Method for producing fibrinogen containing blood coagulation factor XIII

Abstract

To provide a simple and efficient method for producing fibrinogen and coagulation factor XIII from human frozen plasma on an industrial scale.SOLUTION: Disclosed is a method for producing coagulation factor XIII-containing fibrinogen, characterized by using a mixture of a cryoprecipitate-derived processed liquid and a Cohn's cold ethanol fraction I precipitate, both obtained from plasma as a starting material. The production method of the invention comprises the steps of: (a) mixing a cryoprecipitate-derived processed liquid and a Cohn's cold ethanol fraction I precipitate, both of which are obtained from plasma, to form a mixed liquid; (b) centrifuging the mixed liquid obtained in (a) in the presence of glycine to form a precipitate (Glycine fraction); (c) centrifuging the precipitate obtained in (b) in the presence of ethanol to obtain a supernatant (Ethanol fraction 1); and (d) centrifuging the supernatant obtained in (c) in the presence of ethanol to form a precipitate (Ethanol fraction 2).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a biological tissue adhesive, and in particular to a method for the high-yield and simple production of fibrinogen enriched with blood coagulation factor XIII derived from human plasma. [Background technology]

[0002] In surgery, thread sutures have long been used to adhere tissues, but there were problems with this, such as wounds caused by needle holes in the tissue, the time it takes to adhere the tissue, and the effort required to remove the threads after the tissues are adhered. To solve these problems, research began around 1910 on the use of fibrin to strengthen the adhesion of sutures. The first clinical application of modern fibrin glue preparations was in 1944, when Tidrick RT et al. used the two components fibrinogen and thrombin for skin grafts, but the adhesive strength was weak and the effect was insufficient (Non-Patent Document 1).

[0003] Since Cohn et al. conducted research on plasma protein fractionation in 1946 (Non-Patent Documents 2 and 3), protein purification technology has progressed, and it became possible to separate fibrinogen and thrombin from human plasma. In the 1970s, blood coagulation factor XIII, which strengthens the fibrin mesh (blood clot) after the reaction of the two, was discovered (Non-Patent Document 4). With the development of these biotechnologies, research and development of biological tissue adhesives as pharmaceuticals for bonding and covering tissues and severed organs has progressed. These are purified from healthy human plasma and are sometimes called fibrin glue preparations. In 1978, Tisseel (registered trademark: Immuno AG), the world's first fibrin glue preparation, was developed, and in Japan, Veriplast (registered trademark) P (Hoechst Japan Ltd.) was released in 1988, and Bolheal (registered trademark: KM Biologics Co., Ltd.) was released in 1991.

[0004] The fibrin glue is composed of four components: (1) freeze-dried powder of fibrinogen (containing blood coagulation factor XIII), (2) fibrinogen solution (containing bovine lung-derived aprotinin), (3) freeze-dried powder of thrombin, and (4) thrombin solution (containing calcium chloride). Component (1) is dissolved in component (2) to prepare solution A, and component (3) is dissolved in component (4) to prepare solution B. Solution A and solution B are mixed on the adhesive surface of the tissue, and are used for "tissue adhesion and closure." Specific uses of the fibrin glue include reinforcing suture sites, stopping bleeding in damaged tissue, and preventing air and body fluid leakage (Non-Patent Document 5). Some fibrin glues are approved as kit preparations.

