JP2817606B2 - Method for stabilizing endotoxin in aqueous solution - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for stabilizing ET in an aqueous solution, which is used for accurately measuring the concentration of endotoxin (hereinafter abbreviated as ET) in an aqueous solution such as a dialysate for hemodialysis. About the method.

[0002]

BACKGROUND OF THE INVENTION Hemodialysis using an artificial dialysis machine is widely performed mainly for patients with chronic renal failure. In hemodialysis, unnecessary substances in blood are removed or necessary substances are supplied through a dialyzer having a semi-permeable membrane.

[0003] In recent years, in hemodialysis, if not only conventional relatively low molecular weight substances but also relatively high molecular weight harmful substances such as β ₂ -microglobulin are removed, adverse effects on patients may occur. In addition, with the improvement of the technology for fabricating a semipermeable membrane, a highly permeable dialyzer capable of removing substances having a molecular weight of about 10,000 among unnecessary substances in blood has come to be used. However, on the other hand, among the toxic substances that may be present in the dialysate, ET having a molecular weight of several thousands to about 10,000 or a substance similar thereto enters the blood through a semipermeable membrane, and is subjected to reverse filtration and reverse diffusion. The phenomenon is considered problematic. That is,
ET is a lipopolysaccharide (LPS) present in the outer membrane of the cell wall of Gram-negative bacteria, and is known as a strong pyrogen.
This is because preventing contamination of T is considered to be extremely important. In particular, invasion by reverse diffusion cannot be prevented even by devising a dialysis method [S. Takesawa, H. Saito,
H.Hidai, M.Suzuki and K.Sakai: Measurement of Back
Clearance. Trans Am SocIntern Organs. 36 M441-443
(1990)], in a facility using a highly permeable dialyzer, an effort to make the dialysate for hemodialysis ET-free became essential.

[0004] As a method for determining whether or not a dialysate for hemodialysis is ET-free, a method for counting the number of cells or a blood cell component solution of horseshoe crab (hereinafter abbreviated as AL solution) reacts with ET. An ET measurement method utilizing the property of causing an activation reaction or a gelling reaction of an enzyme (such as a protease), a so-called Limulus test, is mainly mentioned. Among them, the bacterial cell counting method is a method that has been established to some extent, but has a problem that detection requires time. For this reason, it is desirable to determine whether or not the dialysate for hemodialysis is ET-free by an ET measurement method capable of quick measurement.

However, ET in a dialysate for hemodialysis
Measurement has not been able to solve an important problem, so its measurement accuracy is a problem. That is, in the ET measurement by the Limulus test, the ET measurement value in the hemodialysis dialysate collected for analysis decreases with time, so that the correct value is obtained unless measurement is performed within a short time after collection. There is a problem that it cannot be obtained [Shingo Takezawa, Nobuki Kikuchi, Hideo Hidai, Yoko Nakamura, Noriko Toda, Masahiko Sugano. Basic study on measurement of dialysate endotoxin. Kidney and Dialysis Separate Volume High Performance Membrane '93 64-66 (1993)].

Therefore, in order to measure ET in an aqueous solution such as a dialysate for hemodialysis by a Limulus test or the like, it is necessary to stabilize the ET in these aqueous solutions. However, it has not yet been found and there is a demand for rapid development.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and has as its object to provide a stabilizing method for suppressing a change with time so that an ET measurement value in a collected aqueous solution can be accurately obtained. Aim.

[0008]

The present invention relates to the coexistence of an aqueous solution containing ET with a water-soluble protein having affinity for ET and not inhibiting or promoting the reaction between ET and AL solution. The invention is characterized by a method for stabilizing ET in an aqueous solution.

In other words, the present inventors have found that in an aqueous solution such as a dialysate for hemodialysis, the measured value of ET decreases with time during storage, and the ET concentration in these aqueous solutions cannot be measured accurately. In order to solve the above problem, intensive research has been conducted on a method for stabilizing ET in an aqueous solution. As a result, the ET has an affinity for ET such as albumin or immunoglobulin and the reaction between ET and AL solution. ET can be stabilized when a water-soluble protein having no property of inhibiting or promoting ET is allowed to coexist in these aqueous solutions.
We found that we could suppress the change over time of the measured values,
The present invention has been completed.

