NZ246865A

NZ246865A - Activated factor xiii (plasma transglutaminase) and its use in food products and compositions

Info

Publication number: NZ246865A
Application number: NZ246865A
Authority: NZ
Inventors: Birger Rostgard Jensen; Bent Riber Petersen; Ivan Diers; Paul Douglas Bishop
Original assignee: Novo Nordisk As
Priority date: 1992-01-22
Filing date: 1993-01-20
Publication date: 1996-10-28
Also published as: CA2128032A1; BR9305768A; EP0726943A1; JPH07506001A; AU665805B2; WO1993015234A1; FI943458A0; FI943458A; AU3449093A

Description

<div class="application article clearfix" id="description"> ZA§ 8 6 5 New Zealand No. . Internationa/No. PCT/DK93/00016 10 BE ENTERED AFTER ACCEPTANCE AND PUBUCATION Priority dates; 22111^1 international fifing date: £©| \ 3 Classification; \ Pclo". G K.38 /4Sj Aa3u» |3I( BiS Publication date: « ^ 2<? OCT 098 Journal No.: IH-O^ NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of invention: Activated factor XIII Name, address and nationality of applicant(s) as in international application form: Novo Nordisk A/S, of Novo Alle, DK-2880 Bagsvaerd, Denmark - WO 93/15234 1 ACTIVATED FACTOR XIII 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 <5-glutamic amide bonds in the fibrin polymer. This crosslinking reaction requires the presence of calcium ions (L. Lorand et al., Proa. 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 fibronectin (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., Proa. 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 WO 93/15234 £.65 2 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., Chirurq. 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 transglutaminases have also been proposed for a variety of industrial purposes, primarly 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 2-20 227057 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 enzyme (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. WO 93/15234 246865 PCT/DK93/00016 3 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 5 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-10 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 15 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 20 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 25 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. 30 The Factor XIII composition may be added to the meat material before, during or after dicing or blending. After incubation, WO 93/15234 ?4 fi PCT/DK93/00016 4 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 5 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 10 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 15 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.). 20 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 25 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 30 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. 2A6 WO 93/15234 ITT/DK93/ 5 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. 5 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. 10 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 15 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. 20 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 25 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 30 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 LP5 WO 93/15234 2468 6 PCI7DK93/00016 6 in K. Mosbach (ed.)> "Immobilized Enzymes" in Methods in Enzvmologv 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 5 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, 10 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). 15 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 20 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 25 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. 30 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 WO 93/15234 246865 PCT/DK93/00016 7 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. 5 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 10 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 15 examples which are not in any way intended to limit the scope of the invention as claimed. Example 1 Preparation of activated stable Factor XIXI Source of rFXIII: recombinant Factor XIII (rFXIII) was 20 expressed substantially as described in P.D. Bishop et al., Biochemistry 2S., 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 25 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 30 centrifugation. The supernatant was further clarified by addition of 0.3% filter aid of the diatomite type and WO 93/15234 ?468 65 PCT/DK93/00016 8 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 11%(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 15 10 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 /im 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. WO 93/15234 246865 PCT/DK93/00016 9 rFXIII was applied with an HPLC-pump (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 5 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 10 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 15 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., 20 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. 