Method of proteolytically cleaving prothrombin to produce thrombin

Prothrombin is cleaved by proteases in the presence of a detergent or certain chaotropic substances to produce thrombin. Under these conditions, controlled and restricted proteolysis occurs such that significant activation without digestion occurs. The chaotropic substances may be urea, guanidinium hydrochloride or a thiocyanate salt. Reversible immobilization of prothrombin on a solid support prior to activation improves the selectivity of the proteolytic activation.

BACKGROUND OF THE INVENTION 
The invention relates to a method for the controlled, limited proteolytic 
cleavage of proteins, in particular proenzymes, for recovering enzymes and 
protein fragments, respectively, wherein the protein is treated with a 
protease. In particular, the invention relates to a method of recovering 
thrombin from prothrombin. 
Such a method has been known from EP-A-0 378 798. According to that method, 
prothrombin derived from plasma or a plasma fraction is adsorbed on a 
solid carrier (immobilized), and the adsorbate is treated with Ca.sup.2+ 
ions. The Ca.sup.2+ ions are used in concentrations up to 30 mM and, 
together with the proteases also derived from the plasma and adsorbed on 
the carrier, cause the thrombin to be cleaved from the adsorbate. As the 
solid carrier, methacrylic and acrylic copolymers are used. 
It has furthermore been known that trypsin degrades dissolved prothrombin 
into fragments of low molecular weight, wherein a complete loss of the 
thrombin activity is to be found (Biochim. Biophys. Act. 329, 221-232 
(1973)); the proteolytic degradation of the prothrombin thus does not end 
with thrombin, and thus this method does not lend itself to the recovery 
of thrombin. 
All the methods of the initially defined kind lead to different prothrombin 
cleavage products, depending on the protease and treatment conditions 
used, which cleavage products in part may be used therapeutically (e.g. 
thrombin) and in part may be used diagnostically and for the recovery of 
specific antibodies. For all these applications it is, however, necessary 
that the protein fragments are highly pure, which necessarily involves a 
lot of work, since all the known cleavage methods lead to a plurality of 
fragments. A further disadvantage of these multi-stage and time-consuming 
purification methods is that they necessarily involve high losses of 
yield. 
SUMMARY OF THE INVENTION 
The invention aims at eliminating these disadvantages and has as its object 
to provide an improved method of recovering enzymes or protein fragments 
from proteins, wherein this method is to be particularly suited for the 
simple recovery of thrombin. 
With the method of the initially defined kind, this object is achieved in 
that the treatment with the protease is effected in the presence of a 
detergent or in the presence of a chaotropic substance, with the exception 
of coagulatively active salts. 
The invention is based on the finding that the proteolytic cleavage of 
proteins, in particular of proenzymes, such as prothrombin, takes place on 
purpose or can be controlled if it is carried out in the presence of a 
detergent or in the presence of a chaotropic substance. It has been shown 
that the cleavage pattern of the proteins is different depending on the 
type and concentration of these substances present. This finding opens up 
the possibility of selecting the reaction environment such that only a few 
and precisely selected protein fragments are formed. 
A preferred variant of the method of the invention consists in that a 
proenzyme is used which has been immobilized on a solid carrier material, 
in particular on a hardly soluble salt or chelate of a bivalent metal, 
preferably of an alkaline earth metal. This variant allows for an easier 
recovery of the protein fragment cleaved from the adsorbed proenzyme, 
since the part of the proenzyme, adsorbed to the carrier, simply may be 
separated from the reaction solution with the carrier.

DETAILED DESCRIPTION 
According to the method described in EP-A-0 378 798 copolymers are used as 
the carriers, which, however, have the disadvantage that they could 
release acrylic and methacrylic monomers into the thrombin-containing 
solution which are difficult to remove again and thus contaminate the 
pharmaceutical preparation to be produced. Such a leakage has been known 
for all types of organic polymeric carriers. It has, however, been shown 
that the inventive controlled cleavage of a proenzyme, in particular 
prothrombin, succeeds not only on copolymers, but also on other carriers, 
e.g. on hardly soluble salts, such as Ca.sub.3 (PO.sub.4).sub.2, 
CaSO.sub.4, CaCO.sub.3, BASO.sub.4, BaCO.sub.3 or barium citrate. The 
salts may either be added to a prothrombin-containing solution, or they 
may be newly formed by precipitation of a prothrombin-containing solution. 
