Process for preparing an ultra-pure thrombin preparation

An ultra-pure, clear thrombin solution having a high specific activity is described as well as a method of manufacture.

BACKGROUND 
Thrombin, a proteolytic enzyme, is essential for hemostasis. It is a 
principle reagent in the formation of blood clots via fibrin production. 
Due to its effectiveness as a clotting aid, thrombin and its preparations 
are useful during surgical procedures to control bleeding. While dry 
thrombin is available, liquid preparations are generally preferred due to 
handling and time considerations. 
Until now, there have been no highly stable, clear liquid thrombin 
preparations which are both storage stable and ready for use during 
surgery. This is because thrombin, when dissolved in water or saline, 
rapidly loses its activity due to denaturation and autolysis of the 
thrombin protein. 
THE INVENTION 
The present invention is directed to a novel modification of a process for 
the preparation of thrombin of ultra-pure quality in solution. This 
solution is completely clear and free of turbidity, and has a 
characteristic of high clotting activity, less inactive protein, and a 
high specific activity, more than any thrombin product hereto described. 
The novelty of the present process, which achieves the goal of a clear 
thrombin solution, is the unique combination of a whole host of steps in a 
particular sequence. 
First of all, the common thrombin, in circulating blood, exists in an 
inactive form called prothrombin and a factor called thromboplastin is 
required in order to convert prothrombin to thrombin. The present 
invention does not involve the usual use of bovine lung extract as a 
source of thromboplastin but uses an isolated, highly purified 
thromboplastin, as described hereinafter. This process eliminates a 
significant amount of contaminating proteins which are the probable source 
of impurities and turbidity often seen in a final product. 
The prothrombin to thrombin conversion mixture, following the usual 
centrifugation step, is passed through an anion exchange chromatography 
column, affording an eluted material which is still turbid. This material 
is then, in the present invention, frozen and then thawed, followed by 
centrifugation to remove most of the turbidity. Removal of this turbidity 
improves the solution flow through the second stage cation-exchange column 
chromatography procedure. 
Following the second passage of the solution through a cation exchanger, 
the improvement in this step comprises eluting the material through this 
column by a normal flow from top to bottom with a salt gradient rather 
than a standard sodium chloride solution. 
The results of these modifications have provided the isolation of a 
water-clear, ultra-pure thrombin, the specific activity being much 
superior to any product available on the market, as shown in Table 1. 
TABLE 1 
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Comparison Between Thrombostat .RTM., Thrombinar .RTM., 
and Ultra-Pure Thrombin 
Ultra- 
Thrombostat .RTM. .sup.a 
Thrombinar .RTM. .sup.b 
Pure 
______________________________________ 
Clotting activity.sup.c 
2164 1372 7820 
(U/mL) 
Protein (mg/mL) 
11.56 0.82 0.82 
Specific activity 
187 1663 9500 
(U/mg) 
______________________________________ 
.sup.a ParkeDavis 
.sup.b Armour 
.sup.c Determined by a modified NIH method hereinafter described. 
Accordingly, the present invention concerns, in its broadest aspects: 
I. An ultra-pure, clear, colorless thrombin solution having specific 
activity from 4000 to 11,000 Units/mg protein. 
II. An ultra-pure, clear, colorless thrombin solution prepared by reacting 
prothrombin with purified thromboplastin and treating the resulting 
thrombin, after centrifugation, by eluting the supernatant through an 
anion-exchange agarose column; freezing, then thawing up to about 
25.degree. C., the desired eluant fractions; centrifuging and eluting the 
supernatant through a cation-exchange agarose column with a salt gradient 
in a buffer. 
III. A process for preparing an ultra-pure, clear, colorless thrombin 
solution comprising: 
reacting prothrombin with purified thromboplastin; centrifuging the 
suspension; eluting the supernatant through an anion-exchange agarose 
column with buffer; freezing then thawing up to about 25.degree. C. the 
desired eluant fractions; centrifuging and eluting the supernatant through 
a cation-exchange agarose column with a salt gradient in a buffer. 
ADVANTAGES 
The thrombin compositions and methods of the invention have several 
advantages over conventional preparations and methods for assisting in 
blood clotting. 
