NOTE—
One USP Insulin Lispro Unit is equivalent to 0.0347 mg of pure Insulin Lispro.
Packaging and storage—
Preserve in tight containers, protected from light, and store in a freezer.
Labeling—
Label it to indicate that it has been prepared by microbial synthesis.
Identification—
A:
The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
B:
Determine the peptide fragments, using the following peptide mapping procedure.
Sulfate buffer, HEPES buffer, Mobile phase, Test digest solution, and Procedure—
Proceed as directed for
Identification test
B under
Insulin Human.
Chromatographic system—
Proceed as directed for
Identification test
B under
Insulin Human, except to use the following elution program.
Time
(minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
| 0–3 |
95 |
5 |
isocratic |
| 3–30 |
95®41 |
5®59 |
linear gradient |
| 30–35 |
41®20 |
59®80 |
linear gradient |
| 35–40 |
20®95 |
80®5 |
return to initial |
| 40–50 |
95 |
5 |
re-equilibration |
The flow rate is about 0.8 mL per minute.
Bioidentity—
Proceed as directed for
Bioidentity Test under
Insulin Assays 
121
, except to obtain the first blood specimen at 45 minutes, instead of 1 hour, after the time of injection: meets the requirements.
Microbial limits 61—
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
Photometric 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 directed in the test for
Limit of high molecular weight proteins under
Insulin: not more than 0.25% is found.
Related compounds—
Solvent—
Proceed as directed in the Assay.
Solution A—
Prepare a filtered and degassed mixture of Solvent and acetonitrile (82:18).
Solution B—
Prepare a filtered and degassed mixture of Solvent and acetonitrile (50:50).
Mobile phase—
Use variable mixtures of
Solution A and
Solution B as directed for
Chromatographic system. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
System suitability solution—
Dissolve an accurately weighed quantity of Insulin Lispro in 0.01 N hydrochloric acid to obtain a solution containing about 3.5 mg per mL. Allow to stand at room temperature to obtain a solution containing between 0.8% and 11% A-21 desamido insulin lispro.
Test solution—
Dissolve about 3.5 mg of Insulin Lispro in 1.0 mL of 0.01 N hydrochloric acid. Store this solution for not more than 56 hours in a refrigerator.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The column temperature is maintained at 40
, and the flow rate is about 1 mL per minute. The chromatograph is programmed as follows.
Time
(minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
| 0–60 |
81 |
19 |
isocratic |
| 60–83 |
81®51 |
19®49 |
linear gradient |
| 83–84 |
51®81 |
49®19 |
linear gradient |
| 84–94 |
81 |
19 |
re-equilibration |
Adjust the
Mobile phase composition and duration of the isocratic elution to obtain a retention time of about 41 minutes for insulin lispro, with A-21 desamido insulin lispro eluting near the start of the linear gradient phase. Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the resolution,
R, between insulin lispro and A-21 desamido insulin lispro is not less than 2.5; and the tailing factor for the insulin lispro peak is not more than 2.0.
Procedure—
Proceed as directed for
Procedure in the test for
Related compounds under
Insulin: not more than 1.00% of A-21 desamido insulin lispro is found; not more than 0.50% of any other individual insulin lispro related compound is found; and not more than 2.00% of total impurities, excluding A-21 desamido insulin lispro, is found.
Zinc content 591—
Determine the zinc content of about 20 mg of Insulin Lispro, accurately weighed: between 0.30% and 0.60% is found, calculated on the dried basis.
Assay—
Solvent—
Dissolve 28.4 g of anhydrous sodium sulfate in 1000 mL of water, mix, and adjust with phosphoric acid to a pH of 2.3.
Mobile phase—
Mix 745 mL of
Solvent and 255 mL of acetonitrile. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
System suitability solution—
Dissolve an accurately weighed quantity of Insulin Lispro in 0.01 N hydrochloric acid to obtain a solution having a concentration of about 1 mg per mL. Allow to stand at room temperature to obtain a solution containing between 0.8% and 11% A-21 desamido insulin lispro.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Insulin Lispro RS in 0.01 N hydrochloric acid to obtain a solution having a known concentration of about 0.7 mg per mL.
Assay preparation—
Dissolve an accurately weighed portion of Insulin Lispro in 0.01 N hydrochloric acid to obtain a solution having a concentration of about 0.8 mg per mL.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm × 10-cm column that contains packing L1. The column temperature is maintained at 40
, and the flow rate is about 0.8 mL per minute. Adjust the
Mobile phase to provide a retention time of about 24 minutes for the main insulin lispro peak. Chromatograph three replicate injections of the
System suitability solution, and record the peak responses as directed for
Procedure: the resolution,
R, between insulin lispro and A-21 desamido insulin lispro is not less than 3.0; the tailing factor for the insulin lispro peak is not more than 1.5; and the relative standard deviation for replicate injections is not more than 1.1%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the potency, in USP Insulin Lispro Units per mg, on the as-is basis by the formula:
(CS / CU)(rU / rS),
in which
CS is the concentration, in USP Insulin Lispro Units per mL, of USP Insulin Lispro RS in the
Standard preparation; CU is the concentration, in mg per mL, of Insulin Lispro in the
Assay preparation; and
rU and
rS are the insulin lispro peak areas obtained from the
Assay preparation and the
Standard preparation, respectively. From the value obtained in the test for
Loss on drying, calculate the potency on the dried basis.
Auxiliary Information—
Staff Liaison :
Larry N. Callahan, Ph.D., Scientist
Expert Committee : (BBPP05) Biologics and Biotechnology - Proteins and Polysaccharides
USP29–NF24 Page 1136
Pharmacopeial Forum : Volume No. 28(4) Page 1125
Phone Number : 1-301-816-8385
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