JP5578390B2 - Method for producing bacterial cell wall skeleton components - Google Patents

Description

  The present invention relates to a method for producing a bacterial cell wall skeletal component, specifically, a cell wall skeletal component (BCG-CWS) of BCG bacteria.

Bacterial cell wall skeletal component [Cell Wall Skeleton may be abbreviated as CWS hereinafter] has an immunostimulatory action, for example, an experimental tumor system using an animal model, and immunotherapy of human cancer It is a substance known to exhibit anti-tumor activity.
For example, in cancer immunotherapy using the cell wall skeleton component of Bacille Calmette-Guerin (BCG) (hereinafter sometimes abbreviated as BCG-CWS), excellent results have been obtained in human cancer treatment. (See Non-Patent Documents 1 and 2). A method for producing BCG-CWS has been reported by To et al. (See Non-Patent Document 3), but there has been a demand for a method for producing BCG-CWS having high purity on an industrial scale as a drug substance.
A. Hayashi et al., Pro. Japan Acad., 70, Ser. B 205-209 (1994) A. Hayashi et al., Pro. Japan Acad., 74, Ser. B 50-56 (1998) I. Azuma et al., Journal of the National Cancer Institute, Vol. 52, NO. 1 95-101 (1974)

  The problem to be solved by the present invention is to provide a method for producing high-purity BCG-CWS.

The inventors of the present invention have completed the present invention as a result of intensive studies to produce high-purity BCG-CWS on an industrial scale.
That is, the present invention
[1] A method for producing BCG-CWS, comprising the following steps (1) to (4):
(1) crushing BCG bacteria and collecting cell walls (WCW),
(2) a step of treating the cell wall (WCW) obtained in (1) with benzonase and pronase;
(3) a step of treating the product obtained in (2) with a surfactant under heating at 55 ° C to 65 ° C;
(4) A step of washing the product obtained in (3) one or more times with one or more organic solvents;
[2] The production method according to [1], wherein the surfactant in the step (3) is a nonionic surfactant;
[3] The production method according to [1] or [2], wherein the nonionic surfactant is polyoxyethylene (10) octylphenyl ether (Triton X-100);
[4] The organic solvent in the step (4) is one or a plurality of the same or different organic solvents selected from alcohol solvents, ether solvents, halogen solvents and mixed solvents thereof. 3] The production method according to any one of
[5] The organic solvent in the step (4) is one or a plurality of the same or different organic solvents selected from methanol, ethanol, tetrahydrofuran, chloroform, dichloroethane and a mixed solvent thereof [1] to [3] The production method according to any one of the above;
[6] The production method according to any one of [1] to [3], wherein the organic solvent in the step (4) is tetrahydrofuran, chloroform-methanol (2: 1), and methanol;
[7] The production method according to any one of [1] to [6], wherein the BCG bacterium is a BCG bacterium Tokyo strain;
[8] High-purity BCG-CWS obtained by the production method according to any one of [1] to [7];
About.

  The present invention has made it possible to provide high-purity BCG-CWS on an industrial scale.

In the present specification, BCG-CWS is a Mycobacterium bovis which is a bacterium of the genus Mycobacterium, more specifically, a cell wall skeleton component derived from BCG (Bacille Calmette-Guerin) (Bacille Calmette-Guerin) Cell Wall Skeleton; CWS). Here, specific examples of BCG bacteria include Tokyo, Russia, Brazil, Sweden, and Australia. More preferable BCG bacteria include BCG bacteria Tokyo 172 strain (ATCC 35737).
“Cell wall skeletal component (CWS)” is a protein obtained by physically pulverizing bacterial cells, followed by purification steps such as nucleic acid removal by nuclease, protein removal by protease, and degreasing by washing with an organic solvent. Represents an insoluble residue substantially free of soluble components such as nucleic acids and fatty acids, and CWS itself is known (J. Nat. Cancer Inst., 52, 95-101 (1974)). It is known that the insoluble residue is a polymer containing mycolic acid, which is a long-chain hydroxy fatty acid, and peptidoglycan.

