JPH01196294A - Elimination of pyrogen from superoxide dismutase - Google Patents

Abstract

PURPOSE:To perform economical elimination of pyrogen in higher efficiency in quick and safe manner, by filtering superoxide dismutase through a specific filter membrane followed by contact of the resultant filtrate with an adsorbent for pyrogen elimination. CONSTITUTION:Firstly, PVA is intramolecularly formalized to produce a ultrafilter membrane consisting of a polymer constituted of recurring unit having a structure of the formula (with such membrane performance as to be 60-80% in the blocking rate for 0.1wt.% physiological saline solution of human serum albumin) (A). Secondly, a superoxide dismutase(SOD) sample solution containing several thousands - ten thousands ng/ml of endotoxin of several W/V% protein concentration (B) is prepared. Thirdly, the solution B is filtered through the membrane A under such conditions as 11.5-27cm<2> of the membrane area based on 50-200ml of said solution B and 2.0-4.5kg/cm<2> of filtering pressure, to obtain a filtrate (C). Thence, a nitrogen-contg. heterocyclic compound such as histidine and a water-insoluble carrier such as cellulose are mutually conjugated to prepare an adsorbent for pyrogen elimination (D). Finally, the solution C and the adsorbent D are brought into contact with each other to adsorb pyrogen on said adsorbent D, thus eliminating said pyrogen.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention is directed to superoxide dismutase (superoxide dismutase).
roxlde dismutase, Ec l15.1
．． l: Superoxide dismutation enzyme, hereinafter abbreviated as SOD.

), which relates to a method for removing pyrogenic substances from
More specifically, an SOD aqueous solution containing no pyrogenic substance is obtained by filtering the SOD solution containing the pyrogenic substance through an ultrafiltration membrane in which polyvinyl alcohol is formalized in the molecule, and then bringing it into contact with a depyrodiene adsorbent. The present invention relates to a method for removing pyrogenic substances which is characterized by excellent protein recovery rate, rapidity, reproducibility, safety and economic efficiency.

This method is useful for purifying SOD as a pharmaceutical or injectable biological product.

[Prior art and its problems] Although oxygen is essential for living organisms, active oxygen, i.e., superoxide, drastic (02τ) (active radical or superoxide ion) is generated by ultraviolet irradiation or biochemical reactions. and hydrogen peroxide derived therefrom (ll, 0.
), hydroxyl radical (・01l).

- Heavyt oxygen (10), hypochloride (001-), etc. are produced. In addition, these active oxygen species have a bactericidal effect on white blood cells, enzyme reactions, and lipid oxidation.

It has become clear that it is deeply related to radiation damage, inflammation, immune system carcinogenesis, cataractosis, arteriosclerosis, etc.

One of the substances that plays a role in processing active oxygen generated in living organisms is SOD. Among these active oxygen species, SOD is an enzyme (superoxide ion degrading enzyme or superoxide dry dismutation enzyme; 20b+2H-020H2O2) that uses superoxide radical as a substrate and disproportionates it. is [
J.M. McCord.

Ai Fridovich (1, Pr1dovich),
Journal of Biological Chemistry (J,
BIol.

Ct1co+, ), 244.8049-6055. (
1969)].

To date, SOD has been isolated from many species, and its amino acid sequence and three-dimensional structure have been clarified.

Currently, SOD is already a highly safe rheumatism drug [W, 1 luber et al. Perspectives in Inflammation (Per-sp').
cetivcs In 1nNaLI1mation)
, Redwood Barn Limited
od Burn Ltd, ), 1977゜527-54
41 and Behcet's disease [Y, N1vaat
al,, Clinical and Experimental Immunology (CIIn, EXp, 1ms+uno1.)
, 49, 247-255, (1982)] and the effect of preventing side effects of radiotherapy [Edsmyr, P., Pathology of Oxygen, Ac.
Ad, Prcss, 315-3213 (1982), etc. have been reported.

