脂肪酸酰化修饰胰岛素的纯化

Purification of Acylated Insulin by Polymer Resin

目的:脂肪酸酰化修饰的胰岛素类似物是常见的获得长效胰岛素的手段,但因其反应位点的偏差易产生多种传统纯化工艺较难以分离的杂质。本文探索以聚苯乙烯类有机聚合物填料为固定相纯化脂肪酸酰化修饰的胰岛素方法。方法:经过填料对比,有机溶剂浓度和pH选择后,再经过一系列正交试验优化,HPLC方法检验纯度和含量,质谱分析定性终产品,从而确定最佳条件。结果:经过对不同厂家不同型号的聚苯乙烯类有机聚合物填料的比较,填料选定采用纳微Uni Ps 30-300为最佳,其目的蛋白吸附量比为6.32 mg/ml,洗脱率为95.27%。在一系列的梯度实验中证实采用30-50%异丙醇浓度最佳。之后的pH对比得出,pH在4.0以下才能分离得到纯度在95%以上的产品。在以上一系列的单因素实验基础上,最后经过正交实验优化证实最佳的洗脱方案为采用A相为含25 mM硫酸铵的0.1 M磷酸盐缓冲液,pH 3.0;B相为异丙醇流动相,洗脱梯度为30-50%B的10 CV洗脱,流速60 cm/h。放大验证可以得到纯度大于97.5%,回收率70%以上的结果。结论:此方法具有分离效果好,成本低,易于放大的优点。

Objective： Modification of insulin with Fattyacid is the most common way to obtain Long-acting Insulin. But some im- purities would be generated during the acylation of insulin, which are hard to separated by traditional methods of purification. The paper aimed to setup an optimal method for purifying acylated insulin by polymer resins. Method： One-factor experiments related to the facts： resins, pH and solvents, and Orthogonal experiment were adopted. The concentration and purity of acylated insulin was measured by HPLC and MALDI-TOF MS. Result： In the experiment related to the selection of resins, Uni Ps 30-300 showed a higher adsorbtion rate of 6.32 mg/mL, and a higher recovery rate of 95.27 %. The experiments also showed that the optimum solvent for elution should be 30-50 % isopropyl alcohol, and pH value should be 4.0 or lower to remove the impurities. And finally the Orthogonal experiment＇s result showed the best protocol as follows： Buffer system consisted of an A-Buffer 0.1 M Phosphate salt buffer, 0.025 M （NH4）2SO4, pH 3.0 and Buffer B consisting of isopropyl alcohol. The crude product was eluted with a linear gradient from 30 % B to 50 % B in 10 CV, and the linear velocity was 60 em/h. The final product＇s purity can reach up to 97.5 %, and recovery rate was more than 70%. Conclusion： This technology is considered simple, cheap and suitable for industrialization.