[0005] Fibrinogen and thrombin, which are the main components of fibrin glue preparations, are purified from human frozen plasma. Fibrinogen is purified from either cryoprecipitate (hereinafter also referred to as "cryoprecipitate") obtained by thawing human frozen plasma at 2 to 4°C and centrifuging it, or precipitate obtained by adding 8 to 10% ethanol to cryoprecipitate-free plasma (supernatant liquid excluding cryoprecipitate) and centrifuging it at -3 to -5°C (Cohn low-temperature ethanol fractionation method I precipitation) (Non-patent documents 6 and 7). In this case, the purified fibrinogen may contain blood coagulation factor XIII at a concentration of 150 units or less per gram of fibrinogen (Patent documents 1, 2, and 3). Fibrinogen is one of the main components of fibrin glue preparations, but fibrinogen preparations are administered alone to improve bleeding tendency in congenital hypofibrinogenemia. The amount of blood coagulation factor XIII contained in fibrinogen preparations is usually 150 units or less per gram of fibrinogen. On the other hand, in fibrin glue preparations, blood coagulation factor XIII purified from human frozen plasma is added to fibrinogen purified from human frozen plasma at a concentration of 150 units or more per gram of fibrinogen, making the blood clot stronger and enhancing the "tissue adhesion and closure" effect (Patent Documents 4 and 5). Blood coagulation factor XIII is purified from human frozen plasma by the above-mentioned Cohn low-temperature ethanol fractionation method I precipitation (Non-Patent Document 3 ). Thrombin, the other major component of fibrin glue preparations, is purified from the supernatant of frozen human plasma by the Cohn cold ethanol fractionation method I described above. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO1995-022316 [Patent Document 2] Patent Publication No. 55-110556 [Patent Document 3] Patent Publication No. 61-087628 [Patent Document 4] JP-A-58-135817 (p.115 right column) [Patent Document 5] Patent Publication No. 2007-182434 [Patent Document 6] JP 10-150980 [Patent Document 7] Patent Publication No. 57-139017 [Non-patent literature]

[0007] [Non-Patent Document 1] Tidrick RT, Warner ED. Fibrin fixation of skin transplants. Surg. 1944;15:90-5 [Non-Patent Document 2] Cohn EJ, Strong LE. Preparation and properties of serum and plasma proteins; a system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids J Am Chem Soc. 1946;68:459-75 [Non-Patent Document 3] Production of Plasma Proteins for Therapeutic Use 2021;John Wiley & Sons, Chapter 32 [Non-Patent Document 4] Japan Blood Products Association website: http: / / www.ketsukyo.or.jp / plasma / fibrin-paste / fib_02.html (verified on April 20, 2021) [Non-Patent Document 5] Japan Blood Products Association website: http: / / www.ketsukyo.or.jp / plasma / fibrin-paste / fib_05_03.html (verified on April 20, 2021) [Non-Patent Document 6] Vox Sang 1997;72:133-143 [Non-Patent Document 7] Production of Plasma Proteins for Therapeutic Use 2021;John Wiley & Sons, Chapter 10 [Non-Patent Document 8] Cytiva website: https: / / www.cytivalifesciences.co.jp / newsletter / biodirect_mail / technical_tips / tips53.html (verified on April 20, 2021) [Non-Patent Document 9] Prep Biochem Biotechnol.゜2003 Nov;33(4):239-52. [Non-Patent Document 10] Int J Biochem.゜1980;11(6):559-64 [Non-Patent Document 11] Ark.Kemi 1956;10:415-443 Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, in order to produce a fibrin glue preparation with enhanced "tissue adhesion and closure" effects, it is necessary to purify fibrinogen, blood coagulation factor XIII, and thrombin separately from human frozen plasma, which is very time-consuming. Therefore, the object of the present invention is to establish an efficient method for purifying fibrinogen and blood coagulation factor XIII contained in a fibrin glue preparation on an industrial scale. That is, the object of the present invention is to provide a method for simply and efficiently purifying fibrinogen and blood coagulation factor XIII from human frozen plasma on an industrial scale. [Means for solving the problem]

[0009] In order to solve the above problems, the inventors have discovered a method for producing fibrinogen rich in blood coagulation factor XIII easily and in high yield by using a mixture of a process solution derived from cryoprecipitation as an intermediate material and a Cohn low-temperature ethanol fractionation method I precipitate as the starting material, rather than purifying fibrinogen and blood coagulation factor XIII separately from human frozen plasma, and have thus completed the present invention.