A protein used in the present invention that has an affinity for ET and does not have the property of inhibiting or promoting the reaction between ET and AL solution (hereinafter abbreviated as ET affinity protein). ) Is not particularly limited as long as it is a protein having such properties. For example, albumin (including ovalbumin and the like), immunoglobulin (IgG, IgM, IgA and the like), lysozyme and the like are preferable. No. The origin of these ET affinity proteins is not particularly limited, and may be derived from, for example, mammals such as humans, cows, horses, sheep, rabbits, rats, mice, etc., plasmas of birds such as chickens, or eggs of birds such as chickens. What you do is good. still,
It goes without saying that, as long as they have properties equivalent to those of the ET affinity proteins, those obtained by genetic engineering and those synthesized are also applicable to the present invention.

[0011] It should be noted that, for example, proteins and such as to present a false positive in Limulus test as serine proteases such as factor Xa, for example, antithrombin III, α ₂ over plasmin inhibitor, antitrypsin, high-density lipoprotein (HD
Proteins that give a false negative in the Limulus test, such as L), even if they have an affinity for ET,
Needless to say, it cannot be used in the present invention.

The ET affinity protein used in the present invention may be a protein having a plurality of components mixed therein or a protein rich in albumin, immunoglobulin and the like, for example, an albumin fraction obtained by corn fraction, An immunoglobulin fraction, for example, an albumin fraction obtained by electrophoresis or an immunoglobulin fraction, for example, in the form of inactivated plasma (eg, 80 ° C., 5 minutes) plasma or serum, such as egg white dilution Good.

The ET affinity protein used in the present invention does not necessarily have to be specially prepared for the purpose. For example, a fresh liquid commercially available from the Japanese Red Cross Society or the like may be used. Plasma and fresh frozen human plasma, or
Nippon Pharmaceutical Co., Ltd., Roller Company, Armor Company, Cutter Company,
Heated human plasma protein preparations and human serum albumin preparations commercially available from Baxter Co., Ltd., Midori-Cross Inc. and Kaketsuken, etc., and also Takeda Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Midori-Cross Inc. , Hoechst, Japanese Red Cross and Fujirebio
Human immunoglobulin preparations commercially available from Co., Ltd., etc., and those prepared using fetal calf serum commercially available from Whitaker, Hycron, Gibco, Boehringer Mannheim, etc. Can be used.

[0014] The ET affinity protein according to the present invention must of course not contain an amount of ET that affects the ET measurement. For this purpose, a substance having a low ET content is selected, or when an ET-containing substance is used, the ET content is previously determined using an autoclave, an ET adsorbent, or the like.
Need to be removed.

If the ET is removed by an autoclave, for example, the above-mentioned ET affinity protein is converted into an aqueous solution of an appropriate concentration, and then, for example, autoclaved at a temperature (about 121 ° C.) for about 15 to 120 minutes. do it.

If ET is removed using an ET adsorbent, a specific trade name of a carrier such as a carrier on which polymyxin B is immobilized or a carrier on which histidine is immobilized via a spacer is detox gel (Pierce). Company),
It is sufficient to use Affiprep polymyxin (manufactured by Bio-Rad) and Pyrosep (manufactured by Tanabe Seiyaku Co., Ltd.), but the invention is not limited to these.

For example, the concentration of the ET affinity protein according to the present invention in an aqueous solution such as a dialysate for hemodialysis varies depending on the type of the ET affinity protein used and the lot, and is not necessarily constant. As a protein concentration in the usual about 0.1 ~ 2500μg / ml, preferably 0.25 ~ 1,000μg
/ ml, more preferably about 2.5 to 500 µg / ml. Further, for example, when the ET affinity protein is human serum albumin, usually about 0.1 to 2,500 μg / ml, preferably about 0.25 to 1,000 μg / ml, more preferably 2.5 to 250 μg
/ ml, and when the ET affinity protein is, for example, a human plasma solution inactivated, the protein concentration is usually
It is about 0.1 to 200 μg / ml, preferably about 0.25 to 100 μg / ml, and more preferably about 2.5 to 70 μg / ml.