Unlike powdered preparations, the compositions of the instant invention 
require no reconstitution prior to use. Thus, measuring, mixing, 
sterilizing, etc. of one or more component(s) or container(s) are not 
important considerations. The instant clear preparations can be used 
without preparation before final use because of the absence of particles 
which cause the turbidity in former liquid preparations. 
Furthermore, the stability of the instant thrombin-containing materials is 
such that the need for stock inventories and/or rotation of products is 
largely eliminated. Unlike most saline or water-solutions of thrombin, 
which are stable for only about one week at 4.degree. C., the instant 
preparations are designed to be stable at normal refrigeration 
temperatures (i.e., about 4.degree. C.) and at room temperature (i.e., 
about 25.degree. C.) for 6 months or more. 
It is known that high concentrations of glycerol, sucrose, and other 
polyols can stabilize proteins in solution. In the case of thrombin, it is 
known that a glycerol concentration of 67% can greatly stabilize a 1,000 
.mu./mL thrombin solution. However, use of high glycerol concentrations is 
not practical in the large scale manufacture of a sterile thrombin 
solution because of the high viscosity of such a preparation. The instant 
compositions, which may contain 30% or less, of glycerol avoid these 
problems. 
Other advantages and aspects of the invention will become apparent from a 
consideration of the following description of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Isolation of Thromboplastin From Lung Tissue 
Bovine/veal lung tissue is trimmed to remove fatty tissue, cut into small 
pieces, and homogenized in a tissue homogenizer with 25-50 mM phosphate 
buffer pH 6.2-6.7, preferably pH 6.5 containing 0.5-1.0% polysorbate 80. 
The surfactant aids in the extraction of the enzyme. Other nonionic 
surfactants can be used, e.g., fatty acid esters of polyoxyethylene 
sorbitan, e.g., Tween.RTM., ICI; polyoxyalkylyne fatty acid esters, e.g., 
MYRJ.RTM., ICI; polyoxyethylene fatty ethers, e.g., Brij.RTM., ICI; 
polyoxypropylene-polyethylene ethers, e.g., Pluronic.RTM., BASF; sucrose 
mono esters; and Triton X-100. The homogenate is centrifuged at 12-14K RPM 
and the supernatant is collected. The pH of the supernatant is adjusted to 
pH 5.2-5.7, preferably pH 5.5 using drops of 10% acetic acid and 
centrifuged at 12-14K RPM. The supernatant from this step is loaded on a 
conventional cation exchange crosslinked agarose column, e.g., 
CM-Sepharose.RTM. (other cationic resins can be used, e.g., 
CM-Sephadex.RTM.) and eluted with 25-50 mM phosphate buffer as described 
above. The eluate is monitored at 280 nM. The activity of thromboplastin 
in the eluate is also monitored indirectly by first converting prothrombin 
to thrombin and the latter activity is then determined by measuring 
clotting activity using a fibrometer. 
The initial fractions eluting from the column form part of the first peak 
which contains thromboplastin. Fractions eluting later which are part of 
the second peak contain nonactive extraneous proteins. This purified 
thromboplastin solution is opalescent without any haze or turbidity. This 
fraction can be clarified further by a freeze/thaw and centrifugation 
cycle without any loss of prothrombin converting activity. 
Preparation, Isolation, and Purification of Ultra-Pure Thrombin 
Assay for Thrombin Activity 
Thrombin clotting activity was determined using a modified NIH method. The 
solutions used consisted of: a) Imidazole buffer, stock, (IBS) made by 
dissolving 1.72 g imidazole in 90 mL of 0.1N hydrochloric acid and then 
made to 100 mL with distilled water (final pH should be about 7.2). b) 
PEG/IBS solution made by diluting IBS, 58.8 mL; sodium chloride, 9.0 g; 
and polyethylene glycol (PEG, molecular weight 8000), 5 g to 1000 mL with 
water. Normal human plasma was used as a source of fibrinogen and was 
diluted with 0.154M sodium chloride (1:1) prior to use. NIH thrombin was 
used as a standard and was diluted with polyethylene glycol 
(8000)/imidazole buffered saline (PEG/IBS) to give 5 U/mL. Clotting assay 
was performed using a fibrometer. Diluted plasma (200 .mu.L) was incubated 
at 37.degree. C. for 3 minutes, then standard thrombin (100 .mu.L) was 
added and the clotting time (in seconds) was recorded (14 to 15 seconds). 
