CA2128032A1

CA2128032A1 - Activated factor xiii

Info

Publication number: CA2128032A1
Application number: CA002128032A
Authority: CA
Inventors: Birger R. Jensen; Bent R. Petersen; Ivan Diers; Paul D. Bishop
Original assignee: Individual
Current assignee: Novo Nordisk AS
Priority date: 1992-01-22
Filing date: 1993-01-20
Publication date: 1993-08-05
Also published as: NZ246865A; JPH07506001A; EP0726943A1; 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

WO93/15234 212 ~ 0 3 2 PC~/DK93/00~16 ACTIV~TED FACTOR XIII

FIELD OF INVENTION

The present invention relat~s to an activated Factor XIII
product with improved properties, a composition comprising the 5 activated ~actor XIII, and ~ process for producing activated Factor XIII.

BACKGROUND OF ~HE INVENTION

Factor XIII (also known as plasma transglu~aminase) is one of the compon~nts of the blood coagulatlon sy~tem, and circulates 10 in the blood in:zymogen ~orm until it is activated by thrombin in the final stages o~ blood coagulation~ Acti~ated Factor XIII
cataly es th~ crosslinking of fibrin polymers by introducing covalent bond~ be~ween non-covalent fibrin polymers. More specifically, activated Factor~XIII catalyses the formation of :: 15 covalen~ b~nds betw~en free ~-NH2-lysine groups and S glutamic ~ amide bond~ in the;fibrin~poIymerO Thi~ crosslinking reaction :~ : r~quire~ the presence~o~ càlcium ions ~L. Lorand et al., Proq.
emos~ h~mk~ 5~ ~1980~ ~pp~ 245-290) n A~tivated Factor XIII i5 al~o ~ wn t~: catalyse crosslinking ~eac:tions between o~her

2 0 protein;m~leaules, è~.g. collagen and~fibronectin (Y0 5aka~a and N~. Aoki,;~ Cli~. ~ In~v~ 65,: 1980, pp.: 29~ 297; V~F. Mosher, . ~
Biol.~Cnem.;~250~ l975,~pp. 6614-6~2l;~D.F. Mosher and P.E.
had,~ J.~ in~ Invest.~64, 1979,~ pp. 78l-787; J.E. Folk and JtSO;Finl~y~on~Ldv.~prot. Ch~m 3l,,l977; pp. 1-133; IJ. Lorand 25 et~al., Prvq~_~emost. Thromb. S, 1980, pp. 245-290).

In ~he blood,~Factor XIII ~circulates as a tet~americ complex onsisting o~ two~a~subunits~(~r of about 83 kD) containing the a~alytic si~e of: the enzyme and ~two b subunits (Mr of about 80 kD~ (S.:I. ChuIlg et al., J.~ siOl. Ch~n. 2~9 , 1~74 , pp. 940-950) .
~:~ 30 On :activation by ~ throm~in and in~ the presence of ca+~, the bsubunits are cleaved of f . Furthsrmore, a 4 kD f rag~nent is WO 93/15234 2 1 2 8 (1 3 2 PCI/DK91/ilD016 cleaved off the N-terminal end of each of the a subunits (5chwarkz 2t 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 ~unction in the coagulation process, Factor XIII has been used for treating patients with postoperative wound healing disorders (Mishima et al., Chirurg 55, 1984, pp. ~03-808) and scleroderma (Delbarre et al., Lancet 2, 1~81, p.204).
Furthermore, Factor XIII has been used as a component of tissue 10 adhesives (US 4,~14,976; US 41453,939î US 4,377,572; US
4,362,567; US 4,298,5~8) and has b~en suggested for use in antifibrinoly~ic therapy for the prevention of postoperative bleeding and in the ~r~atment of subarachnoid haemorrhage, ulcerative colitis and general wound healing.

15 Apart from these medical uses, Factor xIIr and other transglutaminases~ha~e also :been proposed for a variety of industrial purposes, primarly within th~ food industry. For ; example, transglutaminase: has been added to minced meat and i h paste (cf. for instance JP 2-255060 to Ajinomoto, JP 2-20 227057 to:Taiyo Fishery, JP 2-177863 to Ajinomoto) and to milk or the production o~;~che~se (cf. JP 2-131537 to Ajinomoto).
: Transglutaminase~ h~s~ been;added to gelatin to ~make highly ` polymeri~ed ge~latin:~products (cf. JP 2-~6743 to ~jinomoto).

The major di~ad~antage of using Factor XIII for medical or 25 indus~ial purposes~is:that ~he activated enæyme (Factor XIII
a'a)~ is not storage ~stable. This means that in concentrated : solutions or on dxying the enz~me activity is irreversibly : lost. I~ is therefore~an object of the~present invention to ; :pro~ide an activated Factor XIII preparation with imprQved : 30 storagé s~ability. ~: ~
: ~

: ;~ , : : ~ : .
-: :

WO93/152~ . 212 ~ ~-3 2 PCT/DK93/0~0~6 SUMMAR~ OF T~E INV~NTION

Accordingly, in one aspect the present invention relates to activated stable Factor XIII.

