Solution A— Dissolve 6.9g of monobasic sodium phosphate in 1000mLof water,and adjust with phosphoric acid to a pHof 2.85.Filter and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Solution B— Use acetonitrile.

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Dialysis solution— Prepare an aqueous solution containing,in each mL,480mg of urea,44mg of tris(hydroxymethyl)aminomethane,and 0.88mg of edetic acid.Adjust with hydrochloric acid to a pHof 8.6.

Standard preparation— Dissolve an accurately weighed quantity of USP Alteplase RSin water,and dilute quantitatively with water to obtain a solution having a known concentration of about 1.0mg per mL.Dialyze about 2.0mLof this solution into the Dialysis solutionat room temperature for not less than 12hours.Measure the volume of the solution,and transfer it to a clean test tube.For each mLof solution in the tube,add 10µLof 1Mdithiothreitol.Incubate at room temperature for 4hours,then add 25µLof 1Miodoacetic acid per mLof the solution,and incubate in the dark for 30minutes.Quench the reaction by the addition of 50µLof 1Mdithiothreitol per mLof the solution.Dialyze the solution against 0.1Mammonium bicarbonate for 24hours,replacing the 0.1Mammonium bicarbonate twice during the dialysis period.To 2.0mLof the dialyzed solution,add 20µg of trypsin,and incubate for 6to 8hours at room temperature.Again add 20µg of trypsin,and incubate for 16to 18hours for a total of 24hours of incubation of the trypsin-treated solution.[NOTE—Store this Standard preparationin a freezer.]

Test preparation— Using an accurately weighed quantity of Alteplase,proceed as directed for Standard preparation.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm ×10-cm column that contains packing L1.The flow rate is about 1mLper minute.The system is programmed to provide a Mobile phaseconsisting of variable mixtures of Solution Aand Solution B.The system is equilibrated with 100%Solution A.After injection of the solution under test,the proportion of Solution Bis increased linearly from 0%to 30%at a rate of 0.33%per minute.The proportion of Solution Bis then increased linearly at a rate of 1.0%per minute until the proportion of Solution Bis 60%,and is held at that composition for 10minutes.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between peaks 6and 7as defined by the USP Alteplase RSData Sheet is not less than 1.5,and the times of their baseline widths are not more than 0.5minutes.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparation,the Test preparation,and a mixture of the Standard preparationand the Test preparation(1:1)into the chromatograph,record the chromatograms,and measure the responses for not less than 20major peaks as defined in the USP Alteplase RSData Sheet:the retention times of corresponding peaks from the Standard preparationand the Test preparationdo not differ by more than 0.4minutes,and the peak area ratios relative to peak 19(as shown on the USP Alteplase RSData Sheet)do not differ by more than 20%.No additional significant peaks or shoulders are found,a significant peak or shoulder being defined as one having a peak area response of not less than 5%of peak 19.

SDSbuffer— Prepare a solution in sodium dodecyl sulfate solution (8in 100)containing,in each mL,400mg of glycerol,5.52mg of tris(hydroxymethyl)aminomethane hydrochloride,3.28mg of tris(hydroxymethyl)aminomethane,0.20mg of bromophenol blue,and 0.20mg of xylene cyanole FF.

Diluted SDSbuffer— Dilute 1volume of SDSbufferwith 4volumes of water.

Ammoniacal silver nitrate solution— Transfer 105mLof sodium hydroxide solution (0.36in 100)and 7.0mLof ammonium hydroxide to a 500-mLvolumetric flask,and add slowly,with stirring,20.0mLof silver nitrate solution (20in 100).Dilute with water to volume,and mix.[NOTE—Prepare this solution immediately before use and protect it from light.This amount of solution is sufficient for two slab gels.]

Citric acid-formaldehyde solution— To 500mLof water add 25mg of citric acid,0.25mLof formaldehyde,and 0.025mLof methanol,omitting the methanol if the formaldehyde is preserved with methanol.[NOTE—Prepare this solution fresh at the time of use.This amount of solution is sufficient for two slab gels.]

