Antithrombin III Human
(an'' tee throm' bin hue' man).
DEFINITION
Antithrombin III Human is a glycoprotein, which is the major inhibitor of thrombin and other activated clotting factors, including factors IX, X, XI, and XII, and the cofactor through which heparin exerts its effect. It is obtained from human plasma of healthy donors who must, as far as can be ascertained, be free from detectable agents of infection transmissible by transfusion of blood or blood derivatives. The method of manufacturing includes steps that have been shown to remove or inactivate known agents of infection. If substances are used for inactivation of viruses during production, the subsequent purification procedure must be validated to demonstrate that the concentration of these substances is reduced to an acceptable level and that any residues are such as not to compromise the safety of the preparation for patients. The antithrombin III concentrate is passed through a bacteria-retentive filter, filled aseptically into its final, sterile containers, and immediately frozen. It is then freeze-dried, and the containers are closed under vacuum. No antimicrobial preservative is added at any stage of production. Antithrombin III Human complies with the requirements for Biologics 
1041
. When reconstituted in the recommended volume of diluent, the potency is NLT 25 USP Antithrombin III Units/mL.
1041. When reconstituted in the recommended volume of diluent, the potency is NLT 25 USP Antithrombin III Units/mL.
[Note—One USP Antithrombin III Unit is the amount of antithrombin III that forms a complex with one unit of thrombin at 25
in the presence of heparin at a pH of 8.4. ]
in the presence of heparin at a pH of 8.4. ]
IDENTIFICATION
• Meets the requirements of the Assay
ASSAY
• Procedure
Solution A:
Dissolve Tris(hydroxymethyl)aminomethane, edetic acid, and sodium chloride in water containing 0.1% polyethylene glycol 6000 to obtain a solution having concentrations of 0.050 M, 0.0075 M, and 0.175 M, respectively. Adjust with hydrochloric acid or sodium hydroxide solution to a pH of 8.4.
Solution B:
0.05% (w/v) of albumin human in Solution A
Solution C:
10 mg/mL of polybrene in Solution B
Solution D:
15 USP Heparin Units/mL of USP Heparin Sodium for Assays RS in Solution B
Solution E:
Reconstitute thrombin bovine, and dilute with Solution B to obtain a solution having a concentration of 2.0 Thrombin Units/mL.
Solution F:
Prepare a solution of chromogenic substrate for amidolytic test (see Reagents, Indicators, and Solutions—Reagent Specifications) for factor IIa in water to obtain a solution having a concentration of about 5.0 mM, and dilute the solution further with Solution C to 1.0 mM.
Stopping solution:
20% (v/v) of acetic acid in water
Standard solution A:
USP Antithrombin III Human RS in Solution D to obtain a solution containing 1.0 USP Antithrombin III Unit
Standard solutions B, C, D, and E:
Dilute Standard solution A with Solution D 60-, 120-, 180-, and 300-fold.
Sample solution A:
Dissolve a quantity of Antithrombin III Human in Solution D to obtain a solution having the same concentration as Standard solution A.
Sample solutions B, C, D, and E:
Dilute Sample solution A with Solution D 60-, 120-, 180-, and 300-fold.
Analysis:
Pipet 400 µL each of Standard solutions B, C, D, and E and Sample solutions B, C, D, and E into suitable tubes placed in a water bath set at 37. Add 200 µL of Solution E, prewarmed at 37 to each tube, mix, and incubate for 1 min. Add 200 µL of Solution F prewarmed at 37 to each tube, mix, and incubate for 60 s. Stop the reaction by adding 200 µL Stopping solution. To prepare a blank, add the reagents in reverse order, starting with 200 µL of Stopping solution, followed by the addition of 200 µL of Solution F, then adding 200 µL of Solution E, and ending with 400 µL of Solution D. Record the absorbance at 405 nm against the blank.
. Add 200 µL of Solution E, prewarmed at 37 to each tube, mix, and incubate for 1 min. Add 200 µL of Solution F prewarmed at 37 to each tube, mix, and incubate for 60 s. Stop the reaction by adding 200 µL Stopping solution. To prepare a blank, add the reagents in reverse order, starting with 200 µL of Stopping solution, followed by the addition of 200 µL of Solution F, then adding 200 µL of Solution E, and ending with 400 µL of Solution D. Record the absorbance at 405 nm against the blank.
For Standard solutions and Sample solutions, calculate the regression of the absorbance against log concentrations, and calculate the activity of Antithrombin III Human in USP Antithrombin III Units, using a suitable statistical method for parallel-line assays. The four independent relative activity estimates are then combined to obtain the final mean, and the confidence limits are calculated.
Acceptance criteria:
80%–120% of the potency stated on the label. The specific activity is NLT 6.0 USP Antithrombin III Units/mg of total protein. The confidence interval (P = 0.95) is between 90% and 110% of the estimated potency.
IMPURITIES
Organic Impurities
• Procedure: Heparin Content
Solution A:
Dissolve Tris(hydroxymethyl)aminomethane, edetic acid, and sodium chloride in water containing 0.1% polyethylene glycol 6000 to obtain a solution having concentrations of 0.050 M, 0.0075 M, and 0.175 M, respectively. Adjust with hydrochloric acid or sodium hydroxide solution to a pH of 8.4.
Solution B:
Solution of chromogenic substrate for amidolytic test for factor Xa in water to obtain a solution of concentration of 2.5 mM
Solution C:
Dissolve Factor Xa in Solution A to obtain a solution containing 20 nanokatalytic units (nkats).
Solution D:
20% (v/v) of acetic acid in water
Standard solution:
Dissolve USP Antithrombin III Human RS in Solution A to obtain a solution containing 1.0 USP Antithrombin III Unit.
