Insulin

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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To view the Notice from the Expert Committee that posted in conjunction with this accelerated revision, please click

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Change to read:

Insulin (pig) CAS RN®: 12584-58-6.

(RB 1-May-2019)

1 DEFINITION

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Insulin is a two-chain peptide hormone consisting of 51 amino acids, and its structure corresponds to native insulin produced in vivo by the beta cells of the pancreas. The A-chain is composed of 21 amino acids, and the B-chain is composed of 30 amino acids. (USP 1-May-2019) It is obtained from the pancreas of healthy (RB 1-May-2019) porcine animals, (RB 1-May-2019) used for food by humans. Its potency is NLT 26.5 USP Insulin Units/mg, calculated on the dried basis; Insulin labeled as puriffied contains NLT 27.0 USP Insulin Units/mg, calculated on the dried basis. (USP 1-May-2019)

[Note—1 USP Insulin Unit is equivalent to (RB 1-May-2019) 0.0345 mg of pure Insulin derived from pork.]

2 IDENTIFICATION

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A. The retention time of the major peak in the Sample solution corresponds to that

(RB 1-May-2019) of the Identification solution, as obtained in the Assayand no other signi cant peaks are observed. (RB 1-May-2019)

[Note—It may be necessary to inject a mixture of Sample solution and Identification solution.]

Delete the following:

B. Peptide Mapping

Sulfate buffer: 2.0 M ammonium sulfate and 0.5 M sulfuric acid (1:1)

Enzyme solution: 500 units/mL of Staphylococcus aureus V-8 protease activity in water

HEPES buffer: 0.1 M HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid). Adjust with 5 M sodium hydroxide to a pH of 7.5 before diluting with water to a final volume.

Solution A: Acetonitrile, water, and Sulfate buffer (100:700:200)

Solution B: Acetonitrile, water, and Sulfate buffer (400:400:200)

Mobile phase: See Table 1.

Table 1

Standard digest solution: 2 mg/mL of USP Insulin RS of the appropriate species in 0.01 N hydrochloric acid. Transfer 500 μL of the resulting solution to a clean vial. Add 2.0 mL of HEPES buffer and 400 μL of Enzyme solution, and incubate at 25° for 6 h. Quench the digestion by adding 2.9 mL of Sulfate buffer.

Sample digest solution: 2 mg/mL of Insulin in 0.01 N hydrochloric acid, mix to dissolve. Transfer 500 μL of the resulting solution to a clean vial. Add 2.0 mL of HEPES buffer and 400 μL of Enzyme solution, and incubate at 25° for 6 h. Quench the digestion by adding 2.9 mL of

Sulfate buffer.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 4.6-mm × 10-cm; packing L1

Column temperature: 40°

Flow rate: 1 mL/min

System suitability

Sample: Standard digest solution

Suitability requirements

Chromatogram comparability: The chromatogram of the Standard digest solution corresponds to that of the reference chromatogram provided with USP Insulin RS of the appropriate species.

Resolution: NLT 1.9 between digest fragments II and III.

[Note—Fragment I elutes at the same time in insulin derived from pork and Insulin Human; Fragment II elutes at the same time in Insulin

Human and insulin derived from beef and pork; and Fragment III elutes at the same time in insulin derived from beef and pork.]

Tailing factor: NMT 1.5

Analysis

Samples: Standard digest solution and Sample digest solution

Using the gradient program, run a blank. Separately inject equal volumes of the Standard digest solution and the Sample digest solution, and record the responses of each peak.

Acceptance criteria: The chromatographic profile of the Sample digest solution corresponds to that of the Standard digest solution. (USP 1- May-2019)

Add the following:

B. Physicochemical Analytical Procedures for Insulins 〈121.1〉, Peptide Mapping: Proceed as directed in the chapter, except for the Mobile phase and System suitability.

Mobile phase: See Table 1.

Table 1. 

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 1.9 between digest fragments II and III

[Note—Fragment I elutes at the same time in insulin derived from pork and Insulin Human; Fragment II elutes at the same time in Insulin

Human and insulin derived from beef and pork; and Fragment III elutes at the same time in insulin derived from beef and pork.]