A particular advantage of the bivalent ions of the carrier is further that 
they selectively bind prothrombin via their gamma-carboxy-glutamic acid 
terminus. On account of the missing gamma-carboxy-glutamic acid terminus, 
the protein fragments forming during the proteolytic treatment of the 
prothrombin have no affinity to bivalent ions and thus to the carrier. 
As further carriers, also hydroxylapatite, a hydroxylapatite gel or a metal 
chelate-affinity chromatographic carrier loaded with a bivalent cation 
(e.g. Pharmacia Chelating Sepharose.RTM.) are suited. 
A plurality of proteases (e.g. chymotrypsin, dispase, endopeptidase Arg-C, 
endoproteinase Lys-C, endoproteinase Glu-C, endoproteinase Asp-N, factor 
Xa, kallikrein, papain, pepsin, plasmin, pronase, proteinase K, 
staphylocoagulase, subtilisin, thrombin, trypsin (in particular human, 
bovine, porcine), trypsin-like proteases from arthropods or 
microorganisms, such as e.g. Streptomyces griseus--trypsin, 
serine-proteases from venomous snakes, like Angkistrodon rhodostoma, 
Bothrops atrox, Dispholidus typus, Echis carinatus, Naja nigrocollis, 
Oxyuranus scutellatus scutellatus, and others) lend themselves to the 
proteolytic cleavage of proenzymes. Trypsin, chymotrypsin, kallikrein, 
dispase or the endoproteinase Glu-C, Lys-C or Asp-N, are preferably used 
as the protease. Recombinant proteases may be used as well. 
It has been shown that in the presence of a detergent a proenzyme may be 
cleaved so selectively that the enzyme to be recovered forms the main 
portion of the fragment mixture. Deoxycholate (DOC), which preferably is 
used, is suitable as the detergent, and also other substances, such as 
dodecylsulfate (SDS), CHAPS, polyoxyethylene derivatives, such as 
Brij.RTM., Tween.RTM., Triton.RTM. and Pluronic.RTM., are suitable. The 
presence of the detergent further facilitates the desorption of the 
protein-fragments formed, from the carrier. 
Typical chaotropic substances applied in the method according to the 
invention are, e.g., urea, guanidinium hydrochloride as well as 
thiocyanates, yet coagulatively active salts, such as, e.g., calcium 
salts, which also act chaotropically, are not suitable. 
The supernatant obtained, which contains the desired enzyme, may be 
subjected to further purification steps. Advantageously, gel permeation 
chromatography or affinity chromatography may be used therefor. To obtain 
samples as concentrated as possible, the use of affinity chromatographic 
methods is recommended. Dye ligand affinity chromatographic carriers with 
ligands of the Cibacron.RTM.-Blue F3GA-type (produced by Ciba Geigy) or 
Procion.RTM.-Red HE-3B (produced by ICI) or related dyes have proved to be 
suitable. The protein fragments may be adsorbed on the respective affinity 
matrix (e.g. Fractogel.RTM. TSK AF-Blue (produced by Merck), 
Blue-Sepharose.RTM. CL-6B (produced by Pharmacia) in the batch or in the 
packed column directly from the desorption supernatant after the solid 
phase activation has taken place. Subsequently the protein fragments are 
separated from the detergent by washing with a buffer and finally eluted 
with a highly molar (e.g. 1M) chaotropic substance (e.g. KSCN or NH.sub.4 
SCN). 
