EP0726943A1 - Activated factor xiii - Google Patents

Activated factor xiii

Info

Publication number
EP0726943A1
EP0726943A1 EP93903183A EP93903183A EP0726943A1 EP 0726943 A1 EP0726943 A1 EP 0726943A1 EP 93903183 A EP93903183 A EP 93903183A EP 93903183 A EP93903183 A EP 93903183A EP 0726943 A1 EP0726943 A1 EP 0726943A1
Authority
EP
European Patent Office
Prior art keywords
process according
factor xiii
buffer solution
activated
composition according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93903183A
Other languages
German (de)
French (fr)
Inventor
Birger Rostgard Jensen
Bent Riber Petersen
Ivan Diers
Paul Douglas Bishop
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP0726943A1 publication Critical patent/EP0726943A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/1025Acyltransferases (2.3)
    • C12N9/104Aminoacyltransferases (2.3.2)
    • C12N9/1044Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y203/00Acyltransferases (2.3)
    • C12Y203/02Aminoacyltransferases (2.3.2)
    • C12Y203/02013Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Definitions

  • the present invention relates to an activated Factor XIII product with improved properties, a composition comprising the 5 activated Factor XIII, and a process for producing activated Factor XIII.
  • Factor XIII also known as plasma transglutaminase is one of the components of the blood coagulation system, and circulates
  • Activated Factor XIII catalyses the crosslinking of fibrin polymers by introducing covalent bonds between non-covalent fibrin polymers. More specifically, activated Factor XIII catalyses the formation of
  • Factor XIII circulates as a tetrameric complex consisting of two a subunits (Mr of about 83 kD) containing the catalytic site of the enzyme and two b subunits (Mr of about 80 kD) (S.I. Chung et al., J. Biol. Chem. 249. 1974, pp. 940-950).
  • the b subunits are cleaved off.
  • a 4 kD fragment is cleaved off the N-terminal end of each of the a subunits (Schwartz et al., J. Biol. Chem. 248. 1973, pp. 1395-1407).
  • the potential catalytic site is located in the a chain with cysteine at the active centre.
  • Factor XIII Due to its function in the coagulation process, Factor XIII has been used for treating patients with postoperative wound healing disorders (Mishima et al., Chirur ⁇ . 55. 1984, pp. 803- 808) and scleroderma (Delbarre et al., Lancet 2., 1981, p.204) . Furthermore, Factor XIII has been used as a component of tissue 10 adhesives (US 4,414,976; US 4,453,939; US 4,377,572; US 4,362,567; US 4,298,598) and has been suggested for use in antifibrinolytic therapy for the prevention of postoperative bleeding and in the treatment of subarachnoid haemorrhage, ulcerative colitis and general wound healing.
  • transglutaminase has been added to minced meat and fish paste (cf. for instance JP 2-255060 to Ajinomoto, JP 2-
  • Transglutaminase has been added to gelatin to make highly polymerised gelatin products (cf. JP 2-86743 to Ajinomoto) .
  • the present invention relates to activated stable Factor XIII.
  • stable refers to the storage stability of the activated Factor XIII and is intended to indicate that the activated Factor XIII preparation retains at least 60% of its initial activity after about 3 months.
  • the present invention relates to a composition
  • a composition comprising activated stable Factor XIII in freeze- dried form or in the form of a frozen liquid concentrate.
  • the invention relates to a method of producing a processed meat product with improved water-binding and consistency properties, the method comprising mixing the composition of the invention with a meat material and incubating the mixture for a period of time sufficient to let the activated Factor XIII react with proteins present in the meat material.
  • this method may be used in the preparation of restructured meat products, e.g. processed ham, containing finely diced meat, or emulsified meat products such as sausages or chopped beef or pork, optionally together with soy protein.
  • the Factor XIII composition may be added to the meat material before, during or after dicing or blending. After incubation, the mixture may be put into appropriate containers such as sausage casings or tins and boiled.
  • the invention also relates to a method of producing a fish paste product with improved consistency properties, the method comprising mixing composition of the invention with a fish meat material and incubating the mixture for a period of time sufficient to let the activated Factor XIII react with proteins present in the fish meat material.
  • the Factor XIII composition of the invention may be used for the production of sausage casings by crosslinking of collagen, for making gelatin gels, in cheese- making for improving the yield of cheese by crosslinking soluble whey proteins, in baking for strengthening gluten, and in the food industry for making edible protein films for wrapping meat or fish products.
  • the Factor XIII composition may be used generally for the crosslinking of proteins, e.g. for immobilisation, precipitation or modification of the properties of a protein (such as changes in pi, hydrophilicity, hydrophobicity, etc.).
  • activated Factor XIII of the invention for medical purposes.
  • medical applications include, but are not limited to postoperative wound healing, inclusion in tissue adhesives, antifibrinolytic therapy, treatment of ulcerative colitis, cf. EP 268 772 and the references cited therein.
  • the present invention relates to a process for producing activated Factor XIII, the process comprising contacting Factor XIII precursor with an immobilised proteolytic enzyme, and collecting the activated Factor XIII in a buffer solution containing one or more stabilisers, or contacting Factor XIII precursor in a buffer solution containing one or more stabilisers with an immobilised proteolytic enzyme.
  • the term "Factor XIII precursor” is intended to indicate the zymogen form of Factor XIII, i.e. the a 2 dimer (2 x 83 kD) , also known as placental Factor XIII, or the a 2 b 2 tetramer, also known as plasma Factor XIII.
  • the invention relates to a process of producing activated Factor XIII, the process comprising contacting Factor XIII precursor with a proteolytic enzyme in a buffer solution containing one or more stabilisers, followed by addition, after a suitable interval, of a protease inhibitor.
  • the Factor XIII product of the invention may conveniently be provided in the form of a Factor XIII a 2 dimer (i.e. placental Factor XIII) .
  • the Factor XIII product may be in the form of an a'a dimer or a'a' dimer.
  • the a'a dimer form is one in which a 4 kD fragment has been cleaved off the N-terminal end of one of the a monomers
  • the a'a' dimer form is one in which a 4 kD fragment has been cleaved off the N-terminal end of both a monomers.
  • the Factor XIII product of the invention is advantageously a recombinant protein since this is a more reliable and economical source of Factor XIII than plasma.
  • the preparation of recombinant Factor XIII in yeast is described in, for instance, EP 268 772 to ZymoGenetics as well as P.D. Bishop et al., Biochemistry 29. 1990, pp. 1861-1869, the contents of which are incorporated herein by reference.
  • Factor XIII precursor as defined above may be activated with an immobilised proteolytic enzyme.
  • suitable enzymes are thrombin, trypsin or a trypsin-like enzyme (e.g. a protease obtainable from a species of Fusarium. cf. WO 89/06270) .
  • the proteolytic enzyme may suitably be immobilised by one of the procedures described in K. Mosbach (ed.), "Immobilized Enzymes” in Methods in Enzymology 44. Academic Press, New York, 1976, including covalent coupling to insoluble organic or inorganic supports, entrapment in gels and adsorption to ion exchange resins or other adsorbent materials.
  • Suitable support materials for the immobilised enzyme are, for instance, plastics (e.g. polypropylene, polystyrene, polyvinylchloride, polyurethane, latex, nylon, teflon, dacron, polyvinylacetate, polyvinylalcohol or any suitable copolymer thereof) , polysaccharides (e.g. agarose or dextran) , ion exchange resins (both cation and anion exchange resins) , silicon polymers (e.g. siloxane) or silicates (e.g. glass) .
  • plastics e.g. polypropylene, polystyrene, polyvinylchloride, polyurethane, latex, nylon, teflon, dacron, polyvinylacetate, polyvinylalcohol or any suitable copolymer thereof
  • polysaccharides e.g. agarose or dextran
  • ion exchange resins both cation and
  • the Factor XIII precursor may be contacted with a proteolytic enzyme after which a protease inhibitor is added.
  • the protease inhibitor may suitably be a trypsin inhibitor such as aprotinin or soybean trypsin inhibitor.
  • the buffer solution into which the activated Factor XIII is collected is preferably a glycine, alanine or borate buffer.
