EP0726943A1

EP0726943A1 - Activated factor xiii

Info

Publication number: EP0726943A1
Application number: EP93903183A
Authority: EP
Inventors: Birger Rostgard Jensen; Bent Riber Petersen; Ivan Diers; Paul Douglas Bishop
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1992-01-22
Filing date: 1993-01-20
Publication date: 1996-08-21
Also published as: NZ246865A; CA2128032A1; JPH07506001A; FI943458A0; WO1993015234A1; AU3449093A; AU665805B2; BR9305768A; FI943458A

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.