{"id":15558,"date":"2025-10-17T15:33:04","date_gmt":"2025-10-17T08:33:04","guid":{"rendered":"https:\/\/nhathuocngocanh.com\/bp\/?p=15558"},"modified":"2025-10-17T15:33:04","modified_gmt":"2025-10-17T08:33:04","slug":"insulin-glargine","status":"publish","type":"post","link":"https:\/\/nhathuocngocanh.com\/bp\/insulin-glargine\/","title":{"rendered":"Insulin Glargine"},"content":{"rendered":"

C267<\/sub>H404<\/sub>N72<\/sub>O78<\/sub>S6<\/sub>\u00a0 \u00a0 \u00a0 \u00a06063<\/p>\n

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

Hormone; treatment of diabetes mellitus.<\/p>\n

Preparation<\/strong><\/p>\n

Insulin Glargine Injection<\/p>\n

DEFINITION<\/h2>\n

21 –A<\/sup>Glycine-30B<\/sup>a-L-arginine-30 B<\/sup>b-L-arginine-insulin (human).<\/p>\n

2-chain peptide containing 53 amino acids. The A-chain is composed of 21 amino acids and the B-chain is composed of 32 amino acids. It is identical in primary structure to human insulin, only differing in amino acid sequence at position 21 in the A-chain and at the C-terminal end of the B-chain where it contains 2 additional amino acids. Human insulin is Asn(A21), whereas insulin glargine is Gly(A21), Arg(B31), Arg(B32). As in human insulin, insulin glargine contains 2 interchain disulfide bonds and 1 intrachain disulfide bond<\/p>\n

Content<\/h3>\n

94.0 per cent to 105.0 per cent (anhydrous substance).<\/p>\n

By convention, for the purpose of labelling insulin glargine preparations, 0.0364 mg of insulin glargine is equivalent to 1 unit.<\/p>\n

PRODUCTION<\/h2>\n

Insulin glargine is produced by a method based on recombinant DNA (rDNA) technology under conditions designed to minimise the degree of microbial contamination.<\/p>\n

Prior to release, the following tests are carried out on each batch of insulin glargine, unless exemption has been granted by the competent authority.<\/p>\n

Host-cell-derived proteins<\/h3>\n

The limit is approved by the competent authority.<\/p>\n

Single-chain precursor<\/h3>\n

The limit is approved by the competent authority. Use a suitably sensitive method.<\/p>\n

CHARACTERS<\/h2>\n

Appearance<\/h3>\n

White or almost white, hygroscopic powder.<\/p>\n

Solubility<\/h3>\n

Practically insoluble in water and in anhydrous ethanol, soluble in dilute mineral acids.<\/p>\n

IDENTIFICATION<\/h2>\n

A. 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 to the principal peak in the chromatogram obtained with the reference solution.<\/p>\n

B. Peptide mapping (2.2.55).<\/p>\n

SELECTIVE CLEAVAGE OF THE PEPTIDE BONDS<\/p>\n

Test solution: Prepare a 10.0 mg\/mL solution of the substance to be examined in a 1 g\/L solution of hydrochloric acid R and transfer 5 \u03bcL of the solution to a clean tube. Add 1.0 mL of 1 M tris-hydrochloride buffer solution pH 7.5 R and 100 \u03bcL of a 20 U\/mL solution of Staphylococcus aureus strain V8 protease, type XVII-B R in 1 M tris-hydrochloride buffer solution pH 7.5 R. Mix and incubate at 45 \u00b0C for about 2 h. Stop the reaction by adding 2 \u03bcL of phosphoric acid R.<\/p>\n

Reference solution: Prepare at the same time and in the same manner as for the test solution but using insulin glargine CRS instead of the substance to be examined.<\/p>\n

CHROMATOGRAPHIC SEPARATION. Liquid chromatography (2.2.29).<\/p>\n

Buffer solution: Dissolve 11.6 g of phosphoric acid R and 42.1 g of sodium perchlorate R in 1600 mL of water for chromatography R, adjust to pH 2.3 with triethylamine R and dilute to 2000 mL with water for chromatography R.<\/p>\n

Column:<\/p>\n

\u2014 size: l = 0.125 m, \u00d8 = 3.0 mm;<\/p>\n

\u2014 stationary phase: spherical end-capped octadecylsilyl silica gel for chromatography R (4 \u03bcm);<\/p>\n

\u2014 temperature: 35 \u00b0C.<\/p>\n

Mobile phase:<\/p>\n

\u2014 mobile phase A: acetonitrile R1, buffer solution (7:93 V\/V);<\/p>\n

\u2014 mobile phase B: buffer solution, acetonitrile R1 (43:57 V\/V);<\/p>\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 – 30<\/td>\n90 \u2192 20<\/td>\n10 \u2192 80<\/td>\n<\/tr>\n
30 – 35<\/td>\n20<\/td>\n80<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Flow rate: 0.6 mL\/min.<\/p>\n

