechanical integrity. NFS-Protein A demonstrated a high static IgG adsorption capacity of 212.6 ± 6.3   mg/g, which is superior to reported state-of-the-art. Critically, it overcomes the throughput limitations of conventional columns by achieving a hydraulic flux of up to 30.45 ± 0.52   L/(h·bar) and a high dynamic binding capacity of 127.6 ± 4.5   mg/g at 1   mm/min, successfully capturing IgG from a protein mixture with high purity. This scalable, tunable platform integrates convective flow with specific affinity interactions, offering a transformative solution for high-throughput antibody purification.