  BCG bacteria used as starting materials are known and can be produced by methods known to those skilled in the art. That is, BCG bacteria are cultured as a fungal membrane on the surface of the medium in an appropriate medium such as a sorton medium at about 37 ° C. for 7-10 days until the initial stationary phase. The cultured cells are inactivated by heating at about 80 ° C. for 30 minutes, and then the whole culture is centrifuged at 7000 × g for 40 minutes at 4 ° C. to obtain BCG killed bacteria as a precipitate ( J. Nat. Cancer Inst., 52, 95-101 (1974), etc.).

Benzonase (Benzonase (registered trademark) Enzyme Commission (EC) Number 3.1.30.2) used in the present invention is a DNA, RNA, single-stranded, double-stranded, linear, circular, or supercoiled nucleic acid. It is an enzyme that degrades and is an endonuclease derived from Serratia marcescens (classified as a non-specific phosphodiesterase) expressed in recombinant E. coli.
The pronase (Pronase; EC Number 2329666) used in the present invention is an enzyme that nonspecifically hydrolyzes a protein / peptide derived from Streptomyces griseus.

  Examples of the surfactant used in the present invention include nonionic surfactants. Specific examples include polyoxyethylene (10) octyl phenyl ether (Triton X-100).

Each step of the production method of the present invention will be described in detail below.
(1) Step of crushing BCG bacteria and collecting cell walls (WCW):
BCG killed bacteria used as a raw material is usually wet and not dried. BCG bacteria are suspended in about 2 L to 5 L of water per 600 g and crushed. The means for crushing is not particularly limited, but it can be usually crushed under a pressure of 30 to 40 kpsi, preferably about 35 kpsi using a crusher such as a high-pressure homogenizer. The crushed cells can be made into a uniform cell wall (hereinafter sometimes referred to as WCW) from which cell fragments that have not been sufficiently crushed or cells that have not been crushed have been removed by repeating centrifugation one or more times. . The particle size of the cell wall is about 0.5 μm to 0.7 μm.
Here, the conditions for centrifugation for obtaining a cell wall as a raw material for producing CWS can be appropriately selected. For example, after centrifugation at 20-30 ° C., preferably about 25 ° C. at 5000 × g-9000 × g, preferably 7000 × g, for 5-15 minutes, preferably about 10 minutes, the supernatant is 20-30 ° C., preferably about Centrifugation may be performed at 25 ° C. at 16000 × g to 20000 × g, preferably 18000 × g for 30 to 90 minutes, preferably about 60 minutes.

(2) A step of treating the cell wall (WCW) obtained in (1) with benzonase and pronase:
The cell wall (WCW) obtained in (1) is treated with benzonase (Benzonase) and pronase (Pronase).
Here, there is no particular limitation on the order of treatment with both enzymes, but treatment with pronase is preferred after treatment with benzonase.
In the step of treating with benzonase, usually 800 to 1200 U, preferably about 1000 U of benzonase per 1 g of WCW is used, and the reaction is carried out at about 20 to 30 ° C., preferably 25 ° C. for about 15 to 17 hours.
In the step of treating with pronase, 5 to 15 mg, preferably about 10 mg of pronase is usually used per 1 g of WCW, and the reaction is carried out at about 35 to 40 ° C., preferably about 37 ° C. for 15 to 17 hours.
The step of treating with the first enzyme and the second enzyme is not particularly limited and can be appropriately performed by a method well known to those skilled in the art. Usually, WCW is suspended in a buffer of pH 7.5-9 such as Tris buffer, treated with the first enzyme, and then collected by centrifugation. When recovering WCW, a surfactant can be appropriately added. Moreover, after recovering WCW, a step of suspending WCW in water in the presence or absence of a surfactant and centrifuging the WCW may be performed. Examples of the surfactant include those described above, and specifically, 1% Triton X-100 or the like can be used. The washing step is, for example, an operation of stirring at about 23 ° C. to 27 ° C. for about 1 hour and then centrifuging at 16000 × g to 20000 × g, preferably about 18000 × g for about 30 to 90 minutes, preferably about 60 minutes. Alternatively, it may be performed a plurality of times, preferably 1 to 5 times.
Next, the recovered WCW is suspended in a buffer having a pH of 7.5 to 9 such as Tris buffer and treated with the second enzyme, and then the WCW is recovered by centrifugation. When recovering WCW, a surfactant can be appropriately added. Moreover, after recovering WCW, a step of suspending WCW in water in the presence or absence of a surfactant and centrifuging the WCW may be performed. Examples of the surfactant include those described above, and specifically, 1% Triton X-100 or the like can be used. The washing step includes, for example, one or more operations of centrifugation at 16000 × g to 20000 × g, preferably about 18000 × g for 30 to 90 minutes, preferably about 60 minutes at about 20 to 30 ° C., preferably about 25 ° C. Preferably, it may be performed 1 to 5 times. Examples of the surfactant include those described above.
BCG-CWS can be obtained through the above steps.

(3) A step of treating the product obtained in (2) with a surfactant under heating at 55 to 65 ° C:
The BCG-CWS obtained in (2) is suspended in water in the presence of a surfactant and heated at 55 to 65 ° C., preferably about 60 ° C., for about 30 to 90 minutes, preferably about 60 minutes. It can refine | purify by stirring. Examples of the surfactant include those described above, and specifically, 0.9 to 1.1%, preferably 1% Triton X-100 can be used. After stirring, BCG-CWS can be obtained as a precipitate by centrifugation.

(4) The step of washing the product obtained in (3) with an organic solvent:
By washing the BCG-CWS crude product obtained in (3) with an organic solvent, high-purity BCG-CWS can be obtained. Examples of the organic solvent include one or more organic solvents selected from alcohol solvents, ether solvents, halogen solvents, and mixed solvents thereof. Specific examples of the alcohol solvent include methanol and ethanol. Tetrahydrofuran is mentioned as an ether solvent. Examples of the halogen-based solvent include chloroform, 1,1-dichloroethane and the like, and are usually used as a mixed solvent with an alcohol-based solvent. Examples of the mixed solvent include chloroform-methanol (1: 1 to 3: 1, preferably 2: 1).
The washing operation can be appropriately performed one or more times with the same or different solvent selected from the organic solvents described above. The amount of organic solvent used for one washing is usually 1.8 L to 2.0 L per 10 g of BCG-CWS.
Specifically, for example, the following steps 1) to 3):
1) a step of washing with an alcohol solvent such as ethanol,
2) a step of washing with an ether solvent such as tetrahydrofuran and a mixed solvent of a halogen solvent and an alcohol solvent, and 3) a step of washing with an alcohol solvent such as methanol,
including.
In 2), either the washing with an ether solvent such as tetrahydrofuran or the washing with a mixed solvent of a halogen solvent and an alcohol solvent may be performed first.
More preferably, washing is performed in the order of ethanol, tetrahydrofuran, chloroform-methanol (2: 1), and methanol.
After washing, the residue is dried to obtain high-purity BCG-CWS.

High purity BCG-CWS obtained by the production method of the present invention is also included in the category of the present invention. Specifically, according to the production method of the present invention, the protein content is 2.1% (w / w) or less (in terms of dehydrate) as the total amount of non-structural amino acids, non-structural neutral sugars (glucose and mannose) High-purity BCG-CWS having properties such as a content of 0.1% or less (in terms of dehydrated product) is obtained.
More specifically, according to the production method of the present invention, in addition to the above, the content of endotoxin (lipopolysaccharide; LPS) is 0.0015 EU / mg or less (the endotoxin standard product described in the 15th revision Japanese Pharmacopoeia) When used), trehalose dimycolate (TDM) content is less than 0.05% (w / w), nucleic acid content is less than 0.05% (w / w) (λ-Hind III digest is standard) High-purity BCG-CWS having properties such as

  The BCG-CWS of the present invention has an immunostimulatory action and is used as an immunotherapeutic agent in cancer and the like. That is, the BCG-CWS of the present invention is dispersed in an oil component such as squalane, squalene or mineral oil (Drakeol, Moresco Biores, etc.), and a stabilizer such as mannitol and a surfactant are added as appropriate to the water. By emulsifying, an oil-in-water emulsion preparation can be obtained. By freeze-drying the oil-in-water emulsion preparation, a freeze-dried preparation that can be stored for a long period of time can be obtained. For the preparations containing these BCG-CWS, International Publication Pamphlet Nos. 00/03724, 2004/012751 or 2005/102369 can be referred to.

