{"id":30340,"date":"2025-11-11T16:41:06","date_gmt":"2025-11-11T09:41:06","guid":{"rendered":"https:\/\/nhathuocngocanh.com\/bp\/?p=30340"},"modified":"2025-11-11T16:41:06","modified_gmt":"2025-11-11T09:41:06","slug":"somatropin","status":"publish","type":"post","link":"https:\/\/nhathuocngocanh.com\/bp\/somatropin\/","title":{"rendered":"Somatropin"},"content":{"rendered":"

(Ph. Eur. monograph 0951)<\/p>\n

C_{990<\/sub>H_{1528<\/sub>N_{262<\/sub>O_{300<\/sub>S_{7<\/sub>\u00a0 \u00a0 \u00a0 22\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 125<\/p>\n}}}}}

Action and use<\/strong><\/p>\n

Growth hormone.<\/p>\n

Preparations<\/strong><\/p>\n

Somatropin Injection<\/p>\n

Somatropin Powder for Injection<\/p>\n

DEFINITION<\/h2>\n
Protein having the structure (191 amino-acid residues) of the major component of growth hormone produced by the human pituitary.<\/p>\n
Content<\/h3>\n
91.0 per cent to 105.0 per cent (anhydrous substance).<\/p>\n
By convention, for the purpose of labelling somatropin preparations, 1 mg of anhydrous somatropin (C_{990<\/sub>H_{1528<\/sub>N_{262<\/sub>O_{300<\/sub>S_{7<\/sub>) is equivalent to 3.0 IU of biological activity.<\/p>\n}}}}}
PRODUCTION<\/h2>\n
Somatropin is produced by a method based on recombinant DNA (rDNA) technology. In the course of product development, it must be demonstrated that the manufacturing process produces a product having a biological activity of not less than 2.5 IU\/mg, using a validated bioassay based on growth promotion and approved by the competent authority.<\/p>\n
Somatropin complies with the following additional requirements.<\/p>\n
Host-cell-derived proteins (2.6.34)<\/h3>\n
The limit is approved by the competent authority.<\/p>\n
Host-cell- and vector-derived DNA (2.6.35)<\/h3>\n
The limit is approved by the competent authority.<\/p>\n
CHARACTERS<\/h2>\n
Appearance<\/h3>\n
White or almost white powder.<\/p>\n
IDENTIFICATION<\/h2>\n
A. Capillary electrophoresis (2.2.47) as described in the test for charged variants with the following modifications.<\/p>\n
Injection: Test solution (b); under pressure or vacuum, using the following sequence: sample injection for at least 3 s then CZE buffer injection for 1 s.<\/p>\n
Results: In the electropherogram obtained, only 1 principal peak, corresponding to somatropin, is detected: no doubling of this peak is observed.<\/p>\n
B. Examine the chromatograms obtained in the test for related proteins.<\/p>\n
Results: The principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (a).<\/p>\n
C. Peptide mapping (2.2.55).<\/p>\n
SELECTIVE CLEAVAGE OF THE PEPTIDE BONDS<\/p>\n
Test solution Prepare a solution of the substance to be examined in 0.05 M 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 a suitable material such as polypropylene. Prepare a 1 mg\/mL solution of trypsin for peptide mapping R in 0.05 M tris-hydrochloride buffer solution pH 7.5 R and add 30 \u03bcL to the solution of the substance to be examined. Cap the tube and place in a water-bath at 37 \u00b0C for 4 h. Remove from the water-bath and stop the reaction immediately, for example by freezing. If analysed immediately using an automatic injector, maintain at 2-8 \u00b0C.
\nReference 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.<\/p>\n
CHROMATOGRAPHIC SEPARATION Liquid chromatography (2.2.29).<\/p>\n
Column:<\/p>\n
\u2014 size: l = 0.25 m, \u00d8 = 4.6 mm;<\/p>\n
\u2014 stationary phase: octylsilyl silica gel for chromatography R (5-10 \u03bcm) with a pore size of 30 nm;<\/p>\n
\u2014 temperature: 30 \u00b0C.<\/p>\n
Mobile phase:<\/p>\n
\u2014 mobile phase A: dilute 1 mL of trifluoroacetic acid R to 1000 mL with water for chromatography R;<\/p>\n
\u2014 mobile phase B: to 100 mL of water for chromatography R, add 1 mL of trifluoroacetic acid R and dilute to 1000 mL with acetonitrile R1;<\/p>\n\n\n\n\n\n\n\n
Time<\/strong>
\n(min)<\/strong><\/td>\n Mobile phase A<\/strong>
\n(per cent V\/V)<\/strong><\/td>\n Mobile phase B<\/strong>
\n(per cent V\/V)<\/strong><\/td>\n<\/tr>\n
0 – 20<\/td>\n 100 \u2192 80<\/td>\n 0 \u2192 20<\/td>\n<\/tr>\n
20 – 40<\/td>\n 80 \u2192 75<\/td>\n 20 \u2192 25<\/td>\n<\/tr>\n
40 – 65<\/td>\n 75 \u2192 50<\/td>\n 25 \u2192 50<\/td>\n<\/tr>\n
65 – 70<\/td>\n 50 \u2192 20<\/td>\n 50 \u2192 80<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
Flow rate: 1 mL\/min.<\/p>\n
Detection: Spectrophotometer at 214 nm.<\/p>\n
Injection: 100 \u03bcL.<\/p>\n
System suitability: The chromatogram obtained with the reference solution is qualitatively similar to the chromatogram of somatropin digest supplied with somatropin CRS.<\/p>\n
Results The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution.<\/p>\n
D. Examine the chromatograms obtained in the assay.<\/p>\n
Results: The principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (a).<\/p>\n