25 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-5251), lactose monohydrate (Merck 7660), DTT = dithiothreitol (Sigma D-0632) and EDTA = Titriplex III (Merck Art. 8418) . Others are standard 30 laboratory grade. A. Activation WO 93/15234 24686 PCf/DK93/00016 10 Feed composi'tion: 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 5 below (cf. Fig. 1). Table 1 Feed: 5604 mg/1 F435 Flow +THR -THR ml/min % % 9 97. 4 - 74. 3 8 97. 5 77. 9 7 94. 9 79. 1 6 98. 9 85. 8 5 101. 7 90. 5 4 93. 5 87. 3 3 94. 4 89. 7 2 89. 3 88. 2 1 75. 8 74. 4 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 25 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). WO 93/15234 PCT/DK93 11 Table 2 Feed: 10942 mg/1 F435 5 Flow ml/min +THR -TKR 9.9 106.8 65.8 9 106.0 70.7 8 100.1 68.8 7 105.5 75.8 6 104.1 78.1 5 112.4 87.1 4 97.9 83.9 3 97.9 98.9 2 92.2 94.7 1 82.5 88.3 The results show a high activation yield due to the presence of DTT and EDTA. 20 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 25 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 30 (cf. Fig. 3). WO 93/15234 JL4JLP 6 12 Table 3 10 Feed: 5693 mg/l F435 Flow +THR -THR ml/min 2 95.3 34.4 1 89.8 42.5 0.5 90.3 56.6 0.4 87.6 59.3 0.3 84.0 62.9 0.2 75.2 63.8 0.1 34.8 26.4 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. 20 4). Table 25 30 Feed: iri590 mg/l F435 Flow +THR -THR ml/min 6 96.0 82.3 5 98.2 89.5 4 93.6 89.3 3 96.2 90.8 2 94.2 93.0 1 87.2 87.2 0.5 74.3 75.8 35 Results: The high concentration of rFXIXI 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 WO 93/15234 PCI7D103/OOO16 13 sample was diluted within 2 minutes and no precipitation occurred; at 0.5 ml/min precipitation occurred within 3 minutes. E. Stability 5 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 % (CI) were added to the effluent in a factorial set-up. Samples were assayed immediately and after 10 freeze-drying. The results are shown in Table 5 below (in percent yield). 14 Table 5 Feed: 4710 mg/l M57 Flow: 4.0 ml/min +THR -THR 1 E5 LI 62.5 54.6 2 E5 LI CI 83.2 74.1 3 E5 L2 66.3 59.4 4 E5 L2 CI 82.2 77.6 5 E10 LI 67.3 61.3 6 E10 LI CI 89.5 76.8 7 E10 L2 72.3 61.5 8 E10 L2 CI 85.6 77.4 after freeze-drying 1 E5 LI 29.3 21. 8 2 E5 LI CI 39.8 29. 2 3 E5 L2 31.0 23. 1 4 E5 L2 CI 40.2 29. 3 5 E10 LI 29.1 23. 2 6 E10 LI CI 42.8 32. 0 7 E10 L2 33.5 26. 7 8 E10 L2 CI 45.6 34. 2 after one month 1 E5 LI 34.6 25. 4 2 E5 LI CI 44.8 35. 3 3 E5 L2 35.4 25. 9 4 E5 L2 CI 44.6 36. 3 5 E10 LI 34.1 24. 4 6 E10 LI CI 44.6 32. 0 7 E10 L2 35.9 24. 7 8 E10 L2 CI 47.0 34. 1 after two months 1 E5 LI 43.1 34. 2 2 E5 LI CI 0.9 41. 4 3 E5 L2 1.7 37. 0 4 E5 L2 CI 53.3 43. 2 5 E10 LI 45.2 35. 2 6 E10 LI CI 50.7 40. 3 WO 93/15234 24686b PCT/DK93/00016 15 7 E10 L2 48.9 37.5 8 E10 L2 CI 53.5 43.5 after three months 1 E5 LI 42.5 31.6 2 E5 LI CI 48.9 41.6 3 E5 L2 36.7 29.3 4 E5 L2 CI 54.0 46.1 5 E10 LI 43.5 33.5 6 E10 LI CI 52.0 41.8 7 E10 L2 42.3 32.6 8 E10 L2 CI 51.5 41.2 Results: The activation yield is 60%, but when casein is added a yield of 75% is obtained. The freeze-dried preparations 15 containing casein have the highest overall yield regardless of the level of lactose and EDTA. F. 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 20 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. WO 93/15234 PCT/DK93/00016 16 Table 6 10 15 20 25 30 35 Feed: 4745 mg/l M57 Flow: 4.0 ml/min % residual activity of feed +THR -THR E10 L2 CO 84.9 73.4 E10 L2 CI 98.7 87.5 E10 L2 C2 104.7 94.9 E10 L2 C4 111.1 98.3 day 6 45.5 34.0 freeze- 58.9 43.9 dried 57.8 47.5 58.4 46.9 kept 38.1 27.4 cold 32.1 15.6 32.2 15.4 30.7 13. 6 day 12 37.6 29.4 freeze- 48.3 34.7 dried 52.2 39. 6 52.5 40.3 kept 26.6 18.8 cold 24.8 9.9 24.0 9.5 20.7 8.5 day 20 37.9 25.2 freeze- 49.1 34.5 dried 53.6 37.9 56.3 41.5 day 27 21.1 12.8 kept 24.0 5.2 cold 24.5 5.7 22.1 5.5 2.5 months 44.8 33.9 freeze- 57.2 45.9 dried 57.7 46.7 58.0 47.3 40 WO 93/15234 246865 PCT/DK93/00016 17 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. 6. Stability Feed composition: 10.76 g M57 in 300 ml 10 mM glycine buffer pH 10 8.0 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 Tab!