Exenatide Injection

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

Exenatide Injection is a sterile solution of exenatide acetate in a suitable diluent. It may contain suitable preservatives. It possesses, in each milliliter, an activity of NLT 90.0% and NMT 110.0% of the labeled amount of exenatide (C₁₈₄H₂₈₂N₅₀O₆₀S).

2 IDENTIFICATION

A. HPLC

Diluent, Buffer solution A, Solution A, Buffer solution B, Solution B, Mobile phase, Standard solution, System suitability solution, Sample solution, Chromatographic system, and System suitability: Proceed as directed in Product-Related Substances and Impurities.

Identity sample solution: 0.25 mg/mL of USP Exenatide RS in Diluent mixed with 0.25 mg/mL of Injection

Analysis

Samples: Standard solution, Sample solution, and Identity sample solution

Acceptance criteria: The retention time of the exenatide peak of the Sample solution corresponds to that of the Standard solution, as obtained in Product-Related Substances and Impurities. In addition, the major peaks of the Identity sample solution coelute.

B. The average mass by Mass Spectrometry 〈736〉 is 4186.6 ± 1.0 Da.

3 ASSAY

3.1 Procedure

Diluent: A 2-L aqueous solution containing 3.26 g of sodium acetate trihydrate, 2.98 g of L-methionine, and 2.06 mL of glacial acetic acid, pH 4.5. Pass through a membrane filter of 0.2-μm pore size.

[Note—Alternative suitable diluents may also be used.]

Buffer solution: 0.13 M sodium sulfate solution

Mobile phase: Buffer solution, acetonitrile, and trifluoroacetic acid (1500:500:2). Pass through a membrane filter of 0.45-μm pore size.

Standard solution: 0.25 mg/mL of USP Exenatide RS in Diluent

Soybean trypsin inhibitor stock solution: 1 mg/mL of soybean trypsin inhibitor in water. Prepare in duplicate.

System suitability solution: Transfer 10–50 μL of Soybean trypsin inhibitor stock solution to 1 mL of the Standard solution and mix with a vortex mixer for at least 30 s. Determine the soybean trypsin inhibitor peak level before initiating sample analysis. The soybean trypsin inhibitor peak level should be ≥2.0% but ≤5.0% relative to the total peak area. If the peak level is <2.0%, transfer additional Soybean trypsin inhibitor stock solution to the System suitability solution. If the peak level is >5.0%, discard and prepare a new System suitability solution.

Sample solution: Transfer an adequate quantity of each Injection (0.25 mg/mL) from its container directly to an HPLC vial, without contact with any intermediate container or transfer device.

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 7.8-mm × 30-cm; 5-μm packing L20

Flow rate: 0.8 mL/min

Injection volume: 20 μL

3.3 Temperatures

Autosampler: 2°–8°

Column: 25°

3.4 System suitability

Samples: Standard solution and System suitability solution

3.5 Suitability requirements

Resolution: NLT 1.3 between the exenatide peak and soybean trypsin inhibitor peak, System suitability solution

Tailing factor: NLT 0.8 and NMT 1.4, Standard solution

Relative standard deviation: NMT 2.0%, Standard solution

3.6 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of exenatide (C₁₈₄H₂₈₂N₅₀O₆₀S) in the portion of Injection taken:

Result = (r_U /r_S ) × (C_S /C_U ) × 100

r_U = peak response from the Sample solution

r_S = mean peak response from the Standard solution

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

C_{​​​​​​​U} = nominal concentration of exenatide in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–110.0%

4 PRODUCT-RELATED SUBSTANCES AND IMPURITIES

Change to read:

4.1 Procedure

Diluent: A 2-L aqueous solution containing 3.26 g of sodium acetate trihydrate, 2.98 g of L-methionine, and 2.06 mL of glacial acetic acid, pH 4.5. Pass through a membrane filter of 0.2-μm pore size.

[Note—Alternative suitable diluents may also be used.]

