Insulin Glargine 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

Insulin Glargine Injection is a sterile solution of Insulin Glargine in Water for Injection. It has a potency of NLT 95.0 and NMT 105.0 USP Insulin Glargine Units/mL.

2 IDENTIFICATION

A. The retention time of the major peak of the Sample solution corresponds to that of the Standard solutions, as obtained in the Assay.

3 ASSAY

Procedure

Buffer: Dissolve 20.7 g of anhydrous monobasic sodium phosphate in 900 mL of water. Adjust with phosphoric acid to a pH of 2.5, and dilute with water to a ￾nal volume of 1000 mL.

Solution A: Dissolve 18.4 g of sodium chloride in 250 mL of Buffer, add 250 mL of acetonitrile, and mix. Dilute the solution with water to a ￾nal volume of 1000 mL.

Solution B: Dissolve 3.2 g of sodium chloride in 250 mL of Buffer, add 650 mL of acetonitrile, and mix. Dilute the solution with water to a ￾nal volume of 1000 mL.

Mobile phase: See Table 1.

[Note—Adjust the Mobile phase composition and the gradient by a parallel shift to obtain a retention time of 18–23 min for the insulin glargine main peak.]

System suitability solution: Dissolve the contents of 1 vial of USP Insulin Glargine for Peak Identi￾cation RS in 0.3 mL of 0.01 N hydrochloric acid, and add 1.7 mL of water.

Standard solution 1: Dissolve the contents of 1 vial of USP Insulin Glargine RS in 1.5 mL of 0.01 N hydrochloric acid, transfer the solution to a 5-mL volumetric ask, and dilute with water to volume. Dilute 4 mL of this solution with water to 10 mL in a volumetric ￾ask.

Standard solution 2: Dissolve the contents of 1 vial of USP Insulin Glargine RS in 1.5 mL of 0.01 N hydrochloric acid, transfer the solution to a 10-mL volumetric ask, and dilute with water to volume.

Standard solution 3: Dissolve the contents of 1 vial of USP Insulin Glargine RS in 1.5 mL of 0.01 N hydrochloric acid, transfer the solution to a 5-mL volumetric ask, and dilute with water to volume. Dilute 3 mL of this solution with water to 5 mL in a volumetric ￾ask.

Sample solution: Quantitatively dilute a portion of Injection with water to obtain a solution containing about 40 USP Insulin Glargine Units/mL.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 3.0-mm × 25.0-cm; 4-µm packing L1

Column temperature: 35°

Flow rate: 0.55 mL/min

Injection volume: 5 µL

System suitability

Samples: System suitability solution, Standard solution 1, Standard solution 2, and Standard solution 3

Suitability requirements

Resolution: NLT 2.0 for the ratio of the height of the 0A-Arg-insulin glargine peak to the height of the valley between the 0A-Arg-insulin glargine peak and the insulin glargine peak, System suitability solution

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

Relative standard deviation: NMT 2.0%, calculated from six response factors from two duplicate injections each of Standard solution 1, Standard solution 2, and Standard solution 3

Analysis

Samples: Standard solutions and Sample solution

Measure the responses of the major peaks. Prepare a calibration curve based on the peak responses from the Standard solutions versus the concentrations (USP Insulin Glargine Units/mL) using linear regression.

Calculate the potency, in USP Insulin Glargine Units/mL, of the portion of Injection taken:

Result = [(r − b)/a] × D

r = peak response of insulin glargine from the Sample solution

b = y-intercept of the calibration curve

a = slope of the calibration curve

D = dilution factor used to prepare the Sample solution

Acceptance criteria: 95.0–105.0 USP Insulin Glargine Units/mL

4 OTHER COMPONENTS

Zinc Determination

Blank: 0.01 N hydrochloric acid

Standard stock solution: 10 µg/mL of zinc in Blank, from a commercially available zinc standard solution for atomic absorption

Standard solutions: 0.2, 0.4, and 0.6 µg/mL of zinc from the Standard stock solution diluted with Blank

Sample solution: Dilute 1 mL of Injection with Blank to 100 mL.

Instrumental conditions

(See Atomic Absorption Spectroscopy 〈852〉.)

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Zinc absorption line at 213.9 nm

Flame: Air–acetylene ￾ame of suitable composition (for example, 11 L of air and 2 L of acetylene per min)

Lamp: Suitable radiation source, such as zinc hollow-cathode or electrodeless-discharge-lamp (EDL)

System suitability

Samples: Blank and Standard solutions

Using the Standard solutions and Blank, construct a calibration curve by plotting the absorbances of the Standard solutions versus their concentrations, and draw the straight line best ￾tting the three plotted points.

Suitability requirements

Correlation coe￾cient: NLT 0.999

Analysis

Samples: Blank, Standard solutions, and Sample solution

Determine the concentration, C, in µg/mL of zinc in the Sample solution using the calibration curve.

Calculate the quantity of zinc in the portion of Injection taken:

Result = C × D

C = concentration of zinc in the Sample solution (µg/mL)

D = dilution factor, 100

Acceptance criteria: 20–40 µg/mL

5 PRODUCT-RELATED SUBSTANCES AND IMPURITIES

Product-Related Substances

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

Analysis

Sample: Sample solution

Calculate the percentage of each individual insulin glargine related substance (%i) in the portion of Injection taken:

Result = (rᵢ / rₜ) × 100

rᵢ = peak response of the insulin glargine related substance from the Sample solution

rₜ = sum of all the peak responses from the Sample solution

Calculate the total percentage of insulin glargine related substances in the portion of Injection taken:

Result = Σ%i

Σ%i = total percentage of insulin glargine related substances from the Sample solution

Acceptance criteria

Any individual insulin glargine related substance: NMT 0.5%

Total insulin glargine related substances: NMT 2.0%

Delete the following:

▲Limit of High Molecular Weight Proteins▲ (USP 1-Dec-2022)

Add the following:

▲Physicochemical Analytical Procedures for Insulins 〈121.1〉, Limit of High Molecular Weight Proteins: Meets the requirements

Acceptance criteria: NMT 0.5%▲ (USP 1-Dec-2022)

6 SPECIFIC TESTS

pH 〈791〉: 3.5–4.5

Change to read:

Bacterial Endotoxins Test 〈85〉: ▲Meets the requirements▲ (USP 1-Dec-2022)

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

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

7 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in the unopened multiple-dose container provided by the manufacturer. Do not repackage. Store in a refrigerator, protected from sunlight, and avoid freezing.

Labeling: States that it has been prepared with Insulin Glargine produced by methods based on recombinant DNA technology. Label it to state that it is to be stored in a refrigerator and that freezing is to be avoided. The label states the potency in USP Insulin Glargine Units/mL.

USP Reference Standards 〈11〉

USP Insulin Glargine RS

USP Insulin Glargine for Peak Identi￾cation RS

Contains insulin glargine and 0A-Arg-insulin glargine.