TESTS<\/h2>\n
Related proteins<\/h3>\n
Liquid chromatography (2.2.29): use the normalisation procedure. Maintain the solutions at 2-8 \u00b0C and use within 24 h.<\/p>\n
Solution A: Dilute 5 \u03bcL of strong hydrogen peroxide solution R to 100 mL with 0.025 M phosphate buffer solution pH 7.0 R.<\/p>\n
Test solution: Prepare a solution of the substance to be examined in an appropriate buffer, for example 0.025 M phosphate buffer solution pH 7.0 R, to obtain a concentration of 1.6 mg\/mL.<\/p>\n
Reference solution (a): Dissolve the contents of a vial of somatropin CRS in 0.025 M phosphate buffer solution pH 7.0 R to obtain a concentration of 1.6 mg\/mL.<\/p>\n
Reference solution (b): Dissolve the contents of a vial of somatropin CRS in solution A to obtain a concentration of 1.6 mg\/mL. Allow to stand for 1-7 days to obtain a sufficient amount of sulfoxide forms.<\/p>\n
Column:<\/p>\n
\u2014 size: l = 0.25 m, \u00d8 = 4.0 mm;<\/p>\n
\u2014 stationary phase: end-capped octadecylsilyl silica gel for chromatography R (7 \u03bcm) with a pore size of 100 nm;<\/p>\n
\u2014 temperature: 45 \u00b0C.<\/p>\n
Mobile phase:<\/p>\n
\u2014 mobile phase A: dissolve 82.6 g of ammonium sulfate R and 34.5 g of sodium dihydrogen phosphate monohydrate R in 2950 mL of water for chromatography R; add 25.0 mL of perchloric acid R and 2000 mL of acetonitrile R1, and dilute to 5000 mL with water for chromatography R;<\/p>\n
\u2014 mobile phase B: to 4000 mL of acetonitrile R1, add 1000 mL of water for chromatography R;<\/p>\n\n\n\n\n\n\n
Time<\/strong>
\n(min)<\/strong><\/td>\n Mobile phase A<\/strong>
\n(per cent V\/V)<\/strong><\/td>\n Mobile phase B<\/strong>
\n(per cent V\/V)<\/strong><\/td>\n<\/tr>\n
0 – 60<\/td>\n 77 \u2192 72<\/td>\n 23 \u2192 28<\/td>\n<\/tr>\n
60 – 61<\/td>\n 72 \u2192 77<\/td>\n 28 \u2192 23<\/td>\n<\/tr>\n
61 – 72<\/td>\n 77<\/td>\n 23<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
Flow rate: 1.0 mL\/min.<\/p>\n
Detection: Spectrophotometer at 215 nm.<\/p>\n
Autosampler: Set at 2-8 \u00b0C.<\/p>\n
Injection: 20 \u03bcL.<\/p>\n
Relative retention: With reference to somatropin (retention time = about 40 min): [MetO^{125<\/sup> ]somatropin (double peak) = about 0.40; [\u03b2-Asp^{107<\/sup> ]somatropin = about 0.78; [MetO^{14<\/sup>\u00a0]somatropin = about 0.91; 130,131-anhydrosomatropin = about 1.27; [seco-130\/131]somatropin = about 1.31.<\/p>\n}}}
If necessary, adjust the percentage of mobile phase B in the gradient so that the main peak elutes at about 40 min.<\/p>\n
System suitability: Reference solution (b):<\/p>\n
\u2014 peak-to-valley ratio: minimum 2.0, where H_{p<\/sub> = height above the baseline of the peak due to [MetO ]somatropin and H_{v<\/sub> = height above the baseline of the lowest point of the curve separating this peak from the peak due to somatropin.<\/p>\n}}
Limit:<\/p>\n
\u2014 total: maximum 6.0 per cent.<\/p>\n
Dimer and related substances of higher molecular mass<\/h3>\n
Size-exclusion chromatography (2.2.30): use the normalisation procedure.<\/p>\n
Test solution: Prepare a solution of the substance to be examined in 0.025 M phosphate buffer solution pH 7.0 R to obtain a concentration of 1.0 mg\/mL.<\/p>\n
Reference solution (a): Dissolve the contents of a vial of somatropin CRS in 0.025 M phosphate buffer solution pH 7.0 R and dilute with the same solution to obtain a concentration of 1.0 mg\/mL.<\/p>\n
Reference solution (b): Place 1 vial of somatropin CRS in an oven at 50 \u00b0C for a period sufficient to generate 1-2 per cent of dimer (typically 12-24 h). Dissolve its contents in 0.025 M phosphate buffer solution pH 7.0 R and dilute with the same solution to obtain a concentration of 1.0 mg\/mL.<\/p>\n
Column:<\/p>\n
\u2014 size: l = 0.30 m, \u00d8 = 7.8 mm;<\/p>\n
\u2014 stationary phase: hydrophilic silica gel for chromatography R of a grade suitable for fractionation of globular proteins in the relative molecular mass range of 5000 to 150 000.<\/p>\n
Mobile phase: 2-propanol R, 0.063 M phosphate buffer solution pH 7.0 R (3:97 V\/V); filter and degas.<\/p>\n
Flow rate: 0.6 mL\/min.<\/p>\n
Detection: Spectrophotometer at 214 nm.<\/p>\n
Injection: 20 \u03bcL.<\/p>\n
Relative retention: With reference to somatropin monomer (retention time = 12 min to 17 min): related substances of higher molecular mass = about 0.65; somatropin dimer = about 0.9.<\/p>\n
System suitability: Reference solution (b):<\/p>\n
\u2014 peak-to-valley ratio: minimum 2.5, 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.<\/p>\n
Limit:<\/p>\n
\u2014 sum of the peaks with retention times less than that of the principal peak: maximum 4.0 per cent.<\/p>\n
Charged variants<\/h3>\n
Capillary electrophoresis (2.2.47).<\/p>\n
Test solution (a): Prepare a solution of the substance to be examined containing 1 mg\/mL of somatropin.<\/p>\n
