Study Of Process Optimization In Heparin Benzyl Ester Production And Protein Recovery From Heparin Wastewater

Heparin acts as the most commonly used clinical anticoagulant and antithrombotic agent and there are no alternatives.Among various heparins,enoxaparin have been most extensive used and most intensive studied globally,its market share is over50% of heparin ware.The most key processing in enoxaparin production is the preparation of heparin benzyl ester,the molecular weight,yield and consequently benefit depend on the esterificationrate of heparin benzyl ester.In general,the time of esterification is over 20 hours which decreased the efficiency and benefit.Meanwhile,traditional anticoagulant activity of heparin is determined by visible judging,consequently is poor accuracy.Costof other methods is uneconomical because of expensive imported regents.It is very critical to develop an improved test method which iscost-saving,easy-to-operate and accurate.At the same time,the amount of waste-water of heparin production is up to 6000t/d in China.Creation of a waste-free technology for the preparation of heparinis an important problem since the manufacture of these preparations is increasing from year to year.With this increase the amount of by-products which are not utilized grows and these are taken out to an irrigation field.Recovering proteinfrom the manufacture of heparin makes it possible to obtain a further both economic andenvironmental advantage as a result of a more complete usage of raw materials.In this thesis,the purification of crude heparin,new test method of anticoagulant activity,optimization processing in preparation of heparin benzyl ester assistant with ultrasonic waves treatment and the recovering protein from casing-heparin waste-water were systematically investigated.The results indicated:1)Study of ultrasonic processing on heparin benzyl ester preparation: First,purifying crude heparin with methanol,purified yield is 49.25%;and the content of protein and nucleic acid in purified heparin was significant decreased than crude heparin;and it suggested that there was no Oversulfate Chondroitin sulfate detected.Then the effect of ultrasonic power and process time on esterification rate during preparing heparin benzyl ester were investigated using the one-factor-at-a-time method and response surface methodology(RSM)approaches.the optimal operation parameters are following as:ultrasonic power,400 w and reaction time,5.5 hours when temperature is 35-40℃.At this treatment,the esterification rate is up to 5.35%.2)Developed of an improved test method on heparin anticoagulant activity applying half-automated coagulation analyzer along with classic test of sheep plasma activated thromboplastin time: Based on new protocol,a very significant linear correlation was observed between detection dosage and response value(P<0.01),and the linear correlation between standard solution and test solution is paralled(P>0.05).The result of CI detection suggested the accuracy of activity in test solution.Applying this protocol,the activity was90.65 IU/mg when that of rabbit whole blood method was 92.96 IU/mg.Feasible limit rates of methods are complied with pharmacopoeia requirements.The results confirm that the new protocol is flexible.3).Process optimization of recovering protein from casing-heparin waste-water:This thesis deals with the optimization of precipitate conditions for the recovery of protein from heparin waste-water using the one-factor-at-a-time method and response surface methodology(RSM)approaches.The one-factor-at-a-time method was adopted to investigate the effects of flocculation components(i.e.Fe Cl3,FeSO4,alumen,chitosan,bentonite and diatomaceous earth)on protein recovery rate.The subsequent optimization of the four most significant factors viz.FeCl3,pH,bentonite and diamaceous earth,was carried out by employing a central composite rotatable design(CCRD)of RSM.the optimal operation parameters are FeCl30.38%,pH7.5,bentonite 1.9% and diamaceous earth 4.25%,which precipitated 51.13% protein.Under this treatment,the waste-water looked clean and smell clear,COD removal rate is 80.94%.