白细胞介素-2(IL-2)已经用于癌症治疗超过30年。然而,由于它的高毒性,已经开发了新的突变变体。这些变体保留了原始分子的一些生物学特性,但提供了其他治疗优势。在分子免疫学中心,IL-2无α突变蛋白,已设计出一种比WTIL-2毒性更低的IL-2激动剂,produced,目前正在I/II期临床试验中进行评估。突变蛋白在大肠杆菌中作为不溶性材料产生,其必须在体外重折叠以产生完全活性的蛋白质。在大肠杆菌中产生的重组蛋白的纯化过程中,受控的氧化步骤是必需的,以确保分子中二硫键的适当形成。在这种情况下,新的纯化过程包括铜催化的空气氧化步骤,以诱导二硫键的建立。pH的最佳条件,铜,通过筛选确定该步骤的蛋白质和洗涤剂浓度。产生的蛋白质显示出保守的3D结构,纯度更高,并且比通过没有氧化步骤的既定方法获得的生物活性更大。生产并评估了四个批次,证明了新过程的一致性。
Interleukin-2 (IL-2) has been used in cancer treatment for over 30 years. However, due to its high toxicity, new mutant variants have been developed. These variants retain some of the biological properties of the original molecule but offer other therapeutic advantages. At the Center of Molecular Immunology, the IL-2 no-alpha mutein, an IL-2 agonist with lower toxicity than wtIL-2, has been designed, produced, and is currently being evaluated in a Phase I/II clinical trial. The mutein is produced in E. coli as an insoluble material that must be refolded in vitro to yield a fully active protein. Controlled oxidation steps are essential in the purification process of recombinant proteins produced in E. coli to ensure the proper formation of the disulfide bonds in the molecules. In this case, the new purification process includes a copper-catalyzed air oxidation step to induce disulfide bond establishment. The optimal conditions of pH, copper, protein and detergent concentration for this step were determined through screening. The produced protein demonstrated a conserved 3D structure, higher purity, and greater biological activity than the obtained by established process without the oxidation step. Four batches were produced and evaluated, demonstrating the consistency of the new process.