Test solution (b): Mix equal volumes of test solution (a) and the reference solution.<\/p>\n
Reference solution: Dissolve the contents of a vial of somatropin CRS in 0.05 M tris-hydrochloride buffer solution pH 7.5 R and dilute with the same solvent to obtain a concentration of 1 mg\/mL.<\/p>\n
Capillary:<\/p>\n
\u2014 material: uncoated fused silica;<\/p>\n
\u2014 size: effective length = at least 70 cm, \u00d8 = 50 \u03bcm.<\/p>\n
Temperature: 30 \u00b0C.<\/p>\n
CZE buffer: 13.2 g\/L solution of ammonium phosphate R adjusted to pH 6.0 with phosphoric acid R and filtered.<\/p>\n
Detection: Spectrophotometer at 200 nm.<\/p>\n
Set the autosampler to store the samples at 4 \u00b0C during analysis.<\/p>\n
Preconditioning of the capillary: Rinse with 1 M sodium hydroxide for 20 min, with water R for 10 min and with the CZE buffer for 20 min.<\/p>\n
Between-run rinsing: Rinse with 0.1 M sodium hydroxide for 2 min and with the CZE buffer for 6 min.<\/p>\n
NOTE: rinsing times may be adapted according to the length of the capillary and the equipment used.<\/p>\n
Injection: Test solution (a) and the reference solution; under pressure or vacuum, using the following sequence: sample injection for at least 3 s then CZE buffer injection for 1 s.<\/p>\n
The injection time and pressure may be adapted in order to meet the system suitability criteria.<\/p>\n
Migration: Apply a field strength of 217 V\/cm (20 kV for capillaries of 92 cm total length) for 80 min, using the CZE buffer as the electrolyte in both buffer reservoirs.<\/p>\n
Relative migration: With reference to somatropin: deamidated forms = 1.02 to 1.11.<\/p>\n
System suitability: Reference solution:<\/p>\n
\u2014 the electropherogram obtained is similar to the electropherogram of somatropin supplied with somatropin CRS. The peak due to deamidated somatropin, I_{4<\/sub>, elutes after and is completely separated from the peak due to somatropin, with a relative migration time (RMT) of at least 1.02. Other commonly observed peaks may include: I_{1<\/sub> (RMT = 0.93 \u2013 0.96), I_{2<\/sub> (RMT = 0.96 \u2013 0.98) and I_{3<\/sub> (RMT = 1.01 \u2013 1.03).<\/p>\n}}}}
NOTE: peak I_{2<\/sub><\/p>\n}
corresponds to the cleaved form and peak I4<\/p>\n
corresponds to the deamidated forms, eluting as a doublet.<\/p>\n
Limits:<\/p>\n
\u2014 deamidated forms: maximum 5.0 per cent;<\/p>\n
\u2014 any other impurity: for each impurity, maximum 2.0 per cent;<\/p>\n
\u2014 total: maximum 10.0 per cent.<\/p>\n
Water (2.5.32)<\/h4>\n
Maximum 10.0 per cent.<\/p>\n
Bacterial endotoxins (2.6.14)<\/h4>\n
Less than 5 IU\/mg, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for removal of bacterial endotoxins.<\/p>\n
ASSAY<\/h2>\n
Size-exclusion chromatography (2.2.30) as described in the test for dimer and related substances of higher molecular mass with the following modification.<\/p>\n
Injection Test solution and reference solution (a).<\/p>\n
Calculate the content of somatropin (C_{990<\/sub>H_{1528<\/sub>N_{262<\/sub>O_{300<\/sub>S_{7<\/sub>) from the assigned content of C_{990<\/sub>H_{1528<\/sub>N_{262<\/sub>O_{300<\/sub>S_{7<\/sub> in somatropin CRS.<\/p>\n}}}}}}}}}}
STORAGE<\/h2>\n
In an airtight container, at a temperature of 2 \u00b0C to 8 \u00b0C. If the substance is sterile, the container is also sterile and tamper- evident.<\/p>\n","protected":false},"excerpt":{"rendered":"
(Ph. Eur. monograph 0951) C990H1528N262O300S7\u00a0 \u00a0 \u00a0 22\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 125 Action and use Growth hormone. Preparations Somatropin Injection Somatropin Powder for Injection DEFINITION Protein having the structure (191 amino-acid residues) of the major component of growth hormone produced by the human pituitary. Content 91.0 per cent to 105.0 per cent (anhydrous substance)….<\/p>\n","protected":false},"author":2,"featured_media":30355,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[174],"tags":[],"class_list":["post-30340","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-medicinal-substances"],"acf":[],"_links":{"self":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/30340","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/comments?post=30340"}],"version-history":[{"count":2,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/30340\/revisions"}],"predecessor-version":[{"id":30357,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/30340\/revisions\/30357"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media\/30355"}],"wp:attachment":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media?parent=30340"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/categories?post=30340"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/tags?post=30340"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}

