Edition: BP 2025 (Ph. Eur. 11.6 update)
Action and use
Hormone; treatment of diabetes mellitus.
DEFINITION
Insulin Lispro Injection is a sterile, aqueous solution of Insulin Lispro.
The injection complies with the requirements stated under Injectable Insulin Preparations, with the exception of the test for Insulin in the supernatant, and with the modifications described below.
Content of insulin lispro, C257H383N65O77S6
90.0 to 105.0% of the stated amount.
CHARACTERISTICS
A clear and colourless solution, free from turbidity and foreign matter.
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
Impurities with molecular masses greater than that of insulin lispro
The sum of the areas of the peaks in the chromatogram obtained with the test solution with a retention time less than that of the principal peak is not more than 1.5% of the total area of the peaks. Disregard any peak with a retention time greater than that of the peak due to insulin lispro monomer.
Related proteins
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) Dilute, if necessary, the preparation being examined with 0.01M hydrochloric acid to produce a solution containing 0.35% w/v of Insulin Lispro. Add 4 μL of 6M hydrochloric acid per mL of this solution. Maintain the solution at 2° to 8° and use within 56 hours.
(2) Prepare a 0.35% w/v solution of Insulin Lispro in 0.01M hydrochloric acid and add 4 μL of 6M hydrochloric acid per mL. Allow to stand at room temperature to obtain a solution containing between 0.8% and 11% of A21 desamido insulin lispro.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm x 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 μm) with a pore size of 30 nm (Vydac Protein and Peptide C18 is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 1 mL per minute.
(d) Use a column temperature of 40°.
(e) Use a detection wavelength of 214 nm.
(f) Inject 20 μL for each solution.
MOBILE PHASE
Mobile phase A Mix 82 volumes of a 2.84% w/v solution of anhydrous sodium sulfate adjusted to pH 2.3 with orthophosphoric acid and 18 volumes of acetonitrile for chromatography.
Mobile phase B Mix equal volumes of a 2.84% w/v solution of anhydrous sodium sulfate, adjusted to pH 2.3 with orthophosphoric acid, and acetonitrile for chromatography.
Use the following gradient.
| Time (Minutes) | Mobile phase A (% v/v) | Mobile phase B (% v/v) | Comments |
| 0-60 | 81 | 19 | Isocratic |
| 60-83 | 81-51 | 19-49 | Linear gradient |
| 83-84 | 51-81 | 49-19 | Linear gradient |
| 84-94 | 81 | 19 | Re-equilibration |
SYSTEM SUITABILITY
Adjust the mobile phase composition to obtain a retention time of about 41 minutes for insulin lispro; A21 desamido insulin lispro elutes near the start of the gradient elution. The test is not valid unless, in the chromatogram obtained with solution (2), the resolution between the first peak (insulin lispro) and the second peak (A21 desamido insulin lispro) is at least 1.5 and the symmetry factor for the peak due to insulin lispro is less than 2.0.
LIMITS
In the chromatogram obtained with solution (1), the amount of A21 desamido insulin lispro is not more than 1.5%. The total amount of related proteins (excluding A21) is not greater than 4.0%.
Total zinc
14 to 35 μg per 100 units of insulin lispro, determined by atomic absorption spectrometry, Appendix II D, Method I.
Test solution Shake the preparation gently and dilute a volume containing 200 units of insulin lispro to 25.0 mL with 0.01M hydrochloric acid. Dilute if necessary to a suitable concentration of zinc (for example 0.4 μg to 1.6 μg of Zn per mL) with 0.01M hydrochloric acid.
Reference solutions Use solutions containing a suitable range of concentrations, for example 0.40 μg, 0.80 μg, 1.00 μg, 1.20 μg and 1.60 μg of Zn per mL, freshly prepared by diluting zinc standard solution (5 mg per mL 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 endotoxin per 100 units of insulin lispro.
ASSAY
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) Dilute, if necessary, the preparation being examined with 0.01M hydrochloric acid to obtain a 0.08% w/v solution. Add 4 μL of 6M hydrochloric acid per mL of this solution. Maintain the solution at 2 to 8° and use within 48 hours.
(2) Dissolve the contents of a vial of insulin lispro EPCRS in 0.01M hydrochloric acid to obtain a 0.08% w/v solution. Maintain the solution at 2° to 8° and use within 48 hours.
(3) Prepare a 0.1% w/v solution of the preparation being examined in 0.01M hydrochloric acid and add 4 μL of 6M hydrochloric acid per mL. Allow to stand at room temperature to obtain a solution containing between 0.8% and 11% of A21 desamido insulin lispro. Maintain the solution at 2° to 8° and use within 14 days.
CHROMATOGRAPHIC CONDITIONS
(a) Stainless steel column (10 cm x 4.6 mm) packed with octadecylsilyl silica gel for chromatography (3 μm) (Spherisorb ODS 2 is suitable).
(b) Isocratic elution using the mobile phase described below.
(c) Flow rate of 0.8 mL per minute.
(d) Column temperature of 40°.
(e) Detection wavelength of 214 nm.
(f) Injection volume of 20 μL for each solution.
MOBILE PHASE
Mix 255 volumes of acetonitrile for chromatography and 745 volumes of a 2.84% w/v solution of anhydrous sodium sulfate adjusted to pH 2.3 with orthophosphoric acid.
SYSTEM SUITABILITY
When the chromatograms are recorded in the prescribed conditions, the retention time of insulin lispro is approximately 24 minutes. The test is not valid unless, in the chromatogram obtained with solution (3), the resolution between the first peak (insulin lispro) and the second peak (A21 desamido insulin lispro), is greater than 1.8, and the maximum relative standard deviation for replicate injections is 1.1% after 3 injections.
DETERMINATION OF CONTENT
Calculate the content of insulin lispro C257H383N65O77S6 from the chromatograms obtained and the declared content of C257H383N65O77S6 in insulin lispro EPCRS.
LABELLING
The label states the potency in units per mL.
100 IU are equivalent to 3.47 mg of insulin lispro.