Thrombin-containing unknown sample was diluted with PEG/IBS to give 
clotting time values higher and lower than the standard clotting time 
value by about 5 seconds. Enzyme activity was calculated as follows: 
##EQU1## 
Enzyme activity is expressed as Units/mL (up to 8000 U/mL) and as Units/mg 
protein (up to 10,000 U/mg protein). 
Purified or partially purified bovine plasma prothrombin is reacted with 
purified thromboplastin in the presence of 10-40 mM calcium chloride 
solution at a temperature between 10.degree. and 25.degree. C. for 15-45 
minutes as described under Example B, "Isolation of Ultra-Pure Thrombin". 
The amount of thromboplastin activity is two to three times that of 
prothrombin at pH 6.5-7.0. The thrombin produced by this reaction is 
further purified as follows. The resulting protein suspension is 
centrifuged (12K RPM) in a refrigerated centrifuge (2.degree.-10.degree. 
C.) to separate insoluble nonactive proteins. The supernatant is loaded on 
a weak anion-exchange column (DEAE-Sepharose.RTM., DEAE-Sephadex.RTM., 
DE-52.RTM.). The column is eluted with 25-50 mM phosphate buffer (pH 6.5) 
containing 0.1M sodium chloride (2.degree.-10.degree. C.). The eluant is 
monitored at 280 nM. Fractions with high UV absorbance are further checked 
for thrombin clotting activity (using a fibrometer). These pooled 
fractions containing thrombin are turbid and are clarified by freezing the 
suspension overnight, followed by thawing (&lt;25.degree. C.) and 
centrifugation or filtration. The thrombin-containing pool is loaded on a 
cation-exchange column (CM-Sepharose.RTM., CM-Sephadex.RTM.) and eluted 
using a salt gradient (0.1M to 1M sodium chloride in 25-50 mM phosphate 
buffer, pH 6.5). 
The eluant is monitored at 280 nM and the fractions containing thrombin 
activity (as determined by the fibrometer) are pooled (FIG. 1). The 
fractions containing ultra-pure thrombin are water clear and may have 
about 8000 U/mL of activity. The purity of ultra-pure thrombin is 
determined by reversed-phase HPLC, polyacrylamide gel electrophoresis, and 
isoelectric focusing. 
Protein content of the thrombin fraction was determined using the Bradford 
assay (Bradford, M., Anal. Biochem., 72:248, 1976) and the protein reagent 
made by BioRad (Richmond, Calif.). Specific activity of the preparation 
was calculated by dividing the amount of enzyme units per unit volume into 
the amount of proteins per same unit volume. 
The exceptionally high specific activity of thrombin made by the present 
invention is attributed to the following: 
1. The use of a freshly harvested prothrombin yields a thrombin product of 
high specific activity. Also, inactive thrombin can co-elute with active 
thrombin and this can result in decreased specific activity of the final 
product. Therefore, all the isolation steps need to be carried out at a 
temperature between about 2.degree. to 7.degree. C. Furthermore, solutions 
were not allowed to stand, even refrigerated, for extended periods of time 
prior to use. Freezing at about -10.degree. to -20.degree. C. was adequate 
to protect the products. 
2. The use of highly purified thromboplastin as described in the present 
invention diminishes the extent of contaminants or the presence of 
nonspecific proteins in the final thrombin preparation, hence increasing 
the specific activity. 
The Thrombin Preparations 
The preparations made in accordance with the invention must contain, in a 
liquid medium, ultra-pure thrombin, and one or more buffers. They may 
contain saline, and other substances conventionally employed in protein 
preparations. 
While the term "preparations" is employed, it should be noted that 
Applicants contemplate all types of formulations in which thrombin, in 
substantially solubilized form, is present in combination with one or more 
glycols and buffers. 
When a liquid formulation is made, it is generally preferred that the 
solvent(s) or other diluent(s) employed have a suitable miscibility with 
thrombin such that production standards, e.g., uniformity of thrombin 
concentration from batch to batch, can be readily met. 
The thrombin employed is an ultra-pure thrombin obtained by the process of 
the present invention. 
This thrombin solution is, if desired, then mixed with glycerol containing 
either acetate buffer or phosphate buffer and saline, in order to prepare 
a stabilized solution. 
