Insulinum humanum - uspbpep.com

1 insulin , human EUROPEAN PHARMACOPOEIA 01/2005:0838 B. Peptide mapping ( ). SELECTIVE CLEAVAGE OF THE PEPTIDE BONDS. insulin , HUMAN Test solution. Prepare a mg/ml solution of the substance to be examined in M hydrochloric acid Insulinum humanum and transfer 500 l of this solution to a clean tube. Add ml of HEPES buffer solution pH R and 400 l 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 ml of sulphate buffer solution pH 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. CHROMATOGRAPHIC SEPARATION. Liquid chromatography ( ). Column : size : l = m, = mm, C257H383N65O77S6 Mr 5808 stationary phase : octadecylsilyl silica gel for chromatography R (3 m) with a pore size of 8 nm, DEFINITION temperature : 40 C.

2 Human insulin is a 2-chain peptide having the structure of Mobile phase : the antidiabetic hormone produced by the human pancreas. mobile phase A : mix 100 ml of acetonitrile for Content : per cent to per cent of human insulin chromatography R, 200 ml of sulphate buffer solution C257H383N65O77S6 plus A21 desamido human insulin (dried pH R and 700 ml of water R ; filter and degas ;. substance). mobile phase B : mix 200 ml of sulphate buffer solution By convention, for the purpose of labelling insulin pH R, 400 ml of acetonitrile for chromatography R. preparations, mg of human insulin is equivalent to and 400 ml of water R ; filter and degas ;. 1 IU of insulin . Time Mobile phase A Mobile phase B. PRODUCTION (min) (per cent V/V) (per cent V/V). Human insulin is produced either by enzymatic modification 0 - 60 90 30 10 70. and suitable purification of insulin obtained from the 60 - 65 30 0 70 100.

3 Pancreas of the pig or by a method based on recombinant DNA (rDNA) technology. 65 - 70 0 100. Human insulin is produced under conditions designed to Flow rate : 1 ml/min. minimise the degree of microbial contamination. For human insulin produced by enzymatic modification Detection : spectrophotometer at 214 nm. of insulin obtained from the pancreas of the pig, the Equilibration : at initial conditions for at least 15 min. manufacturing process is validated to demonstrate Carry out a blank run using the above-mentioned removal of any residual proteolytic activity. The competent gradient. authority may require additional tests. Injection : 50 l. For human insulin produced by a method based on System suitability : rDNA technology, prior to release the following tests are carried out on each batch of the final bulk product, unless the chromatograms obtained with the test solution exemption has been granted by the competent authority.

4 And the reference solution are qualitatively similar to the chromatogram of human insulin digest supplied Host-cell-derived proteins. The limit is approved by the with human insulin CRS, competent authority. Single chain precursor. The limit is approved by the in the chromatogram obtained with the reference competent authority. Use a suitably sensitive method. solution, identify the peaks due to digest fragments I, II and III : CHARACTERS symmetry factor : maximum for the peaks due to Appearance : white or almost white powder. fragments II and III, Solubility : practically insoluble in water and in ethanol resolution : minimum between the peaks due to (96 per cent). It dissolves in dilute mineral acids and with fragments II and III. decomposition in dilute solutions of alkali hydroxides. Results : the profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram IDENTIFICATION obtained with the reference solution.

5 A. Examine the chromatograms obtained in the assay. NOTE : the retention time of fragment I is the same for Results : the principal peak in the chromatogram obtained porcine insulin and for human insulin . The retention with the test solution is similar in retention time to times of fragments II and IV are the same for all insulins. the principal peak in the chromatogram obtained with The retention time of fragment III is the same for bovine reference solution (a). insulin and for porcine insulin . 1800 See the information section on general monographs (cover pages). EUROPEAN PHARMACOPOEIA insulin , human TESTS Maintain the solutions at 2-8 C and use within 24 h. Perform Impurities with molecular masses greater than that of a system suitability test (resolution, linearity) as described insulin . Size-exclusion chromatography ( ) : use the in the assay. If necessary, the relative proportions of the normalisation procedure.

