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Category Human blood and blood products (antihemorrhagic agent, local).

          Fibrin Sealant Kit is essentially composed of two components, namely fibrinogen concentrate (component 1), a protein fraction containing human fibrinogen and a preparation containing human thrombin (component 2). A fibrin clot is rapidly formed when the two thawed or reconstituted components are mixed. Other ingredients (for example, human coagulation factor XIII, a fibrinolysis inhibitor or calcium ions) and stabilizers (for example, Albumin solution) may be added. No antimicrobial preservative is added.

          Human constituents are obtained from human plasma that complies with the requirements stated under Plasma for fractionation, p. 193. No antibiotic is added to the plasma used.

          When thawed or reconstituted as stated on the label, component 1 contains not less than 40 mg per ml of clottable protein; the thrombin activity of component 2 varies over a wide range (approximately 4 to 1000 IU per ml).

Description

Freeze-dried constituents are hygroscopic, white or pale yellow powders or friable solids.

Frozen constituents are colourless or pale yellow, opaque solids.

Liquid constituents are colourless or pale yellow.

Strengths available 40 to 90 mg of fibrinogen per ml and 400 to 1000 IU of thrombin per ml.

Dose Adults: Topical, to the involved area, as prescribed by the physicians.

Contra-indication

          1. It is contra-indicated in patients with arterial and strong venous bleeding or in individual known to have anaphylactic or severe systemic reaction to human blood and to any other components of the product.

          2. It is contra-indicated for intravascular injection because life-threatening thromboembolic event may occur.

          3. It is contra-indicated for application into the eyeball.

Warning

          1. A fibrin sealant kit containing tranexamic acid may cause fatal neurotoxic reaction in patients during surgical operation where contact with cerebrospinal fluid or dura-mater can occur.

          2. Caution should be exercised if it is to be used for tissue fixation or incisional closure in ocular surgery.

Expiration date When stored under the prescribed conditions, the expiration date is not later than 2 years from the date of manufacture, or as indicated on the label.

Packaging and storage Frozen Fibrin Sealant Kit shall be stored at a temperature of –20º or below, protected from light.

          Freeze-dried Fibrin Sealant Kit shall be stored at a temperature of 2º to 8º, protected from light; avoid freezing.

Labelling Complies with the “General Information for Biological Products” p. 177. In addition, the label on the container states (1) the amount of fibrinogen (mg of clottable protein), thrombin (IU), per container, and coagulation factor XIII, if this is more than 10 IU per ml; (2) the name and volume of solvent to be used to reconstitute the components, where applicable.

Before carrying out the identification and the tests (except those for solubility and water), immediately reconstitute or thaw the preparation to be examined as stated on the label.

 

FOR COMPONENT 1 (FIBRINOGEN CONCENTRATE)

Identification

A. The assay for fibrinogen contributes to the identification of the preparation.

B. The assay for factor XIII contributes to the identification of the preparation (where applicable).

pH 6.5 to 8.0 (Appendix 4.11).

Solubility test Freeze-dried concentrates dissolve within 20 minutes in the volume of solvent for reconstitution and at the temperature stated on the label, forming an almost colourless, clear or slightly turbid solution.

Stability of solution No gel formation appears within 120 minutes of reconstitution or thawing.

Water For freeze-dried contituents, not more than 3.0 per cent w/w (Karl Fischer Method, Appendix 4.12). 

Sterility Complies with the “Sterility Test” (Appendix 10.1).

Assay

          FIBRINOGEN (CLOTTABLE PROTEIN) The estimated content in mg of clottable protein is not less than 70 per cent and not more than 130 per cent of the content stated on the label.

          Mix 0.2 ml of the reconstituted preparation with 2 ml of a suitable buffer solution (pH 6.6 to 7.4) containing sufficient human thrombin (approximately 3 IU per ml) and calcium (0.05 mol per litre). Maintain at 37º for 20 minutes, separate the precipitate by centrifugation (5000 × g, 20 minutes), and wash thoroughly with saline TS. Carry out the “Determination of Nitrogen” (Method II, Appendix 6.7). Calculate the protein content by multiplying the result by 6.0. If for a particular preparation this method cannot be applied, use another validated method for determination of fibrinogen.

          FACTOR XIII Where the label indicates that the human coagulation factor XIII activity is more than 10 IU per ml, the estimated activity is not less than 80 per cent and not more than 120 per cent of the activity stated on the label.

          Make at least three suitable dilutions of thawed or reconstituted component 1 and of normal human plasma (reference preparation) using as a diluent coagulation factor XIII deficient plasma or another suitable diluent. Add to each dilution suitable amounts of the following reagents:

          activator reagent, containing bovine or human thrombin, a suitable buffer, calcium chloride and a suitable inhibitor such as Gly-Pro-Arg-Pro-Ala-NH₂ which inhibits clotting of the sample but does not prevent coagulation factor XIII activation by thrombin, detection reagent, containing a suitable factor XIIIaspecific peptide substrate such as Leu-Gly-Pro-Gly-GluSer-Lys-Val-Ile-Gly-NH₂ and glycine ethyl ester as second substrate in a suitable buffer solution,

          NADH¹ reagent, containing glutamate dehydrogenase, α-ketoglutarate and NADH in a suitable buffer solution.

          After mixing, the absorbance changes (ΔA/minute) are measured at a wavelength of 340 nm (Appendix 2.2), after the linear phase of the reaction is reached.

          One IU of factor XIII is equal to the activity of 1 ml of human normal plasma.

          Calculate the activity of the test preparation as described in the “Statistical Analysis of Results of Biological Assays and Tests” (Appendix 9).

          The confidence limits (P = 0.95) are not less than 80 per cent and not more than 125 per cent of the estimated activity.

FOR COMPONENT 2 (THROMBIN PREPARATION)

Identification The assay for thrombin contributes to the identification of the preparation.

pH 5.0 to 8.0 (Appendix 4.11).

Solubility test Freeze-dried preparations dissolve within 5 minutes in the volume of solvent for reconstitution stated on the label, forming a colourless, clear to slightly turbid solution.

Water For freeze-dried constituents, not more than 3.0 per cent w/w (Karl Fischer Method, Appendix 4.12).

Sterility Complies with the “Sterility Test” (Appendix 10.1).

Assay

          THROMBIN If necessary, dilute the reconstituted preparation to be examined to approximately 2 to 20 IU of thrombin per ml using as diluent a suitable buffer pH 7.3 to 7.5, such as imidazole buffer solution pH 7.3 containing 1 per cent w/v of human albumin or bovine serum albumin. To a suitable volume of the dilution add a suitable volume of fibrinogen solution (a 0.1 per cent w/v solution of clottable protein) warmed to 37º and start measurement of the clotting time immediately. Repeat the procedure with each of at least three dilutions, in the range stated above, of a reference preparation of thrombin, calibrated in International Units. Draw a calibration curve on log-log graph paper using the measured clotting times for the dilutions of the reference preparation and the content in International Units of thrombin; use the curve obtained to determine the content in International Units of thrombin in the preparation to be examined.

          Calculate the activity of the test preparation as described in the “Statistical Analysis of Results of Biological Assays and Tests” (Appendix 9).

          The confidence limits (P = 0.95) are not less than 80 per cent and not more than 125 per cent of the estimated activity.

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¹ β-Nicotinamide Adenine Dinucleotide, Reduced Form.