Thrombin is known to be soluble in physiological saline--i.e., a solution 
containing about 0.9% NaCl in water. However, other saline solutions are 
contemplated as useful herein. Furthermore, the replacement of all or part 
of the NaCl in such solutions with one or more other suitable salts is 
contemplated. 
Water is a preferred medium for the preparations of the invention. However, 
the use of one or more other diluents which do not adversely affect the 
solubility and/or stability of thrombin in the subject preparations is 
desirable. 
One such diluent is glycerol. Other useful polyols include mannitol, 
sorbitol, sucrose, glucose, and the like. Mixtures are operable. Glycerol 
is highly preferred. 
The glycerol or other polyol ingredient(s) will be employed at a total 
concentration of from about 10 to about 40 wt. %, preferably 20 to 30 wt. 
% based on total composition weight. 
Unless stated otherwise, all quantities recited are weight percentages 
based on total compositions weight. 
Suitable buffer systems are those whose aqueous solutions will maintain pH 
of the final thrombin solution between about 5.0 and about 8.0, with a 
preferred pH range of about 5.5 to about 6.5. It is highly preferred that 
when a phosphate buffer is used the final pH of the preparation be about 
6.0 to about 6.5 and when an acetate buffer is used, the final pH be about 
5.0. 
pH measurements are made using an ordinary pH meter with a combination 
electrode. 
Useful buffer systems include acetate, phosphate, succinate, bicarbonate, 
imidazole, TRIS, and the zwitterionic buffers described by N. E. Good and 
S. Izawa, in Methods in Enzymol, 24, Part B, 53 (1972); and W. F. 
Ferguson, K. I. Braunschweiger, W. R. Braunschweiger, J. R. Smith, J. 
McCormick, C. C. Wasmann, N. P. Jarvis, D. H. Bell, and N. E. Good in 
Anal. Biochem 104, 300 (1980). These disclosures are hereby incorporated 
by reference. 
Suitable reagents for use in the instant buffer systems include MES, ACES, 
BES, MOPS, TES, HEPES, and the like. Phosphate should only be used when 
calcium ion is absent or in the presence of EDTA. Mixtures of such 
reagents can be employed. If mixed buffers are used, the final pH should 
be suitably adjusted. 
Buffers containing phosphate ion and acetate ions are preferred. Mixtures 
are operable. 
The buffers will be present in the buffer solution, along with water and/or 
other suitable diluent(s) at total concentrations of about 0.01M to about 
0.2M, preferably about 0.025M to about 0.10M. 
The use of various other conventional additives, e.g., antioxidants, 
colorants, surfactants, and the like, is also contemplated. Glutathione 
may be employed as an optional ingredient. Amino acids may be employed as 
optional ingredients, but their presence must not be in such quantities as 
to interfere with the stabilizing action of the polyol and buffer 
components on the purified thrombin. In general, it is preferred that they 
be used in only minute quantities at concentrations of 0.5% or less, if at 
all. 
Hemostats 
Hemostatic materials, such as GELFOAM.RTM., SURGICEL.RTM., and AVICEL.RTM., 
and collagen, which are presently used alone or in combination with 
thrombin powder or thrombin in saline, can be effectively used with the 
stabilized thrombin formulations of the present invention using a variety 
of techniques. Preferably, the stabilized solution is absorbed onto the 
hemostatic agent and the pad is freeze-dried and packaged in a sterile 
manner. 
Antimicrobial or antibiotic agents can also be incorporated into such pads, 
especially for use on burn patients, where prevention of infection is 
critical. 
One type of bandage suitable in the preparation of coagulants in accordance 
with the invention is set forth in U.S. Pat. No. 4,363,319, the disclosure 
of which is hereby incorporated by reference. 
The following is illustrative of the preparation of an ultra-pure thrombin 
solution. 
EXAMPLE 
A. Isolation of Thromboplastin: 
100 g Veal lung was homogenized in 200 mL, 25 mM sodium phosphate buffer, 
pH 6.5, containing 0.5% polysorbate 80 and centrifuged at 12K RPM for 20 
minutes at 5.degree. C. The supernatant was collected and the pH adjusted 
to 5.72 using 10% acetic acid, and let stand in the refrigerator for 1 
hour. The mixture was centrifuged for 20 minutes as above and the 
supernatant collected. 160 mL of the supernatant was loaded on a 
CM-Sepharose.RTM. column (30.times.20.5 cm), which was saturated with 25 
mM sodium phosphate buffer, pH 6.5, and eluted using the same buffer. 
