Isophane Insulin Suspension

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION 

Isophane Insulin Suspension is a sterile suspension of zinc–insulin crystals and Protamine sulfate in buffered Water for Injection, combined in a manner such that the solid phase of the suspension consists of crystals composed of insulin, protamine, and zinc. The Protamine Sulfate is prepared from the sperm or from the mature testes of sh belonging to the genus Oncorhynchus Suckley, or Salmo L. (Fam. Salmonidae). Its potency, based on the sum of its insulin and desamido insulin components, is NLT 95.0% and NMT 105.0% of the potency stated on the label, expressed in USP Insulin Units/mL. 

2 IDENTIFICATION 

Change to read: 

The retention time of the insulin pork peak of Sample solution A or Sample solution B corresponds to that of the main peak of the Identification solution, as obtained in the Assay, and no other significant peaks are observed.  [Note —It may be necessary to inject a mixture of Sample solution and Identification solution.] 

3 ASSAY 

Change to read: 

Procedure 

Solution A: Dissolve 28.4 g of anhydrous sodium sulfate in 1000 mL of water. Pipet 2.7 mL of phosphoric acid into the solution, and adjust with ethanolamine to a pH of 2.3, if necessary. 

Mobile phase: Acetonitrile and Solution A (26:74). [Note—The acetonitrile is warmed to NLT 20° to avoid precipitation.] System suitability solution: 1.5 mg/mL of   insulin pork in 0.01 N hydrochloric acid.   Allow to stand at room temperature for NLT 3 days to obtain a solution containing NLT 5% of A-21 desamido insulin. 

Identification solution: 0.6 mg/mL of   USP Insulin Pork RS in 0.01 N hydrochloric acid. [Note—The Identification solution may be stored at room temperature for up to 12 h or in a refrigerator for up to 48 h.] 

Standard solution: 1.5 mg/mL of   USP Insulin Pork RS in 0.01 N hydrochloric acid.   Sample solution A (for Suspension labeled as containing 40 USP Insulin Units/mL): Add 2.5 µL of 9.6 N hydrochloric acid for each milliliter of an accurately measured volume of Suspension. Allow the suspension to clarify, and mix. 

Sample solution B (for Suspension labeled as containing 100 USP Insulin Units/mL): Add 2.5 µL of 9.6 N hydrochloric acid for each milliliter of an accurately measured volume of Suspension. Allow the suspension to clarify, and mix. [Note—Pooling several package units may be necessary to obtain sufficient volume of the sample.] Pipet 2 mL of this solution into a 5-mL volumetric flask, dilute with 0.01 N hydrochloric acid to volume, and mix. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 214 nm 

Column: 4.6-mm × 15-cm; packing L1 

Column temperature: 40° 

Flow rate: 1 mL/min 

Injection volume: 20 µL 

System suitability 

Samples: System suitability solution and Standard solution 

Suitability requirements 

Resolution: NLT 2.0 between insulin and A-21 desamido insulin, System suitability solution 

Tailing factor: NMT 1.8 for the insulin peak, System suitability solution 

Relative standard deviation: NMT 1.6%, Standard solution 

Analysis 

Samples: Identification solution, Standard solution, and either Sample solution A or Sample solution B 

Measure the peak responses for insulin and A-21 desamido insulin   , using the chromatogram of the Identification solution to identify the insulin peaks. 

Calculate the potency, in USP Insulin Units/mL, in the portion of Suspension taken: 

Result = (Σr_U/Σr_S) × C_S × D 

r_U = sum of the peak responses of insulin human and A-21 desamido insulin human from the Sample solution 

r_S = sum of the peak responses of insulin human and A-21 desamido insulin human from the Standard solution 

C_S = concentration of USP Insulin Human RS in the Standard solution (USP Insulin Human Units/mL) 

D = dilution factor used to prepare the Sample solution 

Acceptance criteria: 95.0%–105.0% of the potency stated on the label, expressed in USP Insulin Units/mL 

4 OTHER COMPONENTS 

Zinc Determination 〈591〉: 10–40 µg for every 100 USP Insulin Units 

5 PRODUCT-RELATED SUBSTANCES AND IMPURITIES 

Physicochemical Analytical Procedures for Insulins 〈121.1〉, Limit of High Molecular Weight Proteins: Proceed as directed in the chapter, except for the Sample solution. It meets the requirements. 

Sample solution: Quantitatively add 4 µL of 6 N hydrochloric acid to each milliliter of an accurately measured volume of Suspension, and mix. Acceptance criteria: NMT 3.0% 

6 SPECIFIC TESTS 

Insulin in the Supernatant 

Sample solution: Centrifuge 10 mL of the Suspension at 1500 × g for 10 min. Use the supernatant. 

Analysis: Determine the insulin content of the Sample solution by a suitable method. 

Acceptance criteria: NMT 1.0 USP Insulin Unit/mL 

pH 〈791〉: 7.0–7.8 

Bacterial Endotoxins Test 〈85〉: NMT 80 USP Endotoxin Units per 100 USP Insulin Units 

Sterility Tests 〈71〉, Test for Sterility of the Product to Be Examined, Membrane Filtration: Meets the requirements when tested as directed in the chapter and the Suspension being filtered immediately after it has been put into a solution using a validated suitable solvent 

7 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in the unopened, multiple-dose container provided by the manufacturer. Do not repackage. Store in a refrigerator, protect from sunlight, and avoid freezing. 

Change to read: 

Labeling: Label it as   porcine   . If the Isophane Insulin Suspension is made from insulin that is purified, label it as such. The Suspension container label states that the Suspension is to be shaken carefully before use. 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 Units/mL. Change to read: 

USP Reference Standards 〈11〉 

USP Insulin Pork RS