Solution A— Dissolve 6.9 g of monobasic sodium phosphate in 1000 mL of water, and adjust with phosphoric acid to a pH of 2.85. Filter and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Solution B— Use acetonitrile.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

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

Chromatographic system (see Chromatography 621)— 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 1 mL per minute. The system is programmed to provide a Mobile phase consisting of variable mixtures of Solution A and Solution B. The system is equilibrated with 100% Solution A. After injection of the solution under test, the proportion of Solution B is increased linearly from 0% to 30% at a rate of 0.33% per minute. The proportion of Solution B is then increased linearly at a rate of 1.0% per minute until the proportion of Solution B is 60%, and is held at that composition for 10 minutes. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between peaks 6 and 7 as defined by the USP Alteplase RS Data Sheet is not less than 1.5, and the times of their baseline widths are not more than 0.5 minutes.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard preparation, the Test preparation, and a mixture of the Standard preparation and the Test preparation (1:1) into the chromatograph, record the chromatograms, and measure the responses for not less than 20 major peaks as defined in the USP Alteplase RS Data Sheet: the retention times of corresponding peaks from the Standard preparation and the Test preparation do not differ by more than 0.4 minutes, and the peak area ratios relative to peak 19 (as shown on the USP Alteplase RS Data 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.

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

Diluted SDS buffer— Dilute 1 volume of SDS buffer with 4 volumes of water.

Ammoniacal silver nitrate solution— Transfer 105 mL of sodium hydroxide solution (0.36 in 100) and 7.0 mL of ammonium hydroxide to a 500-mL volumetric flask, and add slowly, with stirring, 20.0 mL of silver nitrate solution (20 in 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 500 mL of water add 25 mg of citric acid, 0.25 mL of formaldehyde, and 0.025 mL of 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 (1 in 1000) containing 3.03 mg of tris(hydroxymethyl)aminomethane and 14.26 mg of glycine per mL.

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

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

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

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

Standard solution— Dilute an accurately measured volume of Stock standard solution with 0.02 M Arginine solution to obtain a solution having a concentration of 0.25 mg per mL. Heat 0.5 mL of this solution with 116 µL of SDS buffer and 10 µL of 1 M dithiothreitol at 80 for 2 minutes.

Carboxymethylated standard solution— Dilute 1.0 mL of Stock standard solution with 1 mL of Carboxymethylation buffer, and adjust with 1 M sodium hydroxide to a pH of 8.5. Add 20 µL of 1 M dithiothreitol, and incubate at 37 for 60 minutes. Add 100 µL of 1 M iodoacetic acid, and incubate in the dark for 20 minutes. Desalt by passing the solution through a chromatographic column containing fine gel chromatographic packing equilibrated with a buffer solution containing, in each mL, 20 mg of sodium dodecyl sulfate, 100 mg of glycerol, 1.42 mg of tris(hydroxymethyl)aminomethane hydrochloride, and 0.85 mg of tris(hydroxymethyl)aminomethane. Collect the protein fraction of the preparation by elution with the same buffer, and add 20 µL of 1 M dithiothreitol. Adjust the protein concentration to about 0.2 mg per mL with a buffer solution containing, in each mL, 20 mg of sodium dodecyl sulfate, 100 mg of glycerol, 1.42 mg of tris(hydroxymethyl)aminomethane hydrochloride, 0.85 mg of tris(hydroxymethyl)aminomethane, 1.06 mg of dithiothreitol, 0.05 mg of bromophenol blue, and 0.05 mg 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 (10,000 to 100,000 Da) at about 2 mg per mL. Mix 990 µL of Diluted SDS buffer and 10 µL of the molecular weight standard mixture.

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

Blank— Mix 500 µL of water, 126 µL of SDS buffer, and 10 µL of 1 M dithiothreitol.

