JP3282834B2 - Ultra-pure thrombin preparation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

Background Thrombin, a protein hydrolase, is essential for hemostasis. It is the main agent in clot formation through fibrin formation. Thrombin and its preparations are useful in controlling bleeding during surgery due to their effectiveness as clot forming aids. Dry thrombin is available, but liquid preparations are generally preferred for handling and time considerations.

To date, there is no highly stable and clear liquid thrombin preparation that is storage-stable and can be used at any time during surgery. This is because when thrombin is dissolved in water or saline, the activity of thrombin is rapidly lost due to denaturation and autolysis of the thrombin protein.

[0003]

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

[0004] The novelty of the method of the present invention that achieves the purpose of a clear thrombin solution lies in the unique combination of many steps in a particular order.

First, normal thrombin exists in an inactive form called prothrombin in the circulating blood, and a factor called thromboplastin is required to convert prothrombin into thrombin. The present invention uses isolated and highly purified thromboplastin without the use of a normal bovine lung extract as a thromboplastin source, as described below. This method eliminates significant amounts of contaminating proteins that can be a source of impurities and turbidity commonly found in end products.

The eluate obtained by passing the prothrombin to thrombin conversion mixture 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. Removal of this turbidity improves solution flow through the second stage cation exchange column chromatography procedure.

An improvement in the second pass stage of the solution through the cation exchanger is to elute the substance from top to bottom by normal flow through this column using a salt gradient instead of a standard sodium chloride solution. Consisting 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 much better than any product available on the market.

[0009]

[Table 1]

The invention therefore relates in the broadest aspect to the following: Ultra-pure, clear, colorless thrombin solution with a specific activity of 4000-11,000 units / mg protein. II. Reacting prothrombin with purified thromboplastin and, after centrifugation of the resulting thrombin, elute the supernatant through an anion exchange agarose column; freeze the desired eluate fraction and thaw to about 25 ° C; centrifuge An ultrapure, clear, colorless thrombin solution prepared by treating the supernatant by eluting it through a cation exchange agarose column using a salt gradient in buffer. III. React prothrombin with purified thromboplastin; centrifuge the suspension; elute the supernatant with an anion exchange agarose column using buffer;
The desired eluate fraction is thawed to about 25 ° C. after freezing; centrifuging and eluting the supernatant through a cation exchange agarose column with a salt gradient in buffer, ultra-high purity, clear , A method for preparing a colorless thrombin solution.

[0011]

The thrombin composition and method of the present invention have several advantages over conventional preparations and methods that promote clot formation.

Unlike powdered preparations, the compositions of the present invention do not need to be reconstituted prior to use. That is, consideration of measurement, mixing, sterilization, etc. of one or more components or containers is not important. The clear preparations 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 preparations.

Further, the stability of the thrombin-containing material of the present invention is such that the need for stock inventory and / or rotation of the product is largely eliminated.
Unlike most saline or aqueous solutions of thrombin that are stable for only about one week at 4 ° C., the preparations of the present invention have a normal freezing temperature (ie, about 4 ° C.) and room temperature (ie, about 2 ° C.).
(5 ° C.) for more than 6 months.

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 can greatly stabilize a 1,000 μ / ml thrombin solution. However, using high glycerol concentrations is not practical for large-scale production of sterile thrombin solutions due to the high viscosity of such preparations. The compositions of the present invention, which may contain up to 30% glycerol, do not have these problems.

[0015] Other advantages and aspects of the present invention will become apparent from a consideration of the ensuing description of the invention.