The ET affinity protein used in the present invention is
For example, human serum albumin (HSA), human plasma, human serum,
In the case of fetal bovine serum, bovine serum albumin (BSA), immunoglobulin, etc., when the protein concentration in the aqueous solution increases, an inhibitory effect is produced in the ET measurement of the aqueous solution by the Limulus test method, and the inhibitory effect is caused. Also give low values. Therefore, such a high concentration is not preferable. Therefore, the preferred concentration range of the ET affinity protein according to the present invention, particularly the upper limit value thereof, is one taking such points into consideration. In addition, the concentration of the ET affinity protein that causes inhibition at the time of the measurement by the Limulus test method remarkably differs depending on the type of the ET affinity protein. That is, for example, in the case of HSA, inhibition occurs at a protein concentration of about 6.0 mg / ml.

In order to carry out the present invention, ET as described above is used.
It suffices that the affinity protein is added and dissolved in a test aqueous solution such as a dialysate for hemodialysis to a predetermined concentration. E
The method of adding the T-affinity protein to the test aqueous solution so as to have a predetermined concentration is not particularly limited as long as it is a method that can finally add the ET affinity protein to the test aqueous solution so as to have a predetermined concentration. For example, a method of adding an appropriate amount of an aqueous solution containing an ET affinity protein to a test aqueous solution, a method of collecting a test aqueous solution into a sample collection test tube in which an appropriate amount of an aqueous solution containing an ET affinity protein is previously dispensed, A method in which an appropriate amount of an aqueous solution containing a soluble protein is previously dispensed, and then the test aqueous solution is collected in a freeze-dried sample collection test tube. An appropriate amount of the ET affinity protein powdered by freeze-drying or the like is added to the test aqueous solution. The method of adding is preferable. The aqueous solution containing the ET-affinity protein or the lyophilized solution containing the ET-affinity protein may be a phosphate, a Good (buffer) buffer, or the like as long as it does not inhibit or accelerate the measurement of ET by the Limulus test method. It is needless to say that a buffering agent or a chelating agent such as ethylenediaminetetraacetic acid (EDTA) may be contained. In addition, ET affinity protein
In order to add white matter to the test aqueous solution at a predetermined concentration,
Those prepared in the form of a product may be used. like this
As a stable composition, ET in an aqueous solution state is stabilized.
Which has an affinity for ET and ET
Inhibits or promotes the reaction of limulus with a blood cell component solution of horseshoe crab
Containing water-soluble proteins that do not have
It is sufficient if it is, a preferred embodiment of each component, specific
Examples are as described above. The form is
Water-soluble proteins such as bumin, immunoglobulin, and lysozyme
Aqueous solution containing at least one selected from quality, frozen
Dried products or powdered products thereof can be mentioned. Also this
In such compositions, ET is measured by the Limulus test method.
Within the range that does not inhibit or promote
Buffer such as Good buffer, for example, ethylenediamine.
Contains chelating agents such as amine tetraacetic acid (EDTA)
It goes without saying that you may be there.

E in the aqueous solution treated by the method of the present invention
T is stable for about one week even in the state of a solution, but when stored at room temperature or refrigerated, gram-negative bacteria containing ET in the cell wall outer membrane grow and the apparent ET amount increases. There are cases. Therefore, when the aqueous solution treated by the method of the present invention is used as a sample for ET measurement, it is preferable that the solution be stored frozen until it is used for measurement. Even when stored frozen, the measured value of ET in the aqueous solution does not fluctuate for about one week. Even if the solution is repeatedly frozen and thawed about two or three times, the measured value of ET in the solution does not change.

The AL solution used for measuring the amount of ET in the aqueous solution stabilized by the stabilization method of the present invention using a Limulus test is, for example, Limulus sp.
mulus), a blood cell component of horseshoe crab belonging to the genus Tachypleus or the genus Carcinoscorpius, which is not particularly limited as long as it coagulates by reaction with ET. For example, ACC (Associates of Cape Cod)
Based on lyophilized AL solutions commercially available from Whitaker Bioproducts, Inc., Endosafe, Inc., Seikagaku Corporation and Wako Pure Chemical Industries, Ltd. Preparations can of course be used.

