Antithrombin III Human, chemical structure, molecular formula, Reference Standards

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Antithrombin III Human

»Antithrombin III Human is a glycoprotein,which is the major inhibitor of thrombin and other activated clotting factors,including factors IX,X,XI,and XII,and the cofactor through which heparin exerts its effect.It is obtained from human plasma of healthy donors who must,as far as can be ascertained,be free from detectable agents of infection transmissible by transfusion of blood or blood derivatives.The method of manufacturing includes steps that have been shown to remove or inactivate known agents of infection.If substances are used for inactivation of viruses during production,the subsequent purification procedure must be validated to demonstrate that the concentration of these substances is reduced to an acceptable level and any residues are such as not to compromise the safety of the preparation for patients.The antithrombin IIIconcentrate is passed through a bacteria-retentive filter,filled aseptically into its final,sterile containers,and immediately frozen.It is then freeze-dried,and the containers are closed under vacuum.No antimicrobial preservative is added at any stage of production.Antithrombin III Human complies with the requirements for Biologics á1041ñ.When reconstituted in the recommended volume of diluent,the potency is not less than 25USP Antithrombin III Units per mL.[NOTE—One USP Antithrombin III Unit is the amount of antithrombin IIIthat forms a complex with one unit of thrombin at 25

in the presence of heparin at a pHof 8.4.]

Packaging and storage— Use a Type Iglass container with an appropriate stopper and seal.Store protected from light between 2

and 8

,excursions permitted up to 25

Labeling— The labeling should state the content of antithrombin IIIin USP Antithrombin III Units.The diluent and the volume to be used to reconstitute the preparation are indicated.

USP Reference standards á11ñ— USP Albumin Human RS.USP Antithrombin III Human RS.USP Heparin Sodium RS.

Identification— It meets the requirements of the Assay.

pHá791ñ— Reconstitute with the diluent according to the manufacturer's instruction:between 6.0and 7.5.

Osmolality á785ñ— Reconstitute with the diluent according to the manufacturer's instruction:not less than 240mOsmol per kg for the solution.

Heparin content—

pH8.4Buffer— Dissolve tris(hydroxymethyl)aminomethane,edetic acid,and sodium chloride in water containing 0.1%polyethylene glycol 6000to obtain a solution having concentrations of 0.050M,0.0075M,and 0.175M,respectively.Adjust with hydrochloric acid or sodium hydroxide solution to a pHof 8.4.

Chromogenic substrate solution— Prepare a solution of chromogenic substrate for amidolytic test for factor Xain water to obtain a solution of concentration of 2.5mM.

Factor Xasolution— Dissolve an accurately weighed quantity of Factor Xain pH8.4Bufferto obtain a solution containing about 20nanokatalytic units (nkats).

Stopping solution— Prepare a 20%(v/v)solution of acetic acid in water.

Standard solution— Dissolve an accurately weighed quantity of USP Antithrombin III Human RSin pH8.4Bufferto obtain a solution containing 1.0USP Antithrombin III Unit.

Test solution— Dissolve an accurately weighed quantity of Antithrombin III Human in pH8.4Buffer to obtain a solution containing 1.0USP Antithrombin III Unit.

Procedure— Pipet 250µLeach of pH8.4Buffer,the Standard solution,and the Test solutionto suitable tubes placed in a water bath set at 37

.Add 250µLof Factor Xasolutionprewarmed at 37

to each tube,mix,and incubate for 2minutes.Add 250µLof Chromogenic substrate solutionprewarmed at 37

to each tube,mix,and incubate for 120seconds.Stop the reaction by adding 250µLStopping solution.Record the absorbance at 405nm,using pH8.4Bufferas the blank.

Calculation— Calculate the USP Heparin Unit per USP Antithrombin III Unit using the formula:

PR(AF–AT)/(AF–AR),

in which,AF,AT,and ARare the absorbance values from pH8.4Buffer,the Test solution,and the Standard solution,respectively;and PRis the heparin content of USP Antithrombin III Human RSin USP Heparin Unit per USP Antithrombin III Unit:not more than 0.1USP Heparin Unit per USP Antithrombin III Unit.

Sterility á71ñ— It meets the requirements when tested as directed for Direct Inoculation of the Culture Mediumunder Test for Sterility of the Product to be Examined.

Water,Method Iá921ñ: not more than 3.0%.

Pyrogen á151ñ— Inject per kg of the rabbit's weight 50USP Antithrombin III Units,calculated from the activity stated on the label.It meets the requirements.

General safety— It meets the requirements for biologics as set forth for Safety Tests—Biologicalsunder Biological Reactivity Tests,In Vivo á88ñ.

Molecular weight distribution—

Mobile phase— Prepare a suitable degassed and filtered solution containing 0.1Msodium phosphate,0.15Msodium chloride,and 0.05%sodium azide,having a pHof 6.5.

Vo-Marker solution— Prepare a solution of thyroglobulin in Mobile phase containing 4to 5mg per mL.

Test solution— Prepare a solution of Antithrombin III Human containing 8to 10mg per mL.

