JPH04320683A - Ultra-high-purity thrombin preparation - Google Patents

Abstract

PURPOSE: To obtain a highly stable and clear liquid thrombin preparation having a preservation stability and capable of being used at any time in a surgical operation.

CONSTITUTION: This ultra pure and clear thrombin solution is prepared by reacting a prothrombin with a purified thromboplastin, centrifugally separating the produced thrombin, eluting a supernatant thereof through an anion exchanging agarose column, freezing the desired eluted liquid fraction, then thawing the fraction upto approximately at 25°C, centrifugally separating, and eluting the supernatant thereof through a cation exchanging agarose column by using a 0.1-1 M sodium chloride gradient in a phosphate buffer, and the method of the preparation thereof.

COPYRIGHT: (C)1992,JPO

Description

[Detailed description of the invention]

0001

[Background] Thrombin, a protein hydrolase, is essential for hemostasis. It is the main agent in clot formation via fibrin production. Thrombin and its preparations are useful in controlling bleeding during surgical procedures because of their effectiveness as clot formation aids. Although dry thrombin is available, liquid preparations are generally preferred due to handling and time considerations.

To date, there is no highly stable and clear liquid thrombin preparation that is storage stable and ready for use during surgical procedures. This is because when thrombin is dissolved in water or saline, it quickly loses its activity due to denaturation and autolysis of the thrombin protein.

0003

The present invention relates to a new modification of the method for preparing ultra-high purity grade thrombin in solution. This solution is completely clear and non-turbid, and is characterized by higher clot-forming activity, less inactive protein, and higher specific activity than any previously reported thrombin product.

[0004] The novelty of the process of the present invention in achieving the goal of a clear thrombin solution lies in the unique combination of a number of steps in a specific order.

First, normal thrombin exists in the circulating blood as an inactive form called prothrombin, and a factor called thromboplastin is required to convert prothrombin to thrombin. The present invention, as described below, does not involve the use of conventional bovine lung extract as a source of thromboplastin, but uses isolated and highly purified thromboplastin. This method eliminates significant amounts of contaminating proteins that can be a common source of impurities and turbidity in the final product.

The eluate obtained when the prothrombin to thrombin conversion mixture is passed through a conventional centrifugation step through an anion exchange chromatography column is still cloudy. This material is then frozen in the present invention, then thawed, and then centrifuged to remove most of the turbidity. This removal of turbidity improves solution flow through the second stage cation exchange column chromatography procedure.

An improvement in the step of the second passage of the solution through the cation exchanger is that a salt gradient rather than a standard sodium chloride solution is used to elute the material from top to bottom with normal flow through this column. Consists of.

As a result of these modifications, water-clear and ultra-high purity thrombin was isolated, and its specific activity is shown in Table 1.
As shown, it is far superior to anything available on the market.

[0009]

[Table 1]

The invention thus relates in its broadest aspects to: I. Ultra-pure, clear, colorless thrombin solution with a specific activity of 4000-11,000 units/mg protein. II. After reacting prothrombin with purified thromboplastin and centrifuging the generated thrombin, the supernatant is eluted through an anion exchange agarose column; the desired eluate fractions are frozen and then thawed to about 25°C;
Ultrapure, clear, colorless thrombin solution prepared by centrifugation and treatment of the supernatant by elution through a cation exchange agarose column with a salt gradient in buffer. III. Prothrombin is reacted with purified thromboplastin; the suspension is centrifuged; the supernatant is eluted through an anion exchange agarose column with buffer; the desired eluate fraction is frozen and then thawed to approximately 25°C. A method for preparing an ultrapure, clear, colorless thrombin solution comprising centrifugation and elution of the supernatant through a cation exchange agarose column using a salt gradient in buffer.

[0011]

EFFECTS The thrombin compositions and methods of the present invention have several advantages over conventional preparations and methods of promoting clot formation.

Unlike powdered preparations, the compositions of the present invention do not need to be reconstituted prior to use. That is, considerations such as measuring, mixing, sterilizing one or more ingredients or containers are not important. The clear formulations of the present invention can be used without preparation prior to final use due to the absence of particles that cause turbidity in conventional liquid formulations.

Furthermore, the stability of the thrombin-containing materials of the present invention is such that the need for product stock inventory and/or rotation is largely eliminated. Unlike most saline or aqueous solutions of thrombin, which are stable for only about 1 week at 4°C, the preparations of the present invention are stable at normal freezing temperatures (i.e., about 4°C) and room temperature (i.e., about 2°C).
It is designed to be stable for more than 6 months at 5°C.