Diseases involving active radicals are wide-ranging and diverse, including inflammation, disseminated intravascular coagulation syndrome (
DIG), is expected to be applied to suppress carcinogenesis, prevent aging, and furthermore to treat ischemic diseases and organ preservation.

SOD is the above-mentioned rheumatism drug (orgotein; or -
gotcin) is derived from the bovine blood method, and Zn-8OD
In addition, human-derived Cu, Zn-3OD, Mn-8OD, etc. produced by E. coli using genetic recombination methods are known.

By the way, the problem of removing pyrogenic substances from such injectable drug products is important. Pyrogens are low molecular weight pyrogens (
They are broadly divided into pyretics (Pyrctlcs) and pyrogens (Pyrogan H polymer pyrogens).
Among these, pyrogen is more exogenous (Exo-gen).
There are two types: endogenous and endogenous. Exogenous includes bacterial endotoxin and exotoxin
xin), etc. Especially this endotoxin is Lon
It has such strong activity that even a small amount of g/kg causes distinct fever in rabbits.

Generally speaking, pyrogen refers to a bacterial pyrogen.
Endotoxin, which is a component of the outer membrane of the cell wall of Durham-negative bacteria, is attracting particular attention because it is highly likely to be present in pharmaceuticals and can cause fever even in minute amounts.

More specifically, endotoxin is a component of the capsular membrane of Gram-negative bacilli, lipopolysaccharide (Lipopoly-5a).
ccharldc; hereinafter abbreviated as LPS) as the main component,
The active substance of the pyrogenic substance is the polysaccharide (
PS) and glycolipid part (Llpld A) (M, l*oto et al, Pro
c, Japan Acad, 80, Scr B)
.

LPS has a property of forming aggregate micelles in an aqueous solution. Its molecular weight is said to be 10,000 to 25,000 as a subunit, 500,000 to 1,000,000 when it forms micelles, and even several million when it forms vesicles, making it stable.

To deactivate LPS, heat at 250°C for 30 minutes or more at 200°C.
Heating is required at 60 minutes or more at 180 degrees Celsius, and 120 minutes or more at 180 degrees Celsius. Due to such heat resistance and chemical stability of LPS, it is extremely difficult to inactivate or remove it from preparations contaminated with LPS under physiological conditions.

Contamination with endotoxin, whose main component is LPS, poses a problem in recombinant bacterial cell-producing drugs and biological preparations produced using Durham-negative bacteria. S.O.
As mentioned above, since D is a biological extract or a genetically modified bacterial cell product, the issue of endotoxin removal is important.Also, since it is a biological product, there are various aspects such as the amount and frequency of administration to the living body. It is essential to completely remove endotoxin from

However, a simple, safe, and reproducible method that can sufficiently remove endotoxins from solutions and preparations containing enzymes and antibodies, as well as blood products, has not yet been established, and many problems remain. This is the current situation.

For example, the following methods have been reported for removing pyrogens from protein solutions, etc., by broadly classifying conventional methods.

(1) Removal by passing through the membrane (for example, JP-A-59-14
9901, etc.) (2) Pyrogenic substance removal using an adsorbent, elution separation of the target substance (for example, Japanese Patent Application Laid-Open No. SY-1GO+2, etc.) (
3) Adsorption of target substance by adsorbent 9 Elution of pyrogen substance I4 (for example, JP-A-50-89220, JP-A-Sho 5)
2-99286, etc.) (4) Other methods using surfactants (for example, JP-A-81-93111, U.S. Pat. No. 4,412.985)
etc.) However, in method (1), as mentioned above, L
Since PS takes various molecular aggregation states (aggregates, etc.) in solution, depending on the solution composition, low molecular weight LPS may pass through the membrane and cannot be completely filtered out from the target substance. Examples of removing pyrogenic substances from low molecular weight substances include:
There is a known method for removing pyrodiene from injection solutions using an ultrafiltration membrane with a molecular weight cut-off of approximately 10,000 [(G, K
oppcnstcincrot al,,PharIl
, lnd, , 38, 19.827-931.1978)
] However, when filtering an endotoxin-containing solution originating from the large intestine, the solution composition (e.g. ionic strength, etc.)
Depending on the situation, most of it will pass through the membrane. Furthermore, depending on the solution composition, the recovery rate may be extremely poor due to adsorption of the target substance onto the membrane. Further, depending on the method, the target substance is often diluted, and a concentration operation or the like is required in a later step.

