Method for producing lipopolysaccharide

Abstract

The purpose of the present invention is to provide: a lipopolysaccharide production method that is suitable for mass production and that contributes to environmental conservation and occupational health; and a lipopolysaccharide produced by the method. This lipopolysaccharide production method is for extracting and purifying a lipopolysaccharide from gram-negative bacteria, and comprises: a first step for obtaining an extraction solution containing the lipopolysaccharide by performing extraction on the gram-negative bacteria using hot water; and a second step for obtaining the lipopolysaccharide by refining, using reverse-phase liquid chromatography, the extraction solution or a solution containing LPS in the extraction solution, wherein a reversed-phase column used in the reverse-phase liquid chromatography includes a filler formed of a material that has a functional group having 1-8 carbon atoms.

Description

[Technical field]

[0001] The present invention relates to a method for producing lipopolysaccharide. [Background technology]

[0002] Lipopolysaccharide (hereinafter, also referred to as LPS) is a complex compound consisting of lipids and sugars that is present in the outer membrane surrounding the peptidoglycan in the cell walls of gram-negative bacteria such as Escherichia coli, Salmonella, and Bordetella pertussis, and is known as the active component of endotoxin (Non-patent Document 1). The basic structure of LPS is composed of three components: lipid A, which has a specific lipid, an oligosaccharide called R-core covalently bound to lipid A, and an O-specific polysaccharide (Non-Patent Document 2). It is known that the structure of LPS differs depending on the type of bacteria from which it is derived.

[0003] LPS is known as an endotoxin, and is known to activate immune system cells and increase inflammatory cytokines. Recently, however, the function of LPS to activate immune system cells has attracted attention. As a result, research into natural immunity and host defense functions using LPS has become more active. Among these, LPS derived from Pantoea bacteria is a particularly useful LPS, as it has been widely consumed by humans and has been reported to have potential applications in health foods, vaccine adjuvants, and medicines.

[0004] Thus, although the demand for LPS is increasing, the production of LPS requires the use of large amounts of organic solvents such as trichloroacetic acid, phenol, chloroform, toluene, alkanes, and petroleum ether as extraction solvents in the extraction process, which is harmful to the health of production workers and has an adverse effect on the natural environment due to their emissions.In addition, the introduction of special equipment that can handle the above organic solvents is necessary, which increases the production costs, etc., which is also a problem. For example, a known method for producing LPS is the Galanos method, which involves extraction of LPS with a mixture of phenol, chloroform and petroleum ether (PCP), followed by evaporation of the chloroform and petroleum ether, precipitation of LPS by addition of acetone and water, and recovery of LPS by centrifugation or filtration (Non-Patent Document 3). In the TSANG method, LPS is obtained by extraction with an aqueous phenol solution after extraction with an aqueous trichloroacetic acid solution (Non-Patent Document 4). In addition, the Chen method involves LPS extraction with a mixture of chloroform and methanol (CM), followed by a series of methanol precipitation steps (Non-Patent Document 5).

[0005] The Galanos and Sun methods use a mixture of solvents (phenol, chloroform, petroleum ether, trichloroacetic acid, etc.) in the extraction process, which poses environmental, health and safety concerns, making them unsuitable for large-scale production and unsuitable for commercial production. The Chen method also uses chloroform in the extraction process, which poses environmental, health and safety concerns, and the Chen method produces a CM phase rich in LPS-phospholipids, which requires multiple precipitation steps to obtain LPS of sufficient purity, which increases production time and costs.

[0006] Furthermore, for example, Patent Document 1 discloses that in the process of extracting LPS from Gram-negative bacteria, an alcohol, an organic solvent and water are used instead of the solvent mixture of phenol, chloroform and petroleum ether (PCP) used in the Galanos method. However, it is disclosed that the organic solvent used can be selected from the group consisting of chloroform, alkanes, toluene and petroleum ether. This production method also has environmental, health and safety concerns and is not suitable for large-scale production.

[0007] Thus, conventional LPS manufacturing methods include manufacturing processes that place a heavy burden on the environment and occupational health. In other words, conventional LPS manufacturing methods pose risks to environmental conservation in addition to the risks of occupational health management in terms of work environment management, work management, and health management. Furthermore, LPS manufacturing facilities require equipment to counter the above risks, which increases manufacturing costs.

[0008] Among LPS, Pantoea (hereinafter also referred to as Pantoea bacteria)-derived LPS is useful for health foods, vaccine adjuvants, and pharmaceuticals, and its usefulness has been reported in various ways as described above, and demand is expected to increase. Therefore, there has been a particular demand for the development of a production method suitable for large-scale production that does not use organic solvents in the extraction process. However, the only method reported for producing Pantoea bacteria-derived LPS is a method that combines extraction with hot phenol and purification with an anion exchange column (Non-Patent Document 6), and since this method also uses phenol, there are health and safety concerns for large-scale production.

