Preparation of Stacked Polymyxin B-Functionalized Cryogels for Efficient Endotoxin Removal from Complex Biological Systems

Development of efficient and robust endotoxin removal approaches is vital for safeguarding the safety and functional performance of bacteriophage formulations and recombinant protein bioproducts. Here, a stacked gel-casting strategy combined with carboxymethyl chitosan (CMCS) reinforcement was employed to construct a mechanically robust PMB-functionalized cryogel. The synergistic effects of the stacked architecture and CMCS reinforcement significantly enhanced pore-wall stability and functional site accessibility, resulting in a high endotoxin adsorption capacity (1.88 × 106 EU/g) and excellent reusability (>90.30% removal efficiency after six cycles). The cryogel also demonstrated effective endotoxin removal in complex biological samples, achieving >99.00% removal in bacteriophage preparations with improved phage recovery and >99.99% clearance in recombinant protein solutions. These results highlight a promising strategy for endotoxin control in phage-based applications and biopharmaceutical purification.