Enoxaparin Sodium Injection

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

Enoxaparin Sodium Injection is a sterile solution of Enoxaparin Sodium in Water for Injection. Its appearance is analyzed for clarity and degree of color, using a validated method. Its potency value is NLT 90% and NMT 110% of the potency stated on the label in terms of International Anti-Factor Xa Units (IU). It may contain, in multiple-dose containers, a suitable antimicrobial preservative, such as benzyl alcohol.

2 IDENTIFICATION

A.

Analysis: Transfer the total contents of a single-dose container or 0.4 ml. from a multiple-dose container to a glass test tube, add 2 ml of water and 1 ml of 2% (w/v) Protamine sulfate solution, and mix.

Acceptance criteria: A creamy white precipitate is formed.

Change to read:

B.SPECTROSCOPIC IDENTIFICATION TESTS (197), Ultraviolet-Visible Spectroscopy: 197U 

Medium: 0.01 N hydrochloric acid

Standard solution: 500 µg/ml

Sample solution: Transfer the total content of a single-dose container or 0.4 mi from a multiple-dose container to a 100-ml volumetric flask. Dilute with Medium to volume.

Acceptance criteria: The spectra exhibit maxima at 231 ±2 nm.

C. IDENTIFICATION TESTS GENERAL, Sodiumi (191): Meets the requirements

3 ASSAY

ANTI-FACTOR XA ACTIVITY

Acetic acid solution: Glacial acetic acid and water (42:58)

pH 7.4 polyethylene glycol 6000 buffer: Dissolve 6.08 g of tris(hydroxymethyl)aminomethane and 8.77 g of sodium chloride in 500 ml of water. Add 1.0 1.0 g of polyethylene glycol 6000, adjust with hydrochloric acid to a pH of 7.4, and dilute with water to 1000 mL

pH 7.4 buffer: Dissolve 6.08 g of tris(hydroxymethyl) aminomethane and 8.77 g of sodium chloride in 500 ml of water. Adjust with hydrochloric acid to a pH of 7.4, and dilute with water to 1000 ml

pH 8.4 buffer: Dissolve 3.03 g of tris(hydroxymethyl) aminomethane, 5.12 g of sodium chloride, and 1.40 g of edetate sodium in 250 mL of water. Adjust with hydrochloric acid to a pH of 8.4, and dilute with water to 500 mL.

Human antithrombin III solution: Reconstitute a vial of antithrombin III (see Reagents, Indicators, and Solutions-Reagent Specifications) in water to obtain a solution containing 5 Antithrombin III Units/mL. Dilute this solution with pH 7.4 polyethylene glycol 6000 buffer to obtain a solution having a concentration of 1.0 Antithrombin III Unit/mL.

Factor Xa solution: Reconstitute a weighed quantity of bovine factor Xa (see Reagents. Indicators, and Solutions-Reagent Specifications) in pH 7.4 polyethylene glycol 6000 buffer to obtain a solution that gives an increase in absorbance value at 405 nm of NMT 0.20 absorbarice units/min when assayed as described below but using as an appropriate volume, V, the volume in ul of pH 7.4 buffer instead of V ul. of the enoxaparin solution

Chromogenic substrate solution: Prepare a solution of a suitable chromogenic substrate for an amidolytic test (see Reagents Indicators and Solutions-Reagent Specifications) for Factor Xa in water to obtain a concentration of about 3 mM. Dilute with pH 8.4 buffer to obtain a solution having a concentration of 0.5 mM

Standard solutions: Reconstitute the entire contents of an ampul of USP Enoxaparin Sodium for Bioassays RS with water, and dilute with pH

7,4 buffer to obtain four dilutions in the concentration range between 0.025 and 0.2 Anti-Factor Xa IU/mL.

Sample solutions: Proceed as directed for Standard solutions to obtain concentrations of Injection similar to those obtained for the Standard solutions.