System suitability solution— Dilute USP Albumin Human RS,if necessary,with water to obtain a solution containing approximately 5%.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 7.5-×75-mm guard column and a 7.5-×300-mm analytical column,both containing packing L59,maintained at ambient temperature,and a 280-nm UVdetector.The flow rate is 0.5mLper minute maintained constant to ±1%;the tailing factor is between 0.5and 2.5;and the column efficiency is greater than 1500theoretical plates.

Procedure— Inject 10µLof the System suitability solution,and record the chromatogram.Inject 10µLeach of Vo-Marker solutionand the Test solution.Note the retention times of the major peak in the Vo-Marker solutionchromatogram.The relative peak area of the high molecular weight peak eluting at about the same retention time as the major peak in the Vo-Marker solutionchromatogram,or earlier,is not more than 13%.

Total protein content—

Trichloroacetic acid solution— Prepare a solution of trichloroacetic acid in water containing 100g of trichloroacetic acid per 100mLof the solution.

Test solution— Dissolve an accurately weighed quantity of Antithrombin III Human in 0.15Msodium chloride solution to obtain a solution containing about 7.5mg per mL.

Blank: 0.15Msolution of sodium chloride.

Procedure— To each of 2.0mLof the Test solutionand the Blank in suitable centrifuge tubes add 1.5mLof Trichloroacetic acid solution.Mix,allow to stand for at least 10minutes,centrifuge for 5minutes,and decant the supernatant.Resuspend the precipitates in 1.5mLof Trichloroacetic acid solution,centrifuge for 5minutes,decant the supernatant,and hold the tubes inverted on a filter paper to drain.Quantitatively transfer the residues with a minimum quantity of water to a micro-Kjeldahl flask,and determine the nitrogen content using Method II(see Nitrogen Determination á461ñ).Multiply the result,corrected for the Blank,by 6.25to calculate the quantity of protein.

Assay—

Albumin–pH8.4buffer— Prepare a 0.05%(w/v)solution of Albumin Humanin pH8.4Buffer.

Polybrene buffer— Prepare a 10mg per mLsolution of polybrene in Albumin–pH8.4buffer.

Heparin buffer— Dissolve an accurately weighed amount of USP Heparin Sodium RSin Albumin–pH8.4bufferto obtain a solution containing 15USP Heparin Units per mL.

Thrombin bovine solution— Reconstitute thrombin bovine,and dilute with Albumin–pH8.4bufferto obtain a solution having a concentration of 2.0Thrombin Units per mL.

Chromogenic substrate solution for factor IIa— Prepare a solution of chromogenic substrate for amidolytic test (see Reagent Specificationsunder Reagentsin the section Reagents,Indicators,and Solutions)for factor IIain water to obtain a solution having a concentration of about 5.0mM,and dilute the solution further with Polybrene bufferto 1.0mM.

Stopping solution— Prepare a 20%(v/v)solution of acetic acid in water.

Standard preparation A— Dissolve an accurately weighed quantity of USP Antithrombin III Human RSin Heparin bufferto obtain a solution containing 1.0USP Antithrombin III Unit.

Standard preparations B,C,D,and E— Dilute Standard preparation Awith Heparin buffer60-,120-,180-,and 300-fold.

Test preparation A— Dissolve an accurately weighed quantity of Antithrombin III Human in Heparin bufferto obtain a solution having about the same concentration as Standard preparation A.

Test preparations B,C,D,and E— Dilute Test preparation Awith Heparin buffer60-,120-,180-,and 300-fold.

Procedure— Pipet 400µLeach of Standard preparations B,C,D,and Eand Test preparations B,C,D,and Einto suitable tubes placed in a water bath set at 37

.Add 200µLof Thrombin bovine solution,prewarmed at 37

to each tube,mix,and incubate for 1minute.Add 200µLof Chromogenic substrate solution for factor IIaprewarmed at 37

to each tube,mix,and incubate for 60seconds.Stop the reaction by adding 200µLStopping solution.To prepare a blank,add the reagents in reverse order,starting with 200µLof Stopping solution,followed by the addition of 200µLof Chromogenic substrate solution for factor IIa,then adding 200µLof Thrombin bovine solution,and ending with 400µLof Heparin buffer.Record the absorbance at 405nm against the blank.

Calculations— For Standard preparations and Test preparations,calculate the regression of the absorbance against log concentrations,and calculate the activity of Antithrombin III Human in USP Antithrombin III Units,using a suitable statistical method for parallel-line assays.The four independent relative activity estimates are then combined to obtain the final mean,and the confidence limits are calculated.The estimated potency is not less than 80%and not greater than 120%of the potency stated on the label.The specific activity is not less than 6.0USP Antithrombin III Units per mg of total protein.The confidence interval (P=0.95)is between 90%and 110%of the estimated potency.

USP28

Auxiliary Information— Staff Liaison:Radhakrishna S Tirumalai,Scientist

Expert Committee:(BBP)Blood and Blood Products

USP28–NF23Page 171

Pharmacopeial Forum:Volume No.30(1)Page 56

Phone Number:1-301-816-8339