Content<\/h3>\n91.0 per cent to 105.0 per cent (anhydrous substance).<\/p>\nBy convention, for the purpose of labelling somatropin preparations, 1 mg of anhydrous somatropin (C990<\/sub>H1528<\/sub>N262<\/sub>O300<\/sub>S7<\/sub>) is equivalent to 3.0 IU of biological activity.<\/p>\n

Host-cell-derived proteins (2.6.34)<\/h3>\nThe limit is approved by the competent authority.<\/p>\n

Host-cell- and vector-derived DNA (2.6.35)<\/h3>\nThe limit is approved by the competent authority.<\/p>\n

CHARACTERS<\/h2>\n

Appearance<\/h3>\nWhite or almost white powder.<\/p>\n

TESTS<\/h2>\n

Water (2.5.32)<\/h4>\nMaximum 10.0 per cent.<\/p>\n

Bacterial endotoxins (2.6.14)<\/h4>\nLess than 5 IU\/mg, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for removal of bacterial endotoxins.<\/p>\n

Host-cell-derived proteins (2.6.34)<\/h3>\n
The limit is approved by the competent authority.<\/p>\n

Host-cell- and vector-derived DNA (2.6.35)<\/h3>\n
The limit is approved by the competent authority.<\/p>\n

Appearance<\/h3>\n
White or almost white powder.<\/p>\n

Water (2.5.32)<\/h4>\n
Maximum 10.0 per cent.<\/p>\n

Bacterial endotoxins (2.6.14)<\/h4>\n
Less than 5 IU\/mg, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for removal of bacterial endotoxins.<\/p>\n