USP肝素钠

Heparin Sodium (hep' a rin soe' dee um). DEFINITION Heparin Sodium is the sodium salt of sulfated glycosaminoglycans present as a mixture of heterogeneous molecules varying in molecular weights that retains a combination of activities against different factors of the blood clotting cascade. It is present in mammalian tissues and is usually obtained from the intestinal mucosa or other suitable tissues of domestic mammals used for food by man. The sourcing of heparin material must be specified in compliance with applicable regulatory requirements. The manufacturing process should be validated to demonstrate clearance and inactivation of relevant infectious and adventitious agents (e.g., viruses, TSE agents). See Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin 1050 for general guidance on viral safety evaluation. It is composed of polymers of alternating derivatives of -D-glucosamido (N-sulfated, O- sulfated, or N-acetylated) and O-sulfated uronic acid ( -L-iduronic acid or -D-glucuronic acid). The component activities of the mixture are in ratios corresponding to those shown by USP Heparin Sodium for Assays RS. Some of these components have the property of prolonging the clotting time of blood. This occurs mainly through the formation of a complex of each component with the plasma proteins antithrombin and heparin cofactor II to potentiate the inactivation of thrombin (factor IIa). Other coagulation proteases in the clotting sequence, such as activated factor X (factor Xa), are also inhibited. The ratio of anti-factor Xa activity to anti-factor IIa potency is between 0.9 and 1.1. The potency of Heparin Sodium, calculated on the dried basis, is NLT 180 USP Heparin Units in each mg. IDENTIFICATION • A. 1H NMR SPECTRUM (See Nuclear Magnetic Resonance 761 .) Standard solution: NLT 20 mg/mL of USP Heparin Sodium Identification RS in deuterium oxide with 0.02% (w/v) deuterated trimethylsilylpropionic (TSP) acid sodium salt System suitability solution: Prepare 1% (w/w) USP Oversulfated Chondroitin Sulfate RS in Standard solution. Sample solution: NLT 20 mg/mL of Heparin Sodium in deuterium oxide with 0.02% (w/v) deuterated TSP Spectrometric conditions (See Nuclear Magnetic Resonance 761 .) Mode: NMR, pulsed (Fourier transform) Frequency: NLT 500 MHz (for 1H) Temperature: 25 System suitability Samples: Standard solution and System suitability solution Using a pulsed (Fourier transform) NMR spectrometer operating at NLT 500 MHz for 1H, acquire a free induction decay (FID) using NLT 16 scans using a 90 pulse and 20-s delay. Record the 1H NMR spectra of the Standard solution and System suitability solution at 25 . Collect the 1H NMR spectrum with a spectral window of at least 10 to 2 ppm and without spinning. The number of transients should be adjusted until the signal-to-noise ratio of the N-acetyl heparin signal in the Standard solution is at least 1000/1 in the region near 2 ppm. The Standard solution shall be run at least daily when Sample solutions are being run. For all samples, the TSP methyl signal should be set to 0.00 ppm. The chemical shift for the N-acetyl resonance of heparin and oversulfated 页码，1/9(W)w 2010/11/26file://C:\com_caislabs_ebk\data\v34290\usp34nf29s0_m36690.html chondroitin sulfate in the System suitability solution should be observed at 2.05 ± 0.02 and 2.16 ± 0.03 ppm, respectively. Record the 1H NMR spectrum of the Sample solution at 25 . Draw a baseline from 8.00 ppm to 0.10 ppm. The ppm values for H1 of GlcNAc/GlcNS, 6S (signal 1), H1 of IdoA2S (signal 2), the H2 of GlcNS (signal 3), and the methyl of GlcNAc (signal 4) of heparin are present at 5.42, 5.21, 3.28 (doublet centered at 3.28 ppm), and 2.05 ppm, respectively.1 The ppm values of these signals do not differ by more than ±0.03 ppm. Measure the signal heights above the baseline of signal 1 and signal 2, and calculate the