In method (2), depending on the elution conditions, non-specific adsorption of the target substance to the adsorbent may occur, resulting in a poor recovery rate, or depending on the adsorption capacity and selectivity of the adsorbent, it may not be possible to sufficiently remove the pyrogenic substance. Adsorption removal is often impossible.

In method (3), contrary to method (2), nonspecific adsorption of pyrogenic substances to the adsorbent occurs, and the pyrogenic substances often cannot be removed sufficiently. Furthermore, to separate the target substance from the adsorbent, a solution having higher electrical conductivity than the stock solution to be adsorbed is used. In other words, since the salt concentration of the elution solution is increased, the eluate containing the target substance also contains a high concentration of salt, which may require desalting (concentration) in a later step. There are some drawbacks. Furthermore, it is often necessary to use solutions with different compositions, or to apply a salt concentration gradient to elute the target substance, making the operation complicated in many cases.

In method 4), since the surfactant and target substance are mixed and stirred, there are safety issues such as denaturation of the target substance and residue due to insufficient recovery of the surfactant. .

As described above, conventional methods for removing pyrogens from protein solutions and the like are accompanied by many difficulties, and there is currently a strong demand for the development of techniques to improve these problems.

The present invention has been made from the above viewpoints, and its purpose is to provide a simple, rapid, reproducible, safe, economical and efficient SOD with extremely high protein recovery rate, reliable and practical SOD. The present invention provides a method for removing pyrogenic substances.

[Means for solving the problems] The present invention provides an SOD solution containing a pyrogenic substance in polyvinyl vinyl (n-
65Q to 900) A pyrogenic substance from SOD characterized by comprising a step of filtration with an ultrafiltration membrane made of a polymer based on repeating units of the structure represented by 65Q to 900), and then a step of contacting with a depyrodiene adsorbent. According to the method of the present invention, the protein recovery rate is extremely high, and pyrogens can be efficiently, safely and reliably removed from the SOD solution in a short period of time.

The present invention will be explained in detail below.

The ultrafiltration membrane used in the first step is manufactured, for example, by the following method.

That is, polyvinyl alcohol is reacted with formaldehyde according to a conventional method to obtain an intramolecularly formalized polyvinyl formal resin having a degree of polymerization of preferably about 650 to 900, which is then mixed with a water-compatible polar solvent, preferably N- A dope is prepared by dissolving methyl-2-pyrrolidone (or acetone is added to the pyrrolidone at a ratio of 7:3 to 8:2), and this is uniformly applied onto a polyester nonwoven fabric, for example, to give a dope of 5 to 45%. A film can be formed according to a wet film forming method in which the material is introduced into a coagulation bath containing water at a temperature of 0.degree. C. and coagulated. At this time, the polymer concentration of the film forming solution was 1
．． It is preferably 0 to 30% by weight. In this way, an ultrafiltration membrane consisting of a polymer based on repeating units having the structure represented by the following formula is obtained. The degree of intramolecular formalization of the polyvinyl alcohol is preferably 60 to 85 mol%, and the molecular weight cutoff of the ultrafiltration membrane is preferably 100,000 to 300,000.

The molecular weight cutoff of an ultrafiltration membrane means the lower limit of the molecular weight of solute molecules at which 95% or more is blocked when ultrafiltration is performed using the membrane. In this case, protein molecules are used as standard substances. Since the molecular weight fraction often changes depending on the method used to determine it, here we use human serum γ-globulin (molecules 11u1.6
) and human serum albumin (molecular m8.7 x 10'
) to show membrane performance.

