1.0 Description : A white or almost white powder.
2.0 Identification: A. Examine the electropherograms obtained in the test for isoform distribution. In the electropherogram obtained with test solution (a), the principal band corresponds in position to that in the electropherogram obtained with reference solution (a).
B. 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.
C. Examine by peptide mapping.
Test solution. Prepare a solution of the substance to be examined in 0.05M tris- hydrochloride buffer solution pH 7.5 R to obtain a solution containing 2.0 mg/ml of somatropin and transfer about 1.0 ml to a tube made from suitable material such as polypropylene. Prepare a 1 mg/ml solution of trypsin for peptide mapping R in 0.05M tris-hydrochloride buffer solution pH 7.5 R and add 30 µl to the solution of the substance to be examined. Cap the tube and place in a water-bath at 37°C for 4 h. Remove from the water-bath and stop the reaction immediately, for example by freezing. If analyzed immediately using an automatic injector, maintain the temperature at 2°C to 8°C.
Reference solution. Prepare at the same time and in the same manner as for the test solution but using somatropin CRS instead of the substance to be examined.
Examine by liquid chromatography
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 octylsilyl silica gel for chromatography R (5 µm to 10 µm),
— as mobile phase at a flow rate of 1 ml/min:
Mobile phase A. Dilute 1 ml of trifluoroacetic acid R to 1000 ml with water R,
Mobile phase B. To 100 ml of water R add 1 ml of trifluoroacetic acid R and dilute to 1000 ml with acetonitrile for chromatography R,
following the elution conditions as described in the table below (if necessary, the gradient or the temperature of the column may be modified to improve separation of the digest):
— as detector a spectrophotometer set at 214 nm,
maintaining the temperature of the column at 30°C.
Equilibrate the column with mobile phase A for at least 15 min. Carry out a blank run using the above-mentioned gradient.
Inject 100 µl of the test solution and 100 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 somatropin digest. The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution.
D. Examine the chromatograms obtained in the assay. 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.
3.0 Related proteins. Examine by liquid chromatography
Test solution. Prepare a solution of the substance to be examined in 0.05M tris-hydrochloride buffer solution pH 7.5 R, containing 2.0 mg/ml of somatropin.
Reference solution. Prepare a solution of somatropin CRS in 0.05M tris-hydrochloride buffer solution pH 7.5 R, containing 2.0 mg/ml of somatropin.
Resolution solution (somatropin/desamido-somatropin resolution mixture). Prepare a solution of somatropin CRS in 0.05M tris-hydrochloride buffer solution pH 7.5 R to obtain a 2.0 mg/ml solution of somatropin. Either filter through a sterile filter or add sodium azide R to a concentration of 0.1 mg/ml and allow to stand at room temperature for 24 h.
Maintain the solutions at 2°C to 8°C and use within 24 h. If an automatic injector is used, maintain the temperature at 2°C to 8°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 a suitable singly end-capped butylsilyl silica gel, with a granulometry of 5 µm and a porosity of 30 nm. A silica saturation column is to be placed between the pump and the injector valve,
— as mobile phase at a flow rate of 0.5 ml/min a mixture of 29 volumes of propanol R and 71 volumes of 0.05M tris-hydrochloride buffer solution pH 7.5 R,
— as detector a spectrophotometer set at 220 nm,
maintaining the temperature of the column at 45°C.
Prior to use, rinse the column with 200 ml to 500 ml of a 0.1 per cent V/V solution of trifluoroacetic acid R in a 50 per cent V/V solution of acetonitrile R. Repeat as necessary, to improve column performance.
Inject 20 µl of the reference solution. If necessary, adjust the concentration of propanol R in the mobile phase so that the retention time of the principal peak is about 33 min.
Inject 20 µl of the resolution solution. Desamido-somatropin appears as a small peak at a retention time of about 0.85 relative to the principal peak. The test is not valid unless the resolution between the peaks corresponding to somatropin and desamido-somatropin is at least 1.0 and the symmetry factor of the somatropin peak is 0.9 to 1.8.
Inject 20 µl of the test solution. In the chromatogram obtained, the sum of the areas of all the peaks, apart from the principal peak, is not greater than 6.0 per cent of the total area of the peaks. Disregard any peak due to the solvent.
4.0 Dimer and related substances of higher molecular mass. Examine by size-exclusion chromatography as described under Assay.
Inject 20 µl of the test solution. In the chromatogram obtained with the test solution, the sum of the areas of any peak with a retention time less than that of the principal peak is not greater than 4.0 per cent of the total area of the peaks. Disregard any peak due to the solvent.
