Exenatide

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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C₁₈₄H₂₈₂N₅₀O₆₀S 4186.57

l-Histidylglycyl-l-glutamylglycyl-l-threonyl-l-phenylalanyl-l-threonyl-l-seryl-l-aspartyl-l-leucyl-l-seryl-l-lysyl-l-glutaminyl-l-methionyl-l-glutamyl-l- glutamyl-l-glutamyl-l-alanyl-l-valyl-l-arginyl-l-leucyl-l-phenylalanyl-l-isoleucyl-l-glutamyl-l-tryptophyl-l-leucyl-l-lysyl-l-asparaginylglycylglycyl-l-prolyl-l-seryl-l-serylglycyl-l-alanyl-l-prolyl-l-prolyl-l-prolyl-l-serinamide CAS RN®: 141758-74-9.

1 DEFINITION

Exenatide is a 39 amino acid synthetic peptide agonist for glucagon-like peptide-1 (GLP-1) receptor. Exenatide contains NLT 95% and NMT 105% of exenatide (C H N O S), calculated on the anhydrous, acetic acid-free basis. Exenatide is a white to off-white powder.

[Note-Exenatide is very hygroscopic. Protect from exposure to moisture.]

2 IDENTIFICATION

A. HPLC

Solution A, Solution B, Mobile phase, System suitability solution, Standard solution, Sample solution, Chromatographic system, and

System suitability: Proceed as directed in the Assay.

Identity sample solution: Mix equal volumes of the Standard solution and the Sample solution.

Analysis

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

Examine the chromatograms of the Standard solution, Sample solution, and Identity sample solution.

Acceptance criteria: The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, and the major peak from the Identity sample solution elutes as a single peak.

B. Amino acid Analysis

For further discussion of the theory and applications, see Biotechnology-Derived Articles-Amino Acid Analysis 〈1052〉, which may be a helpful, but not mandatory, resource. [Note—Use a suitable, validated hydrolysis and separation and calculation procedure including amino acids used in the calculation (see Biotechnology-Derived Articles-Amino Acid Analysis 〈1052〉.)]

Standardize the instrument with a mixture containing equal molar per volume amounts (except for L-Cystine which is half the molar amount) of glycine and the l-form of the following amino acids: alanine, Arginine, aspartic acid, cystine, glutamic acid, histidine, isoleucine, leucine, Lysine, Methionine, norleucine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, and valine.

Hydrolysis solution: 6 N hydrochloric acid containing 4% of phenol

Sample solution: Accurately weigh out between 0.4 and 1.0 mg of Exenatide in glass ampuls. Add a minimum of 1.0 mL of Hydrolysis solution, freeze the sample ampul, and flame seal under vacuum. Hydrolyze at 110° for about 22 h. After hydrolysis, dry the test sample under vacuum to remove any residual acid. To the ampul add 2 mL of a buffer solution that is suitable for the amino acid analyzer, and pass through a filter of 0.45-μm pore size.

Procedure: Prepare a co-injection of the Standard solution and the test sample. Inject a suitable volume into the amino acid analyzer, and record and measure the responses for each amino acid peak in the Standard solution. Express the content of each amino acid in nanomoles.

Calculate the mean nanomole of the amino acids:

Result = (nmol found in the Analysis for Ala, Arg, Asx, Glx, Gly, His, Ile, Leu, Lys, Phe, Pro, Trp, Val)/30

Divide the nanomole of each amino acid by the Result to determine the amino acid ratios that must meet the Acceptance criteria.

Acceptance criteria: See Table 1.

Table 1

C. The average mass by Mass Spectrometry 〈736〉 is 4186.6 ± 1.0 mass units.

3 ASSAY

3.1 Procedure

Solution A: 10 mM ammonium hydrogen carbonate, pH 9.5, 0.0375% ammonia

Solution B: Acetonitrile and Solution A (90:10)

Mobile phase: See Table 2.

Table 2

System suitability solution: 1.0 mg/mL of USP Exenatide RS and 0.005 mg/mL each of USP [Glu13]-Exenatide RS and USP [Met(O)14]-Exenatide RS

Standard solutions: 1.0 mg/mL of USP Exenatide RS in water, prepared in duplicate. [Note—The retention time for the exenatide peak is approximately 14–20 min.]