For example, if the membrane surface shear rate is around 6 x 1o3/sec, the membrane permeation rate is 151/rr?・When the inhibition rate is 60-80% for human serum albumin (concentration 0.1%, physiological saline solution), human serum γ-globulin (concentration 0
．． Since the rejection rate against 1% physiological saline solution is 96% or more, the molecular weight cutoff of this ultrafiltration membrane is usually about 10.
It is considered to be X 10'. The ultrafiltration membrane used in the present invention has a blocking rate of 95 to 98% for human serum γ-globulin (concentration 0.1%, physiological saline solution), and has a blocking rate of 95 to 98% for human serum albumin (concentration 0.1%, physiological saline solution). It is preferable that the rejection rate against aqueous solution is 75% or more. The ultrafiltration membrane used in the present invention has a pure permeation rate that is approximately 1.2 to 2 times larger than conventional polysulfone-based or polyethersulfone-based membranes.
．． 1-fold W 96) 7-globe'J, 200 m
1 as the sample solution, the device rUF-LaboJ (manufactured by Tosoh Corporation; trade name; circulation time: 5 hours)) is also characterized in that it exhibits only about half the adsorption of conventional membranes.

Therefore, even when SOD is used as a sample solution, the protein recovery rate in the filtration process using this ultrafiltration membrane is 98%.
The above is the recovery rate when using the conventional membrane: 9096 ~
94%, which is extremely superior, and the processing time under the same filtration pressure is about 1/1 of that using conventional membranes.
It is 2.

In other words, the pyrogen removal process from the SOD solution using the ultrafiltration membrane in which polyvinyl alcohol is intramolecularly formalized in the present invention has an excellent SOD protein recovery rate and can be performed in a short time (quickly) compared to conventional methods. become. In addition, filtration using this membrane has the advantage that it also serves as an operation for removing harmful substances, such as sterilization and particle removal.

The filtration conditions should be determined depending on the amount of sample processed.Usually, the sample solution is 50 to 200 r! For t1, the optimal filtration membrane area is 11.5~27cm, and the filtration pressure is 2.0~27cm.
A weight of 4.5 kg/C is optimal.

As mentioned above regarding the change in the association state of LPS and the loss of protein attachment to the membrane, the composition of the SOD solution in this case has a subtle effect on the recovery rate of SOD and the efficiency of endotoxin removal. Here, in consideration of the second step of contacting the depyrodiene adsorbent, the optimum solution composition for contacting the depyrodiene adsorbent, that is, 20-100 mmojl/
Potassium or sodium (K- or Na-) phosphate buffer (pH 5.0 to 7.5) is preferred. More preferably, the pH is about 20 mmoj2/12 and about 5.5. This phosphate buffer is the best from the standpoint of purifying injections. Furthermore, although SOD may be a partially purified product during purification, the method of the present invention has the great advantage of being able to depyrogenate in the final stage of SOD purification, and removes pyrodiene that could not be removed or is contaminated with conventional purification processes. It is best to apply this method to the final product of conventional purification, since it can be removed. Several thousand to tens of thousands of ng/at a protein concentration of several percent (W/V)
Purified, manufactured SOD solution containing - degree of endotoxin (
For example, bovine or human-derived Cu, Zn-3OD) with a specific activity of 3000 units/a+g or more can be treated with a membrane treatment in the first step of the present invention with a recovery rate of 98% or more and an endotoxin concentration of 1.
~ long/mi, and there is no denaturation or inactivation (Protein quantification is by the Lowry method; activity measurement is by the NBT method or cytochrome C method).

The second step of contacting the depyrodiene adsorbent can be carried out continuously with the above solution composition. This contacting can be carried out by a batch method or a column method or a combination of both.

The batch method is a method in which a depyrodiene adsorbent (approximately 10 to 1/1000 volume) is suspended and mixed in a sample solution, stirred, and then the adsorbent and sample solution are separated by filtration, centrifugation, or the like. In addition, in the column method, for example, a depyrodiene adsorbent is suspended in a solution that dissolves the sample, packed into an appropriate column, and equilibrated with the same solution to prepare a depyrodiene adsorbent column. Space velocity:
sv-i to about 3) to obtain a depyrodienated sample solution as the eluate. The optimum ratio between the amount of protein in the sample solution and the amount of adsorbent (in terms of wet product) is 0.4 to 0.5, although it depends on the ligand content of the adsorbent and its type. Similarly, the ratio between the endotoxin content in the sample solution and the adsorbent amount (wet product equivalent) is 2 x lO ~ 2 x 10
-7 is optimal.

