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Insulin Glargine Injection

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.

IDENTIFICATION

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

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 final 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 final 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 final volume of 1000 mL.

Mobile phase: See Table 1.

Table 1

Time (min)	Solution A (%)	Solution B (%)
0	96	4
20	83	17
30	63	37
40	96	4

[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 one vial of USP Insulin Glargine for Peak Identification 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 one vial of USP Insulin Glargine RS in 1.5 mL of 0.01 N hydrochloric acid, transfer the solution to a 5-mL volumetric flask, and dilute with water to volume. Dilute 4 mL of this solution to 10 mL in a volumetric flask.

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

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

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 and Standard solutions

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 of each of the three Standard solutions 1, 2, and 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 = [(rU

b)/a] × D

rU	=	= 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

OTHER COMPONENTS

• Zinc Determination

Standard stock solution: 10 µg/mL of zinc in 0.01 N hydrochloric acid, 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 0.01 N hydrochloric acid

Sample solution: Dilute 1 mL of Injection with 0.01 N hydrochloric acid to 100 mL.

Blank solution: 0.01 N hydrochloric acid

Instrumental conditions

(See Atomic Absorption Spectroscopy

852

Mode: Atomic absorption spectrophotometry

Analytical wavelength: Zinc absorption line at 213.9 nm

Flame: Air–acetylene flame 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: Standard solutions and Blank solution

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

Suitability requirements

Correlation coefficient: NLT 0.999

Analysis

Samples: Standard solutions, Sample solution, and Blank solution

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

Calculate the amount 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: 27.0–33.0 µg/mL

IMPURITIES

• Related Proteins

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 related protein (%ix) in the portion of Injection taken:

Result = (ri/rT) × 100

ri	=	= peak response of the insulin related protein
rT	=	= sum of the responses for all of the peaks

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

Result = S%ix

Acceptance criteria: NMT 0.5% of any individual insulin glargine related protein; NMT 2.0% of total insulin glargine related proteins

• Limit of High Molecular Weight Proteins

Mobile phase: Prepare a mixture of acetonitrile, water, and glacial acetic acid (300:400:200). Adjust with concentrated ammonia (25%–30%) to a pH of 3.0, and dilute with water to a final volume of 1000 mL.

System suitability solution: Dissolve 15 mg of insulin glargine containing more than 0.4% high molecular weight proteins in 1.5 mL of 0.01 N hydrochloric acid. Dilute with water to a final volume of 10 mL. [Note—Insulin glargine containing the indicated percentage of high molecular weight proteins may be prepared by incubating insulin glargine at 100

for 1.5–3 h.

]

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 276 nm

Column: Two 8.0-mm × 30-cm columns in series; 5-µm packing L20

Column temperature: Ambient

Flow rate: 0.5 mL/min

Injection volume: 100 µL

System suitability

Sample: System suitability solution

[Note—The retention time for the insulin monomer is about 35 min, and the high molecular weight proteins elute earlier.

]

Suitability requirements

Resolution: The ratio of the height of the high molecular weight proteins peak to the height of the valley between the high molecular weight proteins peak and the insulin glargine peak is NLT 2.

Tailing factor: NMT 2.0 for the insulin glargine peak

Analysis

Sample: Sample solution

Measure the areas of the peak responses, disregarding any peaks having retention times greater than that of the insulin monomer.

Calculate the percentage of high molecular weight proteins in the portion of Injection taken:

Result = [SrH/(SrH + rM)] × 100

SrH	=	= sum of the responses for all peaks having retention times less than that of insulin glargine
rM	=	= peak response of insulin glargine

Acceptance criteria: NMT 0.3%

SPECIFIC TESTS

• pH

791

: 3.5–4.5

• Bacterial Endotoxins Test

: NMT 80 USP Endotoxin Units/100 USP Insulin Glargine Units

• Sterility Tests

: Meets the requirements when tested as directed for Test for Sterility of the Product to be Examined, Membrane Filtration

• Particulate Matter in Injections

788

: Meets the requirements for small-volume injections

• Injections

: Meets the requirements

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

USP Endotoxin RS

USP Insulin Glargine RS

USP Insulin Glargine for Peak Identification RS

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

USP38

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question	Contact	Expert Committee
Monograph	Edith Chang, Ph.D. Scientific Liaison (301) 816-8392	(BIO12010) Monographs - Biologics and Biotechnology 1
85	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison (301) 816-8339	(GCM2010) General Chapters - Microbiology
71	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison (301) 816-8339	(GCM2010) General Chapters - Microbiology
Reference Standards	RS Technical Services 1-301-816-8129 rstech@usp.org

USP38–NF33 Page 3876

Pharmacopeial Forum: Volume No. 39(4)