Protein-templated macroporous agarose microspheres as a high-performance chromatographic medium for protein purification

Rapid purification of a targeted protein from a complex medium in advanced biotechnology requires efficient separation platforms. Herein, a new approach for preparing macroporous agarose microspheres (AGs) with enhanced mass transfer stability was developed by a protein-templated strategy. By optimizing the agarose-togelatin ratio (3:1, 3:4, 3:7, and 3:10), interconnected macroporous networks were created, promoting rapid protein diffusion and increasing the accessibility of meso/micropores. Different cross-linking reagents were assessed to enhance the mechanical strength of the AGs, with epichlorohydrin (ECH) demonstrating the highest pressure resistance and epoxy density. To achieve selective protein binding, diethylaminoethyl (DEAE) was directly conjugated to the epoxy-functionalized macroporous AGs (3:4 AGs-ECH and 3:7 AGs-ECH) via reactions involving hydroxyl and chlorine groups. The morphology and chemical composition of the composite AGs were systematically characterized. Benefiting from the macropores, the composites demonstrated significant binding capacities for bovine serum albumin (3:4 AGs-ECH-DEAE: 383.76 mg/g; 3:7 AGs-ECH-DEAE: 369.69 mg/g) as well as bovine hemoglobin (3:4 AGs-ECH-DEAE: 363.94 mg/g; 3:7 AGs-ECH-DEAE: 237.58 mg/g). Besides, the prepared 3: 4 AGs-ECH-DEAE and 3: 7 AGs-ECH-DEAE media exhibited ideal separation effects in chromatographic tests compared to DEAE Sepharose Fast Flow, highlighting their potential as effective chromatographic media for the swift separation and purification of proteins.