Somatropin

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Somatropin is a protein hormone consisting of 191 Amino acid residues, and its structure corresponds to the major component of the growth hormone extracted from human pituitary glands. It is produced as a lyophilized powder or bulk solution by methods based on recombinant DNA technology. The presence of host-cell DNA and host-cell protein impurities in Somatropin is process specific—the limits of these impurities are determined by validated methods. Manufacturers must demonstrate a correlation between the Assay and a validated and approved growth-promotion-based bioassay. It may contain excipients.

[NOTE—One mg of anhydrous Somatropin is equivalent to 3.0 USP Somatropin Units.]

2 IDENTIFICATION

A. The retention time of the somatropin peak of the Sample solution corresponds to that of the Standard solution, as obtained in the test for Chromatographic Purity, except that a Standard solution is also chromatographed and prepared by reconstituting a vial of USP Somatropin RS with Diluent to obtain a known concentration of about 2 mg/mL.

B. PEPTIDE MAPPING

Solution A: Trifluoroacetic acid and water (1:999). Filter, and degas. Solution B: Water, trifluoroacetic acid, and acetonitrile (100:1:899)

Solution C: 0.05 M solution of tris(hydroxymethyl)aminomethane (Tris). Adjust with hydrochloric acid to a pH of 7.5. Mobile phase: See Table 1.

Table 1

Trypsin solution: 1 mg/mL of trypsin in Solution C. Store in a freezer, if necessary. Standard stock solution: 2.0 mg/mL of USP Somatropin RS in Solution C

Standard solution: To 1 mL of the Standard stock solution add 30 µL of Trypsin solution. Cap the tube, and place it in a water bath at 37° for 4h. [NOTE—If this solution is not injected immediately, store it in a freezer.]

Sample stock solution: 2.0 mg/mL of Somatropin in Solution C

Sample solution: To 1 mL of the Sample stock solution add 30 µL of Trypsin solution. Cap the tube, and place it in a water bath at 37° for 4 h. [NOTE—If this solution is not injected immediately, store it in a freezer.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.) Mode: LC

Detector: UV 214 nm

Column: 4.6-mm × 25-cm; packing L7 Column temperature: 30°

Flow rate: 1 mL/min Injection volume: 100 µL

Analysis

Samples: Standard solution and Sample solution, separately injected

[NOTE—Condition the chromatographic system by running a blank gradient program before injecting the digests.] Acceptance criteria: The chromatographic profile of the Sample solution is similar to that of the Standard solution.

Change to read:

C. SOMATROPIN BIOIDENTITY TEST 〈126〉 (CN 1-MAY-2023) : Meets the requirements

[NOTE—The bioidentity test may be performed either on the Somatropin bulk drug substance or on the finished pharmaceutical product.]

3 ASSAY

SOMATROPIN CONTENT

Buffer solution: 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 or sodium hydroxide solution to a pH of 7.0. Dilute with water to 1000 mL.

Mobile phase: Isopropyl alcohol and Buffer solution (3:97). Filter, and degas. Diluent: Buffer solution and water (1:1.5)

System suitability solution: Place 1 vial of USP Somatropin RS in an oven at 50° for 12–24 h. Remove from the oven, and dissolve the contents of the vial in Diluent to obtain a solution with a known concentration of about 1 mg/mL and a dimer content of 1%–2%.

Standard solution: Known concentration of about 1 mg/mL of USP Somatropin RS in Diluent

Sample solution: About 1 mg/mL of accurately weighed Somatropin in Diluent, or by diluting a bulk solution of Somatropin with Diluent. [NOTE—If necessary, the amount of protein in the solution can be determined with the test for Total Protein Content.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 7.8-mm × 30-cm; packing L33

Column temperature: Ambient

Flow rate: 0.6 mL/min

Injection volume: 20 µL

System suitability

Sample: System suitability solution Suitability requirements

Resolution: NMT 0.4 for the ratio of the valley height, between the dimer and the monomer, and the dimer peak height

Tailing factor: NMT 1.7 for the monomer (major) peak

Analysis

Samples: Standard solution and Sample solution, separately injected

Record the chromatograms for NLT twice the retention time of the somatropin monomer (major) peak, and measure the peak responses for the monomer.