mean of these signal heights. Other signals of variable heights and ppm values, attributable to heparin and HOD, may be seen between signal 2 and 4.55 ppm. Residual solvent signals may be observed in the 0.10–3.00 range. Heparin Sodium must meet the requirements stated in Residual Solvents 467 . Suitability requirements Number of transients: Adjust until the signal-to-noise ratio of the N-acetyl heparin signal in the Standard solution is at least 1000/1 in the region near 2 ppm. Chemical shift: The TSP methyl signal should be set to 0.00 ppm for all samples. Chemical shifts (for the N-acetyl resonance of heparin and oversulfated chondroitin sulfate): Should be observed at 2.05 ± 0.02 and 2.16 ± 0.03 ppm, respectively, System suitability solution Analysis Sample: Sample solution Acceptance criteria: No unidentified signals greater than 4% of the mean of signal height of 1 and 2 are present in the following ranges: 0.10–2.00, 2.10–3.20, and 5.70–8.00 ppm. No signals greater than 200% signal height of the mean of the signal height of 1 and 2 are present in the 3.35–4.55 ppm for porcine heparin. • B. CHROMATOGRAPHIC IDENTITY Solution A: Dissolve 0.8 g of monobasic sodium phosphate dihydrate in 2 L of water and adjust with phosphoric acid to a pH of 3.0. Pass the solution through a filter membrane with pore sizes of 0.45 µm and degas before use. Solution B: Dissolve 0.8 g of monobasic sodium phosphate dihydrate and 280 g of sodium perchlorate monohydrate in 2 L of water and adjust with phosphoric acid to a pH of 3.0. Pass the solution through a filter membrane with pore sizes of 0.45 µm and degas before use. Mobile phase: See the gradient table below. Standard solution: NLT 20 mg/mL of USP Heparin Sodium Identification RS in water System suitability solution: Prepare 1% (w/w) USP Oversulfated Chondroitin Sulfate RS and 1% (w/w) USP Dermatan Sulfate RS in Standard solution. Sample solution: NLT 20 mg/mL of Heparin Sodium in water Chromatographic system (See Chromatography 621 , System Suitability.) Mode: LC Detector: UV 202 nm Column: 2-mm × 25-cm; packing L61 Guard column: 2-mm × 50-mm; packing L61 Column temperature: Maintain columns at 40 Time (min) Solution A (%) Solution B (%) Elution 0 80 20 Equilibration 60 10 90 Linear gradient 61 80 20 Linear gradient 75 80 20 Re-equilibration 页码，2/9(W)w 2010/11/26file://C:\com_caislabs_ebk\data\v34290\usp34nf29s0_m36690.html Flow rate: 0.22 mL/min Injection size: 10 µL System suitability Sample: System suitability solution [NOTE—The retention times for dermatan sulfate, heparin, and oversulfated chondroitin sulfate are about 20, 30, and 50 min, respectively. ] Suitability requirements Resolution: NLT 1.0 between the dermatan sulfate and the heparin peaks, and NLT 1.5 between the heparin and the oversulfated chondroitin sulfate Relative standard deviation: NMT 2% for the heparin peak determined from three replicate injections Analysis Samples: Standard solution and Sample solution Record the chromatograms, and measure the retention times for the major peaks. Acceptance criteria: The retention time of the major peak from the Sample solution corresponds to that from the Standard solution. • C. ANTI-FACTOR XA TO ANTI-FACTOR IIA RATIO Anti-factor Xa activity pH 8.4 buffer: Dissolve amounts of tris(hydroxymethyl)aminomethane, edetic acid, and sodium chloride in water containing 0.1% of polyethylene glycol 6000 to obtain a solution having concentrations of 0.050, 0.0075, and 0.175 M, respectively. Adjust, if necessary, with hydrochloric acid or sodium hydroxide solution to a pH of 8.4. Antithrombin solution: Reconstitute a vial of antithrombin (see Reagents, Indicators, and Solutions— Reagent Specifications) as directed by the manufacturer, and further dilute with pH 8.4 buffer to obtain a solution having a concentration of 1.0 Antithrombin IU/mL. Factor Xa solution: Reconstitute bovine