(Ph. Eur. monograph 2085)
C257H383N65O77S6 5808
Action and use
Hormone; treatment of diabetes mellitus.
Preparation
Biphasic Insulin Lispro Injection
DEFINITION
28 -L-Lysine-29 -L-proline insulin (human).
Insulin lispro is a 2-chain peptide containing 51 amino acids. The A-chain is composed of 21 amino acids and the B-chain is composed of 30 amino acids. It is identical in primary structure to human insulin, only differing in amino acid sequence at positions 28 and 29 of the B-chain. Human insulin is Pro(B28), Lys(B29), whereas insulin lispro is Lys(B28), Pro(B29).
As in human insulin, insulin lispro contains 2 interchain disulfide bonds and 1 intrachain disulfide bond.
Content
94.0 per cent to 104.0 per cent (dried substance).
By convention, for the purpose of labelling insulin lispro preparations, 0.0347 mg of insulin lispro is equivalent to 1 unit.
PRODUCTION
Insulin lispro is produced by a method based on recombinant DNA (rDNA) technology under conditions designed to minimise the degree of microbial contamination.
Prior to release, the following tests are carried out on each batch of insulin lispro, unless exemption has been granted by the competent authority.
Host-cell-derived proteins
The limit is approved by the competent authority.
Single-chain precursor
The limit is approved by the competent authority. Use a suitably sensitive method.
CHARACTERS
Appearance
White or almost white powder.
Solubility
Practically insoluble in water and in ethanol (96 per cent). It dissolves in dilute mineral acids and with decomposition in dilute solutions of alkali hydroxides.
IDENTIFICATION
A. Examine the chromatograms obtained in the assay.
Results: The principal peak in the chromatogram obtained with the test solution is similar in retention time to the principal peak in the chromatogram obtained with the reference solution.
B. Peptide mapping (2.2.55).
SELECTIVE CLEAVAGE OF THE PEPTIDE BONDS
Test solution: Prepare a 2.0 mg/mL solution of the substance to be examined in 0.01 M hydrochloric acid and transfer 500 μL of this solution to a clean tube. Add 2.0 mL of HEPES buffer solution pH 7.5 R and 400 μL of a 1 mg/mL solution of Staphylococcus aureus strain V8 protease, type XVII-B R. Cap the tube and incubate at 25 °C for 6 h. Stop the reaction by adding 2.9 mL of sulfate buffer solution pH 2.0 R.
Reference solution Prepare at the same time and in the same manner as for the test solution but using insulin lispro CRS instead of the substance to be examined.
CHROMATOGRAPHIC SEPARATION.
Liquid chromatography (2.2.29).
Column:
— size: l = 0.10 m, Ø = 4.6 mm,
— stationary phase: octadecylsilyl silica gel for chromatography R (3 μm) with a pore size of 8 nm,
— temperature: 40 °C.
Mobile phase:
— mobile phase A: mix 100 mL of acetonitrile for chromatography R, 200 mL of sulfate buffer solution pH 2.0 R and 700 mL of water R; filter and degas;
— mobile phase B: mix 200 mL of sulfate buffer solution pH 2.0 R, 400 mL of acetonitrile for chromatography R and 400 mL of water R; filter and degas;
| Time
(min) |
Mobile phase A
(per cent V/V) |
Mobile phase B
(per cent V/V) |
| 0 – 60 | 90 → 30 | 10 → 70 |
| 60 – 65 | 30 → 0 | 70 → 100 |
| 65 – 70 | 0 | 100 |
Flow rate: 1 mL/min.
Detection: Spectrophotometer at 214 nm.
Equilibration: At initial conditions for at least 15 min. Carry out a blank run using the above-mentioned gradient.
Injection: 50 μL.
System suitability:
— the chromatogram obtained with the reference solution is qualitatively similar to the chromatogram of insulin lispro digest supplied with insulin lispro CRS,
— in the chromatogram obtained with the reference solution, identify the peaks due to digest fragments I, II and III: symmetry factor Maximum 1.5 for the peaks due to fragments II and III, resolution Minimum 8.0 between the peaks due to fragments II and III.