It is known that high concentrations of glycerol, sucrose, and other polyols can stabilize proteins in solution. In the case of thrombin, 67
It is known that a glycerol concentration of 1,000 μ/ml thrombin solution can be significantly stabilized. However, using high glycerol concentrations is impractical for large-scale production of sterile thrombin solutions due to the high viscosity of such preparations. Compositions of the invention, which may contain up to 30% glycerol, do not have these problems.

Other advantages and aspects of the invention will become apparent upon consideration of the following description of the invention.

[0016]

[Detailed description of preferred embodiments]

Isolation of Thromboplastin from Lung Tissue Bovine/calf lung tissue was trimmed to remove adipose tissue, cut into small pieces, and treated with 25-50 mM phosphate containing 0.5-1.0% polysorbate 80 in a tissue homogenizer. Homogenize using a buffer (pH 6.2-6.7, preferably pH 6.5). The surfactant facilitates the extraction of the enzyme. Other nonionic surfactants, such as fatty acid esters of polyethylene sorbitan, such as Twe
enR, ICI; polyoxyalkyline fatty acid esters, e.g. MYRJR, ICI; polyoxyethylene fatty ethers, e.g. BrijR, ICI; polyoxypropylene-polyethylene ethers, e.g. Pluroni
cR, BASF; sucrose monoester; and Tr
iton X-100 or the like can be used. The homogenate is centrifuged at 12-14K RPM and the supernatant collected. The pH of the supernatant was adjusted to pH 5.2-5.7 (preferably pH 5.5) using a 10% acetic acid droplet.
and centrifuge at 12-14K RPM. The supernatant from this step is applied to a conventional cation-exchange cross-linked agarose column, such as CM-Sepharose® (other cationic resins such as CM-Sephadex® can also be used), and 25-50 m
Elute using M phosphate buffer. The eluate is 28
Monitor at 0 nM. The activity of thromboplastin in the eluate is also monitored indirectly by first converting prothrombin to thrombin, and the latter activity is then measured by measuring clot-forming activity using a fibrometer.

The initial fraction eluting from the column forms the portion of the first peak containing thromboplastin. This purified thromboplastin solution exhibits an opalescent color without any haze or turbidity. This fraction can be further clarified by freeze/thaw and centrifugation cycles without any loss of prothrombin converting activity.

Preparation, Isolation and Purification of Ultra-Pure Thrombin Assay of Thrombin Activity Thrombin clot-forming activity was measured using a modified NIH method. The solutions used are: a) imidazole buffer;
Stock, (IBS), 1.72g imidazole 9
It was made by dissolving in 0ml of 0.1N hydrochloric acid and then made up to 100ml with distilled water (the final pH should be about 7.2). b) PEG/IBS solution, 58.8ml
of IBS, 9.0 g of sodium chloride and 5 g of polyethylene glycol (PEG, molecular weight 8000) were made by diluting to 1000 ml with water. Normal human plasma was used as the fibrinogen source and was diluted (1:1) with 0.154 M sodium chloride prior to use.
. NIH thrombin was used as a standard and diluted with polyethylene glycol (8000)/imidazole buffered saline (PEG/IBS) to 5 U/ml. Clot formation assays were performed using a fibrometer. Diluted plasma (200 μl) was incubated for 3 minutes at 37° C., then standard thrombin (100 μl) was added and clot formation time (in seconds) was recorded (14-15 seconds). Thrombin-containing unknown samples were diluted with PEG/IBS to give clot formation time values that were approximately 5 seconds higher and lower than standard clot formation time values. Enzyme activity was calculated as follows:

[0019]

[Math 1]

A: High dilution coefficient of unknown thrombin sample B:
Low dilution factor of unknown thrombin sample C: Mean clot formation time of standard thrombin D: Mean clot formation time of unknown sample at low dilution E: Mean clot formation time of unknown sample at high dilution g: In test mixture of standard thrombin divided by the volume of standard thrombin in the test mixture (0.5/0.1) Enzyme activity as units/ml (up to 8000 U/ml)
, also as units/mg protein (10,000U/
(up to mg protein).