Running buffer— Prepare a buffer solution in sodium dodecyl sulfate (1in 1000)containing 3.03mg of tris(hydroxymethyl)aminomethane and 14.26mg of glycine per mL.

Carboxymethylation buffer— Prepare an aqueous solution containing,in each mL,480mg of urea,44mg of tris(hydroxymethyl)aminomethane,and 1.2mg of edetic acid.Adjust with hydrochloric acid if necessary to a pHof 8.6.

Gel— Prepare a 10%T(total acrylamide)-0.25%C(cross-linked bisacrylamide)resolving gel containing 0.1%sodium dodecyl sulfate,0.375M(tris(hydroxymethyl)aminomethane hydrochloride,and 0.05Mtris(hydroxymethyl)aminomethane.

0.2M Arginine solution— Prepare a solution of arginine in water containing 34.8mg per mL.Adjust with phosphoric acid to a pHof 7.3.

Stock standard solution— Dissolve an accurately weighed quantity of USP Alteplase RSin water,and dilute quantitatively with water to obtain a solution having a known concentration of about 1mg per mL.

Standard solution— Dilute an accurately measured volume of Stock standard solutionwith 0.02M Arginine solutionto obtain a solution having a concentration of 0.25mg per mL.Heat 0.5mLof this solution with 116µLof SDSbufferand 10µLof 1Mdithiothreitol at 80for 2minutes.

Carboxymethylated standard solution— Dilute 1.0mLof Stock standard solutionwith 1mLof Carboxymethylation buffer,and adjust with 1Msodium hydroxide to a pHof 8.5.Add 20µLof 1Mdithiothreitol,and incubate at 37for 60minutes.Add 100µLof 1Miodoacetic acid,and incubate in the dark for 20minutes.Desalt by passing the solution through a chromatographic column containing fine gel chromatographic packing equilibrated with a buffer solution containing,in each mL,20mg of sodium dodecyl sulfate,100mg of glycerol,1.42mg of tris(hydroxymethyl)aminomethane hydrochloride,and 0.85mg of tris(hydroxymethyl)aminomethane.Collect the protein fraction of the preparation by elution with the same buffer,and add 20µLof 1Mdithiothreitol.Adjust the protein concentration to about 0.2mg per mLwith a buffer solution containing,in each mL,20mg of sodium dodecyl sulfate,100mg of glycerol,1.42mg of tris(hydroxymethyl)aminomethane hydrochloride,0.85mg of tris(hydroxymethyl)aminomethane,1.06mg of dithiothreitol,0.05mg of bromophenol blue,and 0.05mg of xylene cyanole FF.

Stock test solution,Test solution,and Carboxymethylated test solution— Using an accurately weighed quantity of Alteplase,proceed as directed for Stock standard solution,Standard solution,and Carboxymethylated standard solution,respectively.

Molecular weight standard preparation— Use a commercially available preparation of low molecular weight protein standards (10,000to 100,000Da)at about 2mg per mL.Mix 990µLof Diluted SDSbufferand 10µLof the molecular weight standard mixture.

Control solutions— Prepare a control solution of bovine serum albumin containing 10µg per mL.For a 10ng per 25µLload,mix 600µLof Diluted SDSbufferand 25µLof the control solution,and heat at 90for 2minutes.For a 2.5ng per 25µLload,mix 594µLof Diluted SDSbufferand 6µLof the control solution,and heat at 90for 2minutes.

Blank— Mix 500µLof water,126µLof SDSbuffer,and 10µLof 1Mdithiothreitol.