Results: The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution.
NOTE: the retention times of fragments I, II and IV are the same as for human insulin. The retention time of fragment III differs from human insulin due to differences in sequence at positions 28 and 29 of the B-chain.
TESTS
Impurities with molecular masses greater than that of insulin lispro
Size-exclusion chromatography (2.2.30): use the normalisation procedure.
Test solution: Prepare a solution containing 4 mg/mL of the substance to be examined in 0.01 M hydrochloric acid.
Maintain the solution at 2-8 °C and use within 48 h.
Resolution solution: Use a solution of insulin (about 4 mg/mL), containing more than 0.4 per cent of high molecular mass proteins. An injectable insulin preparation, whether a solution or a suspension, that has been clarified with a sufficient amount of 6 M hydrochloric acid R, containing the indicated percentage of high molecular mass proteins, or a solution prepared from insulin, dissolved in 0.01 M hydrochloric acid, may be used. Insulin containing the indicated percentage of high molecular mass proteins may be prepared by allowing insulin powder to stand at room temperature for about 10 days.
Maintain the solution at 2-8 °C and use within 8 days.
Column:
— size: l = 0.30 m, Ø = 7.8 mm,
— stationary phase: hydrophilic silica gel for chromatography R (5-10 μm) with a pore size of 12-12.5 nm, of a grade suitable for the separation of insulin monomer from dimer and polymers.
Mobile phase: Mix 15 volumes of glacial acetic acid R, 20 volumes of acetonitrile for chromatography R and 65 volumes of a 1.0 g/L solution of arginine R; filter and degas.
Flow rate: 0.5 mL/min.
Detection: Spectrophotometer at 276 nm.
Equilibration: At least 3 injections of the resolution solution; the column is equilibrated when repeatable results are obtained for 2 subsequent injections.
Injection: 100 μL.
Run time: About 35 min.
Retention time: Insulin lispro polymers = 13-17 min; insulin lispro dimer = about 17.5 min; insulin lispro monomer = about 20 min; salts = about 22 min.
System suitability: Resolution solution:
— peak-to-valley ratio: minimum 2.0, where Hp = height above the baseline of the peak due to the dimer and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to the monomer,
— symmetry factor: maximum 2.0 for the peak due to insulin lispro.
Limits: The sum of the areas of the peaks with a retention time less than that of the principal peak is not more than 0.25 per cent 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
Liquid chromatography (2.2.29): use the normalisation procedure.
Test solution: Dissolve 3.5 mg of the substance to be examined in 1.0 mL of 0.01 M hydrochloric acid. Maintain the solution at 2-8 °C and use within 56 h.
Resolution solution: Dissolve 3.5 mg of the substance to be examined in 1.0 mL of 0.01 M hydrochloric acid. Allow to stand at room temperature to obtain a solution containing between 0.8 per cent and 11 per cent of A21 desamido insulin lispro.
Column:
— size: l = 0.25 m, Ø = 4.6 mm,
— stationary phase: octadecylsilyl silica gel for chromatography R (5 μm) with a pore size of 30 nm,
— temperature: 40 °C.
Mobile phase:
— mobile phase A: mix 82 volumes of a 28.4 g/L solution of anhydrous sodium sulfate R adjusted to pH 2.3 with phosphoric acid R and 18 volumes of acetonitrile for chromatography R; filter and degas;
— mobile phase B: mix equal volumes of a 28.4 g/L solution of anhydrous sodium sulfate R adjusted to pH 2.3 with phosphoric acid R and acetonitrile for chromatography R; filter and degas;
| Time
(min) |
Mobile phase A
(per cent V/V) |
Mobile phase B
(per cent V/V) |
| 0 – 60 | 81 | 19 |
| 60 – 83 | 30 → 0 | 70 → 100 |
| 83 – 84 | 51 → 81 | 49 → 19 |
| 84 – 94 | 81 | 19 |
Flow rate: 1 mL/min.
Detection: Spectrophotometer at 214 nm.
