In vitro Binding Activity Measurement of Insulin-Albumin
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 Rmax and KD values of the affinity of TQ05849 and the original drug were 332 and 346 RU,124.8 and 122.4 μmol/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.
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