<2 7 below. Samples were assayed immediately. Some samples were freeze-dried, others were kept in the 15 refrigerator. Table 7 Feed: 4513 mg/l M57 Flow: 4.0 ml/min 20 Day 1 % +THR % THR 25 CL CLED CLE CS CSED CSE 94.5 100.5 106.5 95.9 102.6 99. 6 79.9 85.7 89.4 80.5 86.9 85.5 Day 4 Freeze-dried 30 35 CL CLED CLE CS CSED CSE 55.7 72.8 53.7 60.8 78.7 56.3 40.5 57.1 37.4 40.0 57.0 35.6 WO 93/15234 PCT/15K9 18 LIS.8 10 15 20 25 30 35 Kept cold CL 34.8 18.2 CLED 56.3 38.3 CLE 35.3 17.0 CS 36.3 18.7 CSED 49.5 38.8 CSE 36.3 18.5 Day 13 Freeze-dried CL 48.8 37.4 CLED 68.9 57.3 CLE 49.3 31.6 CS 54.2 37.7 CSED 70.3 53.6 CSE 53.0 35.0 Day 20 Kept cold CL 27.0 6.1 CLED 34.3 10.9 CLE 27.0 5.7 CS 30.8 6.2 CSED 34.6 12.9 CSE 26.0 6.8 Day 43 Freeze-dried +THR -THR CL 65.1 48.9 CLED 87.1 67.5 CLE 64.0 47.5 CS 70.0 51.8 CSED 81.8 63.0 CSE ? 53.3 Kept cold CL 20.4 4.6 CLED 25.7 6.8 CLE 19.7 3.8 CS 23.2 4.8 CSED 24.8 5.6 CSE 21.4 4.3 WO 93/15234 24686b PCT/DK93/00016 19 Day 70 Freeze-dried 5 CL CLED CLE CS CSED CSE 61.9 75.2 61.7 63.2 75.9 66.1 49.3 63.7 49.4 46.0 60.7 48.9 10 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. 15 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 20 immediately. Some samples were freeze-dried, others were kept in the refrigerator. The results are shown in Table 8 below. WO 93/15234 2468 PCT/DK93/00016 20 Table 8 Feed: 4030 mg/l F435 Flow: 4 ml/min 5 +THR 9. -THR & Day 1 CLE 0 DTT 105.9 94.3 CLE 5 DTT 108.9 96.2 CLE 10 DTT 104.9 92.4 10 CLE 20 DTT 107.0 94.4 Day 15 CLE 0 DTT 26.1 10.4 Cold CLE 5 DTT 40.5 22. 0 CLE 10 DTT 40.2 22.9 CLE 20 DTT 44.0 25.3 15 Day 15 CLE 0 DTT 49.2 32.7 Freeze- CLE 5 DTT 77.1 61.9 dried CLE 10 DTT 84.9 70.1 CLE 20 DTT 79.2 62.2 Day 30 CLE 0 DTT 21.7 7.3 20 Cold CLE 5 DTT 35.3 18.9 CLE 10 DTT 33.2 16.9 CLE 20 DTT 42.0 23.6 Day 30 CLE 0 DTT 69.0 57.8 Freeze- CLE 5 DTT 98.3 84.0 25 dried CLE 10 DTT 88.8 76.2 CLE 20 DTT 89.6 76.1 Day 57 CLE 0 DTT 61.6 51.2 Freeze- CLE 5 DTT 84.1 75.4 dried CLE 10 DTT 87.1 74.3 30 CLE 20 DTT 91.5 78.5 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 35 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. WO 93/15234 24686 PCI7DK93/00016 21 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 5 (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 10 Feed: 6453 mg/l F435 Flow: 4.0 ml/min Day 1 +THR -THR % % % casein 0 82.6 77.8 0.1 81.5 76.6 0.25 91.6 85.3 0.5 90.9 80.6 1 104.0 90.4 1.5 84.7 79.8 2 104.2 97.6 15 20 Day 4 Kept cold 25 % casein 0 36.6 31.0 0.1 34.8 28.0 0.25 37.7 30.3 0.5 39.5 31.5 30 1 45.1 35.2 1.5 46.7 38.8 2 52.6 42.9 Freeze-dried % casein 35 0 47.8 41.3 0.1 47.8 40.6 0.25 49.0 40.2 0.5 56.7 48.8 1 63.8 55.6 40 1.5 72.5 65.0 2 75.8 66.9 WO 93/15234 24 68 6 PCT/DK93/00016 22 10 5 Day 24 Freeze-dried % casein 0 0.1 0.25 0.5 1 1.5 2 46.2 48.2 49.7 53.5 61.2 61.6 61.0 34.7 36.9 41.5 45.8 50.7 54.5 53. 0 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 15 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 20 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 Ca"*) (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 25 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 30 treated sample had solidified into a sausage which was stable at room temperature for several days until it was microbially degraded. WO 93/15234 P 23 Example 3 Activity of activated Factor XIII in minced beef Beef mince was prepared with the following composition: 5 Beef minced twice Water Salt (NaCl) 60% 38% 2% 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: 10 l. 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 25 30"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. WO 93/15234 PCT/DK93/00016 24 Table 10 1 2 3 4 Mince 73 .73% 76 .78% 73 .67% 84 .28% 132 . 21g 138 .42g 139 . 31g 153 . 25g Liquid 26 .27% 23 .22% 26 .33% 15 .72% 47 . lOg 41 .87g 49 . 79g 28 . 58g Colour dark dark dark pale Vol. mince 139 . 14ml 143 .85ml 135 .70ml 156 .37ml Density 1. 052 1. 039 0. 974 1, 020 It appears from these results that an increased amount of water is bound in the mince when the activated Factor XIII is added, 15 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 20 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 25 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 30 shows no peak at all (Fig. 7). WO 93/15234 68 6 PCT/DK93/00016 25 Example 4 Activity of activated Factor XIII in fish paste 400 g of pollack meat, 200 ml of water, 3 ml of caci2 and 12 g of NaCl was mixed in a high speed blender. The pH was measured 5 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 10 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 15 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%. 246 8 6 5 26 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> CLAIMS