The eluate may be freed from the chaotropic substance by gel permeation 
chromatography (e.g. via Sephadex.RTM. G25), diafiltration or dialysis and 
may be brought into a suitable buffer or a salt solution. Further 
purification to homogenicity may be effected in a known manner via reverse 
phase chromatography, affinity chromatography or gel permeation 
chromatography. 
The method according to the invention is particularly suitable for 
obtaining pure thrombin. Therefore, the invention also relates to the 
production of a thrombin-containing pharmaceutical preparation, wherein a 
preferred embodiment is characterised in that 
a prothrombin-containing solution is contacted with a solid carrier, in 
particular with a hardly soluble salt or a chelate of a bivalent metal, 
preferably of an alkali earth metal, so as to immobilize prothrombin on 
the carrier, 
the immobilized prothrombin is treated with a protease, in particular 
trypsin, in the presence of a detergent, preferably deoxycholate, so as to 
obtain a thrombin-containing solution that is separated and purified, and 
is processed to a pharmaceutical preparation. 
It has been found that through the detergent treatment a marked reduction 
of virus activity is achieved, if a starting product is used which has 
come from a virus-contaminated pool. In one case it was found that no 
vaccinia viruses were detectable in a thrombin preparation produced 
according to the method of the invention, although the starting material 
(a fermentation supernatant) had contained vaccinia. Naturally, within the 
scope of the invention also additional measures can be taken to inactivate 
possibly present infectious agents, such as, e.g., a vapour-heat-treatment 
of a lyophilized product. 
The inventive method of recovering thrombin suitably is carried out in the 
following manner: 
At first, a prothrombin-containing solution is contacted with the solid 
carrier, so as to adsorb prothrombin. As the prothrombin-containing 
solution not only plasma or plasma fractions, but also recombinant 
prothrombin-containing cell culture supernatant medium may be used. After 
immobilization has been effected, the prothrombin bound to the carrier 
suitably is separated from the solution and washed so as to remove 
unspecifically bound proteins that might contaminate the later final 
product. Subsequently, the immobilized prothrombin is treated with the 
protease in the presence of deoxycholate so as to cleave the thrombin from 
the prothrombin, a thrombin-containing solution and a solid body being 
obtained, the solid body being separated from the solution. This is 
effected by sedimentation or by filtration. 
The thrombin-containing supernatant may be subjected to the above described 
purification steps. It is preferred to carry out an affinity 
chromatography with Fractogel.RTM. TSK-AF Blue (Merck) with subsequent 
Sephadex.RTM.-G25 chromatography. 
Subsequently, the pure thrombin-containing solution obtained optionally is 
concentrated by ultrafiltration or lyophilization and processed to a 
pharmaceutical preparation. 
EXAMPLES 
By the following Examples, the invention will be explained in more detail: 
Preparation of the Immobilized Prothrombin 
A cell culture supernatant medium according to PCT application WO 91/11519, 
which contains recombinant human prothrombin, is admixed with 5 g of 
powderized Ca.sub.3 (PO.sub.4).sub.2 per 100 IU of prothrombin and is 
slightly stirred at 4.degree. C. for one hour. Subsequently, the solid 
phase is centrifuged off at 5000 g, the pellet obtained is resuspended in 
40 ml 20 mM Tris/HCl buffer (pH 7.4), stirred for 10 min and again 
centrifuged off at 5000 g. This procedure is repeated with 40 ml of 5% 
(W/V) ammonium sulfate in the above-mentioned buffer and finally with 40 
ml of pure buffer. 
In the same manner, e.g. a partial prothrombin complex, i.e. a mixture of 
factors II, IX and X, may be used as the prothrombin-containing starting 
material for preparing the immobilized prothrombin. 