In ~he present context, the term "stable" refers to the storage 5 ~tability of the activated Factor XIII and is intended to indicate that the activated Factor XIII preparation retains at least 60% of its initial activity a~ter 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 previou~ report~, it has surprlsingly been found po~5i~1~ according to the pr~sent in~entio~ to prepare a composition of activated Factor XIII which exhibits a satisfactory stability (as defined above) both in freeze-dried : 15 form and a~ a frozen li~uid concentrate. This makes the composition convenient to use for medical as wbell as industrial purposes as no acti~ation of Factor XIII is re~uired ~:
immediately prior to use.

Thu6, in:a ~urther aspect, the:in~ention relates to a methQd of ; 20 produciny a pr:oce~sed meat product with improv~d wat~- binding : and;consis~ency~propertie5,:~he method co~prising mi~ing the composition o~ the in~ention with a meat material and incubatin~: th~ mix~ure ~or a period of time sufficient to let ,t~e~activated Fa¢tor XIII react with pr~teins present in the 25 meat material.

N~re ~pecificall:y, this; method may be used in the preparation of:~restruct~red meat::products, e.g. processed ham, containing : : ~ finely diced meat~ or emulsi~ied~meat products such as sausages ` ~ : or chopped beef or pork, optionally together with soy protein.
~;~ 30:~he Factor XIII composition may be added to the meat material before, during or after dicing or blending. After incubation, W~93/1~234 . PCT/DK93/00016 .,<~
,r f~

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 b~ used for the production of sausage casings by crosslinking of collagen, for making gelatin yels, 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 crosælin~ing of proteinsr e.g. ~or immobilisation, precipitation or modification of the propertles of a protein . (such a~ changes~in pI, hydrophilicity, hydrophobiciky, etc.).

20 It is al~o contemplated to use activated Factor XIII of the in~ention for~ medical purposes. Examples of... medica~
a~plications include, but~are not limited to postopera~ive wound healing,~Inclusion in tissue ad~esi~es, antifibrinolytic therapy, kreatment~of ulcerative~ colitis, c~. EP 268 772 and 25 the references cited~therein.~

` In a ~urther aspect, the present in~ention rslates to a process ~ for producing activated Factor XIII, the~ pro~ess comprising :~: contacting Factor~ ~XIII~ pre~ursor with an immobilised : proteolytic enzymej and collecting the activated Factor XIII in 30 a buffer solution containing one or more stabilisers, or contacting Factor :XIII precursor in a buf~er solution containing one or more stabiIiser~ with an immobilised proteolytic enzyme.
:

WO93/15234 212 ,~ ~ 3 2 PCT/DK93/00016 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 activa~ed Factor XIII, the process comprising contacting Factor XIXI precursor with a proteolytic enzyme in a buffer solution cont~ining o~e or more stabilisers, followed by addition, after a suitable interval, o~ a protease inhibitor.

The Factor XIII product of the in~ention 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 ~e in t~e form of an a'a dimer or a'a' dimer. The i , : a'a dimer form is one in which a 4 kD fragment has been cleaved o~ the N-terminal end of on~ of the a monomers/ while the a'a' ~: dim~r form is one in which a 4 k~ fra ~ ent has been cleaved of~
;~ the N-terminal end o~ both a monomers.
.
20 The Facts:r XIII product of the :invention is advanta~eously a ~: récombinant protein: ~ since thi~s: is a more r~liable and : economical ~ource~ of Factor ~ II, than plasma. The prep~ration of recombinant~ Factor ~XIII in yeask is described in, for instance:, EP 268 772~o ZymoGenetics as well as P.D. Bishop et 25 al~ s~L~ y~29, 1990, pp. 1861 186~, the contents of : whi~h are inro~orated~herein by referen~e, In~ the ~proce~s~ of~the~inv~ntion, ~Facto~ XIII precurs.or as de*ined ab~ve:may~be activated with an immobilised proteolytic ~; ~ : enzyme. Examples of su~itable enzymes are thrombin, trypsin or 30 a: trypsin-like enz~me (e.g. a protease obtainable from a pecies of Fu_arlum, cf. WO 89/06270). The proteolytic enzyme may suitably be immobili~ed by one of the procedures described . W~93/15234 ~ ; PCT/~K93/00~16 J O 3 ~
`

in K. Mosbach (ed~), "Immobilized Enzymesl' in Methods in EnzYmoloq~ 4~, Academic Press, New York, 1976, including covalent coupling ~o insoluble organic or inorganic supports, entrapment in gels and adsorption to ion exchange resins or 5 other a.dsorbent materials. Coating on a particulate support may also be employed (cf. for instance A.R. Macrae and R.C.
Hammond, BiotechnoloaY and_Genetic Enqineerinq Reviews 3, 1985, p. 193. Suitable support materials for the immobilised enzyme are, for instance, plastics ~e.g. polypropylene, polys~yrene 10 polyvinylchloride, polyurethane, latex, nylon, teflon, dacron, polyvinylacetate, polyvinylalcohol or any suitable copolymer ther~of), polysaccharides (e.g. agarose or dextran), ion exchange resi~s (both c~tion and anion exchange re~ins), silic~n polymers (e.g. siloxane) or silicates (e.g. glass).