Purified or partially purified bovine plasma prothrombin was incubated for 10 minutes at a temperature of 10-25° C. for 15-45 minutes as described in Example B, “Isolation of Ultra-Pure Thrombin.”
React with purified thromboplastin in the presence of ~40mM calcium chloride solution. The amount of thromboplastin activity is 2-3 times that of prothrombin at pH 6.5-7.0.
It's double. The thrombin produced by this reaction is further purified as follows. The resulting protein suspension was centrifuged (12K RPM) in a refrigerated centrifuge (2°-10°C).
) Separate insoluble inactive proteins. The supernatant was transferred to a weak anion exchange column (DEAE-Sepharose®,
DEAE-SephadexR, DE-52R). The column contained 0.1M sodium chloride 25-5
Elute with 0mM phosphate buffer (pH 6.5) (
2°C to 10°C). Monitor the eluate at 280 nM. The fractions showing high UV absorption are further checked for thrombin clot-forming activity (using a fibrometer). These pooled fractions containing thrombin were turbid and the suspension was frozen overnight and then thawed (<
25° C.) and clarified by centrifugation or filtration. The thrombin-containing pool was transferred to a cation exchange column (CM-SepharoseR, CM-Sepha).
dexR) and elute with a salt gradient (0.1M to 1M sodium chloride in 25-50mM phosphate buffer, pH 6.5).

The eluate is monitored at 280 nM and the fractions containing thrombin activity (as measured by a fibrometer) are pooled (FIG. 1). Fractions containing ultra-pure thrombin are clear as water and can have an activity of about 8000 U/ml. The purity of ultra-high purity thrombin is determined by reverse phase HPLC, polyacrylamide gel electrophoresis and isoelectric focusing.

The protein content of the thrombin fraction is Bra
dford assay (Bradford, M., A.
nal. Biochem. , 72:248, 19
(76) and protein reagents manufactured by BioRad (Richmond, Calif.). The specific activity of a preparation is the enzyme units per unit volume,
Calculated by dividing by the amount of protein per unit volume.

The exceptionally high specific activity of the thrombin produced according to the invention is due to the following:1. The use of freshly harvested prothrombin provides a thrombin product with high specific activity. Furthermore, inactive thrombin can elute at the same time as active thrombin, which can lead to a decrease in the specific activity of the final product. Therefore, all isolation steps need to be carried out at a temperature of about 2° to 7°C. Moreover, the solutions could not be left for long periods of time before use, even when refrigerated. Freezing at about -10° to -20°C was sufficient to protect the product. 2. The use of highly purified thromboplastin according to the present invention reduces the degree of contaminants or the presence of non-specific proteins in the final thrombin preparation, thus increasing the specific activity.

Thrombin Preparation Preparations made in accordance with the present invention must contain ultrapure thrombin and one or more buffers in a liquid medium. They may contain saline and other substances customary for protein preparations.

Note that while the term "preparation" is used, any type of composition in which thrombin is present in a substantially solubilized form with one or more glycols and buffers is intended. Should.

When making liquid compositions, the solvent(s) or other diluent(s) used will readily meet production standards, such as uniformity of thrombin concentration from batch to batch. It is generally preferred to have adequate miscibility with thrombin so that it is compatible with thrombin.

The thrombin used is ultra-high purity thrombin obtained by the method of the present invention.

The thrombin solution is optionally then mixed with glycerol and saline containing an acetate or phosphate buffer to form a stabilizing solution.

[0030] Thrombin is dissolved in physiological saline, ie
It is known to be soluble in a solution containing 0.9% NaCl in water. However, other saline solutions are also useful in the present invention. Additionally, all or part of the NaCl in such solutions may be replaced by one or more other suitable salts.

Water is the preferred medium for the preparations of the invention. However, it may be desirable to use one or more other diluents that do not adversely affect the solubility and/or stability of thrombin in the preparations of the invention.

One such diluent is glycerol. Other useful polyols include mannitol, sorbitol, sucrose, glucose, and the like. Mixtures can also be used. Glycerol is highly preferred.

[0033] Glycerol or other polyol component(s) may be present in an amount of about 10 to 10% by weight of the total composition.
A total concentration of about 40% by weight, preferably 20-30% by weight will be used.

Unless otherwise specified, all amounts listed are weight percent based on the total composition weight.

A suitable buffer system is one in which the aqueous solution maintains the pH of the final thrombin solution from about 5.0 to about 8.0 (with a preferred pH range of about 5.5 to about 6.5). When using phosphate buffers, the final pH of the preparation is approximately 6.
．． 0 to about 6.5, with a final pH of about 5.0 especially preferred when an acetate buffer is used.

