Preparation of Polymyxin B-Functionalized Cryogels for Efficient Endotoxin Removal from Protein Solutions

To address the limitations of traditional endotoxin adsorbents, which exhibit poor endotoxin removal efficiency and low sample recovery when processing high-concentration samples, a novel cryogel, CG(HEMA-co-AM), based on acrylamide (AM) and hydroxyethyl methacrylate (HEMA) as the second monomer, was successfully designed and synthesized. After optimizing the epoxidation and polymyxin B (PMB) conjugation processes, leading to the successful preparation of the functionalized cryogel CG(HEMA-co-AM)@ECH@PMB, flow-through experiments showed that in Tris-HCl buffer at a flow rate of 6 mL/min, the endotoxin removal efficiency reached 99.82%, with a maximum adsorption capacity of 1408.38 EU/mg. In a complex protein system containing BSA, HSA, Hb, LYS, and OVA (each at 10 mg/mL), the maximum endotoxin removal efficiency was 99.62%. Further investigations revealed that pH and ionic strength critically influenced the regulation of hydrophobic and electrostatic interactions, thus significantly affecting the endotoxin adsorption efficiency. Additionally, weakly hydrophobic and basic lysozyme exhibited significantly higher recovery rates compared to strongly hydrophobic and acidic proteins such as BSA and OVA. This functionalized cryogel integrates a high adsorption capacity with excellent specificity and features a macroporous structure suitable for dynamic chromatographic separation. It offers a novel, reusable adsorbent material for endotoxin removal in protein preparation, biopharmaceutical production, and clinical blood purification applications.