lS Alternati~ely, the Factor XIII precur~or may be contacted with a proteolytic enzyme after which a protease inhibitor is added.
: The protease i~hibitor may suitably be a trypsin inhibitor such : ~as aprotinin or soybean trypsin inhibitor.
:`: :
The buffer soIution into which the acti~ated Factor XIII is 20 collected is preferably a glycine, alanine or borate buffer.
. .
; ~ ~The stabiliæer or stabilisers present in the bu~fer ~;olution as ~well as:in the final Facl:or XIII~eo~nposition may be a c:helating ;~ agent,~or instance ~DTA~ EGTA or citrate. ~DTA may be present ~ in a concentration o~:2-15 mM, preferably 3-12 mM, more : ~ 25 pre~exab~y-5-lO mM. Another~stabi~liser which may be present in the buffer solution :and Fa~tor XIII composition is a reducing agent or another substance capable of preventing oxidation of the~active -SH at Cyæ314 of Factor XIII, e~g. a cysteine or ~ul~ite, or an antioxidant su~h as:ascor~ic acid or glutathion.
30 hn example of a suitab1e reducing agent is dithiothreitol TT),: which may be:present~ in a concentration of l-lO mM, ; preferably 2-7 ~mM~, more preferably 2.5-5 mMO A further `: stabiliser which may be present in the buffer solution and Factor XIII composition is a sugar. Examples of suitable sugars :

WO93/15234 21 ? ~ O ~ 2 PCT/DK93/00016 are lactose, glucose, sucrose, maltose or trehalose. The sugar may be present in an amount of 0.~-5%, preferably 1-2%, by weight. A still further stabiliser which ma~ be present in the bu~fer 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% ~asein, 10 mM EDTA~ and 5 mM DTT in 10 mM glycine 10 buffer, pH 8Ø

According to the invention, it is particularly preferred that the present composition is in freeze-dried form as this generally results in improved sta~ility.

: The present invention is further illustrated in th~ following 15 exampIes which are not in any way intended to limit he scope of the invention as claimed.

mple l : ~

Pr~pa atio~ og~a~tivate~ ~t~ble Fnctor XIII

& urce of--~E~xII~ recombinant~ Factor XIII (rFXIII) was 20 expressed~sub~stantial~y as describe~ in P.D.~Bishop et al., Biochemis~ 29, 1990t ~pp~;1861-1869. The cells were harvested by.: :centrifugat~on :~:re~ulting: in a wet cell volume of :i.app~oxima~te:ly 20% of th~ broth volume. EDTA 10 mM were added and pH~adju~ed~to ~7.8,`and~ the~cells were ruptured by 2 25 separate~uns through a homogenizer (Rannie, Copenhagen,DK) at :800-9OO~bar. The homogenized ce;lls were diluted approximately

3~times with lysis buffer (50 mM:Tris~,HC1, 10 mM EDTA, pH=7.B
: in~deionized water) and l~O Superfloc C521 was added in order to flocculate :the~ ~e11 ~debris,~ which .was:- then removed by 30 centrifugation. The supernatant was~ urther clarified by addition of ~:0.3% filter aid of the diatomite type and : ~

W~93/l5234 PCT/DK93/00016 filtration through a diatomite filter sheet with an appropiate pore size. A crude precipitate of rFXIII was obtained by adding 18% Na2SO4 at 26-28C and pH 7.0-7.2 in 45 minutes. The filter cake wa~ 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 o~ a solution of 10 mM
Tris,HCl, 5 mM EDTA, pH=7.8 and the filter aid was removed by filtration. Finally rFXIII was isoIated by crystallization with 11%(w/v) Na-formiate at 25C 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 ac~ivity 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 w~s used as the so~vent. 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 di~solution of rFXIII. pH was adjusted se~eral times with NaOH. The solution was clarified by 20 centrifugation (14400 x~ g for 10 minutes) followed ~y fil*ration throug~a 0~4S ~m membr~ne filter.
, ~: ~ A clarified solution of : rFXIII wa :pum~ed:throu~h a trypsin-Sepharose column ~available from Pharmacia~ Sweden~, beginning with the highest flow rate.
, 25 After pas6age~0~60:~ml + 3 bed volumes, samples were taken and immediately diluted to assay concentration and assayed with and ~` wi~hoùt thr~mbin activation. The flow rate was diminlshed and after passa~e of 3~ bed volumes, another sample was taken, and so on. All operations were per~ormed at room temperature.

30 General set~ For ~all experiments, ~rypsin-Sepharose was : packed in a column~with a diameter o~ l cm and a bed-height of : 2.5 cm.
::

- :. ~ : :
:
:

4 212 8 n 3 2 P~T/DK93~0016 g 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 a~ove. 10 ml plastic vials containing 5 ml solution with the reagents to be te~ted were

5 placed in a fraction collector. After passage of 60 ml, the e~fluent from the trypsin-Sepharose was directed to the vials in the fraction collec~or. 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-weig~ed with lid) which was plac~d in a tray containing.solid carbon dioxid~. 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 - 8C.

