STANDARD TESTING PROCEDURE (Human Insulin BP)

TABLE OF CONTENTS

1.0 Description : A white or almost white powder.


2.0 Solubility : Practically insoluble in water, in ethanol and in ether. It dissolves in dilute mineral acids and with decomposition in dilute solutions of alkali hydroxides.

3.0 Identification: A. Examine the chromatograms obtained in the test for related proteins. The retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. ( By HPLC)

B. Examine by peptide mapping : ( By HPLC)
Test solution. Prepare a solution of the substance to be examined in 0.01M hydrochloric acid containing 2.0 mg/ml of human insulin 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 R. Cap the tube and incubate at 25°C for 6 h. Stop the reaction by adding 2.9 ml of sulphate 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 human insulin CRS instead of the substance to be examined.

Examine by liquid chromatography (2.2.29).

The chromatographic procedure may be carried out using:
— a stainless steel column 0.10 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (3 µm),
— as mobile phases at a flow rate of 1 ml/min:

Mobile phase A. Mix 100 ml of acetonitrile for chromatography R, 700 ml of water R and 200 ml of sulphate buffer solution pH 2.0 R; filter and degas,

Mobile phase B. Mix 400 ml of acetonitrile for chromatography R, 400 ml of water R and 200 ml of sulphate buffer solution pH 2.0 R; filter and degas.

Follow the elution conditions as described in the table below (if necessary, the gradient may be modified to improve the separation of the digest):



— as detector a spectrophotometer set at 214 nm,

maintaining the temperature of the column at 40°C.

Equilibrate the column at the initial conditions for at least 15 min. Carry out a blank run using the above-mentioned gradient.

Inject 50 µl of the test solution and 50 µl of the reference solution. The test is not valid unless the chromatogram obtained with each solution is qualitatively similar to the Ph. Eur. reference chromatogram of human insulin digest. In the chromatogram obtained with the reference solution, identify the peaks due to digest fragments I, II and III. The symmetry factor of the peaks due to fragments II and III is not greater than 1.5, and the resolution between the two peaks is at least 3.4.

The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution.

NOTE: the retention time of fragment I is the same for insulin derived from pork as for human insulin. The retention time of fragment II is the same for all insulins. The retention time of fragment III is the same for insulin derived from beef as for insulin derived from pork.


4.0 Impurities with molecular masses greater than that of insulin. Examine by size-exclusion chromatography

Test solution. Dissolve 4 mg of the substance to be examined in 1.0 ml of 0.01M hydrochloric acid.

Resolution solution. Use a solution of insulin (approximately 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 6M hydrochloric acid, containing the indicated percentage of high molecular mass proteins, or a solution prepared from insulin, dissolved in 0.01M 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 ten days.

Maintain the solutions at 2°C to 10°C and use within 7 days. If an automatic injector is used, maintain the temperature at 2°C to 10°C.

The chromatographic procedure may be carried out using:
— a column 0.3M long and at least 7.5 mm in internal diameter packed with hydrophilic silica gel for chromatography R (5 µm to 10 µm), of a grade suitable for the separation of insulin monomer from dimer and polymers,
— as mobile phase at a flow rate of 0.5 ml/min a mixture consisting of 15 volumes of glacial acetic acid R, 20 volumes of acetonitrile R and 65 volumes of a 1.0 g/l solution of arginine R; filter and degas,
— as detector a spectrophotometer set at 276 nm.

Equilibration of the column. Before using a new column for chromatographic analysis, equilibrate by repeated injections of an insulin solution containing high molecular mass proteins. This can be done by at least three injections of the resolution solution. The column is equilibrated when repeatable results are obtained from two subsequent injections.

Inject 100 µl of the resolution solution. When the chromatograms are recorded in the prescribed conditions, the retention times are polymeric insulin complexes: 13 min to 17 min, covalent insulin dimer: about 17.5 min, insulin monomer: about 20 min, salts: about 22 min. The test is not valid unless the resolution, defined by the ratio of the height of the dimer peak to the height above the baseline of the valley separating the monomer and dimer peaks, is at least 2.0.

Inject 100 µl of the test solution. Record the chromatogram for approximately 35 min. In the chromatogram obtained, the sum of the areas of any peak with a retention time less than that of the principal peak is not greater than 1.0 per cent of the total area of the peaks. Disregard any peak with a retention time greater than that of the insulin peak.


5.0 Related Proteins :

Examine by liquid chromatography (2.2.29) as described under Assay, following the elution conditions as described in the table below:


Maintain the solutions at 2°C to 10°C and use within 24 h. Perform a system suitability check (resolution, linearity) as described under Assay. If necessary, the relative proportions of the mobile phases may be adjusted to ensure complete elution of A21 desamido porcine insulin before commencement of the gradient. The profile of the gradient may also be adjusted to ensure complete elution of all insulin related impurities.

Inject 20 µl of reference solution (a), 20 µl of reference solution (b), 20 µl of reference solution (c) and 20 µl of the test solution. If necessary, adjust the injection volume to a volume between 10 µl and 20 µl in accordance with the results obtained in the test for linearity as described under Assay. Record the chromatograms for approximately 50 min. In the chromatogram obtained with reference solution (a), A21 desamido human insulin appears as a small peak after the principal peak and has a retention time of about 1.3 relative to the principal peak. In the chromatogram obtained with the test solution, the area of the peak due to A21 desamido human insulin is not greater than 2.0 per cent of the total area of the peaks; the sum of the areas of all peaks, apart from those due to human insulin and that due to A21 desamido human insulin, is not greater than 2.0 per cent of the total area of the peaks. For semisynthetic human insulin only: in the chromatogram obtained with the test solution, the area of any peak corresponding to the principal peak in the chromatogram obtained with reference solution (b) is not greater than the area of the corresponding peak in the chromatogram obtained with reference solution (c) (1.0 per cent of porcine insulin in human insulin).