  The present invention will be specifically described in the following examples, but the present invention is not limited thereto.

Production of BCG-CWS BCG fungus: M. bovis BCG Tokyo 172 strain (ATCC 35737) was cultured as a fungal membrane on the surface of the medium at 37 ° C. in a Sautton medium until the initial stationary phase. Cultured cells were inactivated by heating to 80 ° C. for 30 minutes and centrifuged. BCG-CWS was prepared using BCG killed bacteria obtained as a precipitate as a raw material.
About 600 g of dead BCG (wet) was suspended in 2 L of water and crushed at 35 kpsi using Mini DeBEE (registered trademark) (manufactured by BEE International). The crushed cells were centrifuged at 6760 × g for 10 minutes at 25 ° C. to remove large cell debris and unbroken cells.
The supernatant was then centrifuged at 18000 × g for 1 hour at 25 ° C. to obtain a pellet (the pellet is referred to as WCW). The yield of WCW obtained from 600 g of dead BCG (wet) was about 190 g. Next, Benzonase (Benzonase; manufactured by Merck Ltd.) was added to WCW and incubated at 25 ° C. for 17 hours to decompose the nucleic acid. The obtained WCW was washed 5 times with 1% Triton X-100 by centrifugation, and then pronase (Pronase; manufactured by Sigma-Aldrich Co.) was added and treated at 37 ° C. for 17 hours to decompose the protein. Cell walls were collected by centrifugation at 18000 × g for 20 minutes at 25 ° C., resuspended in 1% Triton X-100, and gently stirred at 60 ° C. for 2 hours to remove protein degradation products. Cell walls were collected and washed with ethanol.
The free fat component was removed by extraction with tetrahydrofuran, chloroform-methanol (2: 1), and then methanol, and then the residue was dried to obtain BCG-CWS. The yield of dry BCG-CWS was about 10 g.

Results of BCG-CWS analysis The results of analysis of BCG-CWS obtained in Example 1 are shown below. The BCG-CWS obtained in Example 1 has a content of non-constitutive amino acids (specifically, asparagine, valine, arginine, cysteine, glycine, threonine, isoleucine, leucine, phenylalanine, serine, etc.) of 1.3. And the content of non-constitutive neutral sugars (glucose and mannose) was 1.2%, which was highly pure.

  BCG-CWS produced by the production method of the present invention is useful as a drug substance for cancer immunotherapy.

Claims (1)

A method for producing high-purity BCG-CWS, comprising the following steps (1) to (6):
(1) A process of suspending and killing dead bacteria of BCG Tokyo strain in water and collecting cell walls (WCW) by centrifugation;
(2) Incubating the cell wall (WCW) obtained in (1) with a recombinant endonuclease derived from Serratia marcescens (Enzyme Commission Number 3.1.30.2),
(3) treating with nonspecific蛋White degradation enzyme derived from Streptomyces griseus the WCW obtained in (2) (Enzyme Commission Number 2329666 ),
(4) A step of treating the WCW obtained in (3) with a nonionic surfactant polyoxyethylene (10) octylphenyl ether under heating at 55 ° C. to 65 ° C. and washing with ethanol,
(5) A step of washing WCW obtained in (4) with tetrahydrofuran, chloroform-methanol (2: 1), and then methanol,
(6) A step of drying the WCW obtained in (5),
As impurities remaining in high purity BCG-CWS,
a) the content of non-constituent amino acids is 1.3% (w / w);
b) The content of non-constitutive neutral sugar is 1.2% (w / w),
c) The content of bacterial endotoxin (liposaccharide; LPS) is 0.0015 EU / mg or less,
d) the content of trehalose dimycolate (TDM) is less than 0.05% (w / w),
e) a nucleic acid content of less than 0.05% (w / w),
A method for producing high-purity BCG-CWS.