Detection: Spectrophotometer at 214 nm.<\/p>\n

Equilibration: At initial conditions for at least 15 min.<\/p>\n

Injection: 50 \u03bcL.<\/p>\n

Retention time: Insulin glargine fragment II = about 14 min; insulin glargine fragment III = about 15 min.<\/p>\n

System suitability:<\/p>\n

\u2014 the chromatogram obtained with the reference solution is qualitatively similar to the chromatogram of insulin glargine digest supplied with insulin glargine CRS;<\/p>\n

\u2014 in the chromatogram obtained with the reference solution, identify the peaks due to digest fragments II and III: symmetry factor Maximum 1.5 for the peaks due to fragments II and III; resolution Minimum 3.4 between the peaks due to fragments II and III.<\/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

NOTE: the retention times of fragments I and IV are the same as for human insulin; the retention times of fragments II and III differ from human insulin due to the difference in the sequence at position 21 of the A-chain and to the 2 additional amino acids of the B-chain.<\/p>\n

TESTS<\/h2>\n

Impurities with molecular masses greater than that of insulin glargine<\/h3>\n

Size-exclusion chromatography (2.2.30): use the normalisation procedure.<\/p>\n

Test solution: Prepare a 4 mg\/mL solution of the substance to be examined in a 1 g\/L solution of hydrochloric acid R.<\/p>\n

Resolution solution: Dry about 200 mg of the substance to be examined in an oven at 100 \u00b0C for 1.5-3 h. Dissolve 15.0 mg of the dried substance in 1.5 mL of a 1 g\/L solution of hydrochloric acid R and dilute to 10.0 mL with water R.<\/p>\n

Reference solution: Dilute 1.0 mL of the test solution to 100.0 mL with water R. Dilute 3.0 mL of this solution to 20.0 mL with water R.<\/p>\n

Column:<\/p>\n

\u2014 size: l = 0.3 m, \u00d8 = 7.8 mm;<\/p>\n

\u2014 stationary phase: hydrophilic silica gel for chromatography R (10 \u03bcm) with a pore size of 12.5 nm, of a grade suitable for fractionation of globular proteins in the relative molecular mass range of 5000 to 150 000.<\/p>\n

Mobile phase: Mix 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.<\/p>\n

Flow rate: 0.5 mL\/min.<\/p>\n

Detection: Spectrophotometer at 276 nm.<\/p>\n

Injection: 100 \u03bcL.<\/p>\n

Run time: About 35 min.<\/p>\n

Retention time: Insulin glargine = about 18 min.<\/p>\n

System suitability:<\/p>\n

\u2014 signal-to-noise ratio: minimum 10 for the principal peak in the chromatogram obtained with the reference solution;<\/p>\n

\u2014 symmetry factor: maximum 2.0 for the peak due to insulin glargine in the chromatogram obtained with the resolution solution;<\/p>\n

\u2014 peak-to-valley ratio: minimum 2.0, where Hp<\/sub> = height above the baseline of the peak due to high molecular mass proteins and Hv<\/sub> = height above the baseline of the lowest point of the curve separating this peak from the peak due to insulin glargine in the chromatogram obtained with the resolution solution.<\/p>\n

Limits:<\/p>\n

\u2014 total of impurities with a retention time less than that of insulin glargine: maximum 0.3 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 glargine.<\/p>\n

Related proteins<\/h3>\n

Liquid chromatography (2.2.29): use the normalisation procedure. Maintain the solutions at 2-8 \u00b0C.<\/p>\n

Test solution: Dissolve 15.0 mg of the substance to be examined in 1.5 mL of a 1 g\/L solution of hydrochloric acid R and dilute to 10.0 mL with water R.<\/p>\n

Reference solution: Dissolve the contents of a vial of insulin glargine CRS in 1.5 mL of a 1 g\/L solution of hydrochloric acid R, transfer the solution with water R to a 10 mL volumetric flask and dilute to 10.0 mL with water R.<\/p>\n

Resolution solution: Dissolve the contents of a vial of insulin glargine for peak identification CRS (containing 0 -Arg- insulin glargine) in 0.3 mL of a 1 g\/L solution of hydrochloric acid R and add 1.7 mL of water R.<\/p>\n

Buffer solution: Dissolve 20.7 g of anhydrous sodium dihydrogen phosphate R in 900 mL of water for chromatography R, adjust to pH 2.5 with phosphoric acid R and dilute to 1000 mL with water for chromatography R.<\/p>\n

Column:<\/p>\n

\u2014 size: l = 0.25 m, \u00d8 = 3.0 mm;<\/p>\n

\u2014 stationary phase: spherical end-capped octadecylsilyl silica gel for chromatography R (4 \u03bcm);<\/p>\n

