Edition: BP 2025 (Ph. Eur. 11.6 update)
Action and use
Hormone; treatment of diabetes mellitus.
DEFINITION
Insulin Glargine Injection is a sterile, acidic, aqueous solution of Insulin Glargine.
The injection complies with the requirements stated under Injectable Insulin Preparations with the modifications described below.
Content of insulin glargine, C267H404N72O78S6
95.0 to 105.0% of the stated amount.
IDENTIFICATION
In the Assay, the retention time of the principal peak in the chromatogram obtained with solution (1) corresponds to that in the chromatogram obtained with solution (2).
TESTS
Acidity
pH, 3.5 to 4.5, Appendix V L.
Impurities with molecular masses greater than that of insulin glargine
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) Dilute the injection, if necessary, with sufficient water to produce a solution containing 0.15% w/v of Insulin Glargine.
(2) Dry 1 vial of insulin glargine EPCRS in an oven at 100° for 1.5 to 3 hours. Dissolve the contents of the vial in 1.5 mL of 0.01M hydrochloric acid and dilute to 10.0 mL with water.
(3) Dilute 1 volume of solution (1) to 100 volumes with water, dilute 3 volumes of this solution to 20 volumes with water.
CHROMATOGRAPHIC CONDITIONS
(a) Use two stainless steel columns (30 cm × 8.0 mm) coupled in series and packed with hydrophilic silica gel for chromatography (5 μm) with a pore size of 15 nm of a grade suitable for the fractionation of globular proteins in the relative molecular mass range of 2000 to 80,000 (Shodex PROTEIN KW-802.5 is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 0.5 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 276 nm.
(f) Inject 100 μL of each solution. If splitting of the principal peak is observed, the injection volume may be decreased according to the provisions given in Appendix III.
(g) Allow the chromatography to proceed for 1.8 times the retention time of insulin glargine.
MOBILE PHASE
Mix 200 volumes of anhydrous acetic acid, 300 volumes of acetonitrile for chromatography and 400 volumes of water, adjust to pH 3.0 with concentrated ammonia and dilute to 1000 volumes with water.
When the chromatograms are recorded under the prescribed conditions the retention time of insulin glargine is about 35 minutes.
SYSTEM SUITABILITY
The test is not valid unless:
in the chromatogram obtained with solution (2), the symmetry factor of the peak due to insulin glargine is not more than 2.0;
in the chromatogram obtained with solution (2), the peak-to-valley ratio is at least 2, where Hp = height above the baseline due to high molecular mass proteins and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to insulin glargine;
in the chromatogram obtained with solution (3), the signal-to-noise ratio of the principal peak is at least 10.
LIMITS
In the chromatogram obtained with solution (1) the sum of the areas of any secondary peaks with a retention time less than that of the peak due to insulin glargine is not greater than 0.3% by normalisation.
Disregard any peaks with a retention time greater than that of the peak due to insulin glargine.
Related proteins
Carry out the method for liquid chromatography, Appendix III D, using the following solutions. Store the solutions at a temperature of 2° to 8°.
(1) Dilute the injection, if necessary, with sufficient water to produce a solution containing 0.15% w/v of Insulin Glargine.
(2) Dissolve the contents of a vial of insulin glargine for peak identification EPCRS (containing 0 -Arg-insulin glargine) in 0.3 mL of 0.01M hydrochloric acid and add 1.7 mL of water.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm × 3.0 mm) packed with end-capped octadecylsilyl silica gel for chromatography (4 μm) (Merck Superspher 100-RP-18e is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 0.6 mL per minute.
(d) Use a column temperature of 35°.
(e) Use a detection wavelength of 214 nm.
(f) Inject 5 μL of each solution.
MOBILE PHASE
Mobile phase A Dissolve 18.4 g of sodium chloride in 250 mL of buffer solution prepared as described below; add 250 mL of acetonitrile R1 and mix; dilute to 1000 mL with water.
To prepare the buffer solution, dissolve 20.7 g of anhydrous sodium dihydrogen phosphate in 900 mL of water, adjust to pH 2.5 with orthophosphoric acid and dilute to 1000 mL with water.
Mobile phase B Dissolve 3.2 g of sodium chloride in 250 mL of the buffer solution prepared as described above; add 650 mL of acetonitrile R1 and mix; dilute to 1000 mL with water for chromatography.
| Time (Minutes) | Mobile phase A (% v/v) | Mobile phase B (% v/v) | Comment |
| 0-20 | 96-83 | 4-17 | linear gradient |
| 20-30 | 83-63 | 17-37 | linear gradient |
| 30-40 | 63-96 | 37-4 | linear gradient |
| 40-48 | 96 | 4 | re-equilibration |
When the chromatograms are recorded under the prescribed conditions the relative retention time of insulin glargine is about 20 minutes.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (2), the peak-to-valley ratio is at least 2, where Hp = height above the baseline due to 0A-Arg-insulin glargine and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to insulin glargine.
LIMITS
In the chromatogram obtained with solution (1):
the area of any secondary peak is not greater than 0.5% by normalisation;
the sum of the areas of all secondary peaks is not greater than 2.0% by normalisation.
Total zinc
27 to 33 μg per 100 units of insulin glargine, determined by atomic absorption spectrometry, Appendix II D, Method I.
Test solution Dilute, if necessary, to a suitable concentration of zinc (for example 0.2 μg to 0.6 μg of Zn per mL) with 0.01M hydrochloric acid.
Reference solutions Use solutions containing a suitable range of concentrations, for example 0.20 μg, 0.40 μg and 0.60 μg of Zn per mL, freshly prepared by diluting zinc standard solution (10 ppm Zn) with 0.01M hydrochloric acid.
Measure the absorbance at 213.9 nm using a zinc hollow-cathode lamp as source of radiation and an air-acetylene flame of suitable composition (for example 11 litres of air and 2 litres of acetylene per minute).
Bacterial endotoxins
Carry out the test for bacterial endotoxins, Appendix XIV C. The endotoxin limit concentration is less than 80 IU of endoxtoxin per 100 units of insulin glargine.
ASSAY
Carry out the method for liquid chromatography, Appendix III D, using the following solutions. Store the solutions at a temperature of 2° to 8°.
(1) Dilute the injection, if necessary, with sufficient water to produce a solution containing 0.15% w/v of Insulin Glargine.
(2) Dissolve the contents of a vial of insulin glargine EPCRS in 1.5 mL of 0.01M hydrochloric acid and dilute to 10.0 mL with water.
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under Related proteins may be used.
DETERMINATION OF CONTENT
Calculate the content of insulin glargine, C267H404N72O78S6, in the injection from the chromatograms obtained and the declared content of C267H404N72O78S6 in insulin glargine EPCRS.
LABELLING
The label states the potency in units per mL.
100 IU are equivalent to 3.64 mg of insulin glargine.
