A: Proceed as directed in the test for Chromatographic purity, except to prepare a Standard solution by reconstituting a vial of USP Somatropin RS with the Diluent to obtain a solution having a known concentration of about 2.0 mg per mL. Chromatograph the Standard solution and the Test solution as directed for Procedure: the retention time of the somatropin peak in the chromatogram of the Test solution corresponds to that in the chromatogram of the Standard solution.

B: Peptide Mapping (see Biotechnology-Derived Articles—Tests 1047)—

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

Tris buffer— Prepare a 0.05 M solution of tris(hydroxymethyl)aminomethane (Tris), and adjust with hydrochloric acid to a pH of 7.5.

Trypsin solution— Prepare a solution containing 1 mg of trypsin per mL of Tris buffer, and mix. Store in a freezer, if necessary.

Standard solution— Prepare a solution containing 2.0 mg of USP Somatropin RS per mL of the Tris buffer, and mix. Add 1 mL of this solution to a suitable tube, and add 30 µL of Trypsin solution. Cap the tube, and place it in a water bath at 37 for 4 hours. [note—If this solution is not injected immediately, store it in a freezer.]

Test solution— Prepare a solution containing 2.0 mg of Somatropin per mL of Tris buffer, and mix. Add 1 mL of this solution to a suitable tube, and add 30 µL of Trypsin solution. Cap the tube, and place it in a water bath at 37 for 4 hours. [note—If this solution is not injected immediately, store it in a freezer.]

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 packing L7. The flow rate is 1 mL per minute, and the column temperature is maintained at 30. The chromatograph is programmed as follows.

Procedure— [note—Condition the chromatographic system by running a blank gradient program prior to injecting the digests.] Separately inject equal volumes (about 100 µL) of the Standard solution and the Test solution, and record the chromatograms: the chromatographic profile of the Test solution is similar to that of the Standard solution.

Buffer solution— Prepare a solution of 0.1 M ammonium bicarbonate, and adjust with sodium hydroxide to a pH of 8.0.

Standard solutions— Reconstitute the USP Somatropin RS, and dissolve in and dilute quantitatively with Buffer solution to obtain solutions having known concentrations between 10 and 100 µg per mL.

Test solutions— Prepare a solution of Somatropin, and dissolve in and dilute quantitatively with Buffer solution to obtain solutions having concentrations similar to those of the Standard solutions. [note—Do not agitate while mixing; swirl gently.]

Control solution— Use the Buffer solution.

Test animals— Select an appropriate number of only female or only male Sprague Dawley rats hypophysectomized at 25 to 30 days of age. After hypophysectomization, feed the rats on rat chow and 5% dextrose water for at least 72 hours. After 72 hours, feed the rats on rat chow and filtered and deionized water adjusted with 1 N hydrochloric acid to a pH of 3.0 ± 0.25. Weigh the rats when they are 37 to 44 days old, and retain only healthy rats. Reweigh the remaining rats 7 days later, and use only those rats that are in good health and have not gained or lost more than 10% of their body weight in the previous 7-day period.

Procedure— Randomly divide the rats into control, standard, and test groups, each group containing approximately 10 rats. Each day for 10 days inject subcutaneously 0.1 mL of the Control solution, Standard solutions, and Test solutions to the control, standard, and test groups, respectively. Record the body weight of each animal at the start of the test and at approximately 18 hours following the 10th injection. Determine the change in body weight for each rat during the 10-day period, and compute the potency of the Test solution relative to that of the Standard solution using appropriate statistical analysis. Calculate the mean potency in USP Somatropin Units per mg: not less than 2 USP Somatropin Units per mg is found. Using appropriate statistical methods, calculate the width, L, of a 95% confidence interval for the estimated logarithm of the relative potency: L is not more than 0.40, which corresponds to confidence limits between 63% and 158% of the calculated potency. If L is more than 0.40, repeat the test until the results from two or more tests, combined by appropriate statistical methods, meet this criterion.

Diluent— Prepare a solution of 0.05 M Tris in water, and adjust with hydrochloric acid to a pH of 7.5.

Mobile phase— Degas the Diluent, mix with n-propyl alcohol (71:29, v/v), and filter. Make adjustments if necessary (see System Suitability under Chromatography 621).

Resolution solution— Prepare a solution of 2.0 mg of Somatropin per mL of the Diluent, pass through a filter to sterilize or add sodium azide to a final concentration of 0.01%, and allow to stand at room temperature for 24 hours. [note—Use within 48 hours after preparation, or store the solution in a refrigerator until ready to use.]

