Solution A— Prepare a filtered and degassed solution (1:1000, v/v) of trifluoroacetic acid in water.

Solution B— To 350 mL of acetonitrile add 150 mL of water and 425 µL of trifluoroacetic acid.

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).

Tris buffer— Prepare a 0.1 M solution of tris(hydroxymethyl)aminomethane (Tris) in water, and adjust with hydrochloric acid to a pH of 8.0.

Dilute tris buffer— Dilute Tris buffer in water (50:50).

EDTA solution— Prepare a 0.1 M solution of ethylenediaminetetraacetic acid (EDTA) in water.

Sample diluent— To 5.76 g of guanidine hydrochloride add approximately 5 mL of Dilute tris buffer and 200 µL of EDTA solution. Dilute with Dilute tris buffer to a final volume of 10 mL.

Dithiothreitol solution— Prepare a 0.1 M solution of dithiothreitol in water.

Iodoacetamide solution— Prepare a 0.1 M solution of iodoacetamide in Tris buffer.

Trypsin solution— Prepare a solution containing 1 mg of trypsin per mL of 10 mM hydrochloric acid, and mix.

Standard solution— Add 20 µL of USP rAlbumin Human RS to 80 µL of Sample diluent, and mix well. Add 5 µL of Dithiothreitol solution, mix, and incubate at 37 for 75 minutes. Add 10 µL of Iodoacetamide solution, mix, and incubate at 37 for 75 minutes in the dark. Add 100 µL of Dilute tris buffer and 400 µL of water, and mix. Add 10 µL of Trypsin solution, mix, and incubate at 37 with shaking for 24 hours. Separate insoluble material from the supernatant by centrifugation. Dilute the supernatant in Solution A (50:50).

Test solution— Dilute the test sample in water to a final concentration of 50 mg per mL. Add 20 µL of this solution to 80 µL of Sample diluent, and mix well. Add 5 µL of Dithiothreitol solution, mix, and incubate at 37 for 75 minutes. Add 10 µL of Iodoacetamide solution, mix, and incubate at 37 for 75 minutes in the dark. Add 100 µL of Dilute tris buffer and 400 µL of water, and mix. Add 10 µL of Trypsin solution, mix, and incubate at 37 with shaking for 24 hours. Separate insoluble material from the supernatant by pulse centrifugation. Dilute the supernatant in Solution A (50:50).

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The column temperature is maintained at 35. The chromatograph is programmed as shown in Table 1. below.

Procedure— Separately inject equal volumes (100 µL) of the Standard solution and the Test solution into the chromatograph, and record the chromatograms: the chromatographic profiles of the Test solution are similar to those of the Standard solution.

Solution A— Dilute 200 µL of trifluroacetic acid in 200 mL of water.

Solution B— To 140 mL of acetonitrile add 180 µL of trifluroacetic acid and 60 mL of water.

Solution C— Dilute acetonitrile in water (50:50).

Solution D— To 5 mL of Solution C add 10 µL of formic acid.

Test solution— Dilute the test sample with water to obtain a final concentration of 10 mg per mL.

Desalted test solution— Desalt the Test solution as described below under Mass spectrometric system.

Mass spectrometric system (see Mass Spectrometry 736)— The LC/MS spectrometer is equipped with an infusion system connected to an electrospray interface. The spectrometer is operated in the positive ion mode. [Note—The infusion system flow rate can be adjusted as needed. To assist in nebulization, the infusion system can contain a sheathing gas fluid.] The liquid chromatographic system is equipped with a 280-nm detector and a 2.1-mm × 3-cm desalting cartridge.1 The chromatograph is programmed as follows. The flow rate is 0.2 mL per minute. Equilibrate the capillary in Solution C. Inject 20 µL of the Test solution, record the chromatograms, and collect the eluate (i.e., the Desalted test solution). Ensure that a single protein peak elutes.

System suitability solution— Accurately weigh about 2 mg of horse heart myoglobin. For each 2 mg weighed, add 589 µL of Water for Injection. Dilute 25 µL of this solution with 475 µL of Solution D.

System suitability— Inject 50 µL of the System suitability solution, and obtain and transform the spectrogram. A single peak with mass in the range 16,949 to 16,953 Da is found.

Procedure— Inject 50 µL of the Desalted test solution, and obtain and transform the spectrogram. The mass is within 20 Da of the theoretical mass.

Stock sample buffer2 —Dilute a mixture of 4 mL of 0.5 M Tris hydrochloride pH 8.6, 0.5 mL of 0.1% bromophenol blue, and 2.0 mL of glycerol with water to 1 L.

Diluted sample buffer— Dilute the Stock sample buffer with water (1:1).

Native stock running buffer3 — Dissolve 29 g of Tris base and 144 g of glycine in 900 mL of water. Mix well, and adjust with water to 1 L.

Running buffer— Dilute 1 volume of Native stock running buffer with 9 volumes of water.

Gel staining solution— Prepare a suitable Coomassie G-250–based solution.4

Native PAGE gel— Prepare a 14% Tris–Glycine gel.5

Test solution— Prepare a solution containing 4 mg of rAlbumin Human per mL of water. Add 500 µL of this solution to 500 µL of Stock sample buffer to obtain a solution having a concentration of 2 mg of rAlbumin Human per mL.