[0009] As described above, there is a need for a method for producing LPS suitable for large-scale production, i.e., a method that places less strain on occupational health and the environment, particularly in the production of Pantoea LPS.

[0010] As for the method of extracting LPS, Patent Document 2 discloses an example of a method of extracting LPS without using harmful organic solvents, in which hot water extraction is performed on acetic acid bacteria, gluconic acid bacteria, Xanthomonas bacteria, Zymomonas bacteria, or Enterobacter bacteria. However, the lipopolysaccharide (LPS) obtained by this production method contains many impurities other than LPS, and it is merely an extraction procedure to obtain an extract with low LPS purity, and cannot be said to be a practical method of producing LPS. Patent Document 2 also discloses a separate method of producing LPS, but this is a method that combines hot phenol extraction and nuclease treatment. In other words, Patent Document 2 does not disclose or suggest a method of producing LPS without using harmful organic solvents.

[0011] In addition, it has been reported that an extracted and purified LPS product is usually a mixture of low and high molecular weight components, and that an increase in the proportion of low molecular weight LPS increases safety and improves physiological activity (cytokine induction ability) (Patent Document 3, Non-Patent Document 7). Therefore, there has been a demand for an LPS that contains more low molecular weight LPS than LPS produced by conventional methods, and a method for producing the same. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] JP 2016-174601 A [Patent Document 2] International Publication No. 2007 / 061102 [Patent Document 3] Patent No. 4043533 [Non-patent literature]

[0013] [Non-Patent Document 1] J.M. Guysen and R. Hakenbeck, eds., New Comprehensive Biochemistry, Vol. 27, Bacterial Cell Wall, p. 18, Elsevea, 1994. [Non-Patent Document 2] "Nikkei Biotechnology Latest Glossary", page 431, Nikkei McGraw-Hill, 1985 [Non-Patent Document 3] Galanos et al.,Eur.J.Biochem.9:245(1969) [Non-Patent Document 4] JCTSANG et al.,JOURNAL OF BACTERIOLOGY,Feb.1974,p.786-795 [Non-Patent Document 5] Chen et al. Journal of Infectious Desieses.128s,43,1973 [Non-Patent Document 6] ANTICANCER RESEARCH27:3701-3706(2007) [Non-Patent Document 7] Biotherapy 10(3):519-521,March,1996 Summary of the Invention [Problem to be solved by the invention]

[0014] In view of the above-mentioned current situation, an object of the present invention is to provide a method for producing lipopolysaccharide which contributes to occupational hygiene and environmental protection and is suitable for large-scale production, and lipopolysaccharide produced thereby. [Means for solving the problem]

[0015] In order to achieve the above-mentioned object, we have intensively investigated a method for extracting and purifying LPS that can be used in large-scale production without using organic solvents that have a high environmental impact. As a result, we have found that by combining a first step of extracting Gram-negative bacteria with hot water and a second step of purifying the extract obtained in the first step or the solution containing LPS in the extract using reversed-phase liquid chromatography equipped with a reversed-phase column having a specific packing material, we can obtain LPS in a yield that can be used in large-scale production without using organic solvents that have a high environmental impact. Furthermore, we have found that the LPS obtained by this production method is highly pure and contains a high content of low-molecular-weight lipopolysaccharides.

[0016] That is, the present invention is a method for producing lipopolysaccharide by extracting and purifying lipopolysaccharide from gram-negative bacteria, comprising a first step of obtaining an extract containing the lipopolysaccharide by extracting the gram-negative bacteria with hot water, and a second step of obtaining lipopolysaccharide by purifying the extract or a solution containing LPS in the extract using reversed-phase liquid chromatography, wherein the reversed-phase column in the reversed-phase liquid chromatography has a packing material composed of a material having a functional group with 1 to 8 carbon atoms. The above-mentioned filler is preferably composed of a material having a functional group having 2 to 6 carbon atoms. The above-mentioned filler is preferably composed of a material having a functional group having 2 to 4 carbon atoms. The filler is preferably composed of a material having a functional group with four carbon atoms. The functional group is preferably an alkyl group. The temperature of the hot water in the first step is preferably 50 to 150°C. The temperature of the hot water in the first step is preferably 50 to 99°C. The temperature of the hot water in the first step is preferably 70 to 99°C. The temperature of the hot water in the first step is preferably 85 to 95°C. The gram-negative bacterium is preferably at least one species selected from the group consisting of the genera Escherichia, Salmonella, Pantoea, Acetobacter, Zymomonas, Xanthomonas, Enterobacter, Roseomonas and Rhodobactor. The Gram-negative bacterium is preferably of the genus Pantoea.

[0017] The present invention also relates to a lipopolysaccharide produced by the production method of the present invention. Moreover, the lipopolysaccharide of the present invention is preferably obtained from the above-mentioned gram-negative bacteria. Furthermore, the lipopolysaccharide of the present invