Insulin Glargine
Insulin Glargine
Insulin Glargine
2 µL of phosphoric acid.
[160337-95-1]. Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
DEFINITION Mode: LC
Detector: UV 214 nm
Change to read: Column: 3.0-mm × 12.5-cm; 4-µm packing L1
Column temperature: 35°
Insulin Glargine is a two-chain peptide containing 53 Flow rate: 0.6 mL/min
amino acids. The A-chain is composed of 21 amino acids, Injection volume: 50 µL
and the B-chain is composed of 32 amino acids. It is System suitability
identical to the primary structure of Human Insulin ex- Sample: Standard solution
cept for position A21 which has Gly rather than Asn as in Suitability requirements
Human Insulin and two additional amino acids at the C Resolution: NLT 3.4 between the peaks indicated as
terminal of the B-chain Arg (B31) and Arg (B32). Insulin fragments II and III
Glargine is produced by methods based on recombinant Tailing factor: NMT 1.5 for the peaks indicated as
DNA technology. Residual host cell protein (HCP) con- fragments II and III
tent is determined by a validated method and is NMT Chromatogram similarity: In the chromatogram
10 ppm (ng HCP per mg of Insulin Glargine). Insulin from the Standard solution, identify the peaks due to
Glargine contains NLT 94.0% and NMT 105.0% of insu- digest fragments I, II, III, and IV. The chromatogram
lin glargine (C267H404N72O78S6), calculated on the anhy- of the Standard solution corresponds to that of the
drous basis, •or on the dried basis when other volatile typical chromatogram provided with USP Insulin
Glargine RS.
.
System suitability solution: Dissolve the contents of 1 Lamp: Suitable radiation source such as zinc hollow-
vial of USP Insulin Glargine for Peak Identification RS in cathode or electrodeless-discharge-lamp (EDL)
0.3 mL of 0.01 N hydrochloric acid, and add 1.7 mL of System suitability
water. Samples: Standard solutions and Blank
Standard solution: Dissolve the contents of 1 vial of Using the Standard solutions and Blank, construct a cali-
USP Insulin Glargine RS in 1.5 mL of 0.01 N hydrochlo- bration curve by plotting the absorbances of the Stan-
ric acid, transfer the solution to a 10-mL volumetric dard solutions versus their concentrations, and draw
flask, and dilute with water to volume. the straight line best fitting the three plotted points.
Sample solution: Dissolve 15 mg of Insulin Glargine in Suitability requirements
1.5 mL of 0.01 N hydrochloric acid, and dilute with Correlation coefficient: NLT 0.999
water to a final volume of 10 mL. Analysis
Chromatographic system Samples: Standard solutions, Sample solution, and
(See Chromatography 〈621〉, System Suitability.) Blank
Mode: LC Determine the concentration, C, in µg/mL of zinc in
Detector: UV 214 nm the Sample solution using the calibration curve.
Column: 3.0-mm × 25.0-cm; 4-µm packing L1 Calculate the percentage of zinc in the portion of Insu-
Column temperature: 35° lin Glargine taken:
Flow rate: 0.6 mL/min
Injection volume: 5 µL Result = [C × F1 × V × (F2/W)] × 100
System suitability
Samples: System suitability solution and Standard C = concentration of zinc in the Sample solution
solution (µg/mL)
Suitability requirements F1 = conversion factor from µg/mL to mg/mL,
Resolution: NLT 2.0 for the ratio of the height of 0.001
the 0A-Arg-insulin glargine peak to the height of the V = volume of the Sample solution, 100 mL
F2 = sampling factor, 5
.
solution
•
basis or dried basis• (IRA 1-Nov-2016)
.
solution
accurately weighed, in 50 mL of 0.01 N hydrochloric Acceptance criteria
acid. Dilute 10 mL of the solution with 0.01 N hydro- Any individual insulin glargine related
chloric acid to a final volume of 100 mL. •substance:• (IRA 1-Nov-2016) NMT 0.5%
Blank: 0.01 N hydrochloric acid Total insulin glargine related •substances:• (IRA 1-Nov-
.
Instrumental conditions
.
weight proteins in 1.5 mL of 0.01 N hydrochloric acid. • BACTERIAL ENDOTOXINS TEST 〈85〉: NMT 10 USP Endo-
Dilute with water to a final volume of 10 mL. [NOTE— toxin Units/mg of Insulin Glargine
Insulin Glargine containing the indicated percentage of • MICROBIAL ENUMERATION TESTS 〈61〉 and TESTS FOR SPECI-
high molecular weight proteins may be prepared by FIED MICROORGANISMS 〈62〉: The total bacterial count
incubating Insulin Glargine at 100° for 1.5–3 h.] does not exceed 300 cfu/g, the test being performed on
Sample solution: Dissolve 15 mg of Insulin Glargine in a portion of about 0.2 g, accurately weighed.
1.5 mL of 0.01 N hydrochloric acid. Dilute with water
to a final volume of 10 mL.
Chromatographic system Change to read:
(See Chromatography 〈621〉, System Suitability.)
Mode: LC • WATER DETERMINATION 〈921〉, Method Ic: NMT 8.0%.
Detector: UV 276 nm
•[NOTE—Use this test when the drug substance predomi-
.
Column: Two 8.0-mm × 30-cm in series; 5-µm pack- nantly contains water.]• (IRA 1-Nov-2016)
ing L20
Column temperature: Ambient Add the following:
Flow rate: 0.5 mL/min
Injection volume: 100 µL •• LOSS ON DRYING 〈731〉: NMT 10.0%. [NOTE—Use this
System suitability
.