The %-yield cal~ulated in the tables shown below i5 based on the assayed content o~ the feed (activated with thro~bin).
~' ~: ~ The assay for Factor XIII activity was carried out by a fluor~metri~ activity assay (referred to in P.D. Bishop et al., 20 supra~. The wh~le content of:the vials was dissolved in 10~0 ml T~NEP bu~fer (OO1 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 :~ inho~ogenieties in:the~fre~ze dried~prapaxations. Samples were further d~luted~in TANEP buff~r pH 7,5 prior to being assayed.
25 The results:shown~are the average of two determinations.

Chem~icals: Casein is Hammersten casein ~Merck Art. ~24~)~ D-: ~f)-trehalose is ~the dihyd~ate from Si ~ a (T-5251), lactose monohydrate ~Merck;7660), DTT = dithiothreitol (Sigma D-0632) and~EDTA = Titriplex III (Merck Art. 8418). Others are standard ~ 3;0~1aborakory grade.
:;`~ ~ : : ~:
. ~oti~ation : :

WO 93/15234 2 ~2 8 0 3 2 P~T/DK93/00016 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 rasults of rFXIII activation appear from Table 1 5 below ~cf. Fig. 1).

Table 1 _ Feed: 5604 mg/l F435 .
Flow +THR -THR
ml/min %
9 97.4 74.3 8 97.5 77.9 7 94.g 79.1

6 ~8.9 ~5.8 101.7 90~5 4 93.5 87.3 3 94.4 89.7 2 89.3 ~8.2 1 75.8 74.

B. A¢tiv~tion ~ ~

Feed~composition: 8,al g F435 in 300 ml 10 mM glyc~ine buffer pH
8.~wa~ applied~ on a t~ypsin-Sepharose column as described 25 abo~. Trehalose~(1%), EDTA ~lO mM) and DTT (lO mM) were added~
to the e~fluent. The result~ are shown in Tab~e 2 below ~cf.
Fig - 2 ) ~

, ~
: ` :
: .

: ~ :
:~ :
:~ :

WO 93/15234 PCI/D~3/0~16 21~32 Table 2 Feed: 1094~ mg/l F435 Flow ml/min +THR -THR
9.9 106.8 6~.8 9 106.0 70.,7 8 100. 1 ~8 . 8

7 lOSo 5 75 ~ 8 ~i 104.:L 78~1 112 ~ 4 87 ~ 1 4 9709 ~3~9 3 g7~9 51809 2 92 ~ 2 94 ~ 7 B2.5 8~3 . _, ::: The results show a high activation yield due to the presenc:e of DTT and EDTA.

20 CO ~c~v~io~ ~
r Feed co~position: 2:, 6~ g F43~5; in 200 ml lO mM ~lycine bu~fer were applied on a Sepharose c:olumn on which a trypsin-like .
pro~ease derived~ . from Fusarium (:prepared as desc:ribed in Wo 89/06270) had been: i~nobilised by adding 200 mg ~f the active 25 enzyme; mixed: ~:with~ coupling buf~er ts:~ ~ g :of CNBr-ac:~ivated SepharoRe 4B~ :(avail~b~e from Phax~acia, Sweden3 at pH 7.5. An HR5/1~0 column ~was pa¢~ed to a bed height o~ 4 3 mm (bed volume 0,84 ml). ~r~halose (;lg6~, EDrrA (10 m~q) and DTT (10 mM~ were adqed to the ~ffluent. The results are shown in Table; 3 below 30 ~ O Fi~. ~ 3) . ; ~ ~

~ : , WO93/15234 P~/DK93J00016 ` 212~032 ~a~le 3 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 1~ 0.4 87.6 59.3 0.3 8~.0 62.9 0.~ 75.2 ~3.8 0.1 34:.8 26.4 , ,. ~

15 D. ~ctiv~tio~

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%), lO mM~EDTA and :10 mM DTT were added to the ef~luent. The results are shown in Table 4 below (cf. Fig.
20 4). ~ ~ ;

Table 4 ~ t Feed: 135gO mg/l F435 - - ~
: .

2`5 ~ : Flow~ ~ : : ~THR THR

S~ 96~.0 82.3 - : 5: : 9~.2 89.5 4 9:3 . 6 i 89 . 3 ~ : : 3~ 96.2 ~0.8 2~ :94~2 ~3.0 35 Resul~:s:: The high~ooncentration of~rFXIII in the *eed re~uires ~: a:flow rate of 2 ml/min. The~:activation yield i5 excellent, - ~ ~ probably d~e to the presence of~stabilizers. At 1 ml/min the W093/15234 212 8 0 ~ 2 PCT/DKg3/~0016 sample was diluted within 2 minutes and no precipitation occurred; at 0.5 ml/min precipitation occurred within 3 minutes.

