脂肪酸修饰胰岛素与白蛋白结合活性的研究

In vitro Binding Activity Measurement of Insulin-Albumin

文章对胰岛素-白蛋白体外结合活性测定方法进行研究,以判定自制长效胰岛素TQ05849与原研药物的相似性,同时探索脂肪酸链在该结合过程中的作用。通过活性琼脂糖基质固定化白蛋白体外结合长效胰岛素、SPR亲和力及结合/解离动力学测定两种方法对结合率进行测定,并设置长效胰岛素前体为阴性对照组,考察脂肪酸链在长效胰岛素与白蛋白结合过程中发挥的作用。通过固定化白蛋白体外结合长效胰岛素的方法测得TQ05849及原研制剂的体外白蛋白结合率分别为86.52%及86.71%,通过SPR亲和力及结合/解离动力学测定法测得TQ05849及原研制剂体外白蛋白亲和力R_(max)值分别为332及346 RU,KD值分别为124.8及122.4μmol/L,均基本一致,且结合/解离动力学图谱无显著差异。两种方法中阴性对照组长效胰岛素前体与白蛋白基本无结合,自制长效胰岛素TQ05849与原研制剂体外白蛋白结合活性相似,脂肪酸链在长效胰岛素与白蛋白体外结合过程中起主要作用。

This study focus on the measurement methods of in vitro binding activity of insulin-albumin,in order to determine the similarity between the self made long-acting insulin TQ05849 and original drug.The role of the fatty acid chain in the binding between long-acting insulin and albumin was indentified at the same time.The binding rate was detected by two methods:One is active agar immobilized albumin method,this method used the active agar to immobilize the albumin,then binding the long-active insulin in vitro,after the binding process was finished,the content of free insulin in the supernatant was detected by HPLC,in order to determine the binding rate between insulin-albumin;The other is SPR(Surface Plasmon Resonance)affinity and binding/dissoci-ation kinetics measurement method,this method used the CM5 chips to immobilize the albumin,then used the molecular interaction instrument(Biacore T200)to measure the affinity and binding/dissociation kinetics of insulin-albumin,the similarity of the binding rate in vitro between TQ05849 and original drug under this binding mode could be obtained by software analysis.The long-acting insulin precursor was set as the negative control group in order to determine the role of fatty acid chain in the binding between long-acting insulin and albumin.The in vitro binding rates of TQ05849 and the original drug were 86.52%and 86.71%,respectively,by the method of active agar immobilized albumin;The R_(max)and KD values of the affinity of TQ05849 and the original drug were 332 and 346 RU,124.8 and 122.4μmol/L,respectively,by SPR affinity and binding/dissociation kinetics measurement method.The results are basically the same,and there were no significant difference in the map of binding/dissociation kinetics.In the negative control groups,there was no binding between the long-acting insulin precursor and albumin.The self-made long-acting insulin TQ05849 has similar in vitro albumin binding activity as the original drug,and the fatty acid chain plays a major role in the in vitro binding process of long-acting insulin and albumin.