  • the stabiliser or stabilisers present in the buffer solution as well as in the final Factor XIII composition may be a chelating agent, for instance EDTA, EGTA or citrate.
  • EDTA may be present in a concentration of 2-15 mM, preferably 3-12 mM, more preferably- 5-10 mM.
  • Another stabiliser which may be present in the buffer solution and Factor XIII composition is a reducing agent or another substance capable of preventing oxidation of the active -SH at Cys314 of Factor XIII, e.g. a cysteine or sulfite, or an antioxidant such as ascorbic acid or glutathion.
  • a suitable reducing agent is dithiothreitol (DTT) , which may be present in a concentration of 1-10 mM, preferably 2-7 mM, more preferably .2.5-5 mM.
  • DTT dithiothreitol
  • a further stabiliser which may be present in the buffer solution and Factor XIII composition is a sugar.
  • suitable sugars are lactose, glucose, sucrose, maltose or trehalose.
  • the sugar may be present in an amount of 0.5-5%, preferably 1-2%, by weight.
  • a still further stabiliser which may be present in the buffer solution and Factor XIII composition is casein.
  • calcium ions should be present.
  • a currently preferred stabilising solution comprises 2% lactose, 2% casein, 10 mM EDTA, and 5 mM DTT in 10 mM glycine buffer, pH 8.0.
  • the present composition is in freeze-dried form as this generally results in improved stability.
  • rFXIII recombinant Factor XIII
  • the homogenized cells were diluted approximately
  • rFXIII was isolated by crystallization with ll%(w/v) Na-formiate at 25°C in 5 hours. The crystals were 10 harvested by centrifugation (4000 g in 30 minutes) and freezedried. In this way, two preparations of rFXIII were made: M57 with an initial activity of 285 g/kg, and F435 with an initial activity of 580 g/kg.
  • composition of feed Unless otherwise specified, glycine buffer 1510 mM pH 8.0 was used as the solvent. The concentration of rFXIII in the feed was 0.5, 5, 10 and 15 mg/ml. The mixture of glycine buffer and rFXIII was stirred for 1.5 - 2 hours at room temperature to ensure complete dissolution of rFXIII. pH was adjusted several times with NaOH. The solution was clarified by 20 centrifugation (14400 x g for 10 minutes) followed by filtration through a 0.45 ⁇ m membrane filter.
  • the %-yield calculated in the tables shown below is based on the assayed content of the feed (activated with thrombin) .
  • the assay for Factor XIII activity was carried out by a fluorometric activity assay (referred to in P.D. Bishop et al., supra) .
  • the whole content of the vials was dissolved in 100 ml TANEP buffer (0.1 M Tris-acetate, 0.15 M NaCl, 1 mM EDTA, 0.1% PEG 6000) pH 7,5 in order to avoid erroneous results due to inhomogenieties in the freeze-dried preparations. Samples were further diluted in TANEP buffer pH 7,5 prior to being assayed. The results shown are the average of two determinations.
  • Casein is Hammersten casein (Merck Art. 2242) , D-
  • A. Activation Feed composition 4.2 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. 10 mM DTT in TANEP buffer pH 7.5 was added to the effluent. The results of rFXIII activation appear from Table 1 below (cf. Fig. 1) .
  • Feed composition 8,81 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Trehalose (1%) , EDTA (10 mM) and DTT (10 mM) were added to the effluent. The results are shown in Table 2 below (cf. Fig. 2) . Table 2
  • Feed composition 2,6 g F435 in 200 ml 10 mM glycine buffer were applied on a Sepharose column on which a trypsin-like protease derived from Fusarium (prepared as described in WO 89/06270) had been immobilised by adding 200 mg of the active enzyme mixed with coupling buffer to 4 g of CNBr-activated Sepharose 4B (available from Pharmacia, Sweden) at pH 7.5. An HR5/10 column was packed to a bed height of 43 mm (bed volume 0,84 ml). Trehalose (1%), EDTA (10 mM) and DTT (10 mM) were added to the effluent. The results are shown in Table 3 below (cf. Fig. 3) . 10
  • Feed composition 6.99 g F435 in200 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Trehalose (1%) , 10 mM EDTA and 10 mM DTT were added to the effluent. The results are shown in Table 4 below (cf. Fig. 204) .
  • Feed composition 10.04 g M57 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above.
  • EDTA 10 E5 or 20 mM (E10)
  • lactose 2 LI
  • 4 % L2
  • casein 0 or 2 % Cl
  • the activation yield is 60%, but when casein is added a yield of 75% is obtained.
  • the freeze-dried preparations containing casein have the highest overall yield regardless of the level of lactose and EDTA.
  • Feed composition 10.07 g M57 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Lactose (2%) and EDTA (10 mM) and varying amounts of casein (0, 1, 2 and 4%) were added to the effluent. Samples were assayed immediately, and some of them were frozen or freeze-dried, while others were kept in the refrigerator. The results are shown in Table 6 below.
  • Feed composition 10.76 g M57 in 300 ml 10 mM glycine buffer pH 108.0 was applied on a trypsin-Sepharose column as described above. Casein (Hammarsten) 2% (C) , EDTA 10 mM (E) , DTT 5 mM (D) and 2% lactose (L) or sucrose (S) were added to the effluent as indicated in Table 7 below. Samples were assayed immediately. Some samples were freeze-dried, others were kept in the 15 refrigerator.
  • Casein (Hammarsten) 2% (C) , EDTA 10 mM (E) , DTT 5 mM (D) and 2% lactose (L) or sucrose (S) were added to the effluent as indicated in Table 7 below. Samples were assayed immediately. Some samples were freeze-dried, others were kept in the 15 refrigerator.
  • the activation yield is high, 80 - 90%.
  • the freeze- dried preparations containing casein, EDTA, DTT and lactose/sucrose give a high over-all yield of about 60%. They show no sign of loss of activity over two months. The same composition results in the most stable samples when kept cold.
  • Feed composition 2.51 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Casein 2% (C) , lactose 2% (L) , EDTA 10 mM (E) and DTT 0 - 20 mM were added to the effluent. Samples were assayed immediately. Some samples were freeze-dried, others were kept in the refrigerator. The results are shown in Table 8 below.
  • Feed composition 4.82 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Trehalose (1%), EDTA (10 mM) , DTT (10 mM) and casein (0-2%) were added to the effluent from the column. Samples were assayed immediately. Some samples were frozen and freeze-dried, others were kept in the refrigerator. The results are shown in Table 9 below.
  • Example 3 (used as a control) was prepared, and the two samples were placed in a water bath for 2 hours at 30°C followed by cooling at 5°C for 24 hours. After the sausage skins had been removed, the control sample was still liquid, while the enzymatically treated sample had solidified into a sausage which was stable at room temperature for several days until it was microbially degraded.
  • Example 3
  • the ingredients were mixed and blended.
  • the pH of the resulting mince was 5.51.
  • the pH was adjusted to 7.0 with NaOH.
  • the mince was divided into four portions as follows: 101. Control.
  • the amount of Factor XIII in the mince is calculated from a percentage of protein in beef of 20%.
  • the samples were tinned and heat treated in a water bath at 2530°C for 2.5 hours followed by treatment at 80°C for 1 hour.
  • the tins were cooled in a water bath at about 20"C and subsequently in a refrigerator over night.
  • the gel strength of the boiled mince was measured by means of an Instron dynamometer (available from Instron, FRG) , using the method described by P.-G. Klettner, Fleischwirtsch. 69 (2), 1989, pp. 225-226. The results of these experiments is shown in Figs. 5-7.
  • the ordinate indicates the force required to compress a 22 mm slice of the mince to a thickness of 2 mm.
  • the graph showing the gel strength figures for mince treated with 0.5% activated Factor XIII (Fig. 5) shows a distinct peak indicating breaking of the gel (as it should) .
  • the graph for mince treated with inactive Factor XIII at the same dosage level shows a less distinct peak (Fig. 6) , while the graph for mince treated with 0.05% inactive Factor XIII shows no peak at all (Fig. 7) .
  • the PVC skin was removed, and the gel strength af the sausages was measured on samples cut to a height of 25 mm by means of a Bloom gelometer available from Griffin and George Ltd. , Great Britain.
  • the strength of the samples was 234 g and 142 g, respectively, corresponding to a gel strength improvement in the enzymatically treated gel of 65%.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Activated stable Factor XIII may be prepared by a process comprising contacting Factor XIII precursor with an immobilised proteolytic enzyme, and collecting the activated Factor XIII in a buffer solution containing one or more stabilisers, or contacting Factor XIII precursor in a buffer solution containing one or more stabilisers with an immobilised proteolytic enzyme.