factor Xa as directed by the manufacturer (see Factor Xa in Reagents, Indicators, and Solutions—Reagent Specifications), and further dilute in pH 8.4 buffer to obtain a solution that gives an absorbance value between 0.65 and 1.25 at 405 nm when assayed as described below but using 30 µL of pH 8.4 buffer instead of 30 µL of the Standard solutions or the Sample solutions. [NOTE—Factor Xa solution contains about 3 nanokatalytic units/mL, but can vary depending upon the manufacturer of factor Xa or the substrate used. ] Chromogenic substrate solution: Prepare a solution of a suitable chromogenic substrate for amidolytic test (see Reagents, Indicators, and Solutions—Reagent Specifications) specific for factor Xa in water to obtain a concentration of 1 mM. Stopping solution: 20% (v/v) solution of acetic acid Standard solutions: Reconstitute the entire contents of an ampule of USP Heparin Sodium for Assays RS with water, and dilute with pH 8.4 buffer to obtain at least five dilutions in the concentration range between 0.03 and 0.375 USP Heparin Units/mL. Sample solutions: Dissolve or dilute a measured quantity of Heparin Sodium in pH 8.4 buffer, and dilute with the same buffer to obtain solutions having activities approximately equal to those of the Standard solutions. Analysis [NOTE—The procedure can also be performed using alternative platforms. Perform the test with each Standard solution and Sample solution in duplicate. ] To each of a series of suitable plastic tubes placed in a water bath set at 37 , transfer 120 µL of pH 8.4 buffer. Then separately transfer 30 µL of the different dilutions of the Standard solutions or the Sample solutions to the tubes. Add 150 µL of Antithrombin solution, prewarmed at 37 for 15 min, to 页码，3/9(W)w 2010/11/26file://C:\com_caislabs_ebk\data\v34290\usp34nf29s0_m36690.html each tube, mix, and incubate for 2 min. Add 300 µL of Factor Xa solution, prewarmed at 37 for 15 min, to each tube, mix, and incubate for 2 min. Add 300 µL of Chromogenic substrate solution, prewarmed at 37 for 15 min, to each tube, mix, and incubate for exactly 2 min. Add 150 µL of Stopping solution to each tube, and mix. Prepare a blank for zeroing the spectrophotometer by adding the reagents in reverse order, starting with the Stopping solution and ending with the addition of 150 µL of pH 8.4 buffer, and excluding the Standard solutions or the Sample solutions. Record the absorbance at 405 nm against the blank. Calculations: Plot the log of the absorbance values of the Standard solutions and the Sample solutions versus the heparin concentrations in USP Units. Calculate the activity of Heparin Sodium in USP Units/mg using statistical methods for slope ratio assays. Calculate the anti-factor Xa activity of Heparin Sodium: Result = A × (ST/SS) Express the anti-factor Xa activity of the Sample solution as USP Heparin Units/mg, calculated on the dried basis. Calculate the ratio of anti-factor Xa activity against anti-factor IIa potency (see the Assay): Result = anti-factor Xa activity/anti-factor IIa potency Acceptance criteria: 0.9–1.1 • D. IDENTIFICATION TESTS—GENERAL, Sodium 191 : It meets the requirements of the flame test for sodium. ASSAY • ANTI-FACTOR IIA POTENCY pH 8.4 buffer: Dissolve 6.10 g of tris(hydroxymethyl)aminomethane, 10.20 g of sodium chloride, 2.80 g of edetate sodium, and, if suitable, between 0 and 10.00 g of polyethylene glycol 6000 and/or 2.00 g of bovine serum albumin in 800 mL of water. [NOTE—2.00 g of human albumin may be substituted for 2.00 g of bovine serum albumin. ] Adjust with hydrochloric acid to a pH of 8.4, and dilute with water to 1000 mL. Antithrombin solution: Reconstitute a vial of antithrombin (see Reagents, Indicators, and Solutions— Reagent Specifications) in water to obtain a solution of 5 Antithrombin IU/mL. Dilute this solution with pH 8.4 buffer to obtain a solution