Procedure— Separately apply equal volumes (about 25µL)of the Test solution,Standard solution,Carboxymethylated test solution,and Carboxymethylated standard solutionat the 5µg load;apply equal volumes (about 38µL)of the Standard solutionand the Carboxymethylated standard solutionat the 7.5µg load;and apply the Control solutionsat the 10ng and 2.5ng load onto separate lanes of the gel.Apply about 25µLof the Molecular weight standard preparationto each side of the gel,and apply about 25µLof the Blankonto a separate lane.Apply the Test solutionand the Standard solutionon one half and the Carboxymethylated test solutionand Carboxymethylated standard solutionon the other half.Perform the electrophoresis using a constant current of 1.3to 1.5mAmp per cm of gel length and the Running buffer.Remove the gel from the apparatus 10to 20minutes after the tracking dye starts to move.Place the gel in 250mLof a solution of 20%alcohol and 6%glacial acetic acid for not less than 1hour,and change the solution every 20minutes,leaving the gel to soak overnight following the last change.Perform silver staining of the gel by placing the gel in 250mLof a solution (1in 10)in a shallow dish,and shake for about 30minutes.Replace the glutaraldehyde solution with distilled water,allow gel to soak for about 20minutes,and then change the water.Repeat for a total of three washings.Transfer the gel to a dish,and cover with 250mLof Ammoniacal silver nitrate solution.Place the dish on a shaker for about 15minutes.Rinse 4times with 250mLof water,rocking the dish for 1minute between rinses.Continue rocking to prevent the gel from sticking and to facilitate washing.Transfer the gel to a clear dish containing 250mLof Citric acid-formaldehyde solution,and rock the dish.Protein bands become visible.When the gel is visibly stained,wash immediately with water,and rinse it repeatedly with water to remove the Citric acid-formaldehyde solution.Rinse the gel for not less than 1hour,and dry.Soak cellophane membranes in glycerol solution (2in 100).Roll a membrane onto a rigid sheet of plastic.Roll the gel onto the membrane,and cover with another membrane.Lay a frame on the edges of the membranes,and clamp it to the rigid plastic sheet.Dismantle the dryer,and cut off excess cellophane when dry (about 24hours).Visually examine the gel under light.The Test solutionexhibits 3major bands in the region between 66,000Da and 31,000Da,corresponding to the major bands from the Standard solution.The Carboxymethylated test solutionexhibits 6major bands in the region between 92,500Da and 45,000Da,corresponding to the major bands from the Carboxymethylated standard solution.

System suitability— The 2.5ng and 10ng controls must be visible.The nonreduced controls solutions migrate with an apparent molecular weight of slightly less than 66,000Da,as compared with the molecular weight standards.

Mobile phase— Dissolve 27.6g of monobasic sodium phosphate in 1000mLof sodium dodecyl sulfate solution (1in 1000),and adjust with sodium hydroxide to a pHof 6.8.Filter and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

0.02M Dithiothreitol solution— Prepare a solution of dithiothreitol in Mobile phasecontaining 3.12mg per mL.

Standard solution— Dissolve an accurately weighed quantity of USP Alteplase RSin water,and dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 1mg per mL.Pipet 1mLof this solution into a glass tube,add 3mLof 0.02M Dithiothreitol solution,cap the tube,and invert to mix.Heat for 3to 5minutes at about 80.

Test solution— Using an accurately weighed quantity of Alteplase,proceed as directed for Standard solution.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 214-nm detector and a 7.5-mm ×60-cm column that contains packing L25.The flow rate is about 0.5mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the resolution,R,between the single-chain and two-chain alteplase peaks is not less than 1.1.

Procedure— Separately inject equal volumes (about 50µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.[NOTE—The major peaks are from single-chain and two-chain alteplase and from higher and lower molecular weight species.]No peaks or shoulders in the chromatogram of the Test solutionthat are not present in the chromatogram of the Standard solutionare found.Calculate the percentage of single-chain alteplase in the portion of Alteplase taken by the formula: in which rais the peak response for single-chain alteplase,and rSis the sum of the responses of all of the alteplase peaks:not less than 60%is found.