Sample solution:
Dissolve Antithrombin III Human in Solution A to obtain a solution containing 1.0 USP Antithrombin III Unit.
Analysis:
Pipet 250 µL each of Solution A, the Standard solution, and the Sample solution to suitable tubes placed in a water bath set at 37. Add 250 µL of Solution C prewarmed at 37 to each tube, and incubate for 2 min. Add 250 µL of Solution B prewarmed at 37 to each tube, mix, and incubate for 120 s. Stop the reaction by adding 250 µL of Solution D. Record the absorbance at 405 nm, using Solution A as the blank.
. Add 250 µL of Solution C prewarmed at 37 to each tube, and incubate for 2 min. Add 250 µL of Solution B prewarmed at 37 to each tube, mix, and incubate for 120 s. Stop the reaction by adding 250 µL of Solution D. Record the absorbance at 405 nm, using Solution A as the blank.
Calculate the USP Heparin Unit per USP Antithrombin III Unit:
Result = PR (AF 
AU)/(AF AS)
AU)/(AF AS)| PR | = | = heparin content of USP Antithrombin III Human RS in USP Heparin Unit per USP Antithrombin III Unit |
| AF | = | = absorbance value from Solution A |
| AU | = | = absorbance value from the Sample solution |
| AS | = | = absorbance value from the Standard solution |
Acceptance criteria:
NMT 0.1 USP Heparin Unit per USP Antithrombin III Unit
SPECIFIC TESTS
• Sterility Tests 71:
Meets the requirements when tested as directed for Test for Sterility of the Product to Be Examined, Direct Inoculation of the Culture Medium
71:
Meets the requirements when tested as directed for Test for Sterility of the Product to Be Examined, Direct Inoculation of the Culture Medium
• Water Determination, Method I 921:
NMT 3.0%
921:
NMT 3.0%
• Pyrogen Test 151:
Inject 50 USP Antithrombin III Units per kg of the rabbit's weight, calculated from the activity stated on the label: meets the requirements.
151:
Inject 50 USP Antithrombin III Units per kg of the rabbit's weight, calculated from the activity stated on the label: meets the requirements.
• General Safety:
Meets the requirements for biologics as set forth under Biological Reactivity Tests, In Vivo 88, Safety Tests—Biologicals
88, Safety Tests—Biologicals
• Osmolality and Osmolarity, Osmolality 785:
Reconstitute with the diluent according to the manufacturer's instruction: NLT 240 mOsmol/kg for the solution.
785:
Reconstitute with the diluent according to the manufacturer's instruction: NLT 240 mOsmol/kg for the solution.
• pH 791:
Reconstitute with the diluent according to the manufacturer's instruction: 6.0–7.5.
791:
Reconstitute with the diluent according to the manufacturer's instruction: 6.0–7.5.
• Molecular Weight Distribution
Mobile phase:
Solution containing 0.1 M sodium phosphate, 0.15 M sodium chloride, and 0.05% sodium azide, having a pH of 6.5. Degas and filter.
Solution A:
4–5 mg/mL of thyroglobulin in Mobile phase
Sample solution:
8–10 mg/mL of Antithrombin III Human
System suitability solution:
Dilute USP Albumin Human RS, if necessary, with water to obtain a solution containing 5%.
Chromatographic system
Mode:
LC
Detector:
UV 280 nm
Column:
7.5- × 75-mm guard column and a 7.5- × 300-mm analytical column, both containing packing L59
Temperature:
Ambient
Flow rate:
0.5 mL/min maintained constant to ±1%
Injection size:
10 µL
System suitability
Sample:
System suitability solution
Suitability requirements
Column efficiency:
Greater than 1500 theoretical plates
Tailing factor:
0.5–2.5
Analysis
Samples:
Solution A and Sample solution
Acceptance criteria:
Note the retention times of the major peak in the Solution A chromatogram. The relative peak area of the high-molecular weight peak eluting at about the same retention time as the major peak in the Solution A chromatogram, or earlier, is NMT 13%.
• Total Protein Content
Solution A:
1000 mg/mL of trichloroacetic acid in water
Sample solution:
7.5 mg/mL of Antithrombin III Human in 0.15 M sodium chloride solution
Blank:
0.15 M solution of sodium chloride
Analysis:
To each of 2.0 mL of the Sample solution and the Blank in suitable centrifuge tubes, add 1.5 mL of Solution A. Mix, allow to stand for at least 10 min, centrifuge for 5 min, and decant the supernatant. Resuspend the precipitates in 1.5 mL of Solution A, centrifuge for 5 min, decant the supernatant, and hold the tubes inverted on a filter paper to drain. Quantitatively transfer the residues with a minimum quantity of water to a micro-Kjeldahl flask, and determine the nitrogen content using Method II (see Nitrogen Determination 461). Multiply the result, corrected for the Blank, by 6.25 to calculate the quantity of protein.
461). Multiply the result, corrected for the Blank, by 6.25 to calculate the quantity of protein.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Use a Type I glass container with an appropriate stopper and seal. Store protected from light between 2 and 8, excursions permitted up to 25.
and 8, excursions permitted up to 25.
• Labeling:
The labeling should state the content of antithrombin III in USP Antithrombin III Units. The diluent and the volume to be used to reconstitute the preparation are indicated.
• USP Reference Standards 11
11
USP Albumin Human RS 
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USP Antithrombin III Human RS
USP Heparin Sodium for Assays RS
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Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Fouad Atouf, Ph.D.
Senior Scientific Liaison 1-301-816-8365 |
(BIO22010) Monographs - Biologics and Biotechnology 2 |
| 71 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 151 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 2231
Pharmacopeial Forum: Volume No. 30(1) Page 56