Description

ACTIVATED FACTOR XIII
i
FIELD OF INVENTION
The present invention relates to an activated Factor XIII product with improved properties, a composition comprising the 5 activated Factor XIII, and a process for producing activated Factor XIII.
BACKGROUND OF THE INVENTION
Factor XIII (also known as plasma transglutaminase) is one of the components of the blood coagulation system, and circulates
10 in the blood in zymogen form until it is activated by thrombin in the final stages of blood coagulation. Activated Factor XIII catalyses the crosslinking of fibrin polymers by introducing covalent bonds between non-covalent fibrin polymers. More specifically, activated Factor XIII catalyses the formation of
15 covalent bonds between free e-NH2-lysine groups and δ-gl tamic amide bonds in the fibrin polymer. This crosslinking reaction requires the presence of calcium ions (L. Lorand et al., Prog. Hemost. Thromb. 5, 1980, pp. 245-290) . Activated Factor XIII is also known to catalyse crosslinking reactions between other
20 protein molecules, e.g. collagen and fibrbnectm (Y. Sakata and N. Aoki, J. Clin. Invest. 65. 1980, pp. 290-297; D.F. Mosher, J. Biol. Chem. 250. 1975, pp. 6614-6621; D.F. Mosher and P.E. Chad, J. Clin. Invest. 64. 1979, pp. 781-787; J.E. Folk and J.S. Finlayson, Adv. Prot. Chem 31. 1977, pp. 1-133; L. Lorand
25 et al., Prog. Hemost. Thromb. 5 , 1980, pp. 245-290).
In the blood, Factor XIII circulates as a tetrameric complex consisting of two a subunits (Mr of about 83 kD) containing the catalytic site of the enzyme and two b subunits (Mr of about 80 kD) (S.I. Chung et al., J. Biol. Chem. 249. 1974, pp. 940-950). 30 On activation by thrombin and in the presence of Ca++, the b subunits are cleaved off. Furthermore, a 4 kD fragment is cleaved off the N-terminal end of each of the a subunits (Schwartz et al., J. Biol. Chem. 248. 1973, pp. 1395-1407). The potential catalytic site is located in the a chain with cysteine at the active centre.
5 Due to its function in the coagulation process, Factor XIII has been used for treating patients with postoperative wound healing disorders (Mishima et al., Chirurσ. 55. 1984, pp. 803- 808) and scleroderma (Delbarre et al., Lancet 2., 1981, p.204) . Furthermore, Factor XIII has been used as a component of tissue 10 adhesives (US 4,414,976; US 4,453,939; US 4,377,572; US 4,362,567; US 4,298,598) and has been suggested for use in antifibrinolytic therapy for the prevention of postoperative bleeding and in the treatment of subarachnoid haemorrhage, ulcerative colitis and general wound healing.
15 Apart from these medical uses, Factor XIII and other transgluta inases have also been proposed for a variety of industrial purposes, pri arly within the food industry. For example, transglutaminase has been added to minced meat and fish paste (cf. for instance JP 2-255060 to Ajinomoto, JP 2-
20227057 to Taiyo Fishery, JP 2-177863 to Ajinomoto) and to milk for the production of cheese (cf. JP 2-131537 to Ajinomoto). Transglutaminase has been added to gelatin to make highly polymerised gelatin products (cf. JP 2-86743 to Ajinomoto) .
The major disadvantage of using Factor XIII for medical or 25 industrial purposes is that the activated enzym (Factor XIII a*2) is not storage stable. This means that in concentrated solutions or on drying the enzyme activity is irreversibly lost. It is therefore an object of the present invention to provide an activated Factor XIII preparation with improved 30 storage stability. SUMMARY OF THE INVENTION
Accordingly, in one aspect the present invention relates to activated stable Factor XIII.
In the present context, the term "stable" refers to the storage stability of the activated Factor XIII and is intended to indicate that the activated Factor XIII preparation retains at least 60% of its initial activity after about 3 months.
In another aspect, the present invention relates to a composition comprising activated stable Factor XIII in freeze- dried form or in the form of a frozen liquid concentrate.
Contrary to previous reports, it has surprisingly been found possible according to the present invention to prepare a composition of activated Factor XIII which exhibits a satisfactory stability (as defined above) both in freeze-dried form and as a frozen liquid concentrate. This makes the composition convenient to use for medical as well as industrial purposes as no activation of Factor XIII is required immediately prior to use.
Thus, in a further aspect, the invention relates to a method of producing a processed meat product with improved water-binding and consistency properties, the method comprising mixing the composition of the invention with a meat material and incubating the mixture for a period of time sufficient to let the activated Factor XIII react with proteins present in the meat material.
More specifically, this method may be used in the preparation of restructured meat products, e.g. processed ham, containing finely diced meat, or emulsified meat products such as sausages or chopped beef or pork, optionally together with soy protein. The Factor XIII composition may be added to the meat material before, during or after dicing or blending. After incubation, the mixture may be put into appropriate containers such as sausage casings or tins and boiled.
The invention also relates to a method of producing a fish paste product with improved consistency properties, the method comprising mixing composition of the invention with a fish meat material and incubating the mixture for a period of time sufficient to let the activated Factor XIII react with proteins present in the fish meat material.
Apart from this, the Factor XIII composition of the invention may be used for the production of sausage casings by crosslinking of collagen, for making gelatin gels, in cheese- making for improving the yield of cheese by crosslinking soluble whey proteins, in baking for strengthening gluten, and in the food industry for making edible protein films for wrapping meat or fish products. Furthermore, it is contemplated that the Factor XIII composition may be used generally for the crosslinking of proteins, e.g. for immobilisation, precipitation or modification of the properties of a protein (such as changes in pi, hydrophilicity, hydrophobicity, etc.).
It is also contemplated to use activated Factor XIII of the invention for medical purposes. Examples of medical applications include, but are not limited to postoperative wound healing, inclusion in tissue adhesives, antifibrinolytic therapy, treatment of ulcerative colitis, cf. EP 268 772 and the references cited therein.