~. 8t~bilit~

5 Feed composition: 10.04 g M57 in 300 ml 10 mM glycine ~uffer pH
8 a 0 was applied on a t~ypsin-Sepharose column as described above. EDTA 10 (E5) or 20 mM (E10), lactose 2 tLl) or 4 % (L2) and casein 0 or 2 ~ (C1) were added to the effluent in a factorial set-up. Samples were assayed immediately and a~ter 10 freeze-drying. The results are shown in Table 5 below (in percent yield).

~ ~ ' : ~ 1 : :

:

`~: :
: , : : , :, `: : :

W~:) 93/1~234 PCI/DIC93/û0016 2 IL ~ 8 ~ 3 ~ !

Table 5 _.
Feed: 47~0mg/l M57 Flow: 4 . 0 ml/min +THR -THR
1 E5 Ll 62.5 54.6 2 E5 L1 C1 83.2 74,1 3 E5 L2 66.3 59.4 4 ES ~2 Cl 82. 2 77 . 6 E10 Ll 67 . 3 61. 3 6 E10 L1 C189 . 5 76 . 8 7 E~0 L2 72 . 3 61. 5 8 E10 L2 C1 85. 6 77 . 4 after freeze-drying 1 E5 L1 29,.3 21.8 2 E5 ~1 Cl 39.8 29.2 3 E5 L2 31.0 23.1 4 E5 L2 Cl40 . 2 29 . 3 ~0 5 IS10 I,l 29 . 1 23 . 2 6 E10 Ll Cl 42.8 32.0 7 E10 L2 33.5 26.7 8 E10 L2 Cl; ~ 45. 6 ~ 34 ~ 2 af`ter one month 1 E5 L1 : ~34 . 6 25 . 4 2 E5 L1 C1 4 a . 8 3 S . 3 3 E5 L2 3 5 . 4 : 25 . 9 4 E5~ 2: C1 44,.6 : 36.3 ~5: E1O L1 34.1 ~ ~ 24.4 :~0 & E1OA L1 C1 44~,6 : 32.0 ~E~` ~ 7 E10~ L2 ~ ~35.9 :: 24.7 8 :E:1~ L2 C1~ 47 . 0 34 .1 ,~: : : ~ :
aft;er: two month E5 Ll ~ :43~ 34.2 : : ~ 2 E5: Ll::Cl ~:~0.9 :: :: 41,4 3: E5~ L2 ~ : 1.7 : 37.û
- ~ 4 E5 L2 Cl 53 ~ 3 43 0 2 E10 L1 : 4 5. 2 35.2 6 ElO~: Ll ~ C1~ ~: 50 . 7 ~ ~ 40 . 3 ~:, ~ . , :: ::

:
:

::

-: ~: :

WO 93/15234 21 2 8 0 3 ~ P~/DIC93/00016 7 Elo L2 48.9 37.5 8 E10 L2 Cl 53 . 5 43 . 5 after three months 1 E5 Ll 42.5 31.6 2 E5 L1 Cl 48 . 9 41. 6 3 E5 ~2 36.7 29.3 4 E5 L2 Cl 54.0 46.1 ~ E10 Ll 43.5 ` 33"5 6 ElO L1 C1 52 . 0 41. 8 7 Elû L2 42 0 3 32 . 6 8 E10 JJ2 C1 51 ~, 5 41 . 2 .

Results: The actiY~tion yield is 60~6, but when ::asein is added a yield of 75~ is obtained, The freeze-dried preparations 15 containing casein have the highe~;t overall yield reigardless of the level OI lac:tose and EDTA.

y Feed composition: 10.07 g MS7 in 300 ml 10 ~ glycine buffer pH
~: 8. O was applied on a trypsin~Sepharose column as described 2Q above~ Lactose (2%) and E~l~ (lO ~ l) and varying amounts of ca;ein (~0~ ~, 2 and 4%) were added to ~he effluent. Samples were as~;ayed immédiately, and some of them were f roze~ or freeze dried, wh~ile ~other ~ were kept in the refrigerator. The results ar~ sho~n; in ~able ~ ~ below~.
, :

:

. ~ , :
:

WO 93/15234 212 8 0 3 æ P~/DK93/00016 Table 6 Feed: 4745 mg/l M57 Flow: 4 . O ml/min % residual activity of feed _ _ _ +THR -THR
E10 L2 Cû 84.9 73.4 E10 L2 C1 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 ~c p~ 38 . 1 27. 4 cold; 32 . 1 15. 6 3~.2 ~5.4 30.7 13.6 day 1~ ~ 3 7.6 29.4 freeze- : 48.3 34.7 ~: : dried ~ 52 . 2 39 . 6 52:~.5 ~ 40.3 kept 26.6 18n8 : ~5 I::old: ~ :24 . 8 ~ 9 . 9 24 . 0 9~. S
:2 0 . ~ 8 . 5 day 2~0 ~ :37 .9 : 25.2 freez~e~ 4:9 .1 34~. 5 ~: 30 ~ dried~ :: 53o6 ~ 37. 9 56 . 3~ ~ 4~1~. 5 day 27 ~ ~ ~ 21.1 : ~ 12.8 k~pt.~: : 24 . 0: ~ 5~ 2 cold ~ ~ 24.5 ~ 5.7 3 5 ~ 2 2 . 1 ~ ~5 . 5 2.5 months: ~ ~ 44.8 ~33.9 Ereeæ~ 57.2~ ::45.!9 Iried ~ 57~7 ~ 46n7 58 . 0 4 7 ~ 3 :~
.

WO93/1~234 212~ 0 ? PCT/D~93/00016 Results: The activating/stabilising effect of casein is once more apparent, resulting in activation yields near 100%.
Samples whsn assayed with thrombin activation showed 110~
compared to feed~ The freeze-dried preparations with 2 and 4%
5 casein showed the highest activity. All free~e-dried preparations are skable. The samples that were kept cold exhibited up to 27% residual activity after 6 days.

G. ~t~b~lity Feed romposition: 10.76 g M57 in 300 ml 10 mM ~lycine b~ffer 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 ~) 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 refrigerato~

Table 7 Fee~: 4513 mg/1 M57 Flow: 4.0 ml/min :
:~
Day 1 : fTHR -THR--Cl, ~: ~ 94.5 79.9 CLED: : ~ 100.5 85.7 :CLE ~ ; 10~.5 89.~
:: 25 ; ~ S ~ 95.9 80.5 :CSED ; 10~.6 86.9 CSE~ : 99 . 6 B5 . 5 Day ~4 ~ :
Freeze dried ~.. . ; ~ :: :
~ C~ : 55.7 40~
~:~ : : : CLED :: 72.8 57.1 CLE : : :53.7 37.4 :~: CS :: 60.8 40.0 ~ : CSED : 7807 57.0 :~ 35 ~ ~ ~: CSE: 56.3 35.6 . ~

WO93/l5234 21~ ~ 0 3 ~ PCT/DK33/00016 Rept cold CL 34.8 18.2 CLED 56.3 3~.3 C~E 35.3 17.0 ~S 36.3 18.7 CSED 49.5 38.8 CSE 36.3 18.5 Day 13 Freeze-dried ~0 CL 4808 37,4 CLE~ 68.9 57.3 CLE 4~3 31.6 CS 54.2 37.7 CSED 70.3 53.~
CSE 53O0 3S.0 Day 20 Kept cold CL 27.0 6.1 C~ED 34.3 lO~9 ~0 CL~ 2,7.0 5.7 ~: CS 30.8 ~.2 CSED 34 o 6 12 ~ 9 ~SE : 26.0 6.8 Day 43 ~ Freeze~dried ~THR -THR
~ : , CL : :: 65.1 48.9 CLED ; : 87.1 67.5 LE ~ ::64.0 47.5 CS ~ 70.0 51~8 ~ CSED:: 81.8 63qO
E~ ? 53.3 Kept col~d CLED 25.7 6.8 35~ ~19~7 3.8 CS:ED ~ ~ 24 8 5 6 : CS~ 3 .

`

, ~
, ~ : : :

W~93/1~234 212 8 0 ~ 2 PCT/DK93/00016 Day 70 Freeze-dried CL 61.9 49.3 CLED 75.2 63.7 CLE 61.7 49.4 CS 63.2 46.0 CSED 75.9 6007 CSE 66.1 48.9 __ 10 Result~: The activation yield is high, 80 - 90%. The freezP-dried preparations containing casein, EDTA, DTT and lactos~/~ucrose yive a high ~ver-all~yield of abQut 60%~ They show no sign of loss of activi~y over two months. ~he same ~ompositio~ results in the most stable samples when kept.cold.

5 ~ b~ y : ' :
Feed composi~ion: 2051 g F435 in 300 ml lO mM glycine buf~er pH
8.0 was appiied on a trypsin-Sepharosle column as described aboveO ~asein 2% (c~ la~tose 2% (L), EDTA lO ~M (E~ and DTT 0 20 mM were added~; to the effluent~ Samples were assayed 20 imm~diately. Some~samples:were ~r~eze-drie~, others were kept : in the refrigerator. The~result- are shown in Table 8 below.

, .

. , , ~ : ;: ~ :

