Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR PRODUCTION OF RECOMBINANT HUMAN THROMBIN `644
TECHNICAL FIELD
The present application relates to a method for producing recombinant human
thrombin
s from recombinant prothrombin using recombinant ecarin.
BACKGROUND OF THE INVENTION
Thrombin is a key enzyme in the coagulation cascade. By thrombin mediated
proteolytic
digestion of fibrinogen into fibrin monomer, a cascade reaction leading to
clot formation is
started. Clot formation is the first step in wound healing. In addition
thrombin is a chemo
attractant to cells involved in wound healing, and, the fibrin network formed
act as a
scaffold for collagen-producing fibroblasts, increases phagocytosis, promotes
angiogenesis
and binds growth factors thus further supporting the healing process. The rate
of clot
formation is dependent on the concentration of thrombin and fibrinogen.
Because of the
important function in clot formation thrombin has been utilised in a number of
products
intended for haemostasis and/or as tissue sealants or "glues", both as stand-
alone products
(i.e. Thrombin-JMI) or in combination with fibrin or other compounds (i.e.
Tisseel,
Hemaseel, Crosseal). The potential fields of use are numerous; skin grafting,
neuro
surgery, cardiac surgery, toracic surgery, vascular surgery, oncologic
surgery, plastic
surgery, ophthalmologic surgery, orthopedic surgery, trauma surgery, head and
neck
surgery, gynecologic and urologic surgery, gastrointestinal surgery, dental
surgery, drug
delivery, tissue engineering and dental cavity haemostasis.
So far the thrombin in approved thrombin-containing products on the market is
derived
either from human or bovine plasma. Using plasma derived protein confers
several
disadvantages as limited availability and safety concerns such as risk for
transmission of
viruses and prions and the risk of triggering autoantibody formation (bovine
products).
Cases where antibody formation due to bovine thrombin exposure has lead to
significant
bleeding disorders are known.
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In vivo thrombin is obtained from activation of prothrombin through the
coagulation
cascade. Activation through the coagulation cascade is dependent on the
presence of a
functional GLA-domain containing 8-10 glutamic residues converted to gamma-
carboxyglutamate. In vitro, also incomplete gamma-carboxylated prothrombin can
be
s converted to thrombin by the use of prothrombin activators such as ecarin.
Ecarin, a snake
venom derived from the Kenyan viper Echis carinatus is a procoagulant, a
protease which
cleaves human prothrombin between residues Arg320-I1e321 to generate
meizothrombin.
Further autocatalytic processing results in the formation of meizothrombin
desFl and then
alpha-thrombin, which is the mature active form of thrombin.
An ideal commercial thrombin manufacturing process would use a recombinant
thrombin
precursor and a recombinant protease produced at high productivity without
addition of
animal-derived components. Further requirements would be robust performance,
convenience and low cost.
A big obstacle for efficient recombinant human thrombin (rh-thrombin) has been
to obtain
high yields of prothrombin. Although extensive efforts have been spent,
obtaining high
yields of prothrombin under conditions suitable for production of biologicals
has long
remained a challenge. Yonemura et al. (J Biochem 135:577-582, 2004) have used
recombinant GLA-domain-less prethrombin digested with recombinant ecarin to
generate
recombinant human thrombin. The productivity of prethrombin at process scale
was 150-
200 mg/L, which is a modest productivity for commercial scale production.
Recombinant
production of ecarin has also been described in WO 01/04146. In this
publication
generation of rh-thrombin is exemplified by conversion of recombinant
prothrombin
produced in COS cells by a recombinant ecarin produced from CHO cells.
However, the
exemplified methods are not suitable for large-scale production and animal-
derived
components are used.
Recombinant ecarin is produced as a prepro-protein that needs to be activated.
Problems to
efficiently activate the r-ecarin are described in both publications and the
suggested
activation procedures are far from optimal.