[0016]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Isolation of thromboplastin from lung tissue Bovine / calf lung tissue is trimmed to remove adipose tissue, cut into small pieces, and 0.5 with a tissue homogenizer.
25-50m containing ~ 1.0% polysorbate 80
Homogenize with M phosphate 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 Tween ^R, ICI; polyoxyethylene Al giraffe fatty acid esters, e.g. MYRJ ^R, ICI; polyoxyethylene fatty ethers, e.g. Brij ^R, ICI; polyoxypropylene -Polyethylene ethers, such as Pluronic
^R , BASF; sucrose monoester; and Triton
X-100 or the like can be used. The homogenate is centrifuged at 12-14K RPM and the supernatant is collected. The pH of the supernatant was adjusted using a 10% acetic acid droplet.
Adjust to H 5.2-5.7 (preferably pH 5.5) and centrifuge at 12-14K RPM. Cation exchange cross-linked agarose column customary supernatant from this step, for example CM-Sepharose ^R (other cationic resins, for example, CM-Seph
adex ^R ), and elute 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, first by converting prothrombin to thrombin, and the latter is then measured by measuring the clot-forming activity using a fibrometer.

The initial fraction eluted from the column forms part of the first peak containing thromboplastin. The purified thromboplastin solution exhibits an opalescent without any haze or turbidity. This fraction can be further clarified by freeze / thaw and centrifugation cycles without any loss of prothrombin conversion 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 working solutions are as follows: a) imidazole buffer,
Stock, (IBS), 1.72 g of imidazole in 90
Made by dissolving in 0.1 ml of 0.1N hydrochloric acid, then made up to 100 ml with distilled water (final pH should be about 7.2). b) PEG / IBS solution, 58.8 ml of IB
S, 9.0 g of sodium chloride and 5 g of polyethylene glycol (PEG, molecular weight 8000) in water 100
Made by diluting to 0 ml. Normal human plasma was used as a source of fibrinogen, and was used prior to use.
Diluted with 154M sodium chloride (1: 1). NIH
Thrombin was used as a standard and polyethylene glycol (8000) / imidazole buffered saline (PEG
/ IBS) to make 5 U / ml. Clot formation assays were performed using a fibrometer. Diluted plasma (20
0 μl) was incubated at 37 ° C. for 3 minutes, then standard thrombin (100 μl) was added and the clot formation time (in seconds) was recorded (14-15 seconds). The unknown thrombin-containing sample was diluted with PEG / IBS to give clot formation time values about 5 seconds higher and lower than the standard clot formation time value. Enzyme activity was calculated as follows:

[0019]

(Equation 1)

A: High dilution factor of unknown thrombin sample B: Low dilution factor of unknown thrombin sample C: Average clot formation time of standard thrombin D: Average clot formation time of unknown sample in low dilution E: High dilution Average clot formation time of unknown sample in g g: Unit of standard thrombin in test mixture divided by volume of standard thrombin in test mixture (0.5 / 0.5.
1) Enzyme activity is expressed as units / ml (up to 8000 U / ml) and as units / mg protein (up to 10,000 U / mg protein).

The purified or partially purified bovine plasma prothrombin is purified at a temperature of 10 to 25 ° C. for 15 to 45 minutes, as described in Example B, “Isolation of Ultra-Pure Thrombin”.
React with purified thromboplastin in the presence of ４０40 mM calcium chloride solution. The amount of thromboplastin activity is 2-3 times that of prothrombin at pH 6.5-7.0.
It is twice. The thrombin generated by this reaction is further purified as follows. The resulting protein suspension is centrifuged in a refrigerated centrifuge (2 ° -10 ° C.) (12 K RPM) to separate insoluble inactive proteins. The supernatant is applied to a weak anion exchange column (DEAE-Sepharose ^R , DEAE-Sephadex ^R , DE-
52 ^R ). The column is eluted (2 ° C. to 10 ° C.) with 25-50 mM phosphate buffer (pH 6.5) containing 0.1 M sodium chloride. The eluate is monitored at 280 nM. Fractions showing high UV absorption are further checked for thrombin clot forming activity (using a fibrometer). These pooled fractions containing thrombin are cloudy and the suspension is thawed after freezing overnight (<25 ° C.) and clarified by centrifugation or filtration. As thrombin-containing cation exchange column pool ^{(CM-Sepharose R, CM-} Sephadex R) subjected to, and using (0.1M～1M sodium chloride in 25~50mM phosphate buffer (pH 6.5)) salt gradient Elute.