having a concentration of 0.125 Antithrombin IU/mL. Thrombin human solution: Reconstitute thrombin human (factor IIa) (see Reagents, Indicators, and Solutions—Reagent Specifications) in water to give 20 Thrombin IU/mL, and dilute with pH 8.4 buffer to obtain a solution having a concentration of 5 Thrombin IU/mL. [NOTE—The thrombin should have a specific activity of NLT 750 IU/mg. ] Chromogenic substrate solution: Prepare a solution of a suitable chromogenic thrombin substrate for amidolytic test (see Reagents, Indicators, and Solutions—Reagent Specifications) in water to obtain a concentration of 1.25 mM. Stopping solution: 20% (v/v) solution of acetic acid Standard solutions: Reconstitute the entire contents of an ampule of USP Heparin Sodium for Assays RS with water, and dilute with pH 8.4 buffer to obtain at least four dilutions in the concentration range between 0.005 and 0.03 USP Heparin Unit/mL. Sample solutions: Proceed as directed for Standard solutions to obtain concentrations of Heparin Sodium similar to those obtained for the Standard solutions. A = potency of USP Heparin Sodium for Assays RS ST = slope of the line for the Sample solutions SS = slope of the line for the Standard solutions 页码，4/9(W)w 2010/11/26file://C:\com_caislabs_ebk\data\v34290\usp34nf29s0_m36690.html Analysis [NOTE—The procedure can also be performed using alternative platforms. ] For each dilution of the Standard solutions and the Sample solutions, at least duplicate samples should be tested. Label a suitable number of tubes, depending on the number of replicates to be tested. For example, if five blanks are to be used: B1, B2, B3, B4, and B5 for the blanks; T1, T2, T3, and T4 each at least in duplicate for the dilutions of the Sample solutions; and S1, S2, S3, and S4 each at least in duplicate for the dilutions of the Standard solutions. Distribute the blanks over the series in such a way that they accurately represent the behavior of the reagents during the experiments. [NOTE—Treat the tubes in the order B1, S1, S2, S3, S4, B2, T1, T2, T3, T4, B3, T1, T2, T3, T4, B4, S1, S2, S3, S4, B5. ] Note that after each addition of a reagent, the incubation mixture should be mixed without allowing bubbles to form. Add twice the volume (100–200 µL) of Antithrombin solution to each tube containing one volume (50-100 µL) of either the pH 8.4 buffer or an appropriate dilution of the Standard solutions or the Sample solutions. Mix, but do not allow bubbles to form. Incubate at 37 for at least 1 min. Add to each tube 25–50 µL of Thrombin human solution, and incubate for at least 1 min. Add 50–100 µL of Chromogenic substrate solution. Please note that all reagents, Standard solutions, and Sample solutions should be prewarmed to 37 just before use. Two different types of measurements can be recorded: 1. Endpoint measurement: Stop the reaction after at least 1 min with 50–100 µL of Stopping solution. Measure the absorbance of each solution at 405 nm using a suitable spectrophotometer (see Spectrophotometry and Light-Scattering 851 ). The RSD over the blank readings is less than 10%. 2. Kinetic measurement: Follow the change in absorbance for each solution over 1 min at 405 nm using a suitable spectrophotometer (see Spectrophotometry and Light-Scattering 851 ). Calculate the change in absorbance/min (OD/min). The blanks for kinetic measurement are also expressed as OD/min and should give the highest values because they are carried out in the absence of heparin. The RSD over the blank readings is less than 10%. Calculations: The statistical models for Slope ratio assay or Parallel-line assay can be used, depending on which model best describes the correlation between concentration and response. Parallel-line assay: For each series, calculate the regression of the absorbance or change in absorbance/min against log concentrations of the Standard solutions