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Thus there is a need for improved methods to obtain recombinant human
thrombin. During
our efforts to obtain improved productivity of gamma-carboxylated human
prothrombin we
made the surprising discovery that co-expression with gamma-glutamyl
carboxylase
(GGCX) vastly improved also the productivity of incompletely carboxylated
prothrombin
s (see W02005038019).
The present invention describes a process to efficiently produce human
thrombin from
recombinant prothrombin obtained by the expression method as described in
W02005038019. Recombinant carboxylated or incompletely carboxylated
prothrombin
combined with recombinant ecarin has not previously been used for
manufacturing of
recombinant thrombin. Further, the procedure for activating recombinant ecarin
is new.
The methods described would be suitable for large scale rh-thrombin
manufacturing
without the addition of animal-derived components.
is SUMMARY OF THE INVENTION
According to a first aspect of the invention, a method is provided for
producing
recombinant human thrombin from recombinant prothrombin using recombinant
ecarin
having the sequence SEQ ID NO 2 or a homologue thereof.
According to a another aspect, a pharmaceutical composition is provided
comprising a
recombinant thrombin according to said method, in combination with
pharmaceutically
acceptable carriers, vehicles and/or adjuvants.
According to further aspect, an isolated DNA sequence is provided coding for
recombinant
ecarin according to SEQ ID NO 2 or a homologue thereof, having at least 80 %
identity to
SEQ ID NO 2.
According to another aspect, a vector is provided comprising an isolated DNA
sequence
coding for recombinant ecarin according to SEQ ID NO 2 or a homologue thereof,
having
at least 80 % identity to SEQ ID NO 2.
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According to yet another aspect, a cell line is provided comprising a vector
comprising an
isolated DNA sequence coding for recombinant ecarin according to SEQ ID NO 2
or a
homologue thereof, having at least 80 % identity to SEQ ID NO 2.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. FII+GGCX construct
Figure 2. Ecarin construct
Figure 3. Example of a process outline for thrombin manufacturing
Figure 4. Nucleotide sequence alignment of recombinant ecarin used in the
present
io invention and wild type ecarin
Figure 5. Amino acid alignment of recombinant ecarin used in the present
invention and
wild type ecarin
Figure 6. Graph showing the activation of recombinant ecarin during cell death
over time
Figure 7. Activation of recombinant ecarin in cell cultures over time, assayed
by SDS-
1 s PAGE
Figure 8. Chromatogram from CIEX purification of rh-thrombin.
Figure 9. Non-reduced SDS-PAGE analyses of fractions obtained by CIEX
purification
Detailed description of the invention
20 The invention consists in one part of a cell line derived by stable
transfection with a vector
(Figure 1) encoding human prothrombin (FII) associated by suitable control
sequences and
human gamma-glutamyl carboxylase (GGCX) associated by suitable control
sequences.
Control sequences should be chosen so that prothrombin expression is in excess
of the
GGCX expression by at least a factor of 10. The host cell is preferably a
eukaryotic cell.
25 Typical host cells include, but are not limited to insect cells, yeast
cells, and mammalian
cells. Mammalian cells are particularly preferred. Suitable mammalian cells
lines include,
but are not limited to, CHO, HEK, NSO, 293, Per C.6, BHK and COS cells, and
derivatives
thereof. In one embodiment the host cell is the mammalian cell line CHO-S. The
obtained
prothrombin producing cell line is grown under culture conditions optimised
for high yield
30 of prothrombin disregarding gamma-carboxylation. Vitamin K may or may not
be added to
the growth medium.
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It will be appreciated that the invention is not restricted to a particular
prothrombin or
gamma-glutamyl caboxylase or protein encoding sequence of one of these
proteins to be
co-expressed. Moreover, and in particular with respect to blood coagulation
factors,
s numerous mutant forms of the proteins have been disclosed in the art. The
present
invention is equally applicable to prothrombin and gamma-glutamyl caboxylase
mutant
forms, including naturally occurring allelic variants, of the proteins as it
is to wild-type
sequence. In one embodiment the invention can be undertaking with any wild-
type protein
or one with at least 90%, preferably at least 95% sequence identity thereto.