In a further aspect, the present invention relates to a process for producing activated Factor XIII, the process comprising contacting Factor XIII precursor with an immobilised proteolytic enzyme, and collecting the activated Factor XIII in a buffer solution containing one or more stabilisers, or contacting Factor XIII precursor in a buffer solution containing one or more stabilisers with an immobilised proteolytic enzyme. In the present context, the term "Factor XIII precursor" is intended to indicate the zymogen form of Factor XIII, i.e. the a2 dimer (2 x 83 kD) , also known as placental Factor XIII, or the a2b2 tetramer, also known as plasma Factor XIII.
Alternatively, the invention relates to a process of producing activated Factor XIII, the process comprising contacting Factor XIII precursor with a proteolytic enzyme in a buffer solution containing one or more stabilisers, followed by addition, after a suitable interval, of a protease inhibitor.
DETAILED DISCLOSURE OF THE INVENTION
The Factor XIII product of the invention may conveniently be provided in the form of a Factor XIII a2 dimer (i.e. placental Factor XIII) . As it has been found sufficient to activate one of the monomers only to obtain full activity, the Factor XIII product may be in the form of an a'a dimer or a'a' dimer. The a'a dimer form is one in which a 4 kD fragment has been cleaved off the N-terminal end of one of the a monomers, while the a'a' dimer form is one in which a 4 kD fragment has been cleaved off the N-terminal end of both a monomers.
The Factor XIII product of the invention is advantageously a recombinant protein since this is a more reliable and economical source of Factor XIII than plasma. The preparation of recombinant Factor XIII in yeast is described in, for instance, EP 268 772 to ZymoGenetics as well as P.D. Bishop et al., Biochemistry 29. 1990, pp. 1861-1869, the contents of which are incorporated herein by reference.
In the process of the invention, Factor XIII precursor as defined above may be activated with an immobilised proteolytic enzyme. Examples of suitable enzymes are thrombin, trypsin or a trypsin-like enzyme (e.g. a protease obtainable from a species of Fusarium. cf. WO 89/06270) . The proteolytic enzyme may suitably be immobilised by one of the procedures described in K. Mosbach (ed.), "Immobilized Enzymes" in Methods in Enzymology 44. Academic Press, New York, 1976, including covalent coupling to insoluble organic or inorganic supports, entrapment in gels and adsorption to ion exchange resins or other adsorbent materials. Coating on a particulate support may also be employed (cf. for instance A.R. Macrae and R.C. Hammond, Biotechnology and Genetic Engineering Reviews 3. 1985, p. 193. Suitable support materials for the immobilised enzyme are, for instance, plastics (e.g. polypropylene, polystyrene, polyvinylchloride, polyurethane, latex, nylon, teflon, dacron, polyvinylacetate, polyvinylalcohol or any suitable copolymer thereof) , polysaccharides (e.g. agarose or dextran) , ion exchange resins (both cation and anion exchange resins) , silicon polymers (e.g. siloxane) or silicates (e.g. glass) .
Alternatively, the Factor XIII precursor may be contacted with a proteolytic enzyme after which a protease inhibitor is added. The protease inhibitor may suitably be a trypsin inhibitor such as aprotinin or soybean trypsin inhibitor.
The buffer solution into which the activated Factor XIII is collected is preferably a glycine, alanine or borate buffer.
The stabiliser or stabilisers present in the buffer solution as well as in the final Factor XIII composition may be a chelating agent, for instance EDTA, EGTA or citrate. EDTA may be present in a concentration of 2-15 mM, preferably 3-12 mM, more preferably- 5-10 mM. Another stabiliser which may be present in the buffer solution and Factor XIII composition is a reducing agent or another substance capable of preventing oxidation of the active -SH at Cys314 of Factor XIII, e.g. a cysteine or sulfite, or an antioxidant such as ascorbic acid or glutathion. An example of a suitable reducing agent is dithiothreitol (DTT) , which may be present in a concentration of 1-10 mM, preferably 2-7 mM, more preferably .2.5-5 mM. A further stabiliser which may be present in the buffer solution and Factor XIII composition is a sugar. Examples of suitable sugars are lactose, glucose, sucrose, maltose or trehalose. The sugar may be present in an amount of 0.5-5%, preferably 1-2%, by weight. A still further stabiliser which may be present in the buffer solution and Factor XIII composition is casein. Incidentally, it should be noted that when the activated Factor XIII of the invention is used for crosslinking reactions, calcium ions should be present.
A currently preferred stabilising solution comprises 2% lactose, 2% casein, 10 mM EDTA, and 5 mM DTT in 10 mM glycine buffer, pH 8.0.
According to the invention, it is particularly preferred that the present composition is in freeze-dried form as this generally results in improved stability.
The present invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed.
Example 1
Preparation of activated stable Factor XIII
Source of rFXIII: recombinant Factor XIII (rFXIII) was expressed substantially as described in P.D. Bishop et al.,
Biochemistry 29. 1990, pp. 1861-1869. The cells were harvested by. centrifugation resulting in a wet cell volume of approximately 20% of the broth volume. EDTA 10 mM were added and pH adjusted to 7.8, and the cells were ruptured by 2 separate runs through a homogenizer (Rannie, Copenhagen,DK) at
800-900 bar. The homogenized cells were diluted approximately
3 times with lysis buffer (50 mM Tris,HCl, 10 mM EDTA, pH=7.8 in deionized water) and 1% Superfloc C521 was added in order to flocculate the cell debris, which was then removed by centrifugation. The supernatant was further clarified by addition of 0.3% filter aid of the diato ite type and filtration through a diatomite filter sheet with an appropiate pore size. A crude precipitate of rFXIII was obtained by adding 18% Na2S04 at 26-28°C and pH 7.0-7.2 in 45 minutes. The filter cake was harvested by addition of 1% filter aid and filtration 5 on a filter cloth. The filter cake was redissolved by adding 4 times the weight of the wet filter cake of a solution of 10 mM Tris,HCl, 5 mM EDTA, pH=7.8 and the filter aid was removed by filtration. Finally rFXIII was isolated by crystallization with ll%(w/v) Na-formiate at 25°C in 5 hours. The crystals were 10 harvested by centrifugation (4000 g in 30 minutes) and freezedried. In this way, two preparations of rFXIII were made: M57 with an initial activity of 285 g/kg, and F435 with an initial activity of 580 g/kg.
Composition of feed; Unless otherwise specified, glycine buffer 1510 mM pH 8.0 was used as the solvent. The concentration of rFXIII in the feed was 0.5, 5, 10 and 15 mg/ml. The mixture of glycine buffer and rFXIII was stirred for 1.5 - 2 hours at room temperature to ensure complete dissolution of rFXIII. pH was adjusted several times with NaOH. The solution was clarified by 20 centrifugation (14400 x g for 10 minutes) followed by filtration through a 0.45 μm membrane filter.
Determination of optimal flow rate; A clarified solution of rFXIII was pumped through a trypsin-Sepharose column (available from Pharmacia, Sweden) , beginning with the highest flow rate. 25 After passage of 60 ml + 3 bed volumes, samples were taken and immediately diluted to assay concentration and assayed with and without thrombin activation. The flow rate was diminished and after passage of 3 bed volumes, another sample was taken, and so on. All operations were performed at room temperature.
30 General set-up: For all experiments, trypsin-Sepharose was packed in a column with a diameter of 1 cm and a bed-height of 2.5 cm. rFXIII was applied with an HPLC-pu p (Knauer 64) for optimal flow control/constancy and ease of regulation. The optimal flow was determined as described above. 10 ml plastic vials containing 5 ml solution with the reagents to be tested were placed in a fraction collector. After passage of 60 ml, the effluent from the trypsin-Sepharose was directed to the vials in the fraction collector. The fraction collector was run in time mode and 5 ml of effluent was collected. As soon as a vial had been filled to a total of 10 ml it was removed, fitted with a stopper and turned upside down five times. Then the contents were transferred to a T-20 vial (glass, pre-weighed with lid) which was placed in a tray containing solid carbon dioxide. The frozen preparations were freeze-dried (this took two days) . The freeze-dried samples were stored in a refrigerator or in a cold room at 5 - 8°C.
The %-yield calculated in the tables shown below is based on the assayed content of the feed (activated with thrombin) .
The assay for Factor XIII activity was carried out by a fluorometric activity assay (referred to in P.D. Bishop et al., supra) . The whole content of the vials was dissolved in 100 ml TANEP buffer (0.1 M Tris-acetate, 0.15 M NaCl, 1 mM EDTA, 0.1% PEG 6000) pH 7,5 in order to avoid erroneous results due to inhomogenieties in the freeze-dried preparations. Samples were further diluted in TANEP buffer pH 7,5 prior to being assayed. The results shown are the average of two determinations.
Chemicals: Casein is Hammersten casein (Merck Art. 2242) , D-
(+)-trehalose is the dihydrate from Sigma (T-5.251) , lactose monohydrate (Merck 7660), DTT = dithiothreitol (Sigma D-0632) and EDTA = Titriplex III (Merck Art. 8418) . Others are standard laboratory grade.
A. Activation Feed composition: 4.2 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. 10 mM DTT in TANEP buffer pH 7.5 was added to the effluent. The results of rFXIII activation appear from Table 1 below (cf. Fig. 1) .
Table 1
Feed: 5604 mg/1 F435
Flow +THR -THR ml/min % %
B. Activation
Feed composition: 8,81 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Trehalose (1%) , EDTA (10 mM) and DTT (10 mM) were added to the effluent. The results are shown in Table 2 below (cf. Fig. 2) . Table 2
Feed: 10942 mg/1 F435
The results show a high activation yield due to the presence of DTT and EDTA.
C. Activation
Feed composition: 2,6 g F435 in 200 ml 10 mM glycine buffer were applied on a Sepharose column on which a trypsin-like protease derived from Fusarium (prepared as described in WO 89/06270) had been immobilised by adding 200 mg of the active enzyme mixed with coupling buffer to 4 g of CNBr-activated Sepharose 4B (available from Pharmacia, Sweden) at pH 7.5. An HR5/10 column was packed to a bed height of 43 mm (bed volume 0,84 ml). Trehalose (1%), EDTA (10 mM) and DTT (10 mM) were added to the effluent. The results are shown in Table 3 below (cf. Fig. 3) . 10
15 D. Activation
Feed composition: 6.99 g F435 in200 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Trehalose (1%) , 10 mM EDTA and 10 mM DTT were added to the effluent. The results are shown in Table 4 below (cf. Fig. 204) .
Table 4
Feed: 13590 mg/1 F435
25 Flow +THR -THR ml/min
6
5
4
30 3
2
1
0.5
35 Results: The high concentration of rFXIII in the feed requires a flow rate of 2 ml/min. The activation yield is excellent, probably due to the presence of stabilizers. At 1 ml/min the sample was diluted within 2 minutes and no precipitation occurred; at 0.5 ml/min precipitation occurred within 3 minutes.
E. stability
Feed composition: 10.04 g M57 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. EDTA 10 (E5) or 20 mM (E10) , lactose 2 (LI) or 4 % (L2) and casein 0 or 2 % (Cl) were added to the effluent in a factorial set-up. Samples were assayed immediately and after freeze-drying. The results are shown in Table 5 below (in percent yield) .
Table 5
Feed: 4710 mg/1 M57 Flow: 4.0 ml/min
+THR -THR
after freeze-drying
after one month
after two months
Results: The activation yield is 60%, but when casein is added a yield of 75% is obtained. The freeze-dried preparations containing casein have the highest overall yield regardless of the level of lactose and EDTA.
P. Stability
Feed composition: 10.07 g M57 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Lactose (2%) and EDTA (10 mM) and varying amounts of casein (0, 1, 2 and 4%) were added to the effluent. Samples were assayed immediately, and some of them were frozen or freeze-dried, while others were kept in the refrigerator. The results are shown in Table 6 below.
Table 6
Feed: 4745 mg/1 M57 Flow: 4.0 ml/min
% residual activity of feed
+THR -THR
Results: The activating/stabilising effect of casein is once more apparent, resulting in activation yields near 100%. Samples when assayed with thrombin activation showed 110% compared to feed. The freeze-dried preparations with 2 and 4% 5 casein showed the highest activity. All freeze-dried preparations are stable. The samples that were kept cold exhibited up to 27% residual activity after 6 days.
G. Stability