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

The protein content of the thrombin fraction was Bradfo
rd assay (Bradford, M., Anal. Biochem., 72: 248,
1976) and BioRad (Richmond, CA)
Was measured using a protein reagent manufactured by the Company. The specific activity of the preparation was calculated by dividing the enzyme unit per unit volume by the same amount of protein per unit volume.

The exceptionally high specific activity of the thrombin produced according to the invention is due to the following: The use of freshly harvested prothrombin gives a high specific activity thrombin product. In addition, inactive thrombin can elute at the same time as active thrombin, which can lead to reduced specific activity of the final product. Therefore, all isolation steps need to be performed at a temperature of about 2-7 ° C. Furthermore, the solution could not be left for a long time before use, even if it was 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 The preparation made according to the present invention must contain, in a liquid medium, ultrapure thrombin and one or more buffers. They may include saline and other materials commonly used in protein preparations.

The term "preparation" is used, but it is noted that substantially solubilized form of thrombin is intended for any type of composition in which one or more glycols and buffers are present. Should.

When making liquid compositions, the solvent (s) or other diluent (s) used are readily compatible with production standards, eg, uniformity of thrombin concentration from batch to batch. As can be appreciated, it is generally preferred to have suitable miscibility with thrombin.

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

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

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

Water is a preferred medium for the preparations of the present invention. However, it is 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 present 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.

Glycerol or other polyol component (s) may comprise from about 10 to about 10 parts by weight of the total composition.
It will be used in a total concentration of about 40% by weight, preferably 20-30% by weight.

Unless otherwise stated, all amounts stated are weight percentages 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 at about 5.0 to about 8.0 (a preferred pH range is about 5.5 to about 6.5). If a phosphate buffer is used, the final pH of the preparation should be about 6.0.
Particularly preferred is a pH of about 6.5 and a final pH of about 5.0 when using an acetate buffer.

The pH measurement is performed by using the ordinary p
This is performed using an H meter.

Useful buffer systems include acetate, phosphate, succinate, bicarbonate, imidazole,
TRIS, and NE Good and S. Izawa, Methods
in Enzymol, 24 , Part B, 53 (1972); and WF Ferg
uson, KI Braunschweiger, WR Braunschweiger,
JR Smith, J. McCormick, CC Wasmann, NPJar
vis, DH Bell, and NE Good, Anal. Biochem 1
04 , 300 (1980). These disclosures are incorporated herein by reference.

Reagents suitable for use in the buffer system of the present invention include MES, ACES, BES, MOPS, TES, HE
PES and the like are included. The phosphate should only be used in the absence of calcium ions or EDTA. Mixtures of such reagents can be used. If a mixing buffer is used, the final pH should be adjusted appropriately.

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

The buffer may be present in a buffer solution with water and / or other suitable diluent (s) from about 0.01 M to about 0.2 M, preferably about 0.025 M.
M to be present at a total concentration of about 0.10M.

A variety of other conventional additives can also be used, such as antioxidants, colorants, surfactants, and the like. Glutathione may be used as an optional component. Amino acids may be used as optional components, but their presence should not be in an amount that would interfere with the stabilizing effect of the polyol and buffer components on the purified thrombin. In general,
They are preferably used in very small amounts, if any, at concentrations below 0.5%.

The hemostatic currently alone or thrombin powder or hemostats used in combination with thrombin in saline, for example GELFOAM ^R, SURGICEL ^R and AVICE
L ^R, and collagen, using a variety of methods can be effectively used with stabilized thrombin compositions of the present invention. Preferably, the stabilizing solution is absorbed onto the hemostat and the pad is lyophilized and packaged sterilely.

In particular, antimicrobial or antibiotic agents can be incorporated into such pads for use in 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 illustrates the preparation of an ultra-high purity thrombin solution.