Injection: 20 μL.
Retention time: Adjust the mobile phase composition to obtain a retention time of about 41 min for insulin lispro; A21 desamido insulin lispro elutes near the start of the gradient elution.
System suitability: Resolution solution:
— resolution: minimum 1.5 between the 1 peak (insulin lispro) and the 2 peak (A21 desamido insulin lispro),
— symmetry factor: maximum 2.0 for the peak due to insulin lispro.
Limits:
— A21 desamido insulin lispro: maximum 1.0 per cent,\
— any other impurity: maximum 0.50 per cent,
— total (excluding A21): maximum 2.0 per cent.
Zinc
Maximum 1.0 per cent (dried substance).
Atomic absorption spectrometry (2.2.23, Method I).
Test solution: Dissolve at least 50 mg of the substance to be examined in 0.01 M hydrochloric acid and dilute to 25 mL with the same acid. Dilute if necessary to a suitable concentration (for example 0.4-0.6 μg of Zn per millilitre) with 0.01 M hydrochloric acid.
Reference solutions: Use solutions of concentrations which bracket the expected zinc concentration of the samples, for example, 0.2-0.8 μg of Zn per millilitre, freshly prepared by diluting zinc standard solution (5 mg/mL Zn) R with 0.01 M hydrochloric acid.
Source Zinc hollow-cathode lamp.
Wavelength 213.9 nm.
Atomisation device Air-acetylene flame of suitable composition (for example, 11 L of air and 2 L of acetylene per minute).
Loss on drying (2.2.32)
Maximum 10.0 per cent, determined on 0.200 g by drying in an oven at 105 °C for 16 h.
Sulfated ash (2.4.14)
Maximum 2.5 per cent, determined on 0.200 g (dried substance).
Bacterial endotoxins (2.6.14, Method D)
Less than 10 IU/mg, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins.
ASSAY
Liquid chromatography (2.2.29).
Test solution: Dissolve the substance to be examined in 0.01 M hydrochloric acid to obtain a concentration of 0.8 mg/mL.
Maintain the solution at 2-8 °C and use within 48 h.
Reference solution: Dissolve the contents of a vial of insulin lispro CRS in 0.01 M hydrochloric acid to obtain a concentration of 0.8 mg/mL. Maintain the solution at 2-8 °C and use within 48 h.
Resolution solution: Dissolve about 10 mg of the substance to be examined in 10 mL of 0.01 M hydrochloric acid. Allow to stand at room temperature to obtain a solution containing between 0.8 per cent and 11 per cent of A21 desamido insulin lispro. Maintain the solution at 2-8 °C and use within 14 days.
Column:
— size: l = 0.10 m, Ø = 4.6 mm,
— stationary phase: octadecylsilyl silica gel for chromatography R (3 μm) with a pore size of 8 nm,
— temperature: 40 °C.
Mobile phase: Mix 745 volumes of a 28.4 g/L solution of anhydrous sodium sulfate R adjusted to pH 2.3 with phosphoric acid R and 255 volumes of acetonitrile for chromatography R; filter and degas.
Flow rate: 0.8 mL/min.
Detection: Spectrophotometer at 214 nm.
Injection: 20 μL.
Retention time: Insulin lispro = about 24 min.
System suitability:
— resolution: minimum 1.8 between the 1 peak (insulin lispro) and the 2 peak (A21 desamido insulin lispro), in the chromatogram obtained with the resolution solution,
— repeatability: maximum relative standard deviation of 1.1 per cent after 3 injections of the reference solution.
Calculate the content of insulin lispro C257H383N65O77S6 using the chromatograms obtained with the test solution and the reference solution and the declared content of C257H383N65O77S6 in insulin lispro CRS.
STORAGE
In an airtight container, protected from light, at or below -18 °C. When thawed, insulin lispro is stored and weighed under conditions defined by the manufacturer to maintain the quality attributes of the drug substance and is used for manufacturing preparations within a short period of time. To avoid absorption of humidity from the air during weighing,
insulin lispro must be at room temperature before opening the container.