5.0 Isoform distribution. Examine by isoelectric focusing.
Test solution (a). Prepare a solution of the substance to be examined in 0.025M phosphate buffer solution pH 7.0 R, containing 2.0 mg/ml of somatropin.
Test solution (b). Add 0.1 ml of test solution (a) to 1.9 ml of 0.025M phosphate buffer solution pH 7.0 R.
Reference solution (a). Prepare a solution of somatropin CRS in 0.025M phosphate buffer solution pH 7.0 R, containing 2.0 mg/ml of somatropin.
Reference solution (b). Use an isoelectric point calibration solution in the pH range of 2.5 to 6.5, prepared and used according to the manufacturer's instructions.
Operate the apparatus in accordance with the manufacturer's instructions. The isoelectric focusing procedure may be carried out using a pre-cast gel 245 mm × 110 mm × 1 mm, with a pH in the range 4.0 to 6.5. Apply to the gel 15 µl of each solution. Use as the anode solution a 14.7 g/l solution of glutamic acid R in phosphoric acid (50 g/l H3PO4) and as the cathode solution an 89.1 g/l solution of b-alanine R. Adjust the operating conditions to 2000 V and 25 mA. Allow focusing to take place for 2.5 h at a constant voltage and at a power of not more than 25 W. Immerse the gel for 30 min in a solution containing 115 g/l of trichloroacetic acid R and 34.5 g/l of sulphosalicylic acid R, and then for 5 min in a mixture of 8 volumes of acetic acid R, 25 volumes of ethanol R and 67 volumes of deionised water R (de-stain solution). Stain the gel by immersion in a 1.15 g/l solution of acid blue 83 R in de-stain solution at 60°C for 10 min, and then place the gel in de-stain solution until excess stain is removed.
The test is not valid unless the distribution of bands in the electropherogram obtained with reference solution (b) corresponds to the manufacturer's indications. The electropherogram obtained with reference solution (a) contains a major band with an isoelectric point of approximately five, and a slightly more acidic minor band at approximately 4.8. In the electropherogram obtained with test solution (a), no band apart from the major band is more intense than the major band in the electropherogram obtained with test solution (b) (5 per cent).
6.0 Water Lt :Not more than 10.0%
Determined by the micro determination of water.
7.0 Sterility Lt shall be complies
If intended for use in the manufacture of parenteral dosage forms without a further appropriate sterilisation procedure, it complies with the test for sterility.
8.0 Bacterial endotoxins Lt : Shall be Less than 5 IU/mg
if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for removal of bacterial endotoxins.
9.0 Assay Lt: Not less than 91.0% and not more than 105.0%
(on anhydrous basis)
Examine by size-exclusion chromatography
Test solution. Prepare a solution of the substance to be examined in 0.025M phosphate buffer solution pH 7.0 R, containing 1.0 mg/ml of somatropin.
Reference solution. Dissolve the contents of a vial of somatropin CRS in 0.025M phosphate buffer solution pH 7.0 R and dilute with the same solvent to obtain a concentration of 1.0 mg/ml.
Resolution solution. Place one vial of somatropin CRS in an oven at 50°C for a period (typically between 12 and 24 h) sufficient to generate 1 per cent to 2 per cent of dimer. Dissolve its contents in 0.025M phosphate buffer solution pH 7.0 R and dilute with the same solvent to obtain a concentration of 1.0 mg/ml.
The chromatographic procedure may be carried out using:
— a stainless steel column 0.30 m long and 7.8 mm in internal diameter packed with hydrophilic silica gel for chromatography R of a grade suitable for fractionation of globular proteins in the molecular mass range 5000 to 150 000,
— as mobile phase at a flow rate of 0.6 ml/min a mixture (filtered and degassed) consisting of 3 volumes of 2-propanol R and 97 volumes of 0.063M phosphate buffer solution pH 7.0 R,
— as detector a spectrophotometer set at 214 nm.
Inject 20 µl of the resolution solution. In the chromatogram obtained, the main peak elutes at a retention time of approximately 12 min to 17 min and the peaks corresponding to the somatropin dimer and to the higher molecular weight proteins at relative retention times of 0.90 and 0.65 respectively, relative to the main peak. The resolution, defined by the ratio of the height above the baseline of the valley separating the monomer and dimer peaks to the height of the dimer peak, is not greater than 0.4.
Inject 20 µl of the test solution and 20 µl of the reference solution.
Calculate the content of somatropin (C990H1528N262O300S7) from the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of (C990H1528N262O300S7) in somatropin CRS.
10.0 Host-cell-derived proteins. The limit is approved by the competent authority.
11.0 Host-cell- and vector-derived DNA. The limit is approved by the competent authority.
No comments:
Post a Comment