Note: Descriptions are shown in the official language in which they were submitted.
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.
10 DETAILED DISCLOSURE OF THE INVENTION
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
2 1 2 8 0 3 2
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.
::
- :. ~ : :
:
:
WO93/15234 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
12
~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
13
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 ~ !
14
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
16
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
17
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
I9
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
21
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
23
~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
2128032
24
Table lO
__
1 2 3 4
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%.
.
:
~ '~- ' ' ' ~' ' ' ' .