Sample solutions: 1.0 mg/mL of Exenatide in water, prepared in duplicate

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 220 nm

Column: 4.6-mm × 15-cm; 3.5-μm packing L1

Temperatures

Column: 60°

Autosampler: 10°

Flow rate: 0.8 mL/min

Injection volume: 10 μL

3.3 System suitability

Samples: System suitability solution and Standard solutions

3.4 Suitability requirements

Mean peak area: Maximum difference between the mean peak area of the two Standard solutions is ±2%.

Resolution: NLT 1.0 between the [Glu13]-exenatide and [Met(O)14]-exenatide peaks, System suitability solution

Relative standard deviation: NMT 2.0% for the exenatide peak area and retention time from three replicate injections of the two Standard solutions

3.5 Analysis

Samples: Standard solutions and Sample solutions

Calculate the percentage of exenatide (C₁₈₄H₂₈₂N₅₀O₆₀S) in the portion of Exenatide taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of exenatide from the Sample solutions

r_S = peak response of exenatide from the Standard solutions

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

C_U = concentration of Exenatide in the Sample solutions (mg/mL), calculated on the anhydrous, acetic acid-free basis

Acceptance criteria: 95%–105% on the anhydrous, acetic acid-free basis

4 IMPURITIES

4.1 Procedure 1

Exenatide Related Substances and Impurities

[Note—Manufacturers should determine the suitability of their related substances method for their process-related and degradation impurities.

For any impurity peak above the limit for unspecified impurity peaks, identification and appropriate qualification is required.]

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

Acceptance criteria

Individual impurities: See Table 3.

Table 3

Total impurities: NMT 3.0% from Procedure 1

Reporting limit: 0.05%

4.2 Procedure 2

N-Acetyl His¹-Exenatide

[Note-Manufacturers should determine the suitability of their related substances method for their process-related and degradation impurities.

For any impurity peak above the limit for unspecified impurity peaks, identification and appropriate qualification is required.]

Solution A: 0.1 M sodium perchlorate, pH 2.7

Solution B: Acetonitrile

Mobile phase: See Table 4.

Table 4

System suitability solution: 1.0 mg/mL of USP Exenatide RS and 0.005 mg/mL of USP [N-Acetyl-His¹]-Exenatide RS

Standard solution: 1.0 mg/mL of USP Exenatide RS in water, prepared in duplicate

Sample solution: 1.0 mg/mL of Exenatide in water

4.2.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 3.0-mm × 15-cm; 3-μm packing L1

4.2.2 Temperatures

Column: 55°

Autosampler: 10°

Flow rate: 0.6 mL/min

Injection volume: 15 μL

4.2.3 System suitability

Samples: System suitability solution and Standard solution

4.2.4 Suitability requirements

Resolution: NLT 1.0 between the main exenatide peak and the [N-acetyl-His¹]-exenatide impurity peak, System suitability solution

Retention time variability: NMT ±5% of mean for the exenatide peak, Standard solution

Relative standard deviation: NMT 2.0% for the exenatide peak from three replicate injections, Standard solution

4.2.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the portion of Exenatide taken, disregarding any peak with an area less than 0.05% of the main peak:

Result = (r_i/r_T) × 100

r_i = peak area of each individual impurity from the Sample solution, other than the solvent peak and the main exenatide acetate peak

r_T = sum of the areas of all the peaks from the Sample solution, excluding that of the solvent peak

4.2.6 Acceptance criteria

Individual impurities: See Table 5.

Table 5

Total impurities: NMT 3.0% from Procedure 2

4.3 Procedure 3

Limit of d-His¹ Exenatide

System suitability solution A: 100 mg of l-amino acids and 0.5 mg of corresponding d-enantiomers are dissolved in 500 mL of water. An aliquot of 700 μL is dried in a SpeedVac and derivatized as described in the Sample solution.

Sample solution: Hydrolyze 1 mg of the sample in 350 μL of 6 N deuterium chloride (DCl) in heavy water (D O) at 110° for 8 h. After removal of excess reagent by a stream of nitrogen, the sample is esterified with 250 μL of 4 M deuterochloric acid-ethyl alcohol at 110° for 20 min. After cooling to about 40°, open the vial and evaporate the reagent with a gentle stream of nitrogen at moderate temperature. Dissolve the residue in 250 μL of trifluoroacetic anhydride in trifluoroacetic acid ethyl ester (1:2). Tightly close the vials and heat to 130° for 10 min. After cooling to room temperature, remove the excess reagent by a stream of nitrogen. Add 50 μL of isobutyl chloroformate to the sample and heat the closed vial to 110° for 10 min. After removal of the excess reagent under a nitrogen stream, dissolve the residue in 250 μL of dichloromethane.