[0036] pH measurement is carried out using a conventional p
This is done using an H meter.

Useful buffer systems include acetate, phosphate, succinate, bicarbonate, imidazole,
TRIS, and N. E. Good and S. I
zawa, Methods in Enzymol,
24, Part B, 53 (1972); and W. F. Ferguson, K. I. Bra
unschweiger, W. R. Brauns
chweiger, J. R. Smith, J.
McCormick, C. C. Wasmann
, N. P. Jarvis, D. H. Bell
, and N. E. Good, Anal. Bi
Included are the zwitterionic buffers described in Ochem 104, 300 (1980). These disclosures are incorporated by reference into this description.

Reagents suitable for use in the buffer systems of the invention include MES, ACES, BES, MOPS, TES, HE
Includes PES, etc. Phosphates should only be used in the absence of calcium ions or in the presence of EDTA. Mixtures of such reagents can be used. When using mixed buffers, the final pH should be adjusted appropriately.

Buffers containing phosphate and acetate ions are preferred. Mixtures can also be used.

The buffering agent is present in the buffer solution at a concentration of about 0.01 M to about 0.2 M, preferably about 0.025 M, along with water and/or other suitable diluent(s).
It will be present at a total concentration of M to about 0.10M.

Various other conventional additives may also be used, such as antioxidants, colorants, surfactants, and the like. Glutathione may be used as an optional ingredient. Amino acids may be used as optional components, but their presence should not be in such an amount as to interfere with the stabilizing effect of the polyol and buffer components on purified thrombin. In general, it is preferred that they be used in very small amounts, if at all, at concentrations of 0.5% or less.

Haemostasis Currently used hemostatic agents alone or in combination with thrombin powder or saline, such as GELFOAMR, SURGICELR and AVIC.
ELR, and collagen, can be effectively used with the stabilized thrombin compositions of the present invention using a variety of methods. Preferably, the stabilizing solution is absorbed onto the hemostatic agent and the pad is lyophilized and sterilely packaged.

Such pads may also incorporate antimicrobial or antibiotic agents, particularly for use with burn patients where infection control is important.

One type of dressing suitable for preparing the clotting agents of the present invention is described in US Pat. No. 4,363,319, the disclosure of which is incorporated herein by reference.

The following is an illustration of the preparation of an ultrapure thrombin solution.

[0046]

[Example] A. Isolation of Thromboplastin 100 g of calf lung was homogenized in 200 ml of 25 mM sodium phosphate buffer (pH 6.5) containing 0.5% polysorbate 80 and centrifuged for 20 minutes at 5° C., 12K RPM. The supernatant was collected and the pH was adjusted to 5.72 using 10% acetic acid and left in the refrigerator for 1 hour. The mixture was centrifuged for 20 minutes as before and the supernatant was collected. CM-Sepharo with 160 ml of supernatant saturated with 25 mM sodium phosphate buffer (pH 6.5)
It was applied to a seR column (30 x 20.5 cm) and eluted using the same buffer. 230 drop fractions were collected (approximately 13 ml) and the optical density of these fractions at 280 nM was measured.

[0047]

[Table 2]

The pool was assayed for thromboplastin activity by converting prothrombin to thrombin as follows: Prothrombin 1.0 ml saline 0.5
ml pool fraction 0.
Mix 100 μl of 1 ml CaCl2 (0.3 M) and incubate the mixture at 25°C for 25 min.
Centrifuged for 10 minutes at 13K and assayed for thrombin clot-forming activity using a fibrometer:

[0049]

[Table 3]

The two pools contained significant thromboplastin (prothrombin conversion) activity. The first pool was used for the conversion of prothrombin to thrombin due to its low color. However, a second pool was also available.

B. The composition of the isolation conversion mixture of ultrapure thrombin was as follows: prothrombin
97ml saline solution
40m
lCaCl2 (0.3M) 1
0ml thromboplastin (pool 1) 15m
This was incubated at 25°C for 30 minutes and 13
Centrifuged for 20 min at K, 5°C and collected the supernatant.

C. DEAE-Sepharose column chromatography Column (30 x 2.5 cm) with 25 mM sodium phosphate buffer (pH 6.5) containing 0.02% sodium azide
was balanced. Sodium azide was used as a bacteriostatic agent, but this agent cannot be used in the isolation of thromboplastin because it causes browning of the red hemoglobin. Other conventional bacteriostatic agents can be substituted and are preferred. These include phenols, substituted phenols, chlorobutobenzyl alcohol, benzalkonium chloride, benzethonium chloride, thimerosal and phenylmercuric nitrate.