1. Activated stable Factor XIII capable of retaining 60% of the initial activity after about 3 months.

2. Activated stable Factor XIII according to claim l, 5 an a'a dimer.

3. Activated stable Factor XIII according to claim 1, an a'a' dimer.

4 . Activated stable Factor XIII according to any of claims 1-3, which is a recombinant protein. 10

5. Activated stable Factor XIII according to any of claims 1-4, 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. 15

7. A composition according to claim 6 which comprises a chelating agent.

8. A composition according to claim 7, wherein the chelating agent is EDTA, EGTA or citrate. at least which is which is - 27 - 246 8 6 5

9. A composition according to claim 8, wherein EDTA is present in a concentration of 2-15 mM.

10. A composition according to claim 9 wherein EDTA is present in a concentration of 2-15 mM.

11. A composition according to claim 9 wherein EDTA is present in a concentration of 5-10 mM.

12. A composition according to any one of claims 6-11, which comprises a reducing agent.

13. A composition according to claim 12, wherein the reducing agent is dithiothreitol (DTT).

14. A composition according to claim 13, wherein DTT is present in a concentration of 1-10 mM.

15. A composition according to claim 14 wherein DTT is present in a concentration of 2-7 mM.

16. A composition according to claim 14 wherein DTT is present in a concentration of 2.5-5 mM.

17. A composition according to claim 6-11, which comprises a substance capable of preventing oxidation of -SH at Cys314 of Factor XIII.

18. A composition according to claim 17, wherein the substance is cysteine, sulfate or an antioxidant.

19. A composition according to claim 18 in which the antioxidant is ascorbic acid or glutathion. - 28 - 2468 6 5

20. A composition according to any one of claims 6-19, which comprises a sugar.

21. A composition according to claim 20, wherein the sugar is lactose, glucose, sucrose, maltose or trehalose.

22. A composition according to claim 20, wherein the sugar is present in an amount of 0.5-5%, by weight of the composition.

23. A composition according to claim 22 in which the sugar is present in an amount of 1-2% by weight of the composition.

24. A composition according to any one of claims 6-22, which further comprises casein.

25. A composition according to any one of claims 6-24, which further comprises a buffer.

26. A composition according to claim 25 in which the buffer is glycine, alanine or borate buffer.

27. A process for producing activated Factor XIII as claimed in claim 1, 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.