The Limulus test for measuring the amount of ET in an aqueous solution stabilized by the stabilizing method of the present invention using a Limulus test can be used without any particular limitation as long as it is a commonly used method. It is. Examples of commonly used techniques include, for example, the FDA guidelines (Guidelineon validation of the Limulus amoeb).
cell lysate test as an end-productendotoxin tes
t for human and animal parenteral drugs, biol
ogicalproducts, and medical devices, Food and Drug
Adm. (1987)), a gel-overturn method, a synthetic substrate method, a turbidimetric time analysis method, and the like. More specifically, for example, Toxinometer ET-201 (manufactured by Wako Pure Chemical Industries, Ltd.), Toxinometer MT-251 (manufactured by Wako Pure Chemical Industries, Ltd.), LAL-5000 [ACC (ASSOCI
ATES OF CAPE COD) manufactured by a conventional method using an AL solution, such as turbidimetric analysis using a dedicated device. Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[0023]

【Example】

Embodiment 1 FIG. (Reagent) ・ LAL solution A lyophilized product of an AL solution derived from Limulus horseshoe crab (hereinafter abbreviated as LAL; sold by Wako Pure Chemical Industries, Ltd., gelation sensitivity: 0.6 EU / liter, for 5 ml) for injection. The LAL solution obtained by dissolving with distilled water was used. -ET stabilizer solution A solution obtained by dissolving a predetermined ET affinity protein in distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) so as to have a predetermined concentration and then autoclaving (121 ° C., 20 minutes). The ET stabilizer solution was confirmed to be endotoxin-free and not promoting or inhibiting the Limulus test and the like. The following ET affinity proteins were used. Human serum albumin: manufactured by Japanese Red Cross Society. Human immunoglobulin: manufactured by Green Cross Co., Ltd. Bovine serum albumin: manufactured by Wako Pure Chemical Industries, Ltd. Lysozyme: manufactured by Wako Pure Chemical Industries, Ltd. (derived from chicken eggs). Ovalbumin: manufactured by Wako Pure Chemical Industries, Ltd. (derived from chicken eggs). (Sample) To a 2054 Falcon tube (manufactured by Becton Dickinson, volume: about 4 ml), 2 ml of a dialysate for hemodialysis containing about 120 EU / liter of ET and 50 μl of a predetermined ET stabilizer solution were added and mixed. These were used as samples. (Operation method) After stirring and mixing 0.1 ml of the LAL solution and 0.1 ml of the above sample, while maintaining the temperature at 37 ° C, the transmitted light amount of the mixed solution was 5%.
The time until the decrease (hereinafter abbreviated as Tg) was measured using a toxinometer MT-251 (manufactured by Wako Pure Chemical Industries, Ltd.). Separately, the same measurement was performed using distilled water for injection containing a predetermined concentration of ET as a sample, and a calibration curve representing the relationship between the ET concentration and Tg was created. The ET concentration in each sample was calculated based on this calibration curve. The measurement of ET was performed on the sample immediately after preparation and on the sample immediately after preparation and frozen and stored in a freezer at −20 ° C. and thawed after a lapse of a predetermined number of days. (Results) The results obtained are shown in FIG. FIG. 1 is a graph obtained by connecting points obtained by plotting the relative concentration (%) of the ET in the sample obtained for each of the frozen storage days on the horizontal axis along the vertical axis. Indicates the results obtained for a sample (blank) using distilled water for injection instead of the ET stabilizer solution, and-○-indicates the results obtained for a sample using a 0.25% human serum albumin aqueous solution as the ET stabilizer solution. -□-indicates the results obtained for a sample using a 0.02% human serum albumin aqueous solution as the ET stabilizer solution, and-△-indicates the 0.002% human serum albumin aqueous solution as the ET stabilizer solution. , The results obtained for the sample using a 0.1% human immunoglobulin aqueous solution as the ET stabilizer solution, and-◇-for the sample obtained using 0.02% as the ET stabilizer solution. % Bovine serum albumin aqueous solution The results obtained for the samples used were the results obtained for samples using 2.5% inactivated human plasma aqueous solution (total protein concentration; about 0.2 g / dl) as the ET stabilizer solution. ,-+-Indicates the results obtained for a sample using a 0.1% ovalbumin aqueous solution as an ET stabilizer solution, and-☆-indicates a sample using a 0.025% lysozyme aqueous solution as an ET stabilizer solution. Are shown below. The ET relative concentration (%) indicates the ratio of the ET concentration in each sample after cryopreservation for a predetermined number of days, where the ET concentration in each sample immediately after preparation is 100%. As is clear from the results of FIG.
It can be seen that by adding a solution containing various ET affinity proteins to the dialysate for hemodialysis, it is possible to suppress the change over time of the measured ET value in the dialysate. In particular, the measured value of ET in a sample using a 0.02% aqueous human serum albumin solution as the ET stabilizer solution shows no change even on the sixth day after cryopreservation. As an ET stabilizer solution, an aqueous solution containing 0.02% human serum albumin and 10% glucose,
An aqueous solution containing 0.02% human serum albumin and 0.04% polyoxyethylene octyl phenyl ether, or 0.02%
% Of human serum albumin using 10-500 mM phosphate buffer (pH 7.4).
These were also found to be effective as ET stabilizer solutions. In addition, for the sample to which the ET stabilizer solution was added,
When the ET concentration was measured by repeatedly freezing and thawing,
It was also found that no change was observed in the measured values after two or three freeze-thaw cycles.