115 ml of the conversion preparation was applied to the column;
It was then eluted using the same buffer containing 0.1M sodium chloride. Fractions of 230 drops were collected and assayed for thrombin clot activity.

Fractions 7-48 showed significant thrombin clot-forming activity. When they were pooled (510 ml), the material had a cloudy appearance. The pooled material was kept in a plastic bag and frozen overnight. The material was thawed, centrifuged at 13K for 20 minutes at 5°C and the supernatant was collected.

D. CM-Sepharose column chromatography 25mM sodium phosphate buffer (pH 6) containing 0.02% sodium azide and 0.1M sodium chloride
．． 5) The column (30 x 2.5 cm) was equilibrated.

493 ml of the previous supernatant from the DEAE-Sepharose column step was applied.

0.02% sodium azide and 0.1%
225 ml of buffer containing M sodium chloride, and 0
．． The column was eluted with a salt gradient consisting of 225 ml of buffer containing 0.02% sodium azide and 1.0 M sodium chloride. Fractions were collected as described above and assayed for thrombin clot-forming activity using a fibrometer.

Fractions 15-21 contained significant activity and were pooled (volume 90 ml). The pooled material was then assayed for thrombin activity.821
It showed a clot-forming activity of 3 U/ml.

Regarding this pooled fraction, B
Protein content was assayed using the radford method and the Bio-Rad protein assay kit. This pooled material contained 0.82 mg/ml protein.

Final specific activity=8213/0.82=100
15U/mg protein.

[Brief explanation of drawings]

FIG. 1 is a graph showing thrombin purification as a CM-Sepharose column elution profile using 0.1-1.0 M sodium chloride solutions as shown in the Examples.

Claims (13)

[Claims] Claims: 1. Ultra-high purity, clear, colorless thrombin solution. [Claim 2] 4000 to 1 per mg of protein
Ultra-pure, clear, colorless thrombin solution with a specific activity of 1,000 units. 3. An ultrapure, clear, colorless thrombin solution having a specific activity of 4000 to 11,000 units per mg protein as determined by a modified NIH method. [Claim 4] 4000 to 1 per mg of protein
A thrombin solution according to claim 3 having a specific activity of 1,000 units. 5. Reacting prothrombin with purified thromboplastin and eluating the resulting thrombin after centrifugation through an anion exchange agarose column; freezing the desired eluate fraction and then thawing it to about 25°C. ultrapure, clear, prepared by centrifugation and treatment of the supernatant by elution through a cation exchange agarose column with a 0.1M to 1M sodium chloride salt gradient in phosphate buffer. Thrombin solution. 6. The thrombin solution according to claim 5, having a specific activity of 4000 to 11,000 units per mg protein as determined by a modified NIH method. [Claim 7] 4000 to 1 per mg of protein
The thrombin solution according to claim 6, having a specific activity of 1,000 units. Claim 8: (a) React prothrombin with purified thromboplastin; centrifuge the resulting protein suspension; (b) elute the supernatant through an anion exchange agarose column; freeze the desired eluate fraction. (c) by centrifuging and eluting the supernatant through a cation exchange agarose column using a 0.1 M to 1 M sodium chloride salt gradient in phosphate buffer. Method for preparing ultra-high purity, clear thrombin solution. 9. Purified thromboplastin is (a)
Bovine/calf lung tissue was soaked in 25 mM phosphate buffer (pH 6.0) containing about 0.5 to about 1% nonionic surfactant.
(b) Adjust the pH of the supernatant to 5.5 with 10% acetic acid and centrifuge; (c) Homogenize the supernatant with 25 mM phosphate buffer. elute through a cation exchange agarose column using;
9. The method of claim 8, wherein the method is prepared by collecting the desired fractions. 10. Step (a) is performed in a 10-40 mM calcium chloride solution at a temperature of about 10-25°C.
9. The method of claim 8, wherein the method is carried out for minutes. 11. The method of claim 8, wherein the elution in step (b) is carried out using a 25-50 mM phosphate buffer containing 0.1 M sodium chloride. 12. The method of claim 8, wherein the elution of step (c) is carried out using a salt gradient of 0.1M to 1.0M sodium chloride in 25mM phosphate buffer. 13. Freeze the desired fraction, then freeze about 2
10. The method of claim 9, further clarified by 5 DEG thawing; centrifugation and collecting the supernatant.