\u2014 temperature: 35 \u00b0C.<\/p>\n

Mobile phase:<\/p>\n

\u2014 mobile phase A: dissolve 18.4 g of sodium chloride R in 250 mL of the buffer solution, add 250 mL of acetonitrile R1 and mix; dilute to 1000 mL with water for chromatography R;<\/p>\n

\u2014 mobile phase B: dissolve 3.2 g of sodium chloride R in 250 mL of the buffer solution, add 650 mL of acetonitrile R1 and mix; dilute to 1000 mL with water for chromatography R;<\/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>\n96 \u2192 83<\/td>\n4 \u2192 17<\/td>\n<\/tr>\n
20 – 30<\/td>\n83 \u2192 63<\/td>\n17 \u2192 37<\/td>\n<\/tr>\n
30 – 33<\/td>\n63 \u2192 96<\/td>\n37 \u2192 4<\/td>\n<\/tr>\n
33 – 40<\/td>\n96<\/td>\n4<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Flow rate: 0.6 mL\/min.<\/p>\n

Detection: Spectrophotometer at 214 nm.<\/p>\n

Injection: 5 \u03bcL of the test solution and the resolution solution.<\/p>\n

Retention time: Insulin glargine = about 20 min.<\/p>\n

System suitability: Resolution solution:<\/p>\n

\u2014 peak-to-valley ratio: minimum 2, where Hp<\/sub> = height above the baseline of the peak due to 0 -Arg-insulin glargine and Hv<\/sub> = height above the baseline of the lowest point of the curve separating this peak from the peak due to insulin glargine.<\/p>\n

Limits:<\/p>\n

\u2014 any impurity: for each impurity, maximum 0.4 per cent;<\/p>\n

\u2014 total: maximum 1.0 per cent.<\/p>\n

Zinc<\/h3>\n

Maximum 0.80 per cent.<\/p>\n

Atomic absorption spectrometry (2.2.23, Method I).<\/p>\n

Test solution: Dissolve 45.0 mg of the substance to be examined in a 1 g\/L solution of hydrochloric acid R and dilute to 50.0 mL with the same solution. Dilute 10.0 mL of the solution to 100.0 mL with a 1 g\/L solution of hydrochloric acid R.<\/p>\n

Reference solutions: Prepare reference solutions containing 0.2 \u03bcg, 0.4 \u03bcg and 0.6 \u03bcg of zinc per millilitre by diluting zinc standard solution (10 ppm Zn) R with a 1 g\/L solution of hydrochloric acid R.<\/p>\n

Source: Zinc hollow-cathode lamp.<\/p>\n

Wavelength: 213.9 nm.<\/p>\n

Atomisation device: Air-acetylene flame of suitable composition (for example, 11 L of air and 2 L of acetylene per minute).<\/p>\n

Water (2.5.32)<\/h4>\n

Maximum 8.0 per cent, determined on 30.0 mg.<\/p>\n

Bacterial endotoxins (2.6.14, Method D)<\/h4>\n

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.<\/p>\n

ASSAY<\/h2>\n

Liquid chromatography (2.2.29) as described in the test for related proteins with the following modification.<\/p>\n

Injection: 5 \u03bcL of the test solution and the reference solution.<\/p>\n

Calculate the content of insulin glargine (C267<\/sub>H404<\/sub>N72<\/sub>O78<\/sub>S6<\/sub>) taking into account the assigned content of insulin glargine CRS.<\/p>\n

STORAGE<\/h2>\n

In an airtight container, protected from light, at a temperature of -20 \u00b1 5 \u00b0C.<\/p>\n","protected":false},"excerpt":{"rendered":"

C267H404N72O78S6\u00a0 \u00a0 \u00a0 \u00a06063 Action and use Hormone; treatment of diabetes mellitus. Preparation Insulin Glargine Injection DEFINITION 21 –AGlycine-30Ba-L-arginine-30 Bb-L-arginine-insulin (human). 2-chain peptide containing 53 amino acids. The A-chain is composed of 21 amino acids and the B-chain is composed of 32 amino acids. It is identical in primary structure to human insulin, only differing…<\/p>\n","protected":false},"author":2,"featured_media":15576,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[1],"tags":[],"class_list":["post-15558","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-volumes-1-2"],"acf":[],"_links":{"self":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/15558","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=15558"}],"version-history":[{"count":2,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/15558\/revisions"}],"predecessor-version":[{"id":15581,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/posts\/15558\/revisions\/15581"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media\/15576"}],"wp:attachment":[{"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/media?parent=15558"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/categories?post=15558"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/nhathuocngocanh.com\/bp\/wp-json\/wp\/v2\/tags?post=15558"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}