[0010] Thus, the present invention includes the following: [1] A method for producing fibrinogen containing blood coagulation factor XIII, characterized in that the starting materials are a mixture of a process solution derived from cryoprecipitation obtained from plasma and a Cohn low-temperature ethanol fractionation method I precipitation. [2] The method according to [1], comprising the steps of: (a) mixing a process solution derived from cryoprecipitation and a Cohn cold ethanol fractionation method I precipitate, each obtained from plasma, to produce a mixture; (b) centrifuging the mixture obtained in step (a) in the presence of glycine to produce a precipitate (glycine fractionation); (c) centrifuging the precipitate obtained in step (b) in the presence of ethanol to produce a supernatant (ethanol fraction 1); and (d) centrifuging the supernatant obtained in step (c) in the presence of ethanol to produce a precipitate (ethanol fraction 2). [3] The method according to [1] or [2], wherein the process liquid derived from cryoprecipitation obtained from plasma is a flow-through liquid obtained by cold-thawing and centrifuging frozen plasma, treating the precipitate obtained with aluminum hydroxide gel, and then subjecting the precipitate to centrifugation and cation exchange chromatography. [4] The method according to [1] or [2], wherein the Cohn cold ethanol fractionation method I precipitate obtained from plasma is a precipitate obtained by cold thawing and centrifuging frozen plasma, followed by centrifuging the resulting supernatant in the presence of ethanol. [5] The method according to [1] or [2], wherein the Cohn low-temperature ethanol fractionation method I precipitate obtained from plasma is a precipitate obtained by cold-thawing frozen plasma, centrifuging the resulting supernatant, subjecting the resulting supernatant to anion exchange chromatography, and centrifuging the resulting flow-through in the presence of ethanol. [6] The method according to any one of [1] to [5], wherein in step (a), the process liquid derived from cryoprecipitation and the Cohn cold ethanol fractionation method I precipitation are mixed in a ratio of 1:1 to 1:2. [7] The method according to any one of [1] to [6], wherein in step (b), glycine is present at 1.5 mol / L. [8] The method according to any one of [1] to [7], wherein in step (c), the precipitate obtained in step (b) is dissolved in a buffer solution, and then ethanol is added to a concentration of 1%. [9] The method according to any one of [1] to [8], wherein in step (d), ethanol is added to the supernatant obtained in step (c) so that the concentration is 6%.

[10] The method according to any one of [1] to [9], wherein the obtained fibrinogen contains about 490 units or more of blood coagulation factor XIII per gram of fibrinogen.

[11] The method according to

[10] , wherein the obtained fibrinogen contains about 490 to about 700 units of blood coagulation factor XIII per gram of fibrinogen. Effect of the Invention

[0011] According to the method of the present invention, fibrinogen rich in blood coagulation factor XIII can be produced in a high yield and in a simple manner. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram showing an outline of the method for producing fibrinogen enriched with blood coagulation factor XIII of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Preferred embodiments of the present invention will be described in detail below, however, the present invention is not limited to the following embodiments.

[0014] The present invention provides a method for producing fibrinogen containing blood coagulation factor XIII (hereinafter also referred to as "FXIII"), characterized in that the starting materials are a mixture of a process solution derived from cryoprecipitation obtained from plasma and a Cohn low-temperature ethanol fractionation method I precipitation.

[0015] The method for producing fibrinogen containing blood coagulation factor XIII of the present invention comprises the following steps: (a) mixing a process solution derived from cryoprecipitation and a Cohn cold ethanol fractionation method I precipitate, each obtained from plasma, to produce a mixture; (b) centrifuging the mixture obtained in step (a) in the presence of glycine to produce a precipitate (glycine fractionation); (c) centrifuging the precipitate obtained in step (b) in the presence of ethanol to produce a supernatant (ethanol fraction 1); and (d) centrifuging the supernatant obtained in step (c) in the presence of ethanol to produce a precipitate (ethanol fraction 2).

[0016] In the method for producing fibrinogen containing blood coagulation factor XIII of the present invention, first, a process liquid derived from cryoprecipitation obtained from plasma and a Cohn low-temperature ethanol fractionation method I precipitate obtained from plasma are mixed to produce a mixed liquid.