In another
embodiment, sequences listed in Table 1 can be used.
Table 1
CDNA GENE
EMBL SPLICE VARIANTS EMBL
PROTEIN ACC# (PROTEIN) MUTATIONS ACC#
Glutamate gamma 2; BC013979; 1 SNP (EMBL# U65896); 2
carboxylase BC013979 AF253530 SNPs (OMIM# 137167) U65896
approx. 100 SNP's (EMBL#
Prothrombin V00595 1; V00595 AF478696) AF478696
Each of these proteins, including their nucleic acid and amino acid sequences,
are well
is known. Table 2 identifies representative sequences of wild-type and mutant
forms of the
various proteins that can be used in the present invention.
The term "gamma-glutamyl carboxylase" or "GGCX", as used herein, refers to a
vitamin
K dependent enzyme that catalyses carboxylation of glutamic acid residues.
GGCX enzymes are widely distributed, and have been cloned from many different
species
such as the beluga whale Delphinapterus leucas, the toadfish Opsanus tau,
chicken (Gallus
gallus), hagfish (Myxine glutinosa), horseshoe crab (Limulus polyphemus), and
the cone
snail Conus textile (Begley et al., 2000, ibid; Bandyopadhyay et al. 2002,
ibid). The
carboxylase from conus snail is similar to bovine carboxylase and has been
expressed in
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COS cells (Czerwiec et al. 2002, ibid). Additional proteins similar to GGCX
can be found
in insects and prokaryotes such as Anopheles gambiae, Drosophila melanogaster
and
Leptospira with NCBI accession numbers: gi 31217234, gi 21298685, gi 24216281,
gi
24197548 and (Bandyopadhyay et al., 2002, ibid), respectively. The carboxylase
enzyme
s displays remarkable evolutionary conservation. Several of the non-human
enzymes have
shown, or may be predicted to have, activity similar to that of the human GGCX
we have
used, and may therefore be used as an alternative to the human enzyme.
Table 2 identifies representative sequences of predicted proteins homologous
to human
GGXC (sorted after species origin) that can be used in the present invention.
Table 2
Species Data base accession #/ID
Homo sapiens (man) NM000821.2
HUMGLUCARB
HUMHGCA
BC004422
HSU65896
AF253530.1
Papio hamadzyas (red baboon) AC 116665.1
Delphinapterus leucas (white whale) AF278713
Bos taurus (bovine) NM174066.2
BOVCARBOXG
BOVBGCA
Ovis aries (domestic sheep) AF312035
Rattus norvegicus (brown rat) NM_031756.1
AF065387
Mus musculus (mouse) NM019802.1
AF087938
Opsanus tau (bony fishes) AF278714.1
Conus textile (molluscs) AY0044904.1
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AF382823.2
Conus imperialis (molluscs) AF448234.1
Conus episcopatus (molluscs) AF448233.1
Conus omaria (molluscs) AF448235.1
Drosophila melanogaster (fruit fly) NM_079161.2
Anopheles gambiae (mosquito) XM_316389.1
Secale cereale (monocots) SCE314767
Triticum aestivum (common wheat) AF280606.1
Triticum urartu (monocots) AY245579.1
Hordeum vulgare (barley) BLYHORDCA
Leptospira interrogans (spirochetes) AE011514.1
Streptomyces coelicolor (high GC Gram+ SC0939109
bacteria) SC0939124
AF425987.1
Streptomyces lividans (high GC Gram+ bacteria) SLU22894
Streptomyces viginiae (high GC Gram+ bacteria) SVSNBDE
Micrococcus luteus (high GC Gram+ bacteria) MLSPCOPER
Chlamydomonas reinhardtii (green algae) AF479588.1
Dictyostelium discoideum (slime mold) AC115 612.2
Coturnix coturnix (birds) AF364329.1
Bradyrhizobiumjaponicum (a-protoebacteria) AP005937.1
Rhodobacter sphaeroides (a-proteobacteria) RSY14197
Sinorhizobium meliloti (a-proteobacteria) RME603647
AF119834
Mesorhizobium loti (a-proteobacteria) AP003014.2
Chromobacterium violaceum ((3-proteobacteria) AE016910.1
AE016918.1
Pseudomonas aeruginosa (y-proteobacteria) AE004613.1
AF165882
Xanthomonas axonopodis (y-proteobacteria) AE011706.1
Human herpesvirus 8 KSU52064
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KSU75698
AF305694
AF360120
AF192756
Each of the above-identified GGCX proteins and GGCX proteins from other
species can
be used as the carboxylase enzyme in the present invention.