Fractions of 230 drops were collected (about 13 mL) and the optical 
density at 280 nM of the fractions was measured. 
______________________________________ 
Tube No. OD at 280 nM 
______________________________________ 
1 clear 0.0 
2 clear 0.0 
3 clear 0.0 
4 opalescent 1.369 
5 opalescent 3.751 
6 opalescent 3.828 
7 opalescent 0.451 
8 yellowish 3.427 
9 yellowish 3.993 
10 reddish 3.513 
11 reddish 4.060 
12 reddish 4.071 
13 reddish 4.055 
14 reddish 4.073 
15 reddish 3.944 
16 reddish 3.723 
Pool 1 fractions 4-6 
Pool 2 fractions 7-9 
______________________________________ 
The pools were assayed for thromboplastin activity by converting 
prothrombin to thrombin as follows: 
______________________________________ 
Mix prothrombin 1.0 mL 
saline 0.5 mL 
pool fract. 0.1 mL 
CaCl.sub.2 (0.3 M) 
100 .mu.L 
______________________________________ 
incubated the mixture at 25.degree. C. for 25 minutes, centrifuged at 
5.degree. C. at 13K for 10 minutes and assayed for thrombin clotting 
activity using a fibrometer. 
______________________________________ 
Fraction Clotting time (seconds) 
______________________________________ 
Pool 1 10.9 
Pool 2 9.9 
______________________________________ 
The two pools contained significant thromboplastin (prothrombin converting) 
activity. The first pool was used for the conversion of prothrombin to 
thrombin since it has less color. The second pool, however, could be 
utilized also. 
B. Isolation of Ultra-Pure Thrombin 
The conversion mix was made of: 
______________________________________ 
Prothrombin 97 mL 
saline 40 mL 
CaCl.sub.2 (0.3 M) 
10 mL 
Thromboplastin 15 mL 
(Pool 1) 
______________________________________ 
incubated at 25.degree. C. for 30 minutes and centrifuged at 13K for 20 
minutes at 5.degree. C., and collected the supernatant. 
C. DEAE-Sepharose Column Chromatography 
Equilibrated the column (30.times.2.5 cm) with 25 mM sodium phosphate 
buffer pH 6.5 containing 0.02% sodium azide. Sodium azide was used as a 
bacteriostatic agent, however, this agent cannot be used during the 
isolation of thromboplastin since it causes browning of the red 
hemoglobin. Other common bacteriostatic agents can be substituted and are 
preferred. These include phenols and substituted phenols, chlorobutobenzyl 
alcohol, benzalkonium chloride, benzethonium chloride, thimerosal, and 
phenylmercuric nitrate. 
Loaded 115 mL of the converted preparation on the column and eluted using 
the same buffer containing 0.1M sodium chloride. Collected fractions of 
230 drops and assayed for thrombin clotting activity. 
Fractions 7-48 showed significant thrombin clotting activity; they were 
pooled (510 mL) and the material appeared turbid. The pooled material was 
kept in a plastic bag and frozen overnight. The material was thawed, 
centrifuged at 13K for 20 minutes at 5.degree. C. and the supernatant 
collected. 
D. CM-Sepharose Column Chromatography 
Equilibrated the column (30.times.2.5 cm) with 25 mM sodium phosphate 
buffer, pH 6.5 containing 0.02% sodium azide and 0.1M sodium chloride. 
Loaded 493 mL of the previous supernatant from the DEAE-sepharose column 
step. 
Eluted the column with a salt gradient made of 225 mL of the buffer 
containing 0.02% sodium azide and 0.1M sodium chloride and 225 mL buffer 
containing 0.02% sodium azide and 1.0M sodium chloride. Fractions were 
collected as described above and assayed for thrombin clotting activity 
using a fibrometer. 
Fractions 15-21 contained significant activity and were pooled (volume of 
90 mL). The pooled material was then assayed for thrombin activity and 
showed clotting activity of 8213 U/mL. 
This pooled fraction was also assayed for protein content using the 
Bradford method and Bio-Rad protein assay kit. The pooled material 
contained 0.82 mg/mL protein. 
Final specific activity=8213/0.82=10015 U/mg protein.