3509-3510 Insulin Lispro

USP 35

Official Monographs / Insulin 3509

responses as directed for Procedure: the resolution, R, between Standard digest solution—Proceed as directed for Identification human insulin and A-21 desamido human insulin is not less test B under Insulin Human , except to use USP Insulin Lispro RS than 2.0; the tailing factor for the human insulin peak is be-instead of USP Insulin Human RS.

tween 0.8 and 1.5; and the relative standard deviation for repli-Chromatographic system—Proceed as directed for Identifica-cate injections is not more than 1.6%.

tion test B under Insulin Human , except to use the following Procedure—Separately inject equal volumes (about 50 µL) of elution program.

the Soluble insulin test solution and the Total insulin test solution,record the chromatograms, and measure the peak responses for Time Solution A Solution B insulin and A-21 desamido insulin. Calculate the amount of sol-(minutes)

(%) (%)Elution uble insulin human as a per cent of the total human insulin con-0–3955isocratic

tent of the Injection by the formula:

3–3095→41 5→59linear gradient 30–3541→2059→80linear gradient (100D S /D T )(r S /r T )

35–4020→9580→5return to initial in which D S and D T are the dilution factors for the Soluble insulin 40–50

95

5

re-equilibration

test solution and Total insulin test solution, respectively; and r S and r T are the human insulin peak responses obtained from the The flow rate is about 0.8 mL per minute.

Soluble insulin test solution and the Total insulin test solution,Bioidentity—Proceed as directed for Bioidentity Test under In-respectively. The percent of soluble insulin human is in the sulin Assays 〈121〉, except to obtain the first blood specimen at range L ± 5, where L is the per cent of soluble insulin human 45 minutes, instead of 1 hour, after the time of injection: meets stated on the product label.

the requirements.

Assay—Proceed as directed in the Assay under Insulin Human Microbial enumeration tests 〈61〉 and Tests for specified Injection .

microorganisms 〈62〉—The total aerobic microbial count does not exceed 100 per g, a portion of about 0.3 g, accurately weighed, being used.

Bacterial endotoxins 〈85〉: not more than 10 USP Endotoxin Units per mg, the kinetic-chromogenic method under Photomet-Insulin Lispro

ric Techniques being used.

Loss on drying 〈731〉—Dry about 300 mg, accurately

weighed, at 105° for 16 hours: it loses not more than 10.0% of its weight.

Limit of high molecular weight proteins—Proceed as di-C 257H 383N 65O 77S 65807.58

rected in the test for Limit of high molecular weight proteins Insulin (human), 28B -L -lysine-29B -L -proline-.

under Insulin: not more than 0.25% is found.28B -L -Lysine-29B -L -prolineinsulin (human) [133107-64-9].

Related compounds—

» Insulin Lispro is identical in structure to Insulin Solvent—Proceed as directed in the Assay.

Human, except that it has lysine and proline at Solution A—Prepare a filtered and degassed mixture of Sol-positions 28 and 29, respectively, of chain B,vent and acetonitrile (82:18).

whereas this sequence is reversed in Insulin Solution B—Prepare a filtered and degassed mixture of Sol-vent and acetonitrile (50:50).

Human. Insulin Lispro is produced by microbial Mobile phase—Use variable mixtures of Solution A and Solu-synthesis via a recombinant DNA process. Its po-tion B as directed for Chromatographic system. Make adjust-tency is not less than 27.0 USP Insulin Lispro ments if necessar y (see System Suitability under Chromatography Units per mg, calculated on the dried basis. The 〈621〉).

proinsulin content of Insulin Lispro, determined System suitability solution—Dissolve an accurately weighed by an appropriate and validated method, is not quantity of Insulin Lispro in 0.01 N hydrochloric acid to obtain more than 10 ppm. The host cell-derived protein a solution containing about 3.5 mg per mL. Allow to stand at room temperature to obtain a solution containing between content, determined by an appropriate and vali-0.8% and 11% A-21 desamido insulin lispro.

dated method, is not more than 10 ppm.

Test solution—Dissolve about 3.5 mg of Insulin Lispro in 1.0NOTE —One USP Insulin Lispro Unit is equivalent mL of 0.01 N hydrochloric acid. Store this solution for not more to 0.0347 mg of pure Insulin Lispro.

than 56 hours in a refrigerator.

Chromatographic system (see Chromatography 〈621〉)—The Packaging and storage—Preserve in tight containers, pro-liquid chromatograph is equipped with a 214-nm detector and tected from light, and store in a freezer.

a 4.6-mm × 25-cm column that contains packing L1. The col-Labeling—Label it to indicate that it has been prepared by umn temperature is maintained at 40°, and the flow rate is microbial synthesis.

about 1 mL per minute. The chromatograph is programmed as follows.

USP Reference standards 〈11〉—USP Endotoxin RS USP Insulin Lispro RS Time Solution A Solution B (minutes) (%) (%)Elution Identification—

0–608119isocratic

A: The retention time of the major peak in the chromato-60–8381→5119→49linear gradient gram of the Assay preparation corresponds to that in the chro-matogram of the Standard preparation, as obtained in the Assay.83–8451→8149→19linear gradient B: Determine the peptide fragments, using the following 84–94

81

19

re-equilibration

peptide mapping procedure.

Adjust the Mobile phase composition and duration of the iso-Sulfate buffer , HEPES buffer , Mobile phase , Test digest solution ,cratic elution to obtain a retention time of about 41 minutes for and Procedure—Proceed as directed for Identification test B insulin lispro, with A-21 desamido insulin lispro eluting near the under Insulin Human.

start of the linear gradient phase. Chromatograph the System suitability solution , and record the peak responses as directed for