Feed composition: 10.76 g M57 in 300 ml 10 mM glycine buffer pH 108.0 was applied on a trypsin-Sepharose column as described above. Casein (Hammarsten) 2% (C) , EDTA 10 mM (E) , DTT 5 mM (D) and 2% lactose (L) or sucrose (S) were added to the effluent as indicated in Table 7 below. Samples were assayed immediately. Some samples were freeze-dried, others were kept in the 15 refrigerator.
Table 7
Feed: 4513 mg/1 M57 Flow: 4.0 ml/min
20
Day 1 +THR -THR
25
30
35 10
15
20
25
30
35
Results: The activation yield is high, 80 - 90%. The freeze- dried preparations containing casein, EDTA, DTT and lactose/sucrose give a high over-all yield of about 60%. They show no sign of loss of activity over two months. The same composition results in the most stable samples when kept cold.
H. Stability
Feed composition: 2.51 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Casein 2% (C) , lactose 2% (L) , EDTA 10 mM (E) and DTT 0 - 20 mM were added to the effluent. Samples were assayed immediately. Some samples were freeze-dried, others were kept in the refrigerator. The results are shown in Table 8 below.
Results: Activation yields are extremely good; this is probably a combination of the purer F435 preparation and the effect of casein. The freeze-dried preparations have over-all yields of 75% when DTT is present, and they are stable within the time frame. DTT has also a stabilizing effect on the samples which are kept cold: 20% residual activity after one month. I. Stability
Feed composition: 4.82 g F435 in 300 ml 10 mM glycine buffer pH 8.0 was applied on a trypsin-Sepharose column as described above. Trehalose (1%), EDTA (10 mM) , DTT (10 mM) and casein (0-2%) were added to the effluent from the column. Samples were assayed immediately. Some samples were frozen and freeze-dried, others were kept in the refrigerator. The results are shown in Table 9 below.
Table 9
Feed: 6453 mg/1 F435 Flow: 4.0 ml/min
Day 1 +THR -THR
75.8 66.9
Results: the activation yield is fairly good and the effect of casein is once more apparent. Casein also seems to stabilize samples kept in the refrigerator. The over-all yield after freeze-drying is far lower than expected. So far, there is no explanation of this phenomenon.
Example 2
Activity of activated Factor XIII on sodium caseinate
0.4% (w/w of protein) of a freeze-dried, activated Factor XIII composition prepared according to Example 1 (F435 containing 1% trehalose, 10 mM EDTA, 10 mM DTT and 2% casein) was added to a solution of sodium caseinate (11.1% protein, 5 mM CaX)
(Miprodan 30 available from MD Foods, Viby, Denmark) pH 7.0, and a 100 g sample was filled into a PVC sausage skin which was sealed at both ends. A similar sample not containing any enzyme
(used as a control) was prepared, and the two samples were placed in a water bath for 2 hours at 30°C followed by cooling at 5°C for 24 hours. After the sausage skins had been removed, the control sample was still liquid, while the enzymatically treated sample had solidified into a sausage which was stable at room temperature for several days until it was microbially degraded. Example 3
Activity of activated Factor XIII in minced beef
Beef mince was prepared with the following composition:
The ingredients were mixed and blended. The pH of the resulting mince was 5.51. The pH was adjusted to 7.0 with NaOH.
The mince was divided into four portions as follows: 101. Control.
2. Added 0.056% CaCl2 corresponding to 5 mM Ca and 0.5% (of the meat protein) of freeze-dried Factor XIII (M57; not activated) (corresponding to 120 mg of Factor XIII) .
3. Added 0.056% CaCl2 corresponding to 5 mM Ca and 0.05% (of the 15 meat protein) of freeze-dried Factor XIII (M57; not activated)
(corresponding to 12 mg of Factor XIII) .
4. Added 0.056% CaCl2 corresponding to 5 mM Ca and 0.5% (of the meat protein) of freeze-dried, activated Factor XIII prepared as described in Example 1 (F435 containing 1% trehalose, 10 mM
20 EDTA, 10 mM DTT and 2% casein) (corresponding to 116 mg of Factor XIII) .
The amount of Factor XIII in the mince is calculated from a percentage of protein in beef of 20%.
The samples were tinned and heat treated in a water bath at 2530°C for 2.5 hours followed by treatment at 80°C for 1 hour. The tins were cooled in a water bath at about 20"C and subsequently in a refrigerator over night.
The content of mince and liquid in the tins was measured (in %) , as was the volume and density of the mince. The results are 30 shown in Table 10 below. Table 10
It appears from these results that an increased amount of water is bound in the mince when the activated Factor XIII is added, resulting in an increased volume, although some effect of 0.5% inactive Factor XIII is also observed, probably due to activating factors present in the meat.
The gel strength of the boiled mince was measured by means of an Instron dynamometer (available from Instron, FRG) , using the method described by P.-G. Klettner, Fleischwirtsch. 69 (2), 1989, pp. 225-226. The results of these experiments is shown in Figs. 5-7. In the figures, the ordinate indicates the force required to compress a 22 mm slice of the mince to a thickness of 2 mm. The graph showing the gel strength figures for mince treated with 0.5% activated Factor XIII (Fig. 5) shows a distinct peak indicating breaking of the gel (as it should) . The graph for mince treated with inactive Factor XIII at the same dosage level shows a less distinct peak (Fig. 6) , while the graph for mince treated with 0.05% inactive Factor XIII shows no peak at all (Fig. 7) . Example 4
Activity of activated Factor XIII in fish paste
400 g of pollack meat, 200 ml of water, 3 ml of CaCl2 and 12 g of NaCl was mixed in a high speed blender. The pH was measured to 6.86, and was not adjusted. To a 100 g sample of the mixture was added 36 mg of a freeze-dried, activated Factor XIII preparation prepared according to Example 1 (F435 containing 1% trehalose, 10 mM EDTA, 10 mM DTT and 2% casein) and to another 100 g sample (used as a control) was added 36 mg of water. The two samples were filled into PVC sausage skins, diameter 25 mm, which were sealed at both ends. The two samples were placed in a water bath at 30°C for two hours. They were subsequently heated to 90°C for 30 min. and stores at 5"C for 24 hours.
The PVC skin was removed, and the gel strength af the sausages was measured on samples cut to a height of 25 mm by means of a Bloom gelometer available from Griffin and George Ltd. , Great Britain. The strength of the samples was 234 g and 142 g, respectively, corresponding to a gel strength improvement in the enzymatically treated gel of 65%.

