Extraction And Purification Of Heparin Sodium

In this paper,the extraction and purification process of heparin sodium was optimized by using intestinal mucosa as raw material.The key processes include enzymatic hydrolysis,adsorption,desorption and refining.Using the titer of enzymatic hydrolysate,resin adsorption rate,product titer,product yield and so on as evaluation indexes.It can be used as the index of product evaluation in actual production and is the basis of optimizing process.The raw porcine small intestinal mucosa was enzymatically hydrolyzed by 2709 alkaline protease,papain,trypsin and nucleic acid hydrolase,respectively.The optimal three enzyme combinations were selected to form composite enzymes with the titer of the hydrolysate as the index,and the optimal ratio of composite enzymes was studied.Through the optimal proportion of complex enzymes,the relevant influencing factors involved in the enzymatic hydrolysis operation were analyzed,including the temperature,salinity,time and p H of enzymatic hydrolysis etc.Then the single factor test and orthogonal test were used to find the optimal environment for enzymatic hydrolysis.In the process of adsorbing the post-enzymatic treatment solution,multiple types of ion exchange resin were selected,and then the adsorption results were compared,so as to find out the best adsorption material.At the same time,the optimal p H value,adsorption temperature,salt concentration and other factors were analyzed.Then the static desorption and dynamic desorption conditions were explored,including the concentration of eluent,the dosage of eluent,and the eluent time.Sample loading quantity,sample loading flow rate,dynamic elution concentration and flow rate.The refining of crude heparin sodium was realized by direct precipitation of ethanol,and the salt concentration,temperature,alcohol precision,time and other conditions affecting the refining effect were analyzed,and the optimal test conditions were determined after optimization by orthogonal test.Relevant research includes the following four aspects:(1)Compound enzyme selection analysis of 2709 alkaline protease,trypsin,papain three enzymes for enzymatic hydrolysis is better.When the ratio of trypsin to 2709 basic protease was 1:2,its effect was best.The optimal enzymatic hydrolysis indexes of small intestinal mucosa were as follows: the salinity of enzymatic hydrolysis was 3.0%,the temperature of enzymatic hydrolysis was 55?,the p H9.0 of enzymatic hydrolysis,and the enzymatic hydrolysis time was 2.5h.(2)Parameters of ion-exchange resin adsorption mucosal enzymolytic solution: D208 resin was used for adsorption,the best adsorption time of heparin sodium was 8h,the best adsorption temperature was 50?,the best adsorption p H was 9.0,and the best adsorption salt concentration was 3.0%.(3)The optimal static desorption eluent concentration was 4.0mol/L;Resin: Eluent dosage: 1:4;Static elution is 3.5h.4 times resin volume was selected for dynamic adsorption of sample.2.0BV/ h flow rate sampling;Dynamic elution was performed at a flow rate of1.5BV/h of sodium chloride at 4mol/L.(4)Ethanol direct precipitation method was used to realize the refining process of crude heparin sodium.The optimal indexes were as follows: 0.3mol/L salt concentration,40 ?refining temperature,35° alcohol precision,and 40 min refining time.The titer of heparin sodium was significantly increased by direct precipitation of ethanol under optimal conditions.The heparin sodium titer was increased from 98U/mg to 119U/mg,and the increased value was 21U/mg.The yield increased from 90% to 5% and finally to 95%.