Procedure— Separately apply equal volumes (about 25 µL) of the Test solution, Standard solution, Carboxymethylated test solution, and Carboxymethylated standard solution at the 5 µg load; apply equal volumes (about 38 µL) of the Standard solution and the Carboxymethylated standard solution at the 7.5 µg load; and apply the Control solutions at the 10 ng and 2.5 ng load onto separate lanes of the gel. Apply about 25 µL of the Molecular weight standard preparation to each side of the gel, and apply about 25 µL of the Blank onto a separate lane. Apply the Test solution and the Standard solution on one half and the Carboxymethylated test solution and Carboxymethylated standard solution on the other half. Perform the electrophoresis using a constant current of 1.3 to 1.5 mAmp per cm of gel length and the Running buffer. Remove the gel from the apparatus 10 to 20 minutes after the tracking dye starts to move. Place the gel in 250 mL of a solution of 20% alcohol and 6% glacial acetic acid for not less than 1 hour, and change the solution every 20 minutes, leaving the gel to soak overnight following the last change. Perform silver staining of the gel by placing the gel in 250 mL of a solution (1 in 10) in a shallow dish, and shake for about 30 minutes. Replace the glutaraldehyde solution with distilled water, allow gel to soak for about 20 minutes, and then change the water. Repeat for a total of three washings. Transfer the gel to a dish, and cover with 250 mL of Ammoniacal silver nitrate solution. Place the dish on a shaker for about 15 minutes. Rinse 4 times with 250 mL of water, rocking the dish for 1 minute between rinses. Continue rocking to prevent the gel from sticking and to facilitate washing. Transfer the gel to a clear dish containing 250 mL of 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 1 hour, and dry. Soak cellophane membranes in glycerol solution (2 in 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 24 hours). Visually examine the gel under light. The Test solution exhibits 3 major bands in the region between 66,000 Da and 31,000 Da, corresponding to the major bands from the Standard solution. The Carboxymethylated test solution exhibits 6 major bands in the region between 92,500 Da and 45,000 Da, corresponding to the major bands from the Carboxymethylated standard solution.

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

Mobile phase— Dissolve 27.6 g of monobasic sodium phosphate in 1000 mL of sodium dodecyl sulfate solution (1 in 1000), and adjust with sodium hydroxide to a pH of 6.8. Filter and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

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

Chromatographic system (see Chromatography 621)— 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 0.5 mL per 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 50 µL) of the Standard solution and the Test solution into 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 solution that are not present in the chromatogram of the Standard solution are found. Calculate the percentage of single-chain alteplase in the portion of Alteplase taken by the formula: in which ra is the peak response for single-chain alteplase, and rS is the sum of the responses of all of the alteplase peaks: not less than 60% is found.

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

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

Procedure— Obtain an absorption spectrum of the Test solution in a 1-cm cell from 240 nm to 500 nm, and determine the absorbance at 320 nm and at the wavelength of maximum absorbance at about 280 nm, using 0.2 M Arginine solution as the blank. Calculate the protein content in the portion of Alteplase taken by the formula: in which V is the volume of 0.2 M Arginine solution required to prepare the Test solution, Amax is the absorbance value at the wavelength of maximum absorbance, and A320 is the absorbance of the Test solution at 320 nm.

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

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

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

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

Standard preparations— Dissolve an accurately weighed quantity of USP Alteplase RS in water, and dilute quantitatively with water to obtain a solution having a known concentration of about 1.0 mg (580,000 USP Alteplase Units) per mL. Dilute accurately measured volumes of this solution quantitatively and stepwise with water to obtain a series of five Standard preparations having known concentrations ranging from 145 to 9.3 USP 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 1 mg per mL. Dilute an accurately measured volume of this solution quantitatively and stepwise with Buffer to 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 0.5 mL of Human thrombin solution. To separate test tubes add 0.5 mL of each Standard and Assay preparation, mix, and store on ice. To a second set of labeled glass tubes, add 20 µL of Human plasminogen solution and 1 mL of Human fibrinogen solution, mix, and store on ice. Beginning with the Standard/thrombin mixture containing the lowest number of USP Units per mL, record the time, and separately add 200 µL of 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 15 seconds, and carefully place into a rack in a 37 circulating water bath. A visually turbid clot forms within 30 seconds, 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 t is the time, in seconds, to bubble release; US is the activity, in USP Alteplase Units per mL, of the Standard preparation; m is the slope of the line; and b is 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 D is the dilution factor for the Assay preparation. Calculate the specific activity in the portion of Alteplase taken by the formula: in which P is the concentration of protein obtained in the test for Protein content.