Claims

1. Activated stable Factor XIII.
2. Activated stable Factor XIII according to claim 1, which is an a'a dimer.
53. Activated stable Factor XIII according to claim 1, which is an a'a' dimer.
4. Activated stable Factor XIII according to any of claims 1-3, which is a recombinant protein.
5. Activated stable Factor XIII according to any of claims 1-4, 0 which is substantially free from other proteins.
6. A composition comprising activated stable Factor XIII according to any of claims 1-5 in freeze-dried form or in the form of a frozen liquid concentrate.
7. A composition according to claim 6 which comprises a' 5 chelating agent.
8. A composition according to claim 7, wherein the chelating agent is EDTA, EGTA or citrate.
9. A composition according to claim 8, wherein EDTA is present in a concentration of 2-15 mM, preferably 3-12 mM, more 0 preferably 5-10 mM.
10. A composition according to any of claims 6-9, which comprises a reducing agent.
11. A composition according to claim 10, wherein the reducing agent is dithiothreitol (DTT) .
12. A composition according to claim 11, wherein DTT is present in a concentration of 1-10 mM, preferably 2-7 mM, more preferably 2.5-5 mM.
13. A composition according to claim 6-9, which comprises a 5 substance capable of preventing oxidation of -SH at Cys314 of
Factor XIII.
14. A composition according to claim 13, wherein the substance is cysteine, sulfate or an antioxidant such as ascorbic acid or glutathion.
1015. A composition according to any of claims 6-14, which comprises a sugar.
16. A composition according to claim 15, wherein the sugar is lactose, glucose, sucrose, maltose or trehalose.
17. A composition according to claim 15, wherein the sugar is 15 present in an amount of 0.5-5%, preferably 1-2%, by weight of the composition.
18. A composition according to any of claims 6-17, which further comprises casein.
19. A composition according to any of claims 6-18, which 20 further comprises a buffer such as a glycine, alanine or borate buffer.
20. A process for producing activated Factor XIII, the process comprising contacting Factor XIII precursor with an immobilised proteolytic enzyme, and collecting the activated Factor XIII in
25 a buffer solution containing one or more stabilisers, or contacting Factor XIII precursor in a buffer solution containing one or more stabilisers with an immobilised proteolytic enzyme.
21. A process according to claim 20, wherein the proteolytic enzyme is thrombin, trypsin or a trypsin-like enzyme.
22. A process according to claim 20, wherein the buffer solution comprises a glycine, alanine or borate buffer.
523. A process according to claim 20, wherein the buffer solution comprises a chelating agent.
24. A process according to claim 21, wherein the chelating agent is EDTA, EGTA or citrate.
25. A process according to claim 24, wherein EDTA is present in 10 a concentration of 2-15 mM, preferably 3-12 mM, more preferably
5-10 mM.
26. A process according to any of claims 20-25, wherein the buffer solution comprises a reducing agent.
27. A process according to claim 26, wherein the reducing agent 15 is dithiothreitol (DTT) .
28. A process according to claim 27, wherein DTT is present in a concentration of 1-10 mM, preferably 2-7 mM, more preferably 2.5-5 mM.
29. A process according to any of claims 20-25, wherein the 20 buffer solution comprises a substance capable of preventing oxidation of -SH at Cys314 of Factor XIII.
30. A process according to claim 29, wherein the substance is cysteine, sulfate or an antioxidant such as ascorbic acid or
. glutathion.
2531. A process according to any of claims 20-30, wherein the buffer solution comprises a sugar.
32. A process according to claim 31, wherein the sugar is lactose, glucose, sucrose, maltose or trehalose.
33. A process according to claim 31, wherein the sugar is present in an amount of 0.5-5%, preferably 1-2%.
534. A process according to any of claims 20-33, wherein the buffer solution further comprises casein.
35. A process according to any of claims 20-34, which further comprises freeze-drying of the activated Factor XIII.
36. A process of producing activated Factor XIII, the process 10 comprising contacting Factor XIII precursor with a proteolytic enzyme in a buffer solution containing one or more stabilisers, followed by addition, after a suitable interval, of a protease inhibitor.
37. A process according to claim 36, wherein the proteolytic 15 enzyme is thrombin, trypsin or a trypsin-like enzyme.
38. A process according to claim 36 or 37, wherein the protease inhibitor is a trypsin inhibitor.
39. A process according to claim 38, wherein the trypsin inhibitor is aprotinin or soybean trypsin inhibitor.
2040. A process according to claim 36, wherein the buffer solution comprises a glycine, alanine or borate buffer.
41. A process according to claim 36, wherein the buffer solution comprises a chelating agent.
42. A process according to claim 41, wherein the chelating 25 agent is EDTA, EGTA or citrate.
43. A process according to claim 42, wherein EDTA is present in a concentration of 2-15 mM, preferably 3-12 mM, more preferably 5-10 mM.
44. A composition according to any of claims 36-43, wherein the 5 buffer solution comprises a reducing agent.
45. A process according to claim 44, wherein the reducing agent is dithiothreitol (DTT) .
46. A process according to claim 45, wherein DTT is present in a concentration of 1-10 mM, preferably 2-7 mM, more' preferably
102.5-5 mM.
47. A process according to any of claims 36-46, wherein the buffer solution comprises a substance capable of preventing oxidation of -SH at Cys314 of Factor XIII.
48. A process according to claim 47, wherein the substance is 15 cysteine, sulfate or an antioxidant such as ascorbic acid or glutathion.
49. A process according to any of claims 36-48, wherein the buffer solution comprises a sugar.
50. A process according to claim 49, wherein the sugar is 20 lactose, glucose, sucrose, maltose or trehalose.
51. A process according to claim 50, wherein the sugar is present in an amount of 0.5-5%, preferably 1-2%.
52. A process according to any of claims 36-51, wherein the buffer solution further comprises casein.
2553. A process according to any oc claims- 36-52, which further comprises freeze-drying of the activated Factor XIII.
54. A method of producing a processed meat product with improved water-binding and consistency properties, the method comprising mixing a composition according to any of claims 6-19 with a meat material and incubating the mixture for a period of time sufficient to let the activated Factor XIII react with proteins present in the meat material.
55. A method of producing a fish paste product with improved consistency properties, the method comprising mixing a composition according to any of claims 6-19 with a fish meat material and incubating the mixture for a period of time sufficient to let the activated Factor XIII react with proteins present in the fish meat material.
EP93903183A 1992-01-22 1993-01-20 Activated factor xiii Withdrawn EP0726943A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DK9200022 1992-01-22
WOPCT/IB92/00022 1992-01-22
PCT/DK1993/000016 WO1993015234A1 (en) 1992-01-22 1993-01-20 Activated factor xiii

