Research On Standardization Of Reference Substances For Heparin And Low Molecular Weight Heparin

Heparin is the most effective anticoagulant drug widely used in cardiovascular disease and hemodialysis clinical treatment.Except the anticoagulant effect, Heparin has many other biological activities and clinical use.Low molecular weight heparin is a large class of antithrombotic drugs which is developed by heparin.It has more extensive clinical use and has become the first choice of treating acute venous thrombosis and acute coronary syndrome.Low molecular weight heparin shows a continuous increase accounting for antithrombotic drugs.China has the world’s most abundant heparin resources and is heparin API manufacturer and exporter.With the in-depth study of low molecular weight heparin,the domestic LMWH manufacturers are increasing.The quality of heparin and LMWH has a direct impact on the process of industrialization of these two species,which also directly related to medication safety of people.It is very important to control the quality of these two species.Heparin in the new edition of “Chinese Pharmacopoeia” has been amended.The molecular weight and molecular weight distribution measurementitem has been added,while the coagulation method of activity assay has been replaced by chromogenic substrate method. The national drug standardof LMWH have been improved according to Enoxaparin Sodium、Nadroparin Calcium and Dalteparin Sodium, which clearly defined the limits and procedure of activity assay, molecular weight and molecular weight distribution and other projects.The identification and tests of impuries were added,too. Some of the reference standards and standard substance required to these items can only rely on foreign imports.The high cost of experiments and the difficulty in quality control prove that we are in urgent need of domestic standardization.This paper focuses on preparation and standardization of the reference substances used in quality control of heparin and LMWH.To prepare reference substance of these two species through preliminary selection of raw materials and establishment of appropriate calibration method.These reference substance can regulate the operation of experiment and improve the quality of dometic heparin and LMWH,and can alse enhance the competitiveness of dometic heparin and LMWH.This paper is divided into three sections.The first section is about the molecular weight and molecular weight distribution, aiming at preparation and standardization of heparin molecular weight calibration reference standard and LMWH molecular weight calibration reference standard. The molecular and molecular weight of heparin and LMWH has a close relationship with its anticoagulant activity.The commonly used method for measuring the molecular weight is high performance gel chromatography,which needs to use molecular weight reference standard.The heparin molecular weight calibrant candidate was standardized by USP reference standard while 3 molecular weight calibrants of different LMWH depending on different production process were standardized by WHO calibration reference standard in order to ensure the homogeneity between reference standards and sample.This paper used the international molecular reference standard and the candidate molecular reference standardto measure the molecular weight of heparin and LMWH sample,the results showed no significant difference.The molecular weight candidate reference standards of heparin and LMWH can be used as national reference standards.The second section is about the preparation and standardization of chromogenic substrates used in the activity assay.The determination of anti-Ⅹa factor and anti Ⅱa factor methodwas used as activity measurement item of heparin in 2015 version“Chinese Pharmacopoeia ” and activity measurement item of LMWH in new national standard.This method needs two chromogenic substances, respectively S-2765 and S-2238.Chromogenic substratescontain three amino acids with Chromophore p NA attached to end. Based on the property and the application of these two chromogenic substances,this paper set up identification,impurity testing,content determination items to compare the candidate chromogenic substances with the imported chromogenic substance.The chromogenic substances candidates were also used in determination of anti-Ⅹa and anti-Ⅱa activity,compared with the results using imported chromogenic substrate.The results showed that the structure of chromogenic substance candidates is the same with the imported ones.The results of anti--Ⅹa and anti-Ⅱa activity assay showed no significant difference, too.These two chromogenic substances can be used as standard reagents.The third section is bout the standardization of heparanase Ⅰ、Ⅱ、Ⅲused in determination of the amount of 1,6-anhydroderivative in enoxaparin sodium。The activity of heparanase has a great effect on the result of this experiment.Based on the property and usage of heparanase,this paper developed methods to research the molecular weight and purity of the heparanase. A microscale protein concentration determination system was established to measure the protein concentration of the three different heparanase。The absorbance change of enzymatic reaction during unit time of 232 nm was measured to determine the activity of the heparanase.Results showed that the three candidate heparanase can be used as standard reagents.In summary, this paper established methods to standardize the reference standards and reagents used in heparin and LMWH quality control.This is the first time our country standardize the molecular weight calibrants of heparin and LMWH. The standardization of chromogenic substances used in anti-Ⅹa and anti-Ⅱa activity assay and heparinase used in determination of the amount of 1,6-anhydro derivative in enoxaparin sodium can improve the situation of relying on imported reagents.The reference standards standardized in this paper can help to better control the quality of these two species.This research contributes to standardization the regulatory of Heparin and LMWH,while also provides research ideas and inspiration on standardization of reference standards and reagents of other similar species.