Add the following:

Insulin Glargine

(in' su lin glar' jeen).

C267H404N72O78S6

6062.89

Insulin (human), 21A-glycine-30Ba-l-arginine-30Bb-l-arginine

[160337-95-1].

DEFINITION

Insulin Glargine is a two-chain peptide containing 53 amino acids. The A-chain is composed of 21 amino acids, and the B-chain is composed of 32 amino acids. It is identical to the primary structure of Human Insulin except for position A21 which has Gly rather than Asn as in Human Insulin and two additional amino acids at the C terminal of the B-chain Arg (B31) and Arg (B32). Insulin Glargine is produced by methods based on recombinant DNA technology. Residual host cell protein (HCP) content is determined by a validated method and is NMT 10 ppm (ng HCP per mg of Insulin Glargine). Insulin Glargine contains NLT 94.0% and NMT 105.0% of insulin glargine (C267H404N72O78S6), calculated on the anhydrous basis.

[Note—One USP Insulin Glargine Unit is equivalent to 0.0364 mg of pure Insulin Glargine.

]

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.

• B. Peptide Mapping

Phosphate/perchlorate buffer: Dissolve 11.6 g of phosphoric acid and 42.1 g of sodium perchlorate in 1600 mL of water. Adjust with triethylamine to a pH of 2.3, and dilute with water to a final volume of 2000 mL.

Solution A: Prepare a filtered and degassed mixture of acetonitrile and Phosphate/perchlorate buffer (7:93).

Solution B: Prepare a filtered and degassed mixture of acetonitrile and Phosphate/perchlorate buffer (57:43).

Mobile phase: See Table 1.

Table 1

Time (min)	Solution A (%)	Solution B (%)
0	90	10
30	20	80
35	20	80
36	90	10

Tris buffer solution: Dissolve 12.11 g of tris(hydroxymethyl)aminomethane in 90 mL of water. Adjust with hydrochloric acid to a pH of 7.5, and dilute with water to a final volume of 100 mL.

Enzyme solution: Prepare a solution of Staphylococcus aureus V-8 protease in Tris buffer solution having an activity of 20 Units/mL.

Standard solution: Transfer to a vial 35 µL of the Standard solution from the Assay. To this vial, add 1.0 mL of Tris buffer solution and 100 µL of Enzyme solution, and incubate at 45

for 2–3 h. Quench the digestion by adding 2 µL of phosphoric acid.

Sample solution: Transfer to a vial 35 µL of the Sample solution from the Assay. To this vial, add 1.0 mL of Tris buffer solution and 100 µL of Enzyme solution, and incubate at 45

for 2–3 h. Quench the digestion by adding 2 µL of phosphoric acid.

Chromatographic system

(See Chromatography

621

, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 3.0-mm × 12.5-cm; 4-µm packing L1

Column temperature: 35

Flow rate: 0.6 mL/min

Injection volume: 50 µL

System suitability

Sample: Standard solution

Suitability requirements: In the chromatogram from the Standard solution, identify the peaks due to digest fragments I, II, III, and IV. The chromatogram of the Standard solution corresponds to that of the typical chromatogram provided with USP Insulin Glargine RS.

Resolution: NLT 3.4 between the peaks indicated as fragments II and III

Tailing factor: NMT 1.5 for the peaks indicated as fragments II and III

Analysis

Samples: Standard solution and Sample solution

Run a blank, and record the chromatograms.

Acceptance criteria: The chromatographic profile of the Sample solution corresponds to that of the Standard solution. All four fragments, I, II, III, and IV must be present.

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 2.

Table 2

Time (min)	Solution A (%)	Solution B (%)
0	96	4
20	83	17
30	63	37
33	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: 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.

Sample solution: Dissolve 15 mg of Insulin Glargine in 1.5 mL of 0.01 N hydrochloric acid, and dilute with water to a final volume of 10 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: 0.6 mL/min

Injection volume: 5 µL

System suitability

Samples: System suitability solution and Standard solution

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% for six replicate injections, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the potency, in percent, of insulin glargine (C267H404N72O78S6) in the portion of Insulin Glargine taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak response of insulin glargine from the Sample solution
rS	=	= peak response of insulin glargine from the Standard solution
CS	=	= concentration of USP Insulin Glargine RS in the Standard solution (mg/mL)
CU	=	= concentration of the Sample solution (corrected for the water content) (mg/mL)

Acceptance criteria: 94.0%–105.0% on the anhydrous basis

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: Dissolve 45 mg of Insulin Glargine, accurately weighed, in 50 mL of 0.01 N hydrochloric acid. Dilute 10 mL of the solution with 0.01 N hydrochloric acid to a final volume of 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

Sample: 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 percentage of zinc in the portion of Insulin Glargine taken:

Result = [C × F1 × V × (F2/W)] × 100

C	=	= concentration of zinc in the Sample solution (µg/mL)
F1	=	= conversion factor from µg/mL to mg/mL, 0.001
V	=	= volume of the Sample solution, 100 mL
F2	=	= sampling factor, 5
W	=	= weight of Insulin Glargine taken (mg)

Acceptance criteria: NMT 0.80%

IMPURITIES

• Related Proteins

Mobile phase, System suitability solution, Standard solution, 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 Insulin Glargine 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 Insulin Glargine taken:

Result = S%ix

Acceptance criteria: NMT 0.5% of any individual insulin glargine related protein; NMT 1.5% 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: Dissolve 15 mg of Insulin Glargine in 1.5 mL of 0.01 N hydrochloric acid. Dilute with water to a final volume of 10 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 Insulin Glargine 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

• Insulin Assays, Bioidentity Test

121

: Meets the requirements

• Bacterial Endotoxins Test

: NMT 10 USP Endotoxin Units/mg of Insulin Glargine

• Microbial Enumeration Tests

and Tests For Specified Microorganisms

: The total bacterial count does not exceed 300 cfu/g, the test being performed on a portion of about 0.2 g, accurately weighed.

• Water Determination, Method Ic

921

: NMT 8.0%

ADDITIONAL REQUIREMENTS

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

• Labeling: Label it to indicate that the material is produced by methods based on recombinant DNA technology.

• 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
61	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison (301) 816-8339	(GCM2010) General Chapters - Microbiology
62	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 3874

Pharmacopeial Forum: Volume No. 39(4)