0.2M Arginine solution— Prepare a solution of arginine in water containing 34.8mg per mL.Adjust with phosphoric acid to a pHof 7.3.

Test solution— Dissolve an accurately weighed quantity of Alteplase in water to obtain a solution containing about 1mg per mL.Dilute an accurately measured volume of this solution with a volume of 0.2M Arginine solutionto obtain a solution having an absorbance value of 0.5to 1.0at the wavelength of maximum absorbance at about 280nm.Determine the dilution volume,V.

Procedure— Obtain an absorption spectrum of the Test solutionin a 1-cm cell from 240nm to 500nm,and determine the absorbance at 320nm and at the wavelength of maximum absorbance at about 280nm,using 0.2M Arginine solutionas the blank.Calculate the protein content in the portion of Alteplase taken by the formula: in which Vis the volume of 0.2M Arginine solutionrequired to prepare the Test solution,Amaxis the absorbance value at the wavelength of maximum absorbance,and A320is the absorbance of the Test solutionat 320nm.

Buffer— Prepare an aqueous solution containing,in each mL,1.38mg of monobasic sodium phosphate,7.10mg of anhydrous dibasic sodium phosphate,0.20mg of sodium azide,and 0.10mg of polysorbate 80.

Human thrombin solution— Prepare a solution of human thrombin in Buffercontaining 33U.S.Units in terms of the U.S.Standard Thrombin per mL.

Human fibrinogen solution— Prepare a solution of human fibrinogen in Buffercontaining 2mg per mL.

Human plasminogen solution— Prepare a solution of human plasminogen in Buffercontaining 1mg per mL.

Standard preparations— Dissolve an accurately weighed quantity of USP Alteplase RSin water,and dilute quantitatively with water to obtain a solution having a known concentration of about 1.0mg (580,000USP Alteplase Units)per mL.Dilute accurately measured volumes of this solution quantitatively and stepwise with water to obtain a series of five Standard preparationshaving known concentrations ranging from 145to 9.3USP Alteplase Units per mL.

Assay preparations— Dissolve an accurately weighed quantity of Alteplase in water,and dilute with water to obtain a solution having a concentration of about 1mg per mL.Dilute an accurately measured volume of this solution quantitatively and stepwise with Bufferto obtain a series of dilutions of about 1:20,000,1:10,000,and 1:5,000.

Procedure— To a set of labeled glass test tubes,add 0.5mLof Human thrombin solution.To separate test tubes add 0.5mLof each Standardand Assay preparation,mix,and store on ice.To a second set of labeled glass tubes,add 20µLof Human plasminogen solutionand 1mLof Human fibrinogen solution,mix,and store on ice.Beginning with the Standard/thrombin mixturecontaining the lowest number of USP Units per mL,record the time,and separately add 200µLof each of the thrombin mixtures to the test tubes containing the plasminogen-fibrinogen mixture.Using a vortex mixer,intermittently mix the contents of each tube for a total of 15seconds,and carefully place into a rack in a 37circulating water bath.Avisually turbid clot forms within 30seconds,followed by the formation of bubbles within the clot.Record the clot lysis time,tcl,from the first addition of the Alteplase solution to the last bubble to rise to the surface.Using a least squares fit,determine the equation of the line using the log values of the standard concentration,in USP Alteplase Units per mL,versus the log values of their clot lysis times in seconds taken by the formula: in which tis the time,in seconds,to bubble release;USis the activity,in USP Alteplase Units per mL,of the Standard preparation;m is the slope of the line;and bis the y-intercept of the line.The correlation coefficient is not less than -0.9900.From the line equation and using the log of the clot lysis time for the Assay preparation,calculate the log of the activity,UA,by the equation: Calculate the alteplase activity,in USP Alteplase Units per mL,taken by the formula: in which Dis the dilution factor for the Assay preparation.Calculate the specific activity in the portion of Alteplase taken by the formula: in which Pis the concentration of protein obtained in the test for Protein content.