[0017] The "process liquid derived from cryoprecipitation" used as one of the starting materials in the method of the present invention is, for example, a flow-through liquid obtained by cold-thawing frozen plasma, followed by centrifugation (cryocentrifugation), treating the precipitate obtained with aluminum hydroxide gel, followed by centrifugation and cation exchange chromatography. Cryocentrifugation can be carried out by a conventional method known to those skilled in the art. Aluminum hydroxide gel is added after dissolution in order to adsorb the remaining vitamin K-dependent blood coagulation factors. The amount of aluminum hydroxide gel added is 0.24~0.3% Here, the aluminum hydroxide gel for adsorbing the vitamin K-dependent blood coagulation factors may be calcium phosphate, barium sulfate, or hydroxyapatite (Patent Document 7). The aluminum hydroxide gel is then removed by centrifugation to remove the vitamin K-dependent blood coagulation factors. The von Willebrand factor and blood coagulation factor VIII are then removed from the supernatant after centrifugation by cation exchange chromatography. Here, the cation exchanger functional group may be sulfopropyl (SP), methyl sulfonate (S), carboxymethyl (CM), or the like, or any substance commonly used in the field, such as a natural polymer (cellulose, dextran, agarose), a synthetic polymer (styrene / divinylbenzene copolymer, polyacrylic, polyvinyl alcohol, etc.), or an inorganic material (silica gel, alumina, zirconia) on the surface of which an anion exchange group is introduced (Non-Patent Document 8).

[0018] In the method of the present invention, the "Cohn low-temperature ethanol fractionation I precipitation" used as the other starting material refers to a precipitate obtained by thawing frozen plasma in cold and then centrifuging the resulting supernatant (cryoprecipitate-removed plasma) in the presence of ethanol (ethanol fractionation I). However, the precipitate may also be obtained by subjecting the supernatant (cryoprecipitate-removed plasma) to anion exchange chromatography, adsorbing and removing blood coagulation factors VII, IX, X and prothrombin, which are vitamin K-dependent blood coagulation factors, and centrifuging the resulting flow-through liquid in the presence of ethanol. This anion exchange chromatography adsorbs and removes blood coagulation factors VII, IX, X and prothrombin, which are vitamin K-dependent blood coagulation factors, but since these vitamin K-dependent blood coagulation factors are transferred to the supernatant by the subsequent centrifugation of ethanol fraction I, anion exchange chromatography is not necessary. On the other hand, removing vitamin K-dependent blood coagulation factors by anion exchange chromatography at this stage has the advantage of preventing coagulation reactions in the subsequent manufacturing process. Here, the anion exchanger functional group may be quaternary ammonium (Q), diethylaminoethyl (DEAE), diethylaminopropyl (ANX), etc., and may be any substance commonly used in the field, for example, a carrier matrix having an anion exchange group introduced on the surface thereof, such as a natural polymer (cellulose, dextran, agarose), a synthetic polymer (styrene / divinylbenzene copolymer, polyacrylic, polyvinyl alcohol, etc.), or an inorganic material (silica gel, alumina, zirconia) (Non-Patent Document 8).

[0019] The "process liquid derived from cryoprecipitation" and the "Cohn low-temperature ethanol fractionation method I precipitation" can be mixed in a ratio of 1:1 to 1:2, and the resulting mixture can be used as the starting material for the method of producing fibrinogen containing blood coagulation factor XIII of the present invention.

[0020] First, a mixture of the "process solution derived from cryoprecipitation" as the starting material and the "Cohn low-temperature ethanol fractionation method I precipitation" is centrifuged in the presence of glycine to produce a precipitate (glycine fractionation). Here, the concentration of glycine is 1.5~1.8 The concentration may be 1.5 mol / L, preferably 1.5 mol / L. Centrifugation may be performed at about 10,000×g for 15 minutes. The glycine fractionation may be repeated two or more times.

[0021] Next, the precipitate obtained in the glycine fractionation is centrifuged in the presence of ethanol (ethanol fractionation 1) to produce a supernatant. Here, it is preferable to dissolve the precipitate obtained in the glycine fractionation in a buffer solution, and then add ethanol to a concentration of 1%. The buffer solution used may be a citrate buffer solution at pH 7.0, which is adjusted to an absorbance of 30 at a wavelength of 280 nm and held for about 12 hours, after which ethanol is added to a concentration of 1%, and ethanol fractionation 1 is performed by centrifugation at 0°C, 10,000×g, and 20 min to obtain a supernatant.

[0022] Next, the supernatant obtained in ethanol fractionation 1 is centrifuged in the presence of ethanol (ethanol fractionation 2) to produce a precipitate. Here, it is preferable to add ethanol to the supernatant obtained in ethanol fractionation 1 to a concentration of 6%, and then to centrifuge at 1° C., 10,000×g, and 15 min. This precipitate is the desired final product, "fibrinogen containing blood coagulation factor XIII (hereinafter referred to as "FXIII-added fibrinogen").