EXAMPLE 1 
Influence of the Reaction Environment on the Tryptic Cleavage of the 
Immobilized Prothrombin 
To document the influence of chaotropic substances and detergents on the 
tryptic cleavage of prothrombin, at first four solutions (A-D) of a 
Tris/HCl buffer (20 mM; pH 8.3) were prepared with the following additions 
(the fourth buffer solution (D) served for comparison): 
Solution A: urea (0.5M) 
Solution B: Na-deoxycholate (0.05M) 
Solution C: Na-dodecylsulfate (0.05M) 
Solution D: no addition 
To 1 ml of solutions A-D subsequently 200 mg of buffer-moist, washed pellet 
(produced as described above) were each added and shaken at room 
temperature with 20 .mu.l of a 20 mM Tris/HCl buffer containing 1 mg of 
trypsin/ml. After 1, 2 and 3 hours, aliquots of the suspension were each 
drawn, the solid phase was centrifuged off and the supernatants were 
examined for the respective fragment composition by means of Western blot 
analysis. 
In Table 1, the peak area integrals of the fragment bands at 12, 19, 23, 
25,5, 33, 35 and 44 kD after densitometric Western blot analysis are 
entered. It is apparent that tryptic fragments of different sizes can be 
detected in various amounts in dependence on the reaction environment 
selected. Contrary to the 3-hour incubation in pure Tris buffer (solution 
D), fragments in the molecule mass range above 30 kD remain in all the 
other solutions (A-C). The fragments at 33 kD and at 35 kD correspond to 
thrombin, 33 kD being associated with the active form. 
In the presence of deoxycholate (solution B), active thrombin is formed as 
the main fragment, which furthermore is not decomposed to smaller 
oligopeptides even after a 3-hour treatment with trypsin. 
TABLE 1 
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Molecule mass(kD) 
12 19 23 25,5 33 35 44 
Solution 
Reaction time (h) 
Peak Area Integral: 
______________________________________ 
A 1 26 19 22 14 94 95 
2 22 20 13 54 20 
3 24 21 14 51 17 
B 1 31 165 90 
2 15 175 25 
3 18 170 
C 1 53 11 
2 35 2 
3 9 
D 1 25 132 117 22 
2 25 120 34 
3 42 129 
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EXAMPLE 2 
Cleavage of Immobilized Prothrombin by Means of Various Proteases (In the 
Presence of a Detergent) 
From a prothrombin-containing cell culture supernatant medium, prothrombin 
was adsorbed on Ca.sub.3 (PO.sub.4).sub.2 and washed. 
8 samples of 250 mg of buffer-moist adsorbate were each suspended in 1 ml 
of 20 mM Tris/HCl buffer, pH 8.3, containing 200 mM of Na-deoxycholate, 
and 50 .mu.l of the following eight enzyme solutions were added thereto: 
kallikrein from porcine pancreas, 250 U/ml in 20 mM of TBS, pH 8.3, 
dispase I from Bacillus polymyxa, 1 mg/ml in 20 mM of TBS, pH 8.3, 
.alpha.-chymotrypsin from bovine pancreas, 350 U/ml in 20 mM of TBS, pH 
8.3, 
trypsin from porcine pancreas, 0.76 mg/ml in 20 mM of TBS, pH 8.3, 
endoproteinase Glu-C from Staphylococcus aureus V8, 1 mg/ml in 20 mM of 
TBS, pH 8.3, 
endoproteinase Lys-C from Lysobacter enzymogenes, 0.1 mg/ml in 20 mM TBS, 
pH 8.3, 
endoproteinase Asp-N from Pseudomonas fragi, 0.04 mg/ml in 20 mM TBS, pH 
8.3 
factor Xa, human, 20 U/ml 
The term 20 mM TBS (TBS=tris buffered saline) denotes a 20 mM 
tris-HCl-buffer (pH 8.3), which contains 0.9% NaCl. 
The formulations were incubated for 2 hours at room temperature under 
shaking (20 hours in the case of kallikrein). Thereupon, aliquots of the 
formulations were admixed with SDS sample buffer (1:1) and examined for 
their fragment composition by means of Western blot analysis. 
The peak area integrals of the fragment bands at 12, 18, 19, 20, 23, 33, 
34, 35, 44, 47, 50, 52, 54, 55, 71 and 75 kD after densitometric Western 
blot analysis are given in Table 2. 