A second part of the invention is a cell line stably transfected with a
polynucleotide
encoding ecarin and associated control elements (Figure 2). The ecarin
encoding sequence
may be optimised for expression in mammalian cells, but is not limited to such
sequences.
In one embodiment of the invention the sequence according to SEQ ID NO 2 or a
homologue thereof is used to express ecarin. A homologue of SEQ ID NO 2 coding
for
ecarin may have at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the
sequence
SEQ ID NO 2. The host cell is preferably a eukaryotic cell. Typical host cells
include, but
are not limited to insect cells, yeast cells, and mammalian cells. Mammalian
cells are
particularly preferred. Suitable mammalian cells lines include, but are not
limited to, CHO,
is HEK, NSO, 293, Per C.6, BHK and COS cells, and derivatives thereof. In one
embodiment
the host cell is the mammalian cell line CHO-S.
In one embodiment prothrombin and ecarin are produced from cells originating
from the
same parent cell line. This cell line origin may be, but is not limited to,
Chinese Hamster
Ovary cells (CHO) including derivatives and NSO (myeloma BALB/c mouse)
including
derivatives. The purpose of using the same cell line background is to
facilitate purification
and evaluation of purity of the thrombin product.
In another embodiment ecarin and prothrombin are produced from different host
cell line;
i.e. CHO and NSO, respectively.
In one aspect of the invention use of recombinant ecarin is preferred as this
facilitates
detection of non-thrombin product derived components during the thrombin
generation
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process and in the final thrombin product. In a second aspect recombinant
ecarin is
preferred due to reduced risk for exposure to allergenic or toxic components
that may be
present in ecarin derived from snake venom. In a third aspect ecarin from
snake venom is
not preferred due to batch variation and limited batch size of ecarin
preparations.
The crude prothrombin and the crude ecarin are mixed and incubated under
conditions that
allow formation of thrombin, such as described in Example 3. Generated
thrombin is then
purified by methods described in Example 4 or by other methods known by
persons skilled
in the art. Alternatively prothrombin and/or ecarin can first be purified by
methods known
io in the art and then mixed to obtain thrombin. Thrombin is then purified
from non-product
components.
An example of a suitable thrombin manufacturing process is outlined in Figure
3.
A method is provided for producing recombinant human thrombin from recombinant
is prothrombin using recombinant ecarin having the sequence SEQ ID NO 2 or a
homologue
thereof. The recombinant ecarin can be expressed and secreted by a cell
containing the
gene comprising the nucleotide sequence SEQ ID NO 2 or a homologue thereof in
CHO-S
cells, which ecarin has an amino acid sequence equal to that of wild type
ecarin.
In the above method the recombinant protrombin is subjected to recombinant
ecarin, which
20 recombinant ecarin can be isolated in active form after extra-cellular
expression by CHO-S
cells, said cells being left to apoptosis/necrosis for a time sufficient to
activate said ecarin,
whereupon a human recombinant thrombin is isolated.
The recombinant prothrombin can be produced by a cell-line comprising a
prothrombin
expressing gene having a nucleotide sequence comprising the sequence SEQ. ID.
