Human Insulin Isophane Suspension and Human Insulin Injection
» Human Insulin Isophane Suspension and Human Insulin Injection is a sterile buffered suspension of Insulin Human, complexed with Protamine Sulfate, in a solution of Insulin Human. Its potency, based on the sum of its insulin and desamido insulin components, as determined in the Assay, is not less than 95.0 percent and not more than 105.0 percent of the potency stated on the label, expressed in USP Insulin Human Units in each mL.
Packaging and storage—
Preserve in the unopened, multiple-dose container provided by the manufacturer. Store in a refrigerator, protect from sunlight, and avoid freezing.
Labeling—
The Injection container label states that the Injection is to be properly resuspended before use. Label it to indicate that it has been prepared with Insulin Human of semisynthetic origin (i.e., derived by enzyme modification of pork pancreas insulin) or with Insulin Human of recombinant DNA origin (i.e., obtained from microbial synthesis), whichever is applicable. Label it to state that it is to be stored in a refrigerator and that freezing is to be avoided. The label states the potency in USP Insulin Human Units per mL and the percent ratio of human insulin isophane suspension to soluble human insulin injection.
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Identification—
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.
Bacterial endotoxins 
85
—
It contains not more than 80 USP Endotoxin Units per 100 USP Insulin Human Units.
85—
It contains not more than 80 USP Endotoxin Units per 100 USP Insulin Human Units.
Sterility 71—
It meets the requirements of the test for Sterility under Isophane Insulin Suspension.
71—
It meets the requirements of the test for Sterility under Isophane Insulin Suspension.
pH 791:
between 7.0 and 7.8, determined potentiometrically.
791:
between 7.0 and 7.8, determined potentiometrically.
Zinc content 591:
between 0.02 mg and 0.04 mg for each 100 USP Insulin Human Units.
591:
between 0.02 mg and 0.04 mg for each 100 USP Insulin Human Units.
Limit of high molecular weight proteins—
Proceed as directed in the test for Limit of high molecular weight proteins under Insulin Injection: not more than 3.0% is found.
Soluble insulin human content—
[note—Use one of the two methods listed below. ]
method 1—
Mobile phase, System suitability solution, and Chromatographic system—
Proceed as directed in the Assay under Insulin.
Soluble insulin test solution—
Maintain the temperature at 25 ± 1
throughout the procedure. Transfer 5.0 mL of the Injection to a centrifuge tube. Add 20 µL of 1 N sodium hydroxide, and adjust with 0.05 N hydrochloric acid or 0.05 N sodium hydroxide to a pH of 8.20 ± 0.02 if the total zinc concentration is approximately 20 µg per mL or adjust to a pH of 8.35 ± 0.02 if the total zinc concentration is approximately 30 µg per mL. Record the volume, in µL, of acid or base needed to adjust the pH. Mix, and allow to stand for 1 hour. Centrifuge, transfer the supernatant to another centrifuge tube, and repeat the centrifugation. Transfer 2 mL of the supernatant to another tube, add 5 µL of 9.6 N hydrochloric acid, and mix.
throughout the procedure. Transfer 5.0 mL of the Injection to a centrifuge tube. Add 20 µL of 1 N sodium hydroxide, and adjust with 0.05 N hydrochloric acid or 0.05 N sodium hydroxide to a pH of 8.20 ± 0.02 if the total zinc concentration is approximately 20 µg per mL or adjust to a pH of 8.35 ± 0.02 if the total zinc concentration is approximately 30 µg per mL. Record the volume, in µL, of acid or base needed to adjust the pH. Mix, and allow to stand for 1 hour. Centrifuge, transfer the supernatant to another centrifuge tube, and repeat the centrifugation. Transfer 2 mL of the supernatant to another tube, add 5 µL of 9.6 N hydrochloric acid, and mix.
Total insulin test solution—
Transfer 2 mL of Injection to a suitable vessel, add 5 µL of 9.6 N hydrochloric acid, and allow the suspension to clarify. Dilute the resulting solution with 0.01 N hydrochloric acid to the same theoretical concentration of insulin as the Soluble insulin test solution (e.g., if the Injection is labeled to contain 20% soluble insulin, the dilution factor is 100/20 = 5).