The adsorbent used in the present invention is one in which a nitrogen-containing heterocyclic compound is bonded to a water-insoluble carrier directly or via a spacer. The nitrogen-containing polycyclic compound is -killing R-A-
Represented by X, R is an imidazole skeleton, a pyrazole skeleton,
Pyrimidine skeleton, pyridazine skeleton, pyrazine skeleton, purine skeleton, acridine skeleton, triazole skeleton, oxadiazole skeleton, tetrazole skeleton, indazole skeleton.

Benzotriazole skeleton, benzopyridazine skeleton.

A heterocyclic group having a benzopyrimidine skeleton, benzopyrazine skeleton, or naphthyridine skeleton, where A is a single bond, an alkylene group having 1 to 12 carbon atoms, or an alkylene group having 2 to 12 carbon atoms.
is an alkenylene group in which X is a hydrogen atom, an amino group, a hydroxyl group or a carboxyl group, and particularly preferred are histidine, .histamine, urocanic acid and the like. Preferred examples of the water-insoluble carrier include polysaccharides such as cellulose, agarose, and crosslinked dextran, aminoalkylated polysaccharides, and carboxyalkylated polysaccharides. Furthermore, examples of the spacer include those having an alkyl chain having about 1 to 12 carbon atoms and having an amino group, a carboxyl group, a hydroxyl group, or a combination thereof at both ends.

The column method is superior to the batch method in pyrodiene removal efficiency. In particular, results using a column method using an immobilized histidine depyrodiene adsorbent prepared by covalently bonding histidine to aminohexyl sepharose activated with epichlorohydrin show that the first step of membrane treatment yields a recovery rate of over 98%. and the endotoxin concentration is 1-L.
ong/mj! When a slightly reduced SOD sample solution was directly passed through this column, it was hardly diluted, the recovery rate was 96% or more, and the endotoxin content was determined by the highly sensitive Limulus test (for example, Limulus ll5- for endotoxin detection). Single Test Wako; Wako Pure Chemical ■
An SOD sample solution was obtained which was reduced to below the detection limit (0.01 ng/rR1). This sample solution was also negative in the official Japanese Pharmacopoeia pyrogenicity test (based on the method described in Part 1, Section 83 of the 8th revised Japanese Pharmacopoeia), which is determined by rabbit body temperature rise value. .

By the way, the Limulus test is based on the US Pharmacopoeia RU, S.

Phara + acopala XX, 888 (1980
)], and it has also been adopted in Japan during the radiopharmaceutical standard general examination.

The correlation between this limulus test and the fever test using rabbits is difficult because it involves comparing the in vitro and in vlvo results, but LPS (H, Co110111:B4
), the endotoxin concentration that causes a rise in body temperature in the retest was approximately 5 ng/mj based on the Japanese Pharmacopoeia [Il, Usal
lIl.

Ann, Rcp, TokyoMctr, Rcs
, Lab, P, ](, 33, 100-104.

(1982)], i.e., 5 ng/d concentration is 50
This corresponds to a dose of ng/kg, and at this concentration, the maximum body temperature increase was 0.8°C, which is the standard value under the same law. Humans are generally about three times more sensitive to fever than rabbits, so it is said that they may exhibit even higher body temperature increases [Masayoshi Tamakuma et al.

“Endotoxin Shockco,” Chugai Igakusha (1977)
].

Judging from these facts, the human clinical dose of SOD (for example, usually several to several tens of 111 g s, that is, several tens to hundreds of ng/hearing). It has been confirmed that the removal of pyrogenic substances by the method described above sufficiently achieves the purpose.

