Thrombin Product Hybrid Protein Detection, Endotoxin Removal And Stability

Thrombin is an important clinical hemostatic drug with great market demands and broad application prospect.But most thrombin products in the market have mixed proteins and endotoxin,and are not stable,which are easy to result in clinical accidents.Therefore,it is very important to assay mixed proteins in thrombin productions,invent appropriate methods to remove mixed proteins and endotoxin from thrombin productions and improve their stabilities.In our work,BAPNA and effective method that the activity of antithrombinⅢimproves thousand times and inhibits blood coagulation when micro-heparin rests were used to assay the mixed proteins in thrombin samples.Ion-exchange chromatography and gel chromatography were used to remove mixed proteind and endotoxin in thrombin samples.Acid,trehalose and glucose were added into thrombin solution samples,and proper additives to improve stability of thrombin productions were selected.Ⅰ,The methods of assaying alpha-2 Macrogbulin and antithrombinⅢwere found successively,and the result showed there was alpha-2 Macrogbulin in the thrombin.The thrombin sample of batch 060401,060402,060403,060501, 060502,060601,060602 and their specific activity 68.9IU/mg,79.1IU/mg,130IU/mg,142.96IU/mg,339.69IU/mg,420IU/mg, 476.66IU/mg were studied,and the results indicate that trypsase can keep stability in about 40min after reaction begins and the accumulation of hydrolysate varies directly with time and therefore forms linear relationship.With the activity of thrombin increases,its light abstract value becomes smaller.The activity of alpha-2 Macroglobulin gradually reduces from 1.6 to 0.2 U,which indicate that the the thrombin samples with higher activity and purity have fewer mixed proteins and lower alpha-2 Macroglobulin activity.The studys on the thrombin of batch 060401,060402,060403,060501,060502 show that thrombin solutions in different chroma group and additive of heparin group is nearly same when thrombin solutions begin to coagulate.This indicates that there is not antithrombinⅢin the thrombin.The experiment shows there is alpha-2 Macroglobulin in thrombin production and no antithrombinⅢ. A method of assaying alpha-2 Macroglobulin of thrombin was found in our study.ⅡEndotoxin of thrombin were removed effectively by chromatography.Ion-exchange chromatography and gel chromatography were used to remove endotoxin of thrombin samples of batch 060401,060402,060403 and and their specific activity 68.9IU/mg,79.1IU/mg,130IU/mg.Before chromatography,endotoxin in per unit thrombin is greater than 1.1726EU and after chromatography ndotoxin in per unit thrombin is less than 0.7313EU.Our result is that endotoxin in per unit thrombin is less than 0.9758EU,which indicate that ion-exchange chromatography and gel chromatography can remove endotoxin in thrombin samples and the effect of ion-exchange chromatography is more obvious.ⅢThe stability were improved by the additives added to the same thrombin solutions.Different additives were added to the same chroma thrombin solutions.After 37℃,11d,adding glycin and fucose group can keep 30% and 18%activity of enzymes separately,while the control group can only keep 15%activity.After 4℃,13d,adding glycin and fucose group can keep 68%and 55%activity of enzyme separately,while control group is 48%.The results indicate that glycin and fucose can increase stability of thrombin solution,and stability is highest when the ratio of glycin: thrombin is 1:1000.