System suitability solution B: About 100 μg of each d- and l-amino acid is derivatized as described in Sample solution.

4.3.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: GC–MS (see Mass Spectrometry 〈736〉)

Detector: Mass

Column: 20-m × 0.28-mm; 0.28-μm packing G49

4.3.2 Temperatures

Oven: 155°, 3 min isotherm, 4°/min to 190°, 5 min

Injection port: 220°

Carrier gas: Hydrogen

Flow rate: 26 mL/min

Injection volume: 1.0 μL

4.3.3 System suitability

Samples: System suitability solution A and System suitability solution B

4.3.4 Suitability requirements

Resolution: NLT 1.0 for enantiomers of the analyte and for any additional peak next to the analyte, System suitability solution B

Tailing factor: NMT 2.0 for the representative enantiomer, System suitability solution B

Relative standard deviation: NMT ±0.1% from the calculated content of d-Ala, d-Pro, d-Asp, d-Glu, d-Lys, and d-Arg, System suitability solution A

4.3.5 Analysis

Sample: Sample solution. The mass fragment of 379 Da is used for detection.

Calculate the percentage of d-His¹-exenatide impurity in the portion of Exenatide taken:

Result = [A_D /(A_D + A_L )] × 100

A_D = peak response of d-His from the Sample solution

A_L = peak response of l-His from the Sample solution

Acceptance criteria: NMT 1.0%

5 PROCESS-RELATED IMPURITIES

Procedure 4: Limit of Phosphate

[Note-Limit of phosphate should be performed if phosphate is used in manufacture.]

Solution A: 50 mM potassium hydroxide in water

Solution B: Water

Mobile phase: See Table 6.

Table 6

Diluent: 1 mM sodium hydroxide in water

Standard solution: 2 μg/mL of phosphate in Diluent

Sample solution: 2 mg/mL of Exenatide in Diluent

System suitability solution: Standard solution and Sample solution (1:1)

5.1 Chromatographic system

(See Ion Chromatography 〈1065〉.)

Mode: IC

Detector: Conductivity

Column: 2-mm × 25-cm; 10.5-μm packing L83

5.2 Temperatures

Column: 30°

Sample: 15°

Suppressor: Anion self-regenerating suppressor

Suppressant: Autosuppression

Flow rate: 0.25 mL/min

Injection volume: 25 μL

5.3 System suitability

Samples: Standard solution and System suitability solution

5.4 Suitability requirements

Tailing factor: NMT 3%, Standard solution

Relative standard deviation: NMT 10%, Standard solution

5.5 Analysis

Samples: Standard solution and Sample solution

The peak area for the phosphate ion peak in the chromatograms obtained with the Standard solution shows a peak corresponding to phosphate ion at a retention time of 15–21 min. The identity of the phosphate peak in the exenatide sample is confirmed by the System suitability solution showing one single peak. The phosphate content in exenatide is >0.1%, if the mean peak area of the Sample solution is higher than the mean peak area of the Standard solution. The phosphate content in exenatide is ≤0.1%, if the mean peak area of the

Sample solution is equal to or smaller than the mean peak area for the Standard solution.

Acceptance criteria: NMT 0.1%

Procedure 5: Trifluoroacetic Acid (TFA) in Peptides 〈503.1〉: Exenatide must contain NMT 0.25% trifluoroacetic acid.

6 OTHER COMPONENTS

Acetic Acid in Peptides 〈503〉: NMT 5.0%

7 SPECIFIC TESTS

Bacterial Endotoxins Test 〈85〉: It contains NMT 10 USP Endotoxin Units/mg of Exenatide.

Microbial Enumeration Tests 〈61〉andTests for Specified Microorganisms 〈62〉: The total aerobic microbial count is less than 200 cfu/g and the

total combined yeasts and molds count does not exceed 200 cfu/g.

Water Determination 〈921〉, Method I, Method Ic: NMT 7.0%

8 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight, light-resistant containers at −20 ± 5°.

USP Reference Standards 〈11〉

USP [N-Acetyl-His¹]-Exenatide RS

USP Exenatide RS

USP [Glu13]-Exenatide RS

USP [Met(O)14]-Exenatide RS