The SOD solution obtained as described above is generally obtained as one with low ionic strength and high protein content, but if necessary, it may be desalted or concentrated, and an appropriate amount of sugars etc. may be added thereto. By making frozen or freeze-dried products,
Stable and safe for a long time.

It is possible to make a highly pure SOD preparation.

The above series of operations for removing pyrogenic substances can be carried out at a temperature of about 0 to 30°C, but it is not necessarily necessary to carry out the operation at a low temperature, and it can be carried out satisfactorily at room temperature. In addition, the instruments used can be cleaned and sterilized using normal methods, and no special treatment is required.

In addition, the regeneration of the ultrafiltration membrane used is about IN Na0I.
A conventional method such as immersion treatment in I is sufficient.

It is possible to regenerate the depyrodiene suction tube while it is still packed in the column; for example, in the case of the above-mentioned histidine-immobilized adsorbent, a 0.2N aqueous sodium hydroxide solution (10 to 30% , preferably containing 20% ethanol), sequentially regenerated with 30 times the volume of 1.5M aqueous sodium chloride solution and 10 times the volume of pyrogen-free water,
Next, it is sufficient to equilibrate with a solution that dissolves the sample. In this way, it was possible to use it repeatedly at least several thousand times, and the reproducibility was also good.

The above pyrogenic substance removal method uses Cu and Zn-8 derived from the bovine blood method.
It can be applied regardless of the type or origin of SOD, such as human-derived Cu, Zn-3OD, human-derived or bacterial-derived Mn-8OD, and SOD produced by genetically recombinant (E. coli) cells.

[Effects of the Invention] As is clear from the above description, according to the present invention, the following effects can be obtained.

(1) High-concentration (several thousand to tens of thousands of ng/rR1) endotoxin contained in a high-concentration (several w/v%) SOD solution can be detected from at least 0.1 ng/vdl to the detection limit of the highly sensitive Limulus test (0 , 01 ng/vri) or less, and it is possible to obtain SOD from which endotoxin has been sufficiently removed for pharmaceutical use.

(2) It is possible to obtain an SOD solution with a high recovery rate of 96% or higher in at least the entire process without dilution, without any change in the specific activity or i degree of Sol).

(3) Pyrogenic substances can be removed quickly and easily. There is no need to perform complicated operations such as changing the composition of the sample solution or creating a concentration gradient in column processing, and the entire process can be performed continuously at room temperature using the same solution composition.

(4) It has excellent reproducibility and can safely and reliably remove pyrogenic substances. There is no variation due to multiple treatments, and there is no contamination of the sample solution due to leakage of adsorbent ligands, etc.

(5) Pyrogenic substances can be removed economically and efficiently.

The membrane and adsorbent used can be manufactured at low cost, the throughput of the membrane and the amount of pyrodiene adsorbed by the adsorbent are large, and a large amount of sample solution can be processed in a short time. Moreover, by simple regeneration treatment of these membranes and adsorbents, they can be used repeatedly at least ten to several dozen times.

(6) It is easy to scale up and can remove pyrogenic substances from a large amount of sample solution. It can also be adopted as a practical process for large-scale (industrial) mass purification.

(7) The safety of SOD for medical use can be significantly improved by almost complete removal of pyrogens.

[Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Cu, Zn-8 OD solution purified by bovine blood method (SOD specific activity 3500 units/mg: NBT method, same below; protein concentration 35 mg/rrll: Lovr
The endotoxin concentration contained in the Limulus test (“Limulus It for endotoxin detection”)
s-Single Test Wako” manufactured by Wako Pure Chemical Industries ■, product 1
(hereinafter the same) was approximately 10,000 ng/-. This solution was mixed with 0.02M potassium (K-) phosphate buffer (pH 5,5
1001R1 was filtered through an ultrafiltration membrane (molecular weight cut off: about 100,000) made of a polyvinyl alcohol polymer derivative that had been thoroughly washed in a conventional manner. The filtration membrane area is 27cj, the filtration pressure is 2.5
kg/C, and the filtration device is rUIIP-45J.
(Made by Toyo Roshi ■.