Tailing factor: NMT 1.5 for digest fragments II and III

Chromatogram similarity: The chromatogram of the Standard solution corresponds to that of the reference chromatogram provided with

USP Insulin Pork RS.

Acceptance criteria: Meets the requirements (USP 1-May-2019)

Add the following:

C. Insulin Assays 〈121〉, Assay, Bioidentity Test: Meets the requirements (USP 1-May-2019)

3 ASSAY

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Procedure

Solution A: Dissolve 28.4 g of anhydrous sodium sulfate in 1000 mL of water. Pipet 2.7 mL of phosphoric acid into the solution, and adjust with ethanolamine to a pH of 2.3, if necessary.

Mobile phase: Acetonitrile and Solution A (26:74). [Note—The acetonitrile is warmed to a temperature of NLT 20° to avoid precipitation.]

System suitability solution: 1.5 mg/mL of Insulin in 0.01 N hydrochloric acid. Allow to stand at room temperature for NLT 3 days to obtain a solution containing NLT 5% of A-21 desamido insulin.

[Note—The Identification solution, Standard solution, and Sample solution may be stored at room temperature for up to 12 h or in a refrigerator for up to 48 h.]

Identification solution: 0.6 mg/mL of USP Insulin Pork RS (RB 1-May-2019) in 0.01 N hydrochloric acid

Standard solution: 1.5 mg/mL of USP Insulin Pork RS (RB 1-May-2019) in 0.01 N hydrochloric acid

(RB 1-May-2019)

Sample solution: 1.5 mg/mL of Insulin in 0.01 N hydrochloric acid

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 4.6-mm × 15-cm; packing L1

Column temperature: 40°

Flow rate: 1 mL/min

Injection volume: 20 μL

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 2.0 between insulin and A-21 desamido insulin, System suitability solution

Tailing factor: NMT 1.8 for the insulin peak, System suitability solution

Relative standard deviation: NMT 1.6%, Standard solution

Analysis

Samples: Identification solution, Standard solution, and Sample solution

Measure the peak responses for insulin and A-21 desamido insulin, using the chromatogram of the Identification solution to identify the insulin peaks.

(RB 1-May-2019) Calculate the potency on the undried basis, in USP Insulin Units/mg, of Insulin in the Sample solution:

Result = (Σr_U/Σr_S) × (C_S/C_U)

r_U = sum of the peak responses of insulin and A-21 desamido insulin from the Sample solution

r_S = sum of the peak responses of insulin and A-21 desamido insulin from the Standard solution

C_S = concentration of (RB 1-May-2019) USP Insulin Pork RS in the Standard solution (USP Insulin Units/mL)

C_U = concentration of Insulin in the Sample solution (mg/mL) (RB 1-May-2019)

Acceptance criteria: NLT 26.5 USP Insulin Units/mg on the dried basis; Insulin labeled as puriffied contains NLT 27.0 USP Insulin Units/mg on the dried basis.

OTHER COMPONENTS

Change to read:

Zinc Determination 〈591〉

Acceptance criteria: NMT 1.0% on the dried basis (USP 1-May-2019)

PRODUCT-RELATED SUBSTANCES AND IMPURITIES

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Product-Related Substances (USP 1-May-2019)

Solution A: Dissolve 28.4 g of anhydrous sodium sulfate in 1000 mL of water. Pipet 2.7 mL of phosphoric acid into the solution, and adjust with ethanolamine to a pH of 2.3, if necessary.

Solution B: Acetonitrile and Solution A (18:82)

Solution C: Acetonitrile and Solution A (50:50)

Mobile phase: See Table 2.

Table 2

System suitability solution: 1.5 mg/mL of Insulin in 0.01 N hydrochloric acid. Allow to stand at room temperature for NLT 3 days to obtain a solution containing NLT 5% of A-21 desamido insulin.

[Note—Standard solutions A–C may be stored at room temperature for up to 12 h and in a refrigerator for up to 48 h.]

Standard solution A: 3.75 mg/mL of USP Insulin Pork RS (RB 1-May-2019) in 0.01 N hydrochloric acid

(RB 1-May-2019)

Standard solution B: 0.375 mg/mL of

(RB 1-May-2019) USP Insulin Pork RS in 0.01 N hydrochloric acid prepared as follows. Pipet 1 mL of

Standard solution A into a 10-mL volumetric flask, dilute with 0.01 N hydrochloric acid to volume, and mix.