Analysis

Samples: Standard solutions, Sample solutions, Human antithrombin III solution, pH 7.4 buffer, Factor Xasolution, Chromogenic substrate solution, and Acetic acid solution

Label 18 suitable tubes: B1 and B2 for blanks T1, T2, T3, and T4 each in duplicate for the dilutions of the Sample solutions, and S1, S2, S3, and S4 each in duplicate for the dilutions of the Standard solutions. [NOTE-Treat the tubes in the order B1, S1, S2, S3, S4, T1, T2, T3, T4, T1, T2, T3, T4, S1, S2, S3, S4, B2) To each tube add the same volume, V (20-50 µL), of Human antithrombin III solution and an equal volume, V, of either the blank (pH 7.4 buffer) or an appropriate dilution of the Sample solutions or the Standard solutions. Mix, but do not allow bubbles to form. Incubate at 37° for 1.0 min. Add to each tube 2V (40-100 µL) of Factor Xa solution, and incubate for 1.0 min.

Add a 5V (100-250 at) volume of Chromogenic substrate soutan Stop the reaction after 4.0 min with a 5V (100-250 µL) volume of Acetic acid solution. Measure the absorbance of each solution at 405 cm, using a suitable spectrophotometer (see Lau Sorcimamoc/857) against blank 81. The mading of blank B2 relative so blank B1 in NMT ±0.05 absorbence uelt.

Calculations: For each series, calculate the regression of the absorbance against log concentrations of the Sample solutions and of the Standard solutions and calculate the potency of the enoxaparin sodium in the Injection in I of Anti-Factor Xa activity/mi, seing statistical methods for parallelline assays. The four independent log relative potency estimates are then combined to obtain the final geometric mean its confidence limits are calculated.

Acceptance criteria: The potency in NLT 90% and NMT 110% of the potency stated on the label in terms of international Anti-Factor Xa Units (IU).

Anti-Factor Xa to Anti-Factor IIa Ratio:: The ratio of the numencal value of the Anti Factor Xa activity in Anti Factor Xa 1/ml to the numeric Value of the Ant-Factor la activity in Anti-Factor Ba Umi, as determined by Ante-Factor Xe Activity and Ant-Factor ila Activity, nespectively is NLT 3.3 and NMT 5.3.

4 OTHER COMPONENTS

Benzyl Alcohol Content (if Present)

Mobile phase: Acetonitrile, methanol, and water (3:1:16)

Standard solution 1.5 mg/mL of USP Benzyl Alcohol in Mobile phase

Sample solution: Transfer exactly 5.0 ml, of the injection to a 50 ml volumetric flask. Dilute with Mobile phose to volume

Chromatographic system

Mode: LC

Detector: UV 250 nm

Column: 4.6mm 15-cm stainless steet packing L7

Flow rate: 1.0 m/min, maintained constant to ±10%

Injection volume: 20 μL

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage (w/v) of benzyl alcohol in the portion of injection taken

Result = (r_U/(r_S) x C

r_U = peak area of benzyl alcohol from the Sample solution

r_S = peak area of benzyl alcohol from the Standard solution

C = concentration of benzyl alcohol in the Standard solution (mg/mL)

Acceptance criteria: 1.35%-1.65%

5 SPECIFIC TESTS

pH (791): 5.5-7.5

Bacterial Endotoxins Test (85): It contains less than 01 USP Endotoxinfurt of Anti-Factorta activity in Anti-factor Xa

Anti-Factor IIa Activity

Acetic acid solution, pH 7.4 polyethylene glycol 6000 buffer, pH 7.4 buffer, pH 8.4 buffer, and Human antithrombin III solution: Proceed as directed in the Assay for Anti-factor Xa Activity, except that the concentration of the Human antithrombin al aslution is 11.5 Antithrombin

Thrombin human solutions: Reconstitute thrombin human (see Reaperta inabcatoca and Solutions Hempert Specificationiclm waher, and dilute in pH 7. polyethylene glycol 6000 buffer to obtain a solution having a concentration of 5 Thrombin Unite/m

Chromogenic substrate solution: Prepare a solution of a suitable chromogenic substrate for an amidolytic test (see Snoozets

Bolduce Haugere Spectations) for thrombin in water so obtain a concentration of about 3 m. immediately before use, dilute with pH 8.4 buffer to 0.5mM.