[0024]

As is apparent from the above description, the present invention relates to a method for stabilizing ET in an aqueous solution such as a dialysate for hemodialysis and the like. It is possible to accurately measure the ET concentration in an aqueous solution in which the ET measurement value has been remarkably reduced over time over a long period of time. is there.

[Brief description of the drawings]

FIG. 1 is a plot of the relative concentration (%) of endotoxin (hereinafter abbreviated as ET) in a sample obtained for each frozen storage day on the horizontal axis, obtained in Example 1, along the vertical axis. It is the connection between the points.

[Explanation of symbols]

In FIG. 1,-●-indicates the results obtained for a sample (blank) using distilled water for injection instead of the ET stabilizer solution,
-○-indicates the results obtained for the sample using 0.25% human serum albumin aqueous solution as the ET stabilizer solution,
□-indicates the results obtained for the sample using 0.02% human serum albumin aqueous solution as the ET stabilizer solution,
-Indicates the results obtained for a sample using a 0.002% human serum albumin aqueous solution as the ET stabilizer solution,-
-Indicates the results obtained for the sample using a 0.1% human immunoglobulin aqueous solution as the ET stabilizer solution,-、-
Represents the results obtained for a sample using a 0.02% bovine serum albumin aqueous solution as the ET stabilizer solution, and-▽-represents E.
The results obtained for a sample using a 2.5% inactivated human plasma aqueous solution (total protein concentration: about 0.2 g / dl) as the T stabilizer solution are shown.
% Ovalbumin aqueous solution, the results obtained for the sample were as follows.
The results obtained for the samples using the 5% lysozyme aqueous solution are shown respectively.

──────────────────────────────────────────────────続 き Continuation of front page (51) Int.Cl. ⁶ Identification symbol FI // C07K 1/14 C07K 1/14 (56) References JP-A-62-110153 (JP, A) JP-A-2-18763 (JP, A) ACS Symp. Ser. (Anal.chem.Bacillus Thuringiensis) 432 (1990) P.A. 70-77 (58) Field surveyed (Int. Cl. ⁶ , DB name) G01N 33/579 G01N 33/531 CA (STN)

Claims (4)

(57) [Claims] An aqueous solution containing endotoxin is characterized by coexistence of a water-soluble protein having an affinity for endotoxin and not having a property of inhibiting or promoting the reaction between endotoxin and a horseshoe crab blood cell component solution. A method for stabilizing endotoxin in an aqueous solution. 2. The method according to claim 1, wherein the protein is at least one selected from albumin, immunoglobulin and lysozyme. 3. An endotoxin having an affinity for an endotoxin.
Inhibits the reaction between dotoxin and horseshoe crab blood cell components
Contains water-soluble proteins that have no
Composition for stabilizing endotoxin in aqueous solution
Stuff. 4. The method according to claim 1, wherein the protein is albumin, immunoglobulin or protein.
And at least one selected from lysozyme and lysozyme
Item 4. The composition according to Item 3.