(trade name) under pressure with N2 gas. The endotoxin concentration of this membrane permeate is 1~1Ong/mj! Therefore, the 800 recovery rate determined from the protein quantitative value was 98%. Next, a depyrodiene adsorbent (particle size 60-120 microns, ligand content 0.014
~0.020 mM/g [Adsorbent wet product, pyrodiene adsorption capacity ff 10.5 mg or more/g [Adsorbent] wet product; "Pyrosep"; manufactured by Daicel Chemical Industries ■, trade name) 8d (adsorbent 0.7 g ) was suspended in a 1.5 M aqueous sodium chloride solution, then packed into a sterilized column (inner diameter 8 mm, length Loom), and mixed with 1.5 M sodium chloride consisting of pyrogen-free water. It was washed with 250 d of aqueous solution and 100 d of pure water. 0.02MK
- The column was washed with 100 volumes of phosphate buffer (pH 5,5), and the above membrane permeate was allowed to flow down the column at a flow rate of 5V-1.

All of these series of operations were performed at room temperature. The protein concentration of this eluate is 291I1g/r! T1, was barely diluted, and the 800 recovery was over 98%. Furthermore, the endotoxin concentration determined by the high-sensitivity Limulus test was below the detection limit (0.01 ng/ml).

In these series of depyrodiene treatment steps, the specific activity of SOD in the sample solution did not change. Also, SDS
'M pneumophoresis high performance liquid chromatography (rD
EAE 5PWJ, manufactured by Tosoh ■, product name) etc. SOD
Purity analysis also confirmed that there was no change before and after the above depyrodiene treatment.

Furthermore, the depyrogenated SOD eluate was aseptically diluted to 0.10.100 times with physiological saline and used as a test substance. Partly according to the method described in Section 83), the test volume was 10 d/kg (rabbit body weight),
When a pyrogenic substance test was conducted, none of the three birds showed a rise in body temperature exceeding 0°6°C, and was judged to be negative for pyrogenic substances.

Example 2 Cu, Zn-3OD solution CSO purified from bovine blood method
D specific activity 3500 units/mg; protein concentration (i5
mg/rrdl) to a concentration of 0.02 MK-phosphate buffer (pl!5.5). The endotoxin concentration contained in this solution was approximately 10,000 ng/rtdl according to the Limulus test.

This was used as a sample solution, and depyrodiene treatment was performed in the same manner as in Example 1. The endotoxin concentration of the membrane permeate in the first step was 10 to 1100 n/mA, and the 800 recovery rate was 98%. The endotoxin concentration of the eluate from the depyrodiene column in the second step was 0. Below lng/ml, the protein concentration was 53+ng/ml and the 800 recovery rate was 98%. As a result of SOD specific activity measurement and SOD purity analysis similar to those in Example 1, it was confirmed that there were no changes before and after the depyrodiene treatment. Furthermore, Example 1
Similar pyrogen test results were also negative.

Example 3 Human blood cell-derived Cu, Zn-8OD solution (manufactured by Sigma) and human Cu, Zn produced by genetically recombinant Escherichia coli cells
-8°D solution with a protein concentration of 30ag/mj
! The solution was prepared to have a concentration of 0.02M K-phosphate buffer (pH 5.5). Depyrodiene treatment was performed in the same manner as in Example 1 using these solutions as sample solutions.

In both cases, the protein recovery rate is over 96% in all processes, the protein is rarely diluted, and the endotoxin concentration is 0.01ng/
-The following were obtained. Furthermore, as a result of the same analysis as in Example 1, there was no change in purity or physical properties before and after the treatment.

Example 4 Two types of Mn-8OD derived from bacteria [Bacillus stearothermophilus]
hermopbllS)-derived Mn-8OD (manufactured by Unitika) and Escherichia coli-derived Mn-8OD (51g1
Sample solutions (protein concentration 1%) were prepared from each sample (manufactured by IA) and depyrodiene treatment was performed in the same manner as in Example 1. In both cases, the protein recovery rate is over 98% in all processes, the protein is rarely diluted, and the endotoxin concentration is 0.0.
1 ng/mj or less was obtained. Further, no change in purity or physical properties was observed before and after treatment.