Publications (1)

Publication Number Publication Date
EP0726943A1 true EP0726943A1 (en) 1996-08-21

Family

ID=8153787

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93903183A Withdrawn EP0726943A1 (en) 1992-01-22 1993-01-20 Activated factor xiii

Country Status (8)

Country Link
EP (1) EP0726943A1 (en)
JP (1) JPH07506001A (en)
AU (1) AU665805B2 (en)
BR (1) BR9305768A (en)
CA (1) CA2128032A1 (en)
FI (1) FI943458A (en)
NZ (1) NZ246865A (en)
WO (1) WO1993015234A1 (en)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19508192A1 (en) * 1995-03-09 1996-09-12 Behringwerke Ag Stable transglutaminase preparations and process for their preparation
JPH08333277A (en) * 1995-06-05 1996-12-17 Hoechst Japan Ltd Stabilized aqueous solution formulation of human blood coagulating xiii-th factor
JPH10276695A (en) 1997-04-11 1998-10-20 Ajinomoto Co Inc Production of noodle
EP0981630B1 (en) 1997-05-16 2008-11-19 Novozymes, Inc. Polypeptides having prolyl pipeptidyl aminopeptidase activity and nucleic acids encoding same
GB2350046B (en) * 1999-05-20 2002-12-18 British Sugar Plc Edible compositions containing trehalose
AU2001253469A1 (en) * 2000-04-27 2001-11-12 Sunrise Technologies International, Inc. Compositions and methods for stabilizing modified tissue
WO2004007520A2 (en) * 2002-07-12 2004-01-22 Medarex, Inc. Methods and compositions for preventing oxidative degradation of proteins
JP4524076B2 (en) * 2003-04-02 2010-08-11 天野エンザイム株式会社 Stabilized transglutaminase
MY142987A (en) * 2005-06-08 2011-02-14 Hayashibara Biochem Lab Solution for tissue adhesion prevention and method for tissue adhesion prevention
ATE521700T1 (en) * 2005-12-22 2011-09-15 Zymogenetics Inc METHOD FOR ACTIVATION OF PRETHROMBIN-1
EP2004214B1 (en) 2006-03-16 2012-11-07 STELLARIS PHARMACEUTICALS Aps Local treatment with factor vii
JP5166259B2 (en) * 2006-06-22 2013-03-21 キッコーマン株式会社 Methods for stabilizing polyamine oxidase and improving substrate specificity
JP5326324B2 (en) * 2008-04-01 2013-10-30 東洋紡株式会社 Method for stabilizing tyramine oxidase and composition thereof
WO2010074955A1 (en) 2008-12-16 2010-07-01 Novozymes, Inc. Polypeptides having carboxypeptidase activity and polynucleotides encoding same
WO2010074338A1 (en) 2008-12-26 2010-07-01 味の素株式会社 Enzyme preparation for single-ingredient meat product and method for producing single-ingredient meat product
JP5919914B2 (en) * 2012-03-15 2016-05-18 東ソー株式会社 Pretreatment reagent for homocysteine measurement
US11473073B2 (en) 2015-06-26 2022-10-18 Dupont Nutrition Biosciences Aps Aminopeptidases for protein hydrolyzates
WO2022192529A1 (en) * 2021-03-10 2022-09-15 Curie Co. Inc. Activation of zymogens by immobilized protease enzymes

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2705024B2 (en) * 1987-07-02 1998-01-26 マルハ株式会社 Food manufacturing method
DE3734923C1 (en) * 1987-10-15 1989-01-26 Biotest Pharma Gmbh Process for the preparation of a sterile plasma protein solution containing fibrinogen and coagulation factor XIII
CS136091A3 (en) * 1990-05-10 1992-04-15 Zymo Genetics Agents for determining thrombi and their application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9315234A1 *

Also Published As

Publication number Publication date
WO1993015234A1 (en) 1993-08-05
CA2128032A1 (en) 1993-08-05
AU3449093A (en) 1993-09-01
NZ246865A (en) 1996-10-28
JPH07506001A (en) 1995-07-06
FI943458A (en) 1994-09-21
BR9305768A (en) 1997-01-28
FI943458A0 (en) 1994-07-21
AU665805B2 (en) 1996-01-18

Similar Documents

Publication Publication Date Title
AU665805B2 (en) Activated factor XIII
US4377572A (en) Tissue adhesive
EP0123304B1 (en) A method for stabilizing tissue plasminogen activator and a stable aqueous solution or powder containing the same
Ambrus et al. Plasmin-antiplasmin complex as a reservoir of fibrinolytic enzyme
Dahlqvist et al. Hydrolysis of β‐galactosides using polymer‐entrapped lactase. A study towards producing lactose‐free milk
JPH05186369A (en) Method for producing concentrated human thrombin liquid for therapeutic application
FI85335C (en) Process for Preparation of Lyophilized Pharmaceutical Tissue Plasma Minogen Activator (t-PA) Composition
JPH0665280B2 (en) Protein gelling agent and protein gelling method using the same
JPH07501517A (en) topical fibrinogen complex
JP2787317B2 (en) Concentrate of thrombin clotting protein, method for producing the same and therapeutic use thereof
HRP931496A2 (en) Process for the preparation of biological preparations not containing (active) viruses
JP2690944B2 (en) High concentration solution of protein having tissue plasminogen activator activity
US20110114524A1 (en) Storage-stable, functionally intact fibrinogen
US4082612A (en) Plasminogen activator complex
JP4010573B2 (en) Composition for stabilizing plasma during pasteurization and pasteurized plasma solution for therapeutic use
AU4650189A (en) Purification of factor xiii
WO1996041817A1 (en) Collagen peptide fraction and its uses
CA1283047C (en) Stabilized plasminogen activator precursor and method of producing the same
Jamlang et al. Effect of p H, Protein Concentration, and Ionic Strength on Heat Inactivation of Staphylococcal Enterotoxin B
Walker et al. A simplified method for preparation of fibrinogen
Cheftel et al. Effects of high pressure on dairy proteins: a review
EP0047879B1 (en) Process for preparing modified protein compositions
RU2045902C1 (en) Method for stabilization of blood plasma during pasteurization and method for pasteurization of blood plasma
Mahoney et al. Stabilization of lactase (Escherichia coli) by milk components and related compounds
Váradi et al. Purification and properties of human factor IXa

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19940822

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 19990416

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20030606