[0023] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way. EXAMPLES

[0024] Preparation of fibrinogen containing blood coagulation factor XIII (hereinafter referred to as FXIII): (1) Preparation of ethanol fractionation method I precipitate Raw plasma (fresh frozen plasma) stored at -20°C or less was thawed at 10°C or less. Cryoprecipitate was removed by centrifugation at about 8,300×g (cryocentrifugation). Sodium heparin was added to the supernatant to a concentration of about 20 units / mL, and the supernatant was developed on anion exchange chromatography (Cytiva, Q Sepharose Fast Flow, functional group: quaternary aminomethyl group). The resulting flow-through liquid was subjected to ethanol fractionation I, that is, left to stand at an ethanol concentration of 8%, pH 6.8, and a liquid temperature of -3°C, and then centrifuged at about 8,300×g to obtain ethanol fractionation I precipitate (hereinafter referred to as "FI paste").

[0025] (2) Purification of cryoprecipitate The cryoprecipitate obtained in (1) above was dissolved in 3.5 volumes of buffer (NaCl: 10.55 g / L, glycine: 15 g / L, calcium chloride hydrate: 0.088 g / L, pH 7.3), and the volume was increased to 5 volumes of cryoprecipitate. Aluminum hydroxide gel was added to a concentration of 0.3%, and the mixture was centrifuged at approximately 12,000×g to obtain a supernatant. This was developed using a cation exchange chromatography (Tosoh Corporation, SP Toyopearl 550C, functional group: sulfopropyl group) to obtain a cation exchange chromatography flow-through liquid (hereinafter referred to as "SPBT"). The fibrinogen content of the SPBT was approximately 10 mg / mL.

[0026] (3) Mixing SPBT and FI paste The FI paste obtained in (1) above was dissolved in a 9-fold amount of buffer solution (NaCl: 8.77 g / L, trisodium citrate dihydrate: 7.35 g / L, pH 7.0) and mixed with the SPBT obtained in (2) above. The fibrinogen content of the FI paste solution was 19-20 mg / mL. The mixing ratio of SPBT to FI paste was 1:1 or 1:2, calculated based on the amount of plasma from which it was derived. Specifically, when the mixing ratio was 1:1, 27 g of FI paste was used for 500 mL of SPBT. After mixing, polysorbate 80 was added to a concentration of 0.8%.

[0027] (4) Glycine fraction The temperature of the mixture obtained in (3) above was adjusted to 20° C., glycine was added to the mixture to a concentration of 1.5 mol / L, and the mixture was centrifuged at 10,000×g for 15 minutes to obtain a precipitate.

[0028] (5) Ethanol fraction 1 The precipitate obtained in (4) above was dissolved in a buffer solution (NaCl: 4.38 g / L, trisodium citrate dihydrate: 2.94 g / L, pH 7.0), and the OD (absorbance at a wavelength of 280 nm) was adjusted to 30. After holding for about 12 hours, ethanol was added to a concentration of 1%, and the mixture was centrifuged at 0°C, 10,000×g, and 20 min to obtain a supernatant (ethanol fraction 1).

[0029] (6) Ethanol fraction 2 Ethanol was added to the supernatant obtained in (5) above to a concentration of 6%, and the mixture was centrifuged at 1°C, 10,000×g, and 15 min (ethanol fractionation 2) to obtain a precipitate. This precipitate was used as FXIII-added fibrinogen.

[0030] (7) Evaluation of FXIII-added fibrinogen The yield of fibrinogen per plasma of the recovered FXIII-added fibrinogen was 0.83 g / L when the mixture ratio of SPBT and FI paste was 1:1, and 0.67 g / L when the mixture ratio was 1:2 (Table 1), which was higher than that of the conventional technology (Table 2). Furthermore, the degree of purification of the obtained FXIII-added fibrinogen, calculated by dividing the fibrinogen content by the total protein amount, was 90% or higher in all cases, indicating high purity (Table 3). Furthermore, all samples contained sufficient FXIII (Table 4). That is, fibrinogen purified by the conventional manufacturing method contains blood coagulation factor XIII at a concentration of 150 units (IU) or less per 1 g of fibrinogen, whereas the manufacturing method of the present invention obtains a blood coagulation factor XIII concentration of 494 units per 1 g of fibrinogen when the mixing ratio of SPBT and FI paste is 1:1, and 698 units when the mixing ratio is 1:2 (Table 4), which means that fibrinogen much richer in blood coagulation factor XIII than fibrinogen purified by the conventional manufacturing method can be obtained. The activity value of FXIII was calculated using a Sysmex XIII coagulation factor kit (Berichrome FXIII). The fibrinogen content was calculated by the method of Birger Blombaeck et al. (Non-Patent Document 10).