6.0 Proinsulin-like immunoreactivity (PLI). Not more than 10 ppm, calculated with reference to the dried substance and determined by a suitably sensitive immunochemical method (2.7.1) such as radio-immunoassay. For human insulin produced by enzymatic modification of porcine insulin use the International Reference Reagent for porcine proinsulin to calibrate the method. For human insulin produced by rDNA technology use the International Reference Reagent for human proinsulin.

7.0 Zinc. Not more than 1.0 per cent of Zn, determined by atomic absorption spectrometry (2.2.23, Method I), calculated with reference to the dried substance.

Test solution. Dissolve 50.0 mg of the substance to be examined in 0.01M hydrochloric acid and dilute to 25.0 ml with the same acid. Dilute if necessary to a suitable concentration (for example 0.4 µg to 1.6 µg of Zn per millilitre) with 0.01M hydrochloric acid.

Reference solutions. Use solutions containing 0.40 µg, 0.80 µg, 1.00 µg, 1.20 µg and 1.60 µg of Zn per millilitre, freshly prepared by diluting zinc standard solution (5 mg/ml Zn) R 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).

8.0 Loss on drying.: Not more than 10.0 per cent, determined on 0.200 g by drying in an oven at 100°C to 105°C for 24 h.

9.0 Sulphated ash : Not more than 2.5 per cent, determined on 0.200 g and calculated with reference to the dried substance.

10.0 Bacterial endotoxins : less than 10 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for removal of bacterial endotoxins. (LAL test)

11.0 ASSAY

Examine by liquid chromatography

Test solution. Dissolve 40.0 mg of the substance to be examined in 0.01M hydrochloric acid and dilute to 10.0 ml with the same solvent.

Reference solution (a). Dissolve the contents of a vial of human insulin CRS in 0.01M hydrochloric acid to obtain a concentration of 4.0 mg/ml.

Reference solution (b). Dissolve the contents of a vial of porcine insulin CRS in 0.01M hydrochloric acid to obtain a concentration of 4.0 mg/ml.

Reference solution (c). Dilute 1.0 ml of reference solution (b) to 50.0 ml with 0.01M hydrochloric acid. To 1.0 ml of this solution add 1.0 ml of reference solution (a).

Reference solution (d). Dilute 1.0 ml of reference solution (a) to 10.0 ml with 0.01M hydrochloric acid.

Resolution solution. Mix 1.0 ml of reference solution (a) and 1.0 ml of reference solution (b).

Maintain the solutions at 2°C to 10°C and use within 48 h. If an automatic injector is used, maintain the temperature at 2°C to 10°C.

The chromatographic procedure may be carried out using:
— a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm),
— as mobile phase at a flow rate of 1 ml/min the following solutions, prepared and maintained at a temperature not lower than 20°C:

Mobile phase A. Dissolve 28.4 g of anhydrous sodium sulphate R in water R and dilute to 1000 ml with the same solvent; add 2.7 ml of phosphoric acid R; adjust the pH to 2.3, if necessary, with ethanolamine R; filter and degas,

Mobile phase B. Mix 550 ml of mobile phase A with 450 ml of acetonitrile R. Warm the solution to a temperature not lower than 20°C in order to avoid precipitation (mixing of mobile phase A with acetonitrile is endothermic); filter and degas,
— as detector a spectrophotometer set at 214 nm,

maintaining the temperature of the column at 40°C.

Elute with a mixture of 42 volumes of mobile phase A and 58 volumes of mobile phase B, adjusted if necessary.

Inject 20 µl of the resolution solution and 20 µl of reference solution (b). Record the chromatogram of the resolution solution until the peak corresponding to the principal peak in the chromatogram obtained with reference solution (b) is clearly visible. In the chromatogram obtained with the resolution solution, identify the peaks due to porcine insulin and human insulin. The test is not valid unless the resolution between the peaks corresponding to human insulin and porcine insulin is at least 1.2. If necessary, adjust the concentration of acetonitrile in the mobile phase until this resolution is achieved.

Inject 20 µl of the test solution, 20 µl of reference solution (a) and 20 µl of reference solution (d). The test is not valid unless the area of the principal peak in the chromatogram obtained with reference solution (a) is 10 ±0.5 times the area of the principal peak in the chromatogram obtained with reference solution (d). If this test fails, adjust the injection volume between 10 µl and 20 µl, in order to be in the linearity range of the detector.

Calculate the content of human insulin C257H383N65O77S6 plus A21 desamido human insulin from the area of the principal peak and the area of the peak corresponding to A21 desamido human insulin in the chromatograms obtained with the test solution and reference solution (a) and the declared content of human insulin plus A21 desamido human insulin in human insulin CRS.


12.0 Host-cell-derived proteins & Host-cell- and vector-derived DNA :
Limit : NMT 10 ppm

For human insulin produced by enzymatic modification of insulin obtained from the pancreas of the pig, the manufacturing process is validated to demonstrate removal of any residual proteolytic activity. The competent authority may require additional tests.

For human insulin produced by a method based on rDNA technology, prior to release the following test is carried out on each batch of the final bulk product unless exemption has been granted by the competent authority.