Calibration curve solutions— Dilute the Test solution quantitatively, and stepwise if necessary, with Diluted sample buffer to obtain standard curve solutions having known concentrations of 0.1 mg per mL, 0.02 mg per mL, 0.015 mg per mL, 0.01 mg per mL, 0.005 mg per mL, 0.002 mg per mL, and 0.001 mg per mL.

Gel loading scheme— Load 10-µL aliquots of the Calibration curve solutions onto the gel from left to right in the following order:

Running the gel— Perform electrophoresis at 125 V, 35 mA, and 5.0 W, using Running buffer. Remove the gel from the apparatus after approximately 2 hours.

Gel staining— Place the gel in 100 mL of water, and shake gently with circumgyration for about 30 minutes. Pour approximately 50 mL of Gel staining solution into a staining container. Place the gel into the staining container, and allow the stain to completely cover the gel. Place the staining container on an orbital shaker, and stain the gel for 120 minutes with gentle shaking.

Destaining— Drain the Gel staining solution, and add 100 mL of water to the container to cover the gel. Place the container on an orbital shaker, and shake at low speed for about 60 minutes. Change the water, and repeat for a total of two washes.

Gel scan procedure— Set up a gel scanner according to the manufacturer’s instructions. Place the gel in the detector, and obtain a single image of all 10 lanes of the gel.

Data analysis— Perform image analysis of Lanes 1 to 6 to generate a linear calibration curve. Determine the linear regression equation of the standards by the least-squares method, with standard concentrations, in ng, as the dependent variable (x) and the sample band intensity (optical density) as the independent variable (y). Record the linear regression equation and the correlation coefficient, r. A suitable system is one that yields a line having an r2 of not less than 0.990.

Stock sodium azide solution— Dissolve 50 g of sodium azide in sufficient water to obtain a final volume of 250 mL.

Stock buffer— Dissolve 54.2 g of dibasic sodium phosphate dihydrate, 30.0 g of monobasic sodium phosphate dihydrate, and 284.0 g of anhydrous sodium sulfate in 1600 mL of water. Add 50 mL of Stock sodium azide solution, and dilute with water to 2000 mL.

Working buffer— Prepare a filtered and degassed mixture of water and Stock buffer (10:1).

Test solution— Dilute rAlbumin Human with water to a nominal concentration of 40 mg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 7.8-mm × 30-cm column that contains 5-µm packing L59. The flow rate is 1.0 mL per minute.

Procedure— Inject 50 µL of the Sample solution, record the chromatogram, and measure the peak responses. The peak due to the polymer is located in the part of the chromatogram representing the void volume. Calculate the percentage of polymer in rAlbumin Human taken by the formula: in which rP is the peak response for the rAlbumin Human polymer; and rs is the sum of the responses of all of the rAlbumin Human related peaks: not more than 1.0% is found.

Copper sulfate solution— Dissolve 0.6 g of copper sulfate pentahydrate and 6.0 g of potassium sulfate6 in 10.0 mL of sulfuric acid low in nitrogen.

Test solution— Dilute 0.5 g of rAlbumin Human with 2.5 mL of 0.15 M sodium chloride to obtain a solution containing about 3.3 mg of protein per mL.

Blank— Dilute 0.1 g of glycine with 3.0 mL of the 0.15 M sodium chloride used to make the Test solution.

Procedure— To 3.0 mL of the Test solution and the Blank, in suitable distillation tubes, add 5 mL of Copper sulfate solution. Incubate at 420 for a minimum of 2 hours, or until the residues appear white. When the solutions are cool, transfer the residues quantitatively with a minimum quantity of water to a micro-Kjeldahl flask, and determine the residues, using Method II under Nitrogen Determination 461. Multiply the result, corrected for the Blank and for the specific gravity of the Test solution, by 6.25 to calculate the quantity of protein. It contains not less than 95% and not more than 105% of the quantity of protein stated on the label.

Diluent— Dissolve 1.0 g of cesium chloride in sufficient water to obtain a final volume of 1000 mL.

Test solution— Accurately prepare 40 mL of a solution of rAlbumin Human in Diluent having a concentration of approximately 80 µg per mL of rAlbumin Human.

Reference solutions— Prepare four solutions of sodium chloride in Diluent having concentrations of 0.5 mg per mL, 1.00 mg per mL, 1.50 mg per mL, and 2.00 mg per mL.

Procedure— Set a suitable atomic absorption spectrometer capable of analyzing in emission mode to a wavelength of 589 nm. Operate the atomic emission spectrometer in accordance with the manufacturer's instructions. Introduce a blank solution (Diluent) into the atomic generator, and adjust the instrument reading to zero. Determinations are made by comparison with the Reference solutions of known concentration. If the Test solution emission exceeds that of the Reference solution with the highest concentration, dilute the Test solution with Diluent. Introduce the most concentrated Reference solution into the instrument, and adjust the sensitivity to obtain a suitable reading. Introduce the Test solution and each Reference solution into the instrument at least three times, and record the steady reading. Rinse the apparatus with blank solution each time, and ascertain that the reading returns to its initial blank value. Plot the mean of the readings obtained for the Reference solutions against their respective sodium concentrations. From the standard curve, calculate the sodium concentration content in the Sample solution, and adjust for the specific gravity of the rAlbumin Human (see test for Protein content): the concentration of sodium is not less than 120 mM and not greater than 160 mM.NF27