Example 5 Cu purified from the bovine blood method and subjected to depyrodiene treatment,
ZrrSOD solution C3OD specific activity 3500 units/mg
+ Protein concentration 35mg/mi', 0.02M
Endotoxin derived from Escherichia coli (E, coll 0111:B4;
E, purified product from cultured cells of colI UKT-3 strain,
Manufactured by Wako Pure Chemical Industries, Ltd., United States Food and Drug Administration (U.S., FDA)
) of 7 lance (equivalent to endotoxin EC-2) to 1
Mng/mj! This was used as a sample solution, and depyrodiene treatment was performed in the same manner as in Example 1. The results obtained were similar to Example 1.

Reference Example 1 The column used in Example 1 etc. and whose pyrodiene adsorption capacity decreased due to repeated use was a 0.2 M sodium hydroxide aqueous solution (80 mj) (containing 20% ethanol).

1.5M sodium chloride aqueous solution 250d, pyrogen-free water IQOmj! It was washed and regenerated. Thereafter, as a result of repeating the same operations as in Examples 1 to 5, the same reproducible results were obtained even with a small number of repeated uses (at least 10 times).

Comparative Example 1 In the depyrodiene treatment step of Example 1, the same treatment operation as in Example 1 was performed except that the membrane filtration step of the first step was not performed. That is, as a result of performing only the depyrodiene adsorbent column treatment in the second step in Example 1, the endotoxin concentration of the obtained eluate was 10 to 1100n.
/-, resulting in insufficient endotoxin removal.

Similar results were obtained for the sample solutions of Examples 2-5.

A case where the same sample solution as in Example 1 was used to perform the pyrogenic substance removal treatment according to the present invention, a case where only the first step of the membrane filtration treatment among the pyrogenic substance removal processes according to the present invention was performed, and a case where the second step was performed using the same sample solution as in Example 1. Table 1 shows the results of comparing the endotoxin concentration of the sample solution before and after the treatment for three cases in which only contact with the depyrodiene adsorbent was performed in the process.

Table 1 Comparative Example 2 In the depyrodiene treatment step of Example 1, a conventional polyethersulfone ultrafiltration membrane (molecular weight cutoff of about 304°XIO, rUF-300PSJ) was used instead of the membrane used in the present invention. The same sample solution as in Example 1 was used, and the same processing operations were performed except that the same sample solution was used as in Example 1. The 800 protein recovery rate up to the membrane treatment step of the first step was 94%, and the protein recovery rate of the solution obtained after completing all treatment steps was 92%.

Note that the endotoxin removal rate in each step showed almost the same results as in Example 1.

Table 2 shows the results of comparing the 800 recovery rate at each stage between Example 1 and Comparative Example 2. In addition, in the table, the value of the recovery rate at the end of the depyrodiene column treatment in the second step indicates the value of the recovery rate of SOD at the end of all treatment steps including the membrane filtration treatment in the first step.

Table 2 As is clear from Tables 1 and 2, endotoxin removal is insufficient with membrane filtration treatment alone or depyrodiene adsorbent column treatment alone, and conventional polyethersulfone-based ultrafiltration membranes If this occurs, the protein recovery rate of SOD will decrease.

When the pyrogenic substance removal method according to the present invention is performed, the endotoxin concentration can be reduced to below the detection limit of the highly sensitive Limulus test, and the SOD protein recovery rate is also good. It is clear that an SOD solution is obtained with Φ O

Claims (1)

[Claims] (1) A general formula in which polyvinyl alcohol is intramolecularly formalized into a superoxide and dismutase solution containing a pyrogenic substance ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ (n = 850-900) The repeating unit of the structure is 1. A method for removing pyrogenic substances from superoxide dismutase, comprising the steps of filtration with an ultrafiltration membrane made of a basic polymer, and then contacting with a depyrodiene adsorbent.