[0046]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Isolation of thromboplastin 100 g of calf lung were used to prepare 200 ml of 25 mM sodium phosphate buffer containing 0.5% polysorbate 80 (pH 6.
5) Homogenize in and for 20 minutes at 5 ° C, 12K
Centrifuged at RPM. Collect the supernatant and adjust the pH to 10
% Acetic acid was adjusted to 5.72 and left in the refrigerator for 1 hour. The mixture was centrifuged for 20 minutes as described above and the supernatant was collected. CM-Sepharos obtained by saturating 160 ml of the supernatant with 25 mM sodium phosphate buffer (pH 6.5)
e ^R applied to a column (30 × 20.5cm), and eluted with the same buffer. Collect 230 drop fractions (about 13 ml)
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: 1.0 ml prothrombin 0.5 ml saline 0.1 ml CaCl ₂ (0.3 M) mixed with 100 μl. The mixture was incubated at 25 ° C. for 25 minutes, centrifuged at 5 ° C., 13 K for 10 minutes 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 low color. However, a second pool was also available.

B. Isolation of ultrapure thrombin The composition of the conversion mixture was as follows: 97 ml of prothrombin 40 ml of saline 10 ml of 10 ml of CaCl ₂ (0.3 M) 15 ml of thromboplastin (Pool 1)
K, centrifuged at 5 ° C for 20 minutes and the supernatant was collected.

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

115 ml of the conversion preparation was applied to the column and eluted with the same buffer containing 0.1 M sodium chloride. 230 drop fractions 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 hazy 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 25 mM sodium phosphate buffer containing 0.02% sodium azide and 0.1 M sodium chloride (pH 6.0)
The column (30 × 2.5 cm) was equilibrated in 5).

493 ml of the above supernatant from the DEAE-Sepharose column stage were applied.

0.02% sodium azide and 0.1M
225 ml of buffer containing sodium chloride, and 0.0
The column was eluted with a salt gradient consisting of 225 ml of buffer containing 2% 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 they were pooled (volume 90 ml). The pooled substances were then assayed for thrombin activity,
It showed a clot forming activity of U / ml.

This pooled fraction was further subjected to Br
Protein content was assayed using the adford method and the Bio-Rad protein assay kit. This pooled material contained 0.82 mg / ml protein.

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

[Brief description of the drawings]

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

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Salendra Sea Meter New Jersey, USA 07869. Randolph. High View Terrace 34 (72) Inventor Geilen Davlieu Radeborg New Jersey, USA 07930. Thiester. B Southload 183 (56) References JP-A-1-121224 (JP, A) JP-A-62-106028 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 9 / 74 BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. a) reacting prothrombin with purified thromboplastin; centrifuging the resulting protein suspension; b) eluting the supernatant through an anion exchange agarose column; freezing the desired eluate fraction And then about 2
Melt to 5 ° C .; (c) centrifuge and elute the supernatant through a cation exchange agarose column using a 0.1 M to 1 M sodium chloride gradient in phosphate buffer; A method for preparing a clear thrombin solution comprising collecting. 2. Purified thromboplastin comprises: (a) homogenizing bovine / calf lung tissue with 25 mM phosphate buffer (pH 6.5) containing about 0.5 to about 1% nonionic surfactant; (B) adjusting the pH of the supernatant to 5.5 with 10% acetic acid and centrifuging; (c) cation exchange agarose column using 25 mM phosphate buffer. And is prepared by collecting the desired fractions.
The described method. 3. The method of claim 1, wherein step (a) is performed in a 10-40 mM calcium chloride solution at a temperature of about 10-25 ° C. for about 15-45 minutes. 4. The method of claim 1, wherein the elution of step (b) is performed using a 25-50 mM phosphate buffer containing 0.1 M sodium chloride. 5. The method of claim 1, wherein the elution of step (c) is performed using a salt gradient from 0.1 M to 1.0 M sodium chloride in 25 mM phosphate buffer. 6. Freeze the desired fractions and then
3. The method of claim 2, wherein the mixture is further clarified by centrifuging and collecting the supernatant.