Abstract: The low protein adsorption capacity is caused by the low specific surface area of traditional macroporous polymer-based ion exchange chromatography media. A novel preparation method based on a "grafting to" strategy was proposed to enhance the protein adsorption capacity in this study. A pre-polymer containing primary amine groups was synthesized by copolymerizing two monomers, 2-aminoethyl methacrylate (2-AM) and N,N-dimethylaminopropylacrylamide (DMAPAA), using ammonium persulfate as the initiator. The average degree of polymerization of the resulting pre-polymer, pDMAPAA, was controlled between 2 and 10 by adjusting the molar ratio of 2-AM to DMAPAA.This pre-polymer was then grafted onto the surface of macroporous polyacrylate microspheres to fabricate a weak anion exchange chromatography medium.The optimal preparation conditions were determined by optimizing the coupling parameters. Using BSA as a model protein, the effects of ligand density and grafted chain length on the static binding capacity (SBC), the adsorption kinetics of media with different chain lengths, and the influence of ionic strength on adsorption behavior at different ligand densities were investigated. The results showed that increasing the grafted chain length (2~10 units) significantly enhanced the ion exchange capacity (IC) and SBC of the media. The equilibrium adsorption capacity (qe) increased from 130.9±3 mg/mL to 196.5±5 mg/mL, while the pseudo-second-order adsorption rate constant (k?) decreased. A higher ligand density increased the SBC from 95.6±2 mg/mL to 174.0±3 mg/mL. Increasing the salt concentration (0~0.25 mol/L) led to a decrease in SBC, with media of higher ligand density exhibiting stronger salt tolerance. This study provides a valuable reference for the controlled preparation and application of high-performance weak anion exchange chromatography media.

Key words: Macroporous anion exchange chromatography media, Grafting to, DMAPAA, Ligand density, Protein adsorption