[0031] [Table 1]

[0032] [Table 2]

[0033] [Table 3]

[0034] [Table 4] EXAMPLES

[0035] Assessment of fibrinogen clotting activity: The coagulation ability was evaluated by the titer test of dried human fibrinogen listed in the Biological Products Standards (Ministry of Health, Labor and Welfare Notification No. 274). Specifically, the fibrinogen of Example 1 was adjusted to a concentration of 2%, and mixed with an equal amount of a buffer solution (containing 3.4 mmol / L calcium chloride) to adjust the fibrinogen concentration to 1%. 0.9 mL of this fibrinogen solution was mixed with 0.1 mL of a 10 U / mL thrombin solution. After mixing, the time until the cloudiness started was measured, and the coagulation ability of each sample was evaluated (repeated number of times=3). As a result, the coagulation initiation time was equivalent, suggesting that all samples had sufficient coagulation ability (Table 5).

[0036] [Table 5] [Industrial Applicability]

[0037] INDUSTRIAL APPLICABILITY The present invention can be used as a method for producing fibrinogen containing blood coagulation factor XIII from a plasma-derived fraction.

Claims

1. A method for producing fibrinogen containing blood coagulation factor XIII, comprising using as starting materials a mixture of a cryoprecipitation-derived process solution obtained from plasma and a Cohn low-temperature ethanol fractionation method I precipitation, Mixing the cryoprecipitation-derived process liquid with the Cohn cold ethanol fractionation I precipitate in a ratio of 1:1 to 1:2, wherein the cryoprecipitation-derived process liquid is a flow-through liquid obtained by subjecting a precipitate obtained by cold-thawing frozen plasma and centrifuging the precipitate to an aluminum hydroxide gel treatment, followed by centrifugation and cation exchange chromatography; and the Cohn low-temperature ethanol fractionation method I precipitation is a precipitate obtained by cold-thawing frozen plasma and centrifuging the resulting supernatant in the presence of ethanol; The fibrinogen obtained contains about 490 units or more of blood coagulation factor XIII per gram of fibrinogen. Manufacturing method.

2. The Cohn low-temperature ethanol fractionation method I precipitation is a precipitate obtained by thawing frozen plasma in a cold state, centrifuging the resulting supernatant, subjecting the supernatant to anion exchange chromatography, and centrifuging the resulting flow-through in the presence of ethanol. The method of claim 1 .

3. The method according to claim 1 or 2, comprising the steps of: (a) mixing a process solution derived from cryoprecipitation and a Cohn cold ethanol fractionation method I precipitate, each obtained from plasma, to produce a mixture; (b) centrifuging the mixture obtained in step (a) in the presence of glycine to produce a precipitate (glycine fractionation); (c) centrifuging the precipitate obtained in step (b) in the presence of ethanol to produce a supernatant (ethanol fraction 1); and (d) centrifuging the supernatant obtained in step (c) in the presence of ethanol to produce a precipitate (ethanol fraction 2).

4. 4. The method according to claim 1, wherein in step (b) glycine is present at 1.5 mol / L.

5. The method according to any one of claims 1 to 4, wherein in the step (c), the precipitate obtained in the step (b) is dissolved in a buffer solution, and then ethanol is added to the solution to a concentration of 1%.

6. The method according to any one of claims 1 to 5, wherein in step (d), ethanol is added to the supernatant obtained in step (c) so that the concentration of ethanol becomes 6%.

7. The method according to any one of claims 1 to 6, wherein the fibrinogen obtained contains from about 490 to about 700 units of blood clotting factor XIII per gram of fibrinogen.

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