6 Mobile phases may be adjusted to ensure complete elution of A21 desamido porcine insulin before commencement of the Test solution. Prepare a solution containing 4 mg/ml of the gradient. The profile of the gradient may also be adjusted to substance to be examined in M hydrochloric acid. ensure complete elution of all insulin related impurities. Resolution solution. Use a solution of insulin Inject 20 l of reference solution (a), 20 l of reference (about 4 mg/ml), containing more than per cent solution (b), 20 l of reference solution (c) and 20 l of the of high molecular mass proteins. An injectable insulin test solution. If necessary, adjust the injection volume to preparation, whether a solution or a suspension, that has a volume between 10 l and 20 l in accordance with the been clarified with a sufficient amount of 6 M hydrochloric results obtained in the test for linearity as described in the acid, containing the indicated percentage of high molecular assay.

7 Record the chromatograms for approximately 50 min. mass proteins, or a solution prepared from insulin , dissolved In the chromatogram obtained with reference solution (a), in M hydrochloric acid, may be used. insulin containing A21 desamido human insulin appears as a small peak the indicated percentage of high molecular mass proteins after the principal peak and has a retention time of about may be prepared by allowing insulin powder to stand at room relative to the principal peak. In the chromatogram temperature for about 10 days. obtained with the test solution, the area of the peak due to Maintain the solutions at 2-8 C and use within 7 days. If A21 desamido human insulin is not greater than per an automatic injector is used, maintain the temperature cent of the total area of the peaks ; the sum of the areas of at 2-8 C. all peaks, apart from those due to human insulin and that Column : due to A21 desamido human insulin , is not greater than per cent of the total area of the peaks.

8 For semisynthetic size : l = m, = minimum mm, human insulin only : in the chromatogram obtained with stationary phase: hydrophilic silica gel for the test solution, the area of any peak corresponding to chromatography R (5-10 m) with a pore size of the principal peak in the chromatogram obtained with nm, of a grade suitable for the separation of reference solution (b) is not greater than the area of the insulin monomer from dimer and polymers. corresponding peak in the chromatogram obtained with reference solution (c) ( per cent of porcine insulin in Mobile phase : mix 15 volumes of glacial acetic acid R, human insulin ). 20 volumes of acetonitrile R and 65 volumes of a g/l solution of arginine R ; filter and degas. The following test applies only to human insulin produced by enzymatic modification of porcine insulin . Flow rate : ml/min. Proinsulin-like immunoreactivity (PLI) : maximum 10 ppm, Detection : spectrophotometer at 276 nm.

9 Calculated with reference to the dried substance and Equilibration: before using a new column for determined by a suitably sensitive immunochemical method chromatographic analysis, equilibrate by repeated injections ( ) such as radio-immunoassay. Use the International of an insulin solution containing high molecular mass Reference Reagent for porcine proinsulin to calibrate the proteins. This can be done by at least 3 injections of the method. resolution solution. The column is equilibrated when repeatable results are obtained from 2 subsequent injections. Zinc: maximum per cent (dried substance). Atomic absorption spectrometry ( , Method I). Injection : 100 l. Test solution. Dissolve mg of the substance to be Run time : about 35 min. examined in M hydrochloric acid and dilute to Retention time : polymeric insulin complexes = 13-17 min ; ml with the same acid. Dilute if necessary to a suitable covalent insulin dimer = about min ; insulin concentration (for example, g of Zn per millilitre).

10 Monomer = about 20 min ; salts = about 22 min. with M hydrochloric acid. System suitability : resolution solution : Reference solutions. Use solutions containing g, peak-to-valley ratio : minimum , where Hp = height g, g, g and g of Zn per millilitre, above the baseline of the peak due to the dimer and freshly prepared by diluting zinc standard solution (5 mg/ml Hv = height above the baseline of the lowest point of Zn) R with M hydrochloric acid. the curve separating this peak from the peak due to the Source : zinc hollow-cathode lamp. monomer. Wavelength : nm. Limits : the sum of the areas of any peaks with a retention Atomisation device : air-acetylene flame of suitable time less than that of the principal peak is not greater than composition (for example, 11 litres of air and 2 litres of per cent of the total area of the peaks. Disregard any acetylene per minute). peak with a retention time greater than that of the peak due to insulin .