Procedure—
Separately inject equal volumes (about 20 µL) of the Soluble insulin test solution and Total insulin test solution into the chromatograph, record the chromatograms, and measure the peak responses for insulin and A-21 desamido insulin. Calculate the amount of soluble insulin human as a percent of the total insulin content of the Injection by the formula:
(100/D)[(5020 + VA)/5000](rS / rT)
in which D is the dilution factor for the Total insulin test solution; VA is the number of µL added to adjust the pH of the Soluble insulin test solution; and rS and rT are the responses of the Soluble insulin test solution and the Total insulin test solution, respectively. The percent of soluble insulin human is in the range L ± 5, where L is the percent of soluble insulin human stated on the product label.
method 2—
Mobile phase—
Proceed as directed in the Assay under Insulin.
0.1 M Tris buffer solution—
Dissolve 3.54 ± 0.01 g of Tris(hydroxymethyl)aminomethane hydrochloride and 3.34 ± 0.01 g of Tris(hydroxymethyl)aminomethane in 500 mL of water. The pH of the 0.1 M Tris buffer solution must be between 8.15 and 8.35. If the pH is outside of this range, discard the solution and prepare fresh; do not adjust the pH.
System suitability solution—
Dissolve about 0.14 mg of Insulin in 1.0 mL of 0.01 N hydrochloric acid. Allow to stand at room temperature for not less than 3 days to obtain a solution containing not less than 5% of A-21 desamido insulin.
Soluble insulin test solution—
Dilute a suitable volume of Injection with 0.1 M Tris buffer solution to obtain a solution containing about 6 USP Insulin Human Units of soluble insulin per mL (e.g., 2 mL of 70/30 Human Insulin Isophane Suspension and Human Insulin Injection containing 100 USP Insulin Human Units per mL would be diluted with 8 mL of 0.1 M Tris buffer solution to obtain a filtrate that contains 6 USP Insulin Human Units of soluble insulin per mL). Immerse the container in a water bath at 25 ± 1 for 30 ± 2 minutes. Immediately pass this solution through a 0.2-µm filter using a disposable syringe. Transfer 2 parts of the filtrate to a suitable vessel, and add 1 part 0.2 N hydrochloric acid (e.g., the dilution factor for the Soluble insulin test solution that contains 30% soluble insulin is 5 × 3/2 = 7.5).
for 30 ± 2 minutes. Immediately pass this solution through a 0.2-µm filter using a disposable syringe. Transfer 2 parts of the filtrate to a suitable vessel, and add 1 part 0.2 N hydrochloric acid (e.g., the dilution factor for the Soluble insulin test solution that contains 30% soluble insulin is 5 × 3/2 = 7.5).
Total insulin test solution—
For each mL of Injection, add 3.0 µL of 9.6 N hydrochloric acid, mix, and allow the suspension to clarify. Dilute the resulting solution with 0.01 N hydrochloric acid to 4 USP Insulin Human Units per mL (e.g., if the product is labeled to contain a total of 100 USP Insulin Human Units per mL, the dilution factor is 25).
Chromatographic system—
Proceed as directed in the Assay under Insulin. Make adjustments as necessary (see Chromatography 621) to obtain a retention time for human insulin between 10 and 17 minutes. Chromatograph five replicate injections of the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between human insulin and A-21 desamido human insulin is not less than 2.0; the tailing factor for the human insulin peak is between 0.8 and 1.5; and the relative standard deviation for replicate injections is not more than 1.6%.
621) to obtain a retention time for human insulin between 10 and 17 minutes. Chromatograph five replicate injections of the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between human insulin and A-21 desamido human insulin is not less than 2.0; the tailing factor for the human insulin peak is between 0.8 and 1.5; and the relative standard deviation for replicate injections is not more than 1.6%.
Procedure—
Separately inject equal volumes (about 50 µL) of the Soluble insulin test solution and the Total insulin test solution, record the chromatograms, and measure the peak responses for insulin and A-21 desamido insulin. Calculate the amount of soluble insulin human as a percent of the total human insulin content of the Injection by the formula:
(100DS / DT)(rS / rT)
in which DS and DT are the dilution factors for the Soluble insulin test solution and Total insulin test solution, respectively; and rS and rT are the human insulin peak responses obtained from the Soluble insulin test solution and the Total insulin test solution, respectively. The percent of soluble insulin human is in the range L ± 5, where L is the percent of soluble insulin human stated on the product label.
Assay—
Proceed as directed in the Assay under Insulin Human Injection.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Thomas A. Sigambris, M.S.
Scientific Liaison 1-301-998-6789 |
(BIO12010) Monographs - Biologics and Biotechnology 1 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
|
| 85 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 71 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
USP35–NF30 Page 3508
Pharmacopeial Forum: Volume No. 34(4) Page 941