Test solution— Prepare a solution of 2.0 mg of Somatropin per mL of the Diluent immediately before use. [note—Maintain the solutions between 2 and 8, and use within 24 hours. If an automatic injector is used, maintain the temperature between 2 and 8.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains packing L26 and is maintained at 45. The flow rate is about 0.5 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between somatropin and its adjacent peak is not less than 1.0; and the tailing factor of the somatropin peak (major peak) is between 0.9 and 1.8.

Procedure— Inject about 20 µL of the Test solution, record the chromatograms, and measure the peak responses. Calculate the percentage of impurities in the portion of Somatropin taken by the formula: in which AI is the sum of the responses of all the peaks other than the somatropin peak (major peak) and disregarding any peak due to the solvent; and AS is the response of the somatropin peak: not more than 6.0% of total impurities is found.

Phosphate buffer, Mobile phase, Diluent, Resolution solution, and Chromatographic system— Proceed as directed in the Assay.

Test solution— Prepare as directed for the Assay preparation.

Procedure— Inject about 20 µL of the Test solution, record the chromatogram, and measure the areas of the main peak and of the peaks eluting prior to the main peak, excluding the solvent peaks. Calculate the percentage of high molecular weight proteins in the portion of Somatropin taken by the formula: in which AH is the sum of the areas of the high molecular weight peaks, and AM is the area of the monomer peak in the chromatogram of the Test solution: not more than 4% of high molecular weight proteins is found.

Phosphate buffer— Prepare a 0.025 M solution of monobasic potassium phosphate in water, and adjust with sodium hydroxide to a pH of 7.0.

Test solution— Dissolve an accurately weighed quantity of Somatropin in Phosphate buffer to obtain a solution having an absorbance value between 0.5 and 1.0 at the wavelength of maximum absorbance at about 280 nm.

Procedure— Determine the absorbance of the Test solution using a spectrophotometric cell of path length 1-cm, at the wavelength of maximum absorbance at around 280 nm and at 320 nm, using Phosphate buffer as the blank. Calculate the protein content, in mg, in the portion of Somatropin taken by the formula: in which V is the volume of the Test solution; and Amax and A320 are the absorbance values of the Test solution at the wavelength of maximum absorbance and at 320 nm, respectively.

Phosphate buffer— Dissolve 5.18 g of dibasic sodium phosphate and 3.65 g of monobasic sodium phosphate in 950 mL of water, adjust with phosphoric acid to a pH of 7.0, and dilute with water to 1000 mL.

Mobile phase— Prepare a filtered and degassed mixture of the Phosphate buffer and isopropyl alcohol (97:3, v/v). Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluent— Prepare a mixture of water and Phosphate buffer (1.5:1).

Resolution solution— Place 1 vial of USP Somatropin RS in an oven at 50 for 12 to 24 hours. Remove from the oven, and dissolve the contents of the vial in Diluent to obtain a solution having a known concentration of about 1 mg per mL with the content of the dimer between 1% and 2%.

Standard preparation— Reconstitute a vial of USP Somatropin RS with the Diluent to obtain a solution having a known concentration of about 1.0 mg per mL.

Assay preparation— Dissolve an accurately weighed quantity of Somatropin in Diluent, or dilute a bulk solution of Somatropin with Diluent, to obtain a solution having a concentration of about 1 mg per mL. [note—If necessary, the amount of protein in solution can be determined by the test for Total protein.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 214-nm detector and a 7.8-mm × 30-cm column that contains packing L33 and is maintained at ambient temperature. The flow rate is 0.6 mL per minute. Chromatograph the Resolution solution as directed for Procedure: the resolution, R, (determined as the ratio of the valley height, between the dimer and the monomer, and the dimer peak height) is not more than 0.4; and the tailing factor of the monomer peak (major peak) is not more than 1.7.

Procedure— Separately inject equal volumes (about 20 µL) of the the Standard preparation and the Assay preparation, record the chromatograms for not less than twice the retention time of the somatropin monomer peak (major peak), and measure the peak responses for the monomer. Calculate the concentration, in mg per mL, of somatropin in the Assay preparation by the formula: in which CS is the concentration, in mg per mL, of USP Somatropin RS in the Standard preparation; and rU and rS are the peak responses of the monomer in the Assay preparation and the Standard preparation, respectively.