Standard solutions: Reconstitute the entire contents of an ampul of LS Bronowin Sodian for Blossays RS with water, and dilute with per 74 buffer to obtain four dilutions having concentrations in the range between 1018 and 0.075 I of Anti-Factor lis activity/mL.

Sample solutions: Proceed as directed under Standard solutions to obtain concentrations of Injection similar to those obtained for the Standard solutions.

Analysis: Proceed as directed in the Assay for Anti Factor Xe Activity, except to use Thrombin human solution instead of Factor Ka solution and to use Human anatomber solution as described above

Calculations: For each series, calculate the regression of the absorbance againat log concentrations of the Sample solutions and of the Standard solutions, and calculate the potency of the enoxaparin sodium in the injection in I of Anti-Factor la activity/im, using statistical methods for paralel line assays. The four independent dilution estimates are then combined to obtain the final weighted mean. Then calculate the confidence limits.

Acceptance criteria: The Ant-Factor la activity (or U/mL) is NLT 20.0% and NMT 35.0% of the poterity stated on the label in terms of international Anti-Factor Xa Units (IU or IU/mL).

Free Sulfate Content

Mobile phane: 3.0 mM sodium carbonate solution

Bystem sultability solution: 3 µg/mL of sulfate anion and 5 µg/ml of oxalate anion

Standard sulfate stock solution: Prepare a solution of sodium sulfate in Mobile phase in a suitable sulfate-free container such that the concentration of sulfate is acconately nown at about 1 mg/ml. Transfer 5 g of the sulution to a similar container, and add Mobile phase to obtain 25 g of solution

Standard solution A: 0.1 μg/g of sulfate from Standard sulfate stock solution in Mobile phase

Standard solution B: 0.5 μg/g of sulfate from Standard sulfate stock solution in Mobile phase

Standard solution C: 1 μg/g of sulfate from Standard sulfate stock solution in Mobile phase

Standard solution D: 2 μg/g of sulfate from Standard sulfate stock solution in Mobile phase

Standard solution E: 4 μg/g of sulfate from Standard sulfate stock solution in Mobile phase

Standard solution F: 5 μg/g of sulfate from Standard sulfate stock solution in Mobile phase

Sample solution: Transter a known quantity, m. of Enoxaparin Sodium Injection, accurately weighed, to a suitable previously tared sulfate-free val. Add Mobile phase to obtain a solution having a known concentration of about 10 mg/g

Chromatographic system

(See Chromatography (621), System suitability.)

Detector: Conductivity

Column

Guard: 4-mm × 5-cm; packing L61

Analytical: 4-mm × 25-cm; packing L61

[Note—Use a micromembrane anion autosuppressor² or a suitable chemical suppression system.]

Flow rate: 2.0 mL/min

Injection size: 25 μL

System suitability

Sample: System suitability solution

Suitability requirements

Resolution: NLT I between the sulfate and oxalate peaks

Analysis

Samples: Standard solutions A-F and Sample solution

Pict the standard curve of sulfate peak height at a function of sulfate concentration in ug/g) in Standard zolutions A-F. From the sulfate peak height determine the concentration of suffiste, C, in ugig, in the Sample solution, using the standard curve.

Calculate the percentage of free sulfate content (w/w) in the portion of injection taken:

Result = [[(C x M_s/10m)]]

M_s = total mass of the Sample solution (g)

m = mass of injection taken to prepare the Sample solution (mg)

Acceptance criteria: The percentage of free sulfate in NMT 0.12% (w/w)

Sterility Tests (71): Meets the requirements

Particulate Matter in Injections (788): Meets the requirements

Other Requirements: It meets the requirements under Injections and Implanted Drug Products (1).

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in single-dose or multiple-dose containers in Type I glass. Store between 20° and 25°, excursions permitted between 15° and 30°.

Labeling: Label it to indicate the amount (mg) of Enoxaparin Sodium in the total volume of contents. The label states also that the Enoxaparin Sodium starting material is porcine derived.