Calixarene-immobilized monolithic cryogels for preparative protein chromatography

New cation exchanger monolithic stationary phases were prepared by immobilization of three different calixarene derivatives (i.e. tetracarboxylate calix[4]arene, CLX-COO, tetrasulfonate calix[4]arene, CLX-SO₃, and tetraphosphonate calix[4]arene, CLX-PO₄) onto a monolithic cryogel support (i.e. poly(2-hydroksyethylmethacrilate-co-glycidyl methacrylate, P) and investigated with respect to preparative protein chromatography. The obtained monoliths were characterized through various techniques such as FTIR spectroscopy, isoelectric point measurements, titrimetric analyses, and mercury intrusion porosimetry. Protein retention was investigated using some model proteins (i.e. lysozyme, cytochrome c, and ɑ-chymotrypsinogen A, human serum albumin, and myoglobin), and the role of modifier (i.e. NaCl) concentration and pH was thoroughly analyzed under isocratic and gradient elution conditions. Overloading experiments were also conducted to study dynamic adsorption capacity and the obtained values were found to be ranging between 3 and 8 mg/mL depending on the type of calixarene molecule. Hence, higher or comparable protein adsorption capacities were seen to be applicable on calixarene-immobilized cryogels when compared to any other functionalized cryogels in the literature. Combined with the favorable properties of these monoliths, with respect to mass transport of large molecules, these results qualify calixarene functionalized monolithic cryogels as promising stationary phases for protein preparative purification.