Recently, the importance of biopharmaceuticals in medical treatments has been increasing, and effective protein purification methods are strongly required for their production. In the present study, a temperature-responsive solid-phase extraction (SPE) column was developed for the purification of proteins without affecting their bioactivity. A temperature-responsive polymer hydrogel-modified stationary phase was prepared by coating aminopropyl silica beads (average diameter, 40–64 μm) with poly(N-isopropylacrylamide) (PNIPAAm)-based thermoresponsive hydrogels. n-Butyl methacrylate and acrylic acid were copolymerized with PNIPAAm as hydrophobic and anionic monomers, respectively. Using these temperature-responsive SPE columns, targeted proteins were retained on the thermoresponsive hydrogel at 40 °C through hydrophobic and electrostatic interactions. After the temperature was reduced from 40 °C to 4 °C, the retained proteins were successfully eluted from the column. Using the temperature-responsive SPE system, lysozyme was successfully separated from ovalbumin without any loss in bioactivity (99.7 ± 0.1%). Rituximab, a monoclonal antibody, was also purified from BSA or hybridoma cell culture medium using the prepared SPE column. Denaturation of rituximab was not observed in the rituximab fraction eluted from the SPE column. These results demonstrate that temperature-responsive polymer-based SPE can be applied in biomedical purifications, while maintaining the biological activity of the proteins.

近来，生物药物在医学治疗中的重要性已经提高，并且强烈需要有效的蛋白质纯化方法来生产它们。在本研究中，开发了一种温度响应固相萃取（SPE）色谱柱，用于纯化蛋白质而不影响其生物活性。通过用聚（N-异丙基丙烯酰胺）（PNIPAAm）为基础的热响应水凝胶包被氨基丙基硅胶珠（平均直径为40-64μm）来制备温度响应聚合物水凝胶修饰的固定相。n-分别将甲基丙烯酸丁酯和丙烯酸与PNIPAAm分别作为疏水和阴离子单体共聚。使用这些对温度敏感的SPE色谱柱，目标蛋白通过疏水和静电相互作用在40°C的条件下保留在热响应水凝胶上。将温度从40°C降至4°C后，保留的蛋白质成功地从色谱柱上洗脱下来。使用温度响应型SPE系统，溶菌酶成功地从卵白蛋白中分离出来，而没有任何生物活性损失（99.7±0.1％）。利妥昔单抗（一种单克隆抗体）也使用制备的SPE色谱柱从BSA或杂交瘤细胞培养基中纯化。从SPE柱洗脱的利妥昔单抗馏分中未观察到利妥昔单抗变性。