, WO 93/15234 Pff~/DK93/00016 ~1230~2 Table 8 Feed: 4030 mg/l F435 Flow: 4 ml/min _ ~THR -THR
% %
Day 1 CLE 0 DTT 105 . 9 94 . 3 CLE S l)TT 108 . 9 96 . 2 CLE 10 DTT 104 . 9 92 . 4 CLE 20 DTT 107 . 0 94 . ~
Day 15 CLE O 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 Day 15 CLE 0 DTT 49 . 2 32 . 7 Freeze- CLE : 5 DTT 77 .1 61. 9 dried CIJE 10 DTT 84 . 9 70 .1 CLE 2 0 lDTT :7 9 . 2 6 2 . 2 ; Day 3 0 : CIE 0 DTT 21. 7 7 . 3 Cold ~ C~E 5 DTT 35 . 3 18 ~ 9 : CLE 10 DTT 3 3 . 2 16 . 9 CLE 2 0 ~fTT 4 2 . 0 2 3 . 6 Day 30 ~ ~ CLE~ 0 DTT 69f . 0 ~7:4 8 Freeze- ffr'LE 5 DTT: 9~ . 3 ~:4 . 0 : dried ffiqLE10 DTT :88 . 8 76. 2 :: : : : CId3: 20 DTT : ~89.6 76.1 Day 57 ~ ~CI~E 0 DTT ~~ 61. 6 51~ 2 Freeze- CLE 5 ~DTT 84 .1 75 . 4 ~
dried:~ CLE~ 10 I:~TT ~ 87.~ 74O3 ~ CLE ~20 ~ DTfT ~ ~ ; 9l. 5: 78.5~ ~

Results~:: Activati~on;~yields ~are~extremely good; ~this is probably a combination ~of::the~ purer~ F435 preparation and the cffffsff-t of casain- The ~réeze-dried ~ preparatiorls have over-all yields of 3 5~75% when DTT ~is~pre~;ent~,: and~ they:~are stable with~in the time frame. ~ DTT ha~; ~ also: a~ ta:b;ilizing effect on the samplfes which arfe 3~ept cold:: 20%: ~ residual acti~rity a~ter one month.

, , ~,, ~ . .. .... . . .... . .

WO93/152~ 212 8 0 3 2 PCT/DK~3/00016 I. 8tability Feed composition: 4.82 g F435 in 300 ml lO mM glycine buffer pH
~.0 was applied on a trypsin-Sepharose column as described above. Trehalose (1%)l EDTA ~lO mM), DTT ~lO mM) and casein 5 (0-2~) were added to the effluent from the column. Samples were assayed immediately. Some samples were ~rozen and freeze-dried, others were kept in the refrigerator. The results are shown in Tablz 9 below.

Table 9 1 0 ~
Feed: 64S3 mgjl F435 Flow: 4.0 ml/min Day l ~THR -THR
% casein 0 82.6 77.8 0.~ ~1.5 76.6 : 0.25 ~1.6 ~5.3 0-5: gO.g 80.6 ~0 l 104.0 90.4 1.5 84.7 79.8 : 2 l04.2 97.6 Day 4 Kept :~old :~ æ~ ~ casein .
: :: 0: 36.~ 31.0 O.l:: ~ 34.8 : 2B~0 : 0.25 37.7 3G~3 ~5~ 39.5 31.S
: 30 l 45.l 3S.2 1.5 46.7 38.8 2 52.6 ~2.9 Freeze~dried l : % casein `: ~ 35 0 : 47.~ 41~3 .: O.l ~ 47.8 : 40~6 ` 0.25 : 49.0 40.2 0.5 56.7 48.8 l ~ 63.8 55.6 1~5 : 72.5 ~5.
- 2 75.8 66.9 : :

W~ 93/15234 2128 0 3 2 PCT/DK93/00016 Day 2 4 Freeze-dried % s::asein 46.2 34.7 0.1 48.2 36.g 0.25 49.7 41.5 .5 53.5 45~8 61.2 50.7 1.5 61.6 ~5 2 61 . 0 53 . 0 _ Pc~su1ts: the ac~ivation yield is fairly good and the ef fect of casein is once more apparent. Ca~;ein also seems to stabi1ize samples kept in the re~rigerator. Th~ o~rer-all yield after 15 fre~ze-drying is far lower than expected4 So far! there is no explanation of this pherlomenon.

Ex~mpl~ 2 :
Activ~ t~ o~ tivat~q Factor XIII OD: 80dlillIll c:asei~ate :
.4% ~w/w of prot~in)~ o~ a f~eeze-dried, activated Factor XIII
20 compo ition ~repared according t~ Example 1 (F43~ containing 1~
~: treha10ser 10 mM~:EDTA,~10 mM DTT:and 2~ casein~ was added to a so1ution of sod~i~m:~ aseinate:~1141% protein, 5 mM C~') (Miprodan 30 a~ai1able from ~I~D Fooas~ Viby, Dem~ark) p:EI 7.0, ~: ~ and:~a~ lOO g ~s~mple~ wàs fil~led~ înto a~ PVC sausage skin ~hic:h was 25 sealed~at both:ends~. A similar sample ~ot containing any enzyme used~:as~ :control)~was prepared,~:~and the two samp}es were : placed~in~:a:water~bath:for:;2 h~urs~at 30C fo110~ed ~y coo1ing at 5C :~or 24 :hours. A~ter the ~sausage ~kins had been removed, `'~ th~ control sample~:was ~still~liguid, while the enzymatically 3~0~treated~samp1e::had:~s~olidified into a~sausage which was stable at room~:temperature~for several~days; until it was microbially degraded. ~ : ~

W093/152~ 2 1 ~: 8 0 3 ~ PCr/DK93/0~016 ~xampl~ 3 Acti~ity o~ ~ctlv~t~ F~ctor XIII i~ minced beef Beef mince was prepared with the following composition:

Beef minced twice 60%
Water 38%
Salt (NaC13 2%
The ingredients were mixed and blended. The pH of the resulting mince was 5.51. The pH was adjusted to 7.0 with NaOHn The mince was divided into four portions as ~ollows:
10 1. Control~
2. Added 0.056% CaCl2 corresponding to 5 mM Ca and 0.5% (of the : meat protein) o~ freeze-dried Factor XIII (M57; not acti~ated) corresponding to 120 mg of Factor XIII).
~:~ 3. Added 0.056% aCl2 correspondin~ to 5 mM Ca ~nd 0.05% (of the 15 meat protein) o~ freeze-dried Factor XIII (M57; not activated) (corresponding to 12~mg of Fact~r XIII).
4~ ~dded 0.056% ~aC12 corresponding to 5 mM Ca and 0.5% (of the ~eat proteinj of~:freeze-dried,:activated Factor XIII prepared . as desaribed in~Example 1 (F435 containing 1~ trehalose, lO,mM
20:EDT~, 10 ~M DTT;~and; 2~% ca~ein) (corresponding to 116 mg of iactor XIII).

~he ~amount of~Factor ~XIII in the:mince is calculated from a peroeneag~e of~protein;in~beef o~ ~0~. ~

helsàmple~ were:;tinned~and heat~treated in a water bath at : ~ 25 300C ~or 2.5 h~rs~:followed by;treatment at 80~C for 1 hour.
The::~ins ~wer~ cooled in a ~water :bath at about Z0C and subsequently in:a~re~-igerator over~night.

~The~content of miDce and:li ~ id in the:tin~was measured (in ~)j as~was~the volume and~;density of the~mince. The results are 30 shown in Table lO~below. ~ ; :

W093/~5234 . PCT/DK93/00016 Table lO

__ Mince 73.73~ 76.78% 73~67% 84.28%
1~2.21g 138.42g 139.3lg 153.25g Liquid 26.27% 23.22~ 26.33% 15~72%
~7~10g 41.87g 49O79g 28.58g Colour dark dark daxk 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 act:ivated Factor XIII is added, ~: ~ 15 resulting in an increased ~olume, although some effect of 0.5~ ~ : inactive Factor XIII is a~so observed, probably due to activating actors:present in the m~at, The:~gel~strsngth~of the boiled~mince was meas~red by means of an Instron dynamometer (a~ai~lable from Instron, YRG), using the 0 metho~ described by P.-G. Klettner, Fleischwirtsch. 69 ~2~, 1989, pp. 225~226O::The resul~s of these experiment~ is shown in Figs~. 5-7.~ In ~thé~ figures:, the ordinate indicates ~he force re~ui~ed to~compress~a 2Z~mm~slice o~ the mince to a thickness of~2~ The g~aph showing~the gel~strength figures for mince 25~trea~ed~with O.~5% aotivated F~ctor XIII (Fig. 5) shows a : dis~inct:peak indicating ~reaking of~:the gel (as it should)~
;: :~ The graph for :mince treat~d with inactive Factor XIII at ~ the same~:dosage level shows~:a~less diatinct peak ~Fig. 6), while the~graph ~for~m:ince~ treated with: 0,05% inactive Factor XIII
30 sh4ws no peak at ~ll:(Fig. 7).~

:
-wo g3/l5~3~ 2 1 ^ g~ K93/~0016 2 8 0 ~ 2 ~

Bxample 4 Acti~ity of ~ctiv~te~ Factor ~II in fi~h pa~te 400 g of pollack meat, 200 ml of water, 3 ml of CaCl2 and 12 gof NaCl was mixed in a high speed blender. The pH was measured 5 to 6.86, and was not ad~usted. To a 100 g samr~le of the mixture was added 36 mg of a freeze-dried, acti~ ed Factor XIII
preparation prepared according to Example 1 ( ~ conr.aining 1~
trehalose, lO mM EDTA, lO mM DTT and 2% case and t~ another lO0 g ~iample (used as a control) was added 36 mg of water. The 10 two samples were filled into PVC sausage s~ins, diameter 25 mm, which wer~ sealed at both ends. The two samples were placed in a water bath at 30C for two hours. They were subs2quently heated to 90C for 30 min. and stores at 5C for ~4 hours.
' The PVC skin was removed, and the gel strength af the sausages 15 wa~ measured on ~amples 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, respec~ively, corresponding to a gel strength improvement in the ~nzymatically treated gel of 65%.
.

:

~ '~- ' ' ' ~' ' ' ' .

Claims

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

2. Activated stable Factor XIII according to claim 1, which is an a'a dimer.

3. 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, 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 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 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 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.

15. 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 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 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 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.

23. 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 a concentration of 2015 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 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 buffer solution comprises a substance capable of preventing oxidation of -SH a 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.

31. 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%.

34. 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 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 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.

40. 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 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 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 2.5-5 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 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 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.

53. A process according to any of 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.