Buffer solution A: Transfer 27.20 g of anhydrous monobasic potassium phosphate to a 2-L flask or beaker. Add approximately 1900 mL of water and mix well until dissolved. Adjust with phosphoric acid to a pH of 3.30. Transfer the buffer solution to a 2-L volumetric flask, dilute with water to volume, and mix well.

Solution A: Transfer 1670 mL of Buffer solution A to a glass container, add 1673 mL of acetonitrile, and mix well. Pass through a membrane filter of 0.45-μm pore size.

Buffer solution B: Transfer 27.20 g of anhydrous monobasic potassium phosphate and 56.20 g of sodium perchlorate monohydrate to a 2-L flask or beaker. Add approximately 1950 mL of water, heat to 65° for 5 min, and mix well until dissolved. Adjust with phosphoric acid to a pH of 3.30. Transfer the buffer solution to a 2-L volumetric flask, dilute with water to volume, and mix well.

Solution B: Transfer 1083 mL of Buffer solution B to a glass container, add 1115 mL of acetonitrile, and mix well. Pass through a membrane filter of 0.45-μm pore size.

Mobile phase: See Table 1.

Table 1

0.3% Hydrogen peroxide: Transfer 1 mL of 3% Hydrogen peroxide to a 10-mL volumetric flask and dilute with water to volume. Stopper and mix well.

Standard solution: 0.25 mg/mL of USP Exenatide RS in Diluent

System suitability solution: Transfer 5 μL of 0.3% Hydrogen peroxide to 1 mL of Standard solution and mix well by mixing with a vortex mixer for at least 30 s. Incubate the tightly capped vial in a boiling water bath for 30 min. Cool the vial rapidly to room temperature and transfer the contents to a glass HPLC vial without any contact with an intermediate transfer device.

Sample solution: Transfer an adequate quantity of each Injection (0.25 mg/mL) from its container directly to an HPLC vial, without contact with any intermediate container or transfer device.

4.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 4.6-mm × 10-cm; 3-μm packing L52; two columns in series (ERR 1-Aug-2024)

Flow rate: 1.2 mL/min

Injection volume: 40 μL

4.3 Temperatures

Autosampler: 2°–8°

Column: 40°

4.4 System suitability

Samples: Standard solution and System suitability solution

4.5 Suitability requirements

Resolution: NLT 2.0 between the exenatide peak and the peak at an approximate relative retention time of 1.09, System suitability solution

Column efficiency: NLT 15,000 theoretical plates, Standard solution

Tailing factor: Between 0.8 and 1.5 for the exenatide peak, Standard solution

Relative standard deviation: NMT 2.0% for 5 replicate injections, Standard solution

4.6 Analysis

Samples: Standard solution and Sample solution

Record the chromatograms and measure the response for each peak. Determine the corrected total peak area by excluding system peaks and peaks below 0.10% of the total peak area.

Calculate the percentage of each impurity in the portion of Injection taken by using the corrected total peak area:

Result = (r_i /r_T ) × 100

r_i = peak response of each impurity from the Sample solution

r_T = corrected total peak area

Acceptance criteria: See Table 2.

Table 2

^a Peptide fragment peak resulting from degradation.

5 SPECIFIC TESTS

Bacterial Endotoxins Test 〈85〉: NMT 50 USP Endotoxin Units/mL

Sterility Tests 〈71〉, Test for Sterility of the Product to Be Examined, Membrane Filtration: Meets the requirements

Particulate Matter in Injections 〈788〉: Meets the requirements for small-volume injections

Subvisible Particulate Matter in Therapeutic Protein Injections 〈787〉: NMT 6000 counts/container ≥10 μm, NMT 600 counts/container ≥25 μm

pH 〈791〉: 4.2–4.8

Injections and Implanted Drug Products 〈1〉: Meets the requirements

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Store at 2°–8°. Do not freeze.

Labeling: Meets the requirements in Labeling 〈7〉.

USP Reference Standards 〈11〉

USP Exenatide RS