Insulin Lispro

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

Insulin Lispro is identical in structure to Insulin Human, except that it has Lysine and proline at positions 28 and 29, respectively, of the B-chain, whereas this sequence is reversed in Insulin Human. Insulin Lispro is produced by methods based on recombinant DNA technology. The presence of host cell DNA in Insulin Lispro is process-speci￾c. The capability of the process to clear host-derived DNA requires validation and is determined by validated methods. Its potency is NLT 27.0 USP Insulin Lispro Units/mg, calculated on the dried basis.

[Note—1 USP Insulin Lispro Unit is equivalent to 0.0347 mg of pure Insulin Lispro.]

2 IDENTIFICATION

A. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

B. Physicochemical Analytical Procedures for Insulins 〈121.1〉, Peptide Mapping

Proceed as directed in the chapter, except for the Flow rate in the Chromatographic system and System suitability.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Flow rate: 0.8 mL/min

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 3.4 between digest fragments II and III

Tailing factor: NMT 1.5 for digest fragments II and III

Chromatogram similarity: Identify the peaks due to digest fragments I, II, III, and IV in the Standard solution. The chromatogram of the Standard solution corresponds to that of the typical chromatogram provided with USP Insulin Lispro RS.

Acceptance criteria: Meets the requirements

3 ASSAY

Procedure

Solution A: 28.4 g of anhydrous sodium sulfate in 1000 mL of water. Adjust with phosphoric acid to a pH of 2.3.

Mobile phase: Acetonitrile and Solution A (51:149)

System suitability solution: 1 mg/mL of Insulin Lispro in 0.01 N hydrochloric acid. Allow to stand at room temperature to obtain a solution containing 0.8%–11% of A-21 desamido insulin lispro.

Standard solution: About 0.7 mg/mL of USP Insulin Lispro RS in 0.01 N hydrochloric acid

Sample solution: About 0.8 mg/mL of Insulin Lispro in 0.01 N hydrochloric acid

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 4.6-mm × 10-cm; packing L1

Column temperature: 40°

Flow rate: 0.8 mL/min

Injection volume: 20 µL

System suitability

Adjust the Mobile phase to obtain a retention time of about 24 min for the main insulin lispro peak.

Sample: System suitability solution (3 replicate injections)

Suitability requirements

Resolution: NLT 3.0 between insulin lispro and A-21 desamido insulin lispro

Tailing factor: NMT 1.5 for the insulin lispro peak

Relative standard deviation: NMT 1.1% for the insulin lispro peak

Analysis

Samples: Standard solution and Sample solution

Calculate the potency on the undried basis, in USP Insulin Lispro Units/mg, of insulin lispro in the Sample solution:

Result = (rᵤ / rₛ) × (Cₛ / Cᵤ)

rᵤ = peak response from the Sample solution

rₛ = peak response from the Standard solution

Cₛ = concentration of USP Insulin Lispro RS in the Standard solution (USP Insulin Lispro Units/mL)

Cᵤ = concentration of the Sample solution (mg/mL)

Acceptance criteria: NLT 27.0 USP Insulin Lispro Units/mg on the dried basis

4 OTHER COMPONENTS

Change to read:

▲Zinc Determination 〈591〉▲ (IRA 1-Jan-2019)

Acceptance criteria: 0.30%–0.60% on the dried basis

PRODUCT-RELATED SUBSTANCES AND IMPURITIES

Related Substances

Solvent: 28.4 g of anhydrous sodium sulfate in 1000 mL of water. Adjust with phosphoric acid to a pH of 2.3.

Solution A: Acetonitrile and Solvent (18:82)

Solution B: Acetonitrile and Solvent (50:50)

Mobile phase: See Table 1.

Table 1

System suitability solution: 3.5 mg/mL of Insulin Lispro in 0.01 N hydrochloric acid. Allow to stand at room temperature to obtain a solution containing 0.8%–11% of A-21 desamido insulin lispro.

Sample solution: 3.5 mg/mL of Insulin Lispro in 0.01 N hydrochloric acid.

[Note—Store this solution for NMT 56 h in a refrigerator.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 4.6-mm × 25-cm; packing L1

Column temperature: 40°

Flow rate: 1 mL/min

Injection volume: 20 µL

System suitability

Adjust the Mobile phase composition and the duration of the isocratic elution to obtain a retention time of about 41 min for the main insulin lispro peak, with A-21 desamido insulin lispro eluting just before the start of the gradient elution phase.

Sample: System suitability solution

Suitability requirements

Resolution: NLT 2.5 between insulin lispro and A-21 desamido insulin lispro

Tailing factor: NMT 2.0 for the insulin lispro peak

Analysis

Sample: Sample solution

Calculate the percentage of insulin lispro, A-21 desamido insulin lispro, and other impurities in the portion of Insulin Lispro taken.

Calculate the percentage of insulin lispro (%I):

Result = (rᵢ / rₜ) × 100

rᵢ = peak response of insulin lispro from the Sample solution

rₜ = sum of the responses of all the peaks from the Sample solution

Calculate the percentage of A-21 desamido insulin lispro (%D):

Result = (rᵈ / rₜ) × 100

rᵈ = peak response of A-21 desamido insulin lispro from the Sample solution

Calculate the percentage of other insulin lispro-related substances:

Result = 100 − (%I + %D)

Acceptance criteria

Individual impurities: NMT 1.00% of A-21 desamido insulin lispro

Other individual impurities: NMT 0.50% of any insulin lispro-related substance

Total impurities: NMT 2.00%, excluding A-21 desamido insulin lispro

Physicochemical Analytical Procedures for Insulins 〈121.1〉, Limit of High Molecular Weight Proteins: Meets the requirements

Acceptance criteria: NMT 0.25%

5 PROCESS-RELATED IMPURITIES

Single-Chain Precursor Content: The single-chain precursor content of Insulin Lispro is NMT 10 ng/mg, determined by a validated method.

Host Cell Protein: The residual host cell protein content is NMT 10 ng/mg, determined by a validated method or demonstrated by a validated process.

6 SPECIFIC TESTS

Insulin Assays 〈121〉, Assay, Bioidentity Test

Analysis: Proceed as directed in the chapter, except obtain the ￾rst blood specimen at 45 min, instead of 1 h, after the time of injection.

Acceptance criteria: Meets the requirements

Loss on Drying 〈731〉

Sample: 300 mg

Analysis: Dry the Sample at 105° for 16 h.

Acceptance criteria: NMT 10.0%

Bacterial Endotoxins Test 〈85〉, Photometric Quantitative Techniques, Chromogenic Technique:

NMT 10 USP Endotoxin Units/mg of Insulin Lispro, using the kinetic-chromogenic assay

Microbial Enumeration Tests 〈61〉 and Tests for Specified Microorganisms 〈62〉:

The total aerobic microbial count does not exceed 10² cfu/g, the test being performed on a portion of about 0.3 g, accurately weighed.

7 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers. Store in a freezer and protect from light.

Labeling: Label it to indicate that it has been produced by methods based on recombinant DNA technology.

USP Reference Standards 〈11〉

USP Insulin Lispro RS