It is apparent that a series of fragments in the molecule mass range of 
from 12 to 71 kD can be produced with the above-mentioned proteases. The 
fragment corresponding to active thrombin (33 kD) can be obtained in a 
particularly high yield by means of tryptic degradation of prothrombin. 
TABLE 2 
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Molecule mass (kD) 
12 
18 
19 
20 
23 
33 
34 
35 
44 
47 
50 
52 
54 
55 
71 
75 
Protease Peak Area Integral 
__________________________________________________________________________ 
Kallikrein 12 31 15 
16 16 59 
Dispase 36 27 
8 39 
.alpha.-Chymotrypsin 7 57 9 18 
44 
Trypsin 20 14 
56 
Endoproteinase Glu-Cl 
13 
28 39 17 
13 
17 78 
Endoproteinase Lys-Cl 6 51 21 
Endoproteinase Asp-N 21 83 11 11 19 
Factor Xa 12 
10 31 98 
__________________________________________________________________________ 
EXAMPLE 3 
Cleavage of Immobilized Prothrombin in the Presence of Deoxycholate in 
Various Concentrations 
As described above, at first recombinant prothrombin was adsorbed on 
Ca.sub.3 (PO.sub.4).sub.2 from a fermentation supernatant. 
5 samples of moist adsorbate of 0.6 g each were incubated for three hours 
at room temperature with 3 ml of 20 mM Tris/HCl buffer each, pH 8.3, 
containing Na-deoxycholate in the concentrations 50 mM, 100 mM, 250 mM, 
350 mM and 500 mM, after the addition of 60 .mu.l of a solution of 0.76 
mg/ml of trypsin/ml under shaking. Thereupon the buffers of the media are 
changed against 0.9% NaCl. Then the samples were examined for their 
thrombin activity. Therein, the thrombin time in the coagulation test was 
determined with normal plasma and the amidolytic activity was determined 
with the chromogenic substrate TH-1 (2 AcOH.-D-CHG-ala-arg-p-nitroanilide) 
each photometrically, against an international thrombin standard (FIG. 1). 
FIG. 1 shows that the yield of active thrombin (33 kD-fragment) has a 
maximum at 350 mM deoxycholate. 
Furthermore, it became apparent that the thrombin formed was not further 
degraded over a period of at least 20 hours despite the presence of 
trypsin. 
Purification of Protein Fragments of the Cleaved Prothrombin 
1.5 ml of a prothrombin fragment-containing solution derived in Example 3 
(350 mM DOC) were adsorbed with 0.1% trifluoroacetic acid in H.sub.2 O 
(solvent A) on a reverse phase HPLC column (Nucleosil 100-5C18, 
125.times.4 mm) (flow rate: 1.7 ml/min). Thereupon it was eluted with 0.1% 
trifluoroacetic acid in acetonitrile (solvent B) with a linear gradient of 
from 30 to 70% B in 30 min at a flow rate of 1.7 ml/min. By detection at 
220 nm 6 main peaks (retention times: 10.01 min; 11.96 min; 12.68 min; 
13.47 min; 13.94 min; 14.47 min) could be identified; they were collected 
separately and lyophilized. Further analysis of the separated fragments 
was effected via SDS-PAGE with detection through Coomassie-staining, as 
well as by means of Western blot analysis with a polyclonal 
rabbit-anti-human prothrombin-antiserum. The molecule masses of the 
fragments were determined for the six main peaks with 9 kD, 16 kD, 21 kD, 
23 kD, 12 kD and with 33 kD. 
EXAMPLE 4 
Recovery of Thrombin 
A buffer-moist pellet (approximately 10 g) was incubated for one hour at 
room temperature and under slight stirring with 50 ml of a solution of 
0.76 mg of porcine trypsin (Sigma T-0134) per ml in 20 mM Tris/HCl buffer 
(pH 8.3) which contained 200 mM of Na-deoxycholate. Thereafter, the 
calcium phosphate was separated by centrifugation. The supernatant 
contained primarily thrombin beside a few other fragments of prothrombin. 