and the Sample solutions, and calculate the potency of Heparin Sodium in USP Units/mL using statistical methods for parallel-line assays. Express the potency of Heparin Sodium/mg, calculated on the dried basis. Slope ratio assay: For each series, calculate the regression of the log absorbance or the log change in absorbance/min against concentrations of the Standard solutions and the Sample solutions, and calculate the potency of Heparin Sodium in USP Units/mL using statistical methods for slope ratio assays. Express the potency of Heparin Sodium/mg, calculated on the dried basis. Acceptance criteria: The potency of Heparin Sodium, calculated on the dried basis, is NLT 180 USP Heparin Units in each mg. OTHER COMPONENTS • NITROGEN DETERMINATION, Method I 461 : 1.3%–2.5%, calculated on the dried basis, using the procedure for Nitrates and Nitrites Absent IMPURITIES Inorganic Impurities 页码，5/9(W)w 2010/11/26file://C:\com_caislabs_ebk\data\v34290\usp34nf29s0_m36690.html • RESIDUE ON IGNITION 281 : 28.0%–41.0% • HEAVY METALS, Method II 231 : NMT 30 ppm Organic Impurities • PROCEDURE 1: LIMIT OF GALACTOSAMINE IN TOTAL HEXOSAMINE (a measure of dermatan sulfate and other galactosamine containing impurities) Mobile phase: 14 mM potassium hydroxide Glucosamine standard solution: 1.6 mg/mL of USP Glucosamine Hydrochloride RS in 5 N hydrochloric acid Galactosamine standard solution: 16 µg/mL of USP Galactosamine Hydrochloride RS in 5 N hydrochloric acid Standard solution: Mix equal volumes of Glucosamine standard solution and Galactosamine standard solution. Hydrolyzed standard solution: Transfer 5 mL of the Standard solution to a 7-mL screw-cap test tube, cap, and heat for 6 h at 100 . Cool to room temperature, quantitatively transfer the solution to a 500- mL volumetric flask, and dilute with water to volume. Sample solution: Transfer 12 mg of Heparin Sodium to a 7-mL screw-cap test tube, dissolve in 5 mL of 5 N hydrochloric acid, and cap. Hydrolyzed sample solution: Heat the Sample solution for 6 h at 100 . Cool to room temperature, and dilute with water (1 in 100). Chromatographic system (See Chromatography 621 , System Suitability.) Mode: HPIC Detector: Pulsed amperometric detector, set to the following waveform: Column: 3-mm × 30-mm amino acid trap column in series with a 3- × 30-mm guard column and a 3- mm × 15-cm column that contains packing L69 Column temperature: Maintain columns at 30 . Flow rate: 0.5 mL/min Pre-equilibration: At least 60 min with Mobile phase Injection size: 10 µL Elution: 10 min with Mobile phase Column cleaning: At least 10 min with 100 mM potassium hydroxide Equilibration: At least 10 min with Mobile phase before each injection System suitability Sample: Hydrolyzed standard solution Step Time (s) Potential (V) Integration 1 0.00 +0.1 — 2 0.20 +0.1 Begins 3 0.40 +0.1 Ends 4 0.41 2.0 — 5 0.42 2.0 — 6 0.43 +0.6 — 7 0.44 0.1 — 8 0.50 0.1 — 页码，6/9(W)w 2010/11/26file://C:\com_caislabs_ebk\data\v34290\usp34nf29s0_m36690.html Suitability requirements Resolution: NLT 2 between the galactosamine and glucosamine peaks Column efficiency: NLT 2000 theoretical plates for glucosamine Tailing factor: Between 0.8 and 2.0 for the galactosamine and glucosamine peaks Analysis Samples: Hydrolyzed standard solution and Hydrolyzed sample solution Record the chromatograms, and measure the responses for the peaks at the retention time of galactosamine and glucosamine. Calculate the response ratio of galactosamine to glucosamine (GalNR) in the Hydrolyzed standard solution: Result = (GalNB/GalNW) × (GlcNW/GlcNB) Calculate the percentage of galactosamine in the portion of total hexosamine taken: Result = {[(GalNU/GalNR)]/[(GalNU/GalNR) + GlcNU]} × 100 Acceptance criteria: The percent galactosamine peak area of the total hexosamine of the Hydrolyzed sample solution must be NMT 1%. • PROCEDURE 2: NUCLEOTIDIC IMPURITIE