Temperature-modulated antibody drug separation using thermoresponsive mixed polymer brush-modified stationary phase

Recently, antibody drugs have been investigated for medical treatments of intractable diseases. An effective antibody drug separation method is in demand for production of such drugs. We developed an antibody drug separation material using a mixed polymer brush composed of poly(N-isopropylacrylamide) (PNIPAAm), which is a thermoresponsive polymer, and poly(4-vinylpyridine) (P4VP), which is an antibody-affinity polymer, for temperature modulation and adsorption of the antibody drug rituximab that results in its separation. A mixed polymer brush was prepared on silica bead surfaces by radical polymerization to modify P4VP, followed by atom transfer radical polymerization to modify PNIPAAm. The prepared mixed polymer brush was characterized by CHN elemental analysis, chloride analysis, nitrogen adsorption, thermogravimetric analysis, differential thermal analysis, differential scanning calorimetry, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscopy, and zeta potential measurement. Temperature-modulated adsorption of rituximab on the mixed polymer brush was achieved by concealing and exposing P4VP through the extension and shrinkage of PNIPAAm in the brush, and was investigated using mixed polymer brush-modified silica beads as chromatographic packing materials. By utilizing the specific properties of mixed polymer brushes, rituximab and bovine serum albumin could be separated by simply changing the column temperature. Thus, the mixed polymer brush-modified beads prepared in this study may be useful for purifying rituximab by changing the temperature.