NO. 1 or
25 an homologue thereof. A homologue of SEQ ID NO 1 coding for prothrombin may
have at
least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the sequence SEQ ID NO
1. The
recombinant prothrombin can be a mixture of fully carboxylated prothrombin and
incompletely carboxylated prothrombin. In one embodiment, the recombinant
prothrombin
is a fully carboxylated prothrombin and in another embodiment, the recombinant
30 prothrombin is an incompletely carboxylated prothrombin.
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A further aspect of the invention relates to the recombinant thrombin obtained
by the
method according to the invention. A pharmaceutical composition can be
designed
comprising the recombinant thrombin obtained be the method according to the
invention,
in combination with pharmaceutically acceptable carriers, vehicles and/or
adjuvants.The
s pharmaceutical composition can be in an applicable form.
In one embodiment thrombin produced by the described method can be used in the
manufacturing of tissue sealants ("glues") in combination with other proteins,
i.e. fibrin
originating from recombinant cells, transgenic animals or human plasma. In
another
10 embodiment thrombin produced by the described method can be used as a stand-
alone
product, freeze dried as single active component or in combination with a non-
protein
matrix, or, in solution as single active component or in combination with
other active
components.
is Suitable mix-in components would be, but is not limited to, collagen,
chitin, degradable
polymers, cellulose, recombinant coagulation factors and fibrinogen from
transgenic or
recombinant sources.
The potential fields of use for the tissue sealants ("glues") are numerous;
skin grafting,
neuro surgery, cardiac surgery, toracic surgery, vascular surgery, oncologic
surgery, plastic
surgery, ophthalmologic surgery, orthopedic surgery, trauma surgery, head and
neck
surgery, gynecologic and urologic surgery, gastrointestinal surgery, dental
surgery, drug
delivery, tissue engineering and dental cavity haemostasis.
A further aspect of the invention relates to a method for obtaining
coagulation by
administering a therapeutically effective amount of a recombinant human
thrombin
obtained using the method according to the invention to a patient.
Another aspect of the present invention is an isolated DNA sequence according
SEQ ID
NO 2 or homologues therof coding for a recombinant ecarin. A homologue of SEQ
ID NO
2 coding for ecarin may have at least 80%, 85%, 90%, 95%, 97%, 98% or 99%
identity to
the sequence SEQ ID NO 2. SEQ ID NO 2 is a designed sequence that has been
optimised
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for optimal expression. The sequence is particularly suited for expression in
mammalian
cell systems.
According to another aspect a vector comprising SEQ ID NO 2 or a homologue
thereof is
provided. Said vector can be designed to overexpress SEQ ID NO 2 or a
homologue
s thereof and is operably linked to expression control sequences permitting
expression of
ecarin encoded by SEQ ID NO 2 or a homologue thereof. According to a third
aspect a
host cell comprising said vector is provided that is capable of expressing
ecarin encoded by
SEQ ID NO 2 or a homologue thereof. This host cell is preferably a eukaryotic
cell.
Typical host cells include, but are not limited to insect cells, yeast cells,
and mammalian
cells. Mammalian cells are particularly preferred. Suitable mammalian cells
lines include,
but are not limited to, CHO, HEK, NSO, 293, Per C.6, BHK and COS cells, and
derivatives
thereof. In one embodiment the host cell is the mammalian cell line CHO-S.
According to another embodiment of the present invention a polypeptide
comprising an
is amino acid sequence encoded by SEQ ID NO: 2 or a homologue thereof and
obtained by
the method described in Example 2.
The sequence identity between two sequences can be determined by pair-wise
computer
alignment analysis, using programs such as, BestFit, PILEUP, Gap or
FrameAlign. The
preferred alignment tool is BestFit. In practise, when searching for
similar/identical
sequences to the query search, from within a sequence database, it is
generally necessary to
perform an initial identification of similar sequences using suitable
algorithms such as
Blast, Blast2, NCBI Blast2, WashU Blast2, FastA, or Fasta3, and a scoring
matrix such as
Blosum 62. Such algorithms endeavour to closely approximate the "gold-
standard"
alignment algorithm of Smith-Waterman. Thus, the preferred software/search
engine
program for use in assessing similarity, i.e., how two primary polypeptide
sequences line
up is Smith-Waterman. Identity refers to direct matches, similarity allows for
conservative
substitutions.
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Experimental section
The invention will be further described by means of the following examples
which shall
not be interpreted as limiting the scope of the appended claims.
s Example 1
High yield production of recombinant human prothrombin in CHO cells.
The P I E2 cell line containing the construct PN32 shown in Figure 1 having
the nucleotide
sequence SEQ ID NO: 1, was grown in a fermentor according to the method
described in
W02005038019, using a protein and animal component free growth medium in order
to
io produce prothrombin for use in thrombin manufacturing. The cells were grown
either by
batch or perfusion culture methods (Table 1) and the amount of prothrombin
produced was
measured by an ecarin assay. This ecarin assay was performed essentially as
the
Chromogenix assay (M61nda1, Sweden) using purified plasma-derived human
prothrombin
(Haematologic Technologies Inc., Vermont, USA) as standard.
Table 1. Examples of yield of prothrombin in experimental fermentor runs
Experiment ID Culture method & Viable cells Prothrombin mg/L
time (million cells/mL)
CC2LC (272-8) Batch, 238 h 5.9 281
CC2LD (272-8) Batch, 238 h 6.2 276
326-11B Perfusion, 259 h 18 722
The fermentor experiments showed that both batch and perfusion culture methods
can be
used to produce prothrombin suitable for production of recombinant thrombin
(Table 1).
The share of fully carboxylated prothrombin obtained in these fermentor runs
was about
55-87 %, the rest being incompletely carboxylated prothrombin.
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Example 2
Production of recombinant ecarin in CHO cells.
An ecarin encoding sequence having the nucleotide sequence SEQ ID NO: 2
optimised for
s expression in mammalian cells was synthesized and cloned into the Invitrogen
vector
pCDNA 3.1+ (Figure 2). An alignment of the nucleotide sequence of the
recombinant
ecarin used in the present invention to the sequence of wild type ecarin
(GI:717090) is seen
in Figure 4. As can be seen in Figure 5 this recombinant ecarin is 100 %
homologous to the
amino acid sequence for wild type ecarin. This construct, AZ ecarin (SEQ ID
NO. 3), was
io used to stably transfect CHO-S cells (Invitrogen). Ecarin is secreted by
the host cell to the
extra-cellular space, and in order to screen for ecarin producing clones,
culture supematant
samples were removed and mixed with recombinant human prothrombin (rhFII) to a
final
concentration of 1 mg rhFII /L in assay buffer (50 mM Tris-HC1, pH 7.4
containing 0.1%
BSA). This mix was incubated 20-40 minutes at 37 C. The thrombin generated by
the
is action of ecarin present in the sample was then detected by adding a 1-2 mM
solution of
the chromogenic thrombin substrate S-2238 (Chromogenix, M61nda1). Colour
development
was monitored and stopped when suitable using 20% acetic acid. To estimate the
activity
of the recombinant ecarin produced, snake venom derived ecarin with a declared
activity
was purchased from Sigma and used as standard. The best producing cell line
obtained
20 produced up to 7000 U ecarin per litre culture in lab scale shaker cultures
grown in animal
component free medium.
Activation of recombinant ecarin
The above method produces the recombinant ecarin as a pro protein Thus,
activation by
25 removal of the pro-part is necessary for optimal activity. To our surprise,
we found that
activation was most conveniently obtained by continued incubation of the
culture for at
least 7 days after the death of the ecarin producing cells (Figure 6). The
culture medium
used was CD-CHO supplemented with HT-supplement, non-essential amino acids and
Glutamax I (as recommended by Invitrogen for CHO-S), and growth conditions
were
30 shaker bottles at 37 C in an atmosphere containing 5% carbon dioxide.
Culture samples
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were assayed for activity as described above. As can be seen from Figure 6,
the activity of
recombinant ecarin increased during the activation period.
Samples from culture supematants were also separated by SDS-PAGE and blotted
to
s nitrocellulose mebranes. Labelling of the membrane was performed with
polyclonal rabbit
serum directed towards the mature part of ecarin expressed as inclusion bodies
in E. coli.
"M" indicates the molecular weight marker and numbers refer to day of sample
collection.
As can be seen from Figure 7 the recombinant ecarin remains stable for more
than a week
after the death of the cells. Activation of ecarin may also take place at
lower temperatures,
for instance as low as room temperature, but will then require longer times
for activation.
Ecarin will remain stable for severable months in room temperature in the
presence of dead
host cells. The activity will increase gradually until it levels out. A
decrease in activity has
not been observed except in the presence of bacterial infections or high
temperatures.
Efforts to use trypsin for activation of ecarin were made, but were not
successful.
Example 3
Conversion of prothrombin to thrombin by ecarin.
The ecarin protease converts prothrombin to meizothrombin, an intermediate
form of
thrombin that has thrombin catalytic activity. Further processing into
thrombin is achieved
by auto-catalyses. To determine the estimated amount of ecarin culture needed
for
converting prothrombin into thrombin, we performed a series of test digests.
Different
amounts of ecarin-containing culture supematants as obtained in Example 2,
were mixed
with 1 mg/ml prothrombin (as obtained in example 1) in PBS buffer (Cambrex).
Incubation
of the mixtures was done at 37 C for 1-3 hours. Samples were then analysed by
SDS-
PAGE to identify the amount of recombinant ecarin needed for complete
conversion of
prothrombin into thrombin. By this procedure we found that the recombinant
ecarin was
very potent; one litre of ecarin culture supematant at 7000 U/L is capable of
complete
conversion of 64 grams of prothrombin into thrombin in less than 3 hours at 37
C.
Normally recombinantly produced prothrombin has to be purified in order to
separate
fully-carboxylated prothrombin from the incompletely carboxylated prothrombin.
However
this is not necessary for the present invention as the recombinant ecarin is
able to
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efficiently activate both the fully carboxylated and the incompletely
carboxylated
prothrombins.
Example 4
s Purification of thrombin
Thrombin obtained by the procedure described in example 3 was purified by
cation-
exchange chromatography (CIEX) using AKTA-FPLC (GE Healthcare) and an SP-
Sepharose HP column (GE Healthcare) equilibrated with 25 mM sodium-phosphate
buffer,
pH 6.5. Ecarin-digested prothrombin prepared as in example 3 was adjusted to
pH 6.2 and
10 a conductivity of approximately 8 mS/cm. Thrombin was eluted with a 1M
sodium
chloride gradient in column equilibration buffer over 20 column volumes
(Figure 8).
Selected fractions were analysed by SDS-PAGE (Figure 9). Thrombin activity was
confirmed by incubation with the chromogenic thrombin substrate S-2238
(Chromogenix,
M61nda1).
Example 5
Analyses of rh-thrombin obtained.
To further analyse the obtained thrombin, kinetic parameters were determined
using the
chromogenic thrombin substrate S-2366 (Chromogenix). Activity was estimated by
titration with hirudin. The rh-thrombin was for all parameters; Activity, Kkat
and Vmax,
similar to plasma-derived human a-thrombin from Haematologic Technologies Inc.
(Vermont, USA).
Purified thrombin was also subjected to N-terminal sequencing: Reduced
thrombin heavy
and light polypeptide chains were separated by SDS-PAGE and blotted to
Immobilon P
membrane (Millipor). The excised bands were sequenced by the Edman degradation
method. Heavy chain N-terminal first five amino acids were confirmed to be
IVEGS, and
the light chain five N-terminal amino acids were TFGS as expected.