28. A process according to claim 27, wherein the proteolytic enzyme is thrombin, trypsin or a trypsin-like enzyme.

29. A process according to claim 27, wherein the buffer solution comprises a glycine, alanine or borate buffer. - 29 - 2468 6 5

30. A process according to claim 27, wherein the buffer solution comprises a chelating agent.

31. A process according to claim 28, wherein the chelating agent is EDTA, EGTA or citrate.

32. A process according to claim 31, wherein EDTA is present in a concentration of 2-15 mM.

33. A process according to claim 32 in which EDTA is present in a concentration of 3-12 mM.

34. A process according to claim 32 in which EDTA is present in a concentration of 5-10 mM.

35. A process according to any one of claims 27-34, wherein the buffer solution comprises a reducing agent.

36. A process according to claim 35, wherein the reducing agent is dithiothreitol (DTT).

37. A process according to claim 36, wherein DTT is present in a concentration of 1-10 mM.

38. A process according to claim 37 wherein DTT is present in a concentration of 2-7 mM.

39. A process according to claim 37 wherein DTT is present in a concentration of 2.5-5 mM.

40. A process according to any one of claims 27-34, wherein the buffer solution comprises a substance capable of preventing oxidation of -SH at Cys314 of Factor XIII.

41. A process according to claim 40, wherein the substance is cysteine, sulfate or an antioxidant. - 30 - 246 8 6 5

42. A process according to claim 41 wherein the antioxidant is ascorbic acid or glutathion.

43. A process according to any one of claims 27-42, wherein the buffer solution comprises a sugar.

44. A process according to claim 43, wherein the sugar is lactose, glucose, sucrose, maltose or trehalose.

45. A process according to claim 43, wherein the sugar is present in an amount of 0.5-5%.

46. A process according to claim 45 in which the sugar is present in an amount of 1-2%.

47. A process according to any one of claims 27-46, wherein the buffer solution further comprises casein.

48. A process according to any one of claims 27-47, which further comprises freeze-drying of the activated Factor XIII.

49. A process of producing activated Factor XIII as claimed in claim 1, 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.

50. A process according to claim 49, wherein the proteolytic enzyme is thrombin, trypsin or a trypsin-like enzyme.

51. A process according to claim 49 or 50, wherein the protease inhibitor is a trypsin inhibitor.

52. A process according to claim 51, wherein the trypsin inhibitor is aprotinin or soybean trypsin inhibitor. - 31 - 246 8 6 5

53. A process according to claim 49, wherein the buffer solution comprises a glycine, alanine or borate buffer.

54. A process according to claim 49, wherein the buffer solution comprises a chelating agent.

55. A process according to claim 54, wherein the chelating agent is EDTA, EGTA or citrate.

56. A process according to claim 55, wherein EDTA is present in a concentration of 2-15 mM.

57. A process according to claim 56 wherein EDTA is present in a concentration of 3-12 mM.

58. A process according to claim 56 wherein EDTA is present in a concentration of 5-10 mM.

59. A composition according to any one of claims 49-58, wherein the buffer solution comprises a reducing agent.

60. A process according to claim 59, wherein the reducing agent is dithiothreitol (DTT).

61. A process according to claim 60, wherein DTT is present in a concentration of 1-10 mM.

62. A process according to claim 61 wherein DTT is present in a concentration of 2-7 mM.

63. A process according to claim 61 wherein DTT is present in a concentration of 2.5-5 mM.

64. A process according to any one of claims 49-63, wherein the buffer solution comprises a substance capable of preventing oxidation of -SH at Cys314 of Factor XIII. - 32 - 246 8 6 5

65. A process according to claim 64, wherein the substance is cysteine, sulfate or an antioxidant.

66. A process according to claim 65 wherein the antioxidant is ascorbic acid or glutathion.

67. A process according to any one of claims 49-66, wherein the buffer solution comprises a sugar.

68. A process according to claim 67, wherein the sugar is lactose, glucose, sucrose, maltose or trehalose.

69. A process according to claim 68, wherein the sugar is present in an amount of 0.5-5%.

70. A process according to claim 69 wherein the sugar is present in an amount of 1-2%.

71. A process according to any one of claims 49-70, wherein the buffer solution further comprises casein.

72. A process according to any one of claims 49-71, which further comprises freeze-drying of the activated Factor XIII.

73. A method of producing a process meat product with improved water-binding and consistency properties, the method comprising mixing a composition according to any one of claims 6-26 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.

74. A method of producing a fish paste product with improved consistency properties, the method comprising mixing a composition according to any one of claims 6-26 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. </div>