Standard solution C: 0.0375 mg/mL of

(RB 1-May-2019) USP Insulin Pork RS in 0.01 N hydrochloric acid prepared as follows. Pipet 1 mL of

Standard solution B into a 10-mL volumetric flask, dilute with 0.01 N hydrochloric acid to volume, and mix.

Sample solution: 3.75 mg/mL of Insulin in 0.01 N hydrochloric acid. Prepare the solution in a capped vial, cap the vial, and shake gently to dissolve. Store the solution for NMT 2 h at room temperature or for NMT 12 h in a refrigerator.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 4.6-mm × 25-cm; packing L1

Column temperature: 40°

Flow rate: 1 mL/min

Injection volume: 20 μL

System suitability

Samples: System suitability solution, Standard solution A, Standard solution B, and Standard solution C

[Note—Adjust the Mobile phase composition and the duration of the isocratic elution to obtain a retention time of about 31 min for insulin,

with the A-21 desamido insulin eluting just prior to the start of the gradient elution phase.]

Suitability requirements for the System suitability solution

Resolution: NLT 2.0 between insulin and A-21 desamido insulin

Tailing factor: NMT 1.8 for the insulin peak

Suitability requirements for the Standard solutions

Calculate the factor X :

X₁ = (r_B/r_A) × D

r_B = peak response from Standard solution B

r_A = peak response from Standard solution A

D = dilution factor, 10

Result: Between 0.91 and 1.09

Calculate the factor X :

X₂ = (r_C/r_A) × D

r_C = peak response from Standard solution C

r_A = peak response from Standard solution A

D = dilution factor, 100

Result: Between 0.7 and 1.3

Analysis

Sample: Sample solution

Calculate the percentage of insulin, A-21 desamido insulin, and other insulin-related substances (USP 1-May-2019) in the portion of

Insulin taken:

Calculate the percentage of Insulin (%I):

Result = (r_l/r_T) × 100

r_l = peak response of insulin from the Sample solution

r_T = sum of the responses of all the peaks from the Sample solution

Calculate the percentage of A-21 desamido insulin (%D):

Result = (r_D/r_T) × 100

r_D = peak response of A-21 desamido insulin from the Sample solution

r_T = sum of the responses of all the peaks from the Sample solution

Calculate the percentage of other insulin-related substances: (USP 1-May-2019)

Result = 100 − (%I + %D)

Acceptance criteria: NMT 10.0% of A-21 desamido insulin, and NMT 5.0% of other insulin-related substances (USP 1-May-2019)

(RB 1-May-2019)

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Physicochemical Analytical Procedures for Insulins 〈121.1〉, Limit of High Molecular Weight Proteins: Meets the requirements (USP 1-May-2019)

Acceptance criteria: NMT 1.0%

PROCESS-RELATED IMPURITIES

Add the following:

Proinsulin Content: NMT 10 ng/mg, determined by a validated method (USP 1-May-2019)

4 SPECIFIC TESTS

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Insulin Assays 〈121〉, Assay, Bioidentity Test: Meets the requirements (USP 1-May-2019)

Loss on Drying 〈731〉

Sample: 200 mg

Analysis: Dry the Sample at 105° for 16 h.

Acceptance criteria: NMT 10.0%

Delete the following:

Zinc Determination 〈591〉, Procedure, Dithizone Method

Sample: 10 mg

Acceptance criteria: NMT 1.0% on the dried basis (USP 1-May-2019)

Bacterial Endotoxins Test 〈85〉: NMT 10 USP Endotoxin Units/mg of insulin

Microbial Enumeration Tests 〈61〉 and Tests for Specified Microorganisms 〈62〉: The total bacterial count does not exceed 3 × 10² cfu/g, the test being performed on a portion of about 0.2 g, accurately weighed.

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers. Store, protected from light, in a freezer.

Change to read:

Labeling: Label it

(RB 1-May-2019) as pork

(RB 1-May-2019) . If the Insulin is puriffied, label it as such.

Change to read:

USP Reference Standards 〈11〉

(RB 1-May-2019)

USP Insulin Pork RS