For purifying the supernatant, a column having a cross-sectional area of 8 
cm.sup.2 was packed with Fractogel.RTM. TSK-AF Blue (Merck) at a height of 
12 mm (gel volume--9.6 ml) in 20 mM of Tris/HCl buffer (pH 8.3) and washed 
with the same buffer. These and all subsequent steps were effected at 
4.degree. C. 
The thrombin-containing supernatant (approximately 50 ml) was pumped over 
the gel at a flow rate of 2 ml/min to effect adsorption. Thereafter, 
unspecifically bound prothrombin fragments were eluted with 20 ml of 0.5 M 
NaCl solution, 40 ml of 1.0 M NaCl solution and 20 ml of 20 mM Tris/HCl 
buffer (pH 7.4) at a flow rate of 6 ml/min. In reverse flow direction, 
prothrombin fragments were then eluted at 1 ml/min with 20 mM Tris/HCl 
buffer, which contained 1M of KSCN. The eluate was photometrically 
measured via a flow cell at 280 nm, a total of 15 ml was collected. 
The protein content of the eluate was 188 mg/ml according to Bradford. FIG. 
2. shows the fragment composition (densitometric scan of Western blot) 
with dominant 33 kD-fragment (active thrombin). About 30% thereof are 
thrombin, based on the protein content. By determining the thrombin time, 
a specific activity of approximately 2200 IU/mg of protein could be 
determined for the thrombin. Thus, approximately 100 IU of thrombin could 
be obtained from 1 IU of prothrombin by tryptic solid phase activation. 
The thrombin-containing solution obtained can be further purified in a 
known manner, concentrated and further processed to a pharmaceutical 
preparation. 
EXAMPLE 5 
Cleavage of Dissolved Prothrombin 
Two samples of 1 ml each of prothrombin-containing fermentation supernatant 
were admixed with 1 ml of a 40 mM Tris/HCl buffer, pH 8.3, which contained 
0.1M Na-deoxycholate and 0.1M Na-dodecyl sulfate, respectively, so that 
the detergent concentration in the solution amounted to 0.05M. For reasons 
of comparison, a third sample of 1 ml of prothrombin-containing 
fermentation supernatant was admixed with 40 mM Tris/HCl buffer without a 
detergent. After addition of 15 ml each of a solution of 1 mg trypsin/ml 
in 20 mM Tris/HCl buffer, pH 8.3, which contained 0.9% NaCl, it was 
incubated at room temperature for 4 hours under shaking. 
After 1, 2, 3 and 4 hours, aliquots of the samples of 50 .mu.l were each 
diluted with Laemmli-buffer (1:1), boiled and electrophoretically 
analyzed. The Western blot analysis of a 12% SDS polyacrylamide gel (1st 
antibody: anti-human-factor II rabbit serum; 2nd antibody: 
goat-anti-rabbit IgG peroxidase conjugate; developed by means of 4 
chloro-1-naphtole) was densitometrically examined in the impinging light 
for quantifying the fragments. The results are entered in Table 3. 
TABLE 3 
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Molecule mass (kD) 19 25,5 33 35 
Addition Reaction Time (h) 
Peak Area Integral 
______________________________________ 
Deoxy- 1 10 
cholate 2 9 
3 7 
4 6 
Do- 1 8 68 
decyl- 2 6 67 
sulfate 3 5 69 
4 4 67 
Blank 1 4 9 4 
Value 2 3 1 0 
3 0 0 0 
______________________________________ 
Although the method according to the invention has been described in detail 
by way of the recovery of thrombin from prothrombin, the skilled artisan 
will understand that similar cleavages of proenzymes other than 
prothrombin may be carried out in an analogous manner; in this way, it is, 
e.g., possible to recover plasmin from plasminogen or to recover activated 
blood coaguation factors from their proenzymes.