Calculate the concentration, in mg/mL, of somatropin in the Sample solution taken:

Result = (r_U/r_S) × C_S

r_U = peak response of the monomer from the Sample solution

r_S = peak response of the monomer from the Standard solution

C_S = concentration of USP Somatropin RS in the Standard solution (mg/mL)

Acceptance criteria: NLT 910 µg of somatropin/mg on the anhydrous basis. When prepared as a bulk solution, it contains NLT 910 µg of somatropin/mg of total protein.

4 IMPURITIES

4.1 CHROMATOGRAPHIC PURITY

Diluent: 0.05 M Tris in water. Adjust with hydrochloric acid to a pH of 7.5.

Mobile phase: n-Propyl alcohol and degassed Diluent (29:71). Filter.

System suitability solution: 2.0 mg/mL of Somatropin in 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 h. [NOTE—Use within 48 h of preparation, or store the solution in a refrigerator until ready to use.]

Sample solution: 2.0 mg/mL of Somatropin in Diluent. [NOTE—Maintain the solutions between 2° and 8°, and use within 24 h. If an automatic injector is used, maintain the temperature between 2° and 8°.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.) Mode: LC

Detector: UV 220 nm

Column: 4.6-mm × 25-cm; packing L26 Column temperature: 45°

Flow rate: 0.5 mL/min Injection volume: 20 µL

System suitability

Sample: System suitability solution Suitability requirements

Resolution: NLT 1.0 between somatropin and its adjacent peak Tailing factor: 0.9–1.8 for the somatropin (major) peak

Analysis

Sample: Sample solution

Calculate the percentage of impurities in the portion of Somatropin taken:

Result = [A_U/(A_U + A_S)] × 100

A_U = sum of all the peak responses other than the somatropin (major) peak and disregarding any peak due to the solvent from the Sample solution

A_S = peak response of somatropin from the Sample solution

Acceptance criteria: NMT 6.0% of total impurities

4.2 LIMIT OF HIGH MOLECULAR WEIGHT PROTEINS

Buffer solution, Mobile phase, Diluent, System suitability solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in the Assay.

Analysis

Sample: Sample solution

Measure the areas of the main peak and of the peaks eluting before the main peak, excluding the solvent peaks. Calculate the percentage of high molecular weight proteins in the portion of Somatropin taken:

Result = [A_H/(A_H + A_M)] × 100

A_H = sum of the areas of the high molecular weight peaks

A_M = peak area of the monomer from the Sample solution

Acceptance criteria: NMT 4% of high molecular weight proteins

5 SPECIFIC TESTS

5.1 TOTAL PROTEIN CONTENT

(See Ultraviolet-Visible Spectroscopy 〈857〉.)

The method is used in the calculation of total protein in the assay of bulk solution.

Buffer solution: 0.025 M solution of monobasic potassium phosphate in water. Adjust with sodium hydroxide to a pH of 7.0.

Sample solution: Mix a weighed quantity of Somatropin bulk solution with a weighed quantity of Buffer solution to obtain a solution with an absorbance value between 0.5 and 1.0 at the wavelength of maximum absorbance at 280 nm.

Analysis: Determine the absorbance of the Sample solution using a spectrophotometric cell of path length 1 cm, at the wavelength of maximum absorbance around 280 nm and at 320 nm, using Buffer solution as the blank.

Calculate the protein content, in mg, in the portion of Somatropin taken:

Result = (A_max − A₃₂₀) × (V/0.82)

A_max = absorbance value of the Sample solution at the wavelength of maximum absorbance

A₃₂₀ = absorbance value of the Sample solution at 320 nm

V = volume of the Sample solution

MICROBIAL ENUMERATION TESTS 〈61〉 and TESTS FOR SPECIFIED MICROORGANISMS 〈62〉: The total aerobic microbial count is NMT 3 × 102 cfu/g, the test being performed on 0.2–0.3 g of powder, accurately weighed.

WATER DETERMINATION 〈921〉 , Method I, Method Ic: NMT 10%, when prepared as a lyophilized powder

BACTERIAL ENDOTOXINS TEST 〈85〉: NMT 10 USP Endotoxin Units/mg of Somatropin

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight containers, and store between −10° and −25°.

LABELING: The labeling states that the material is of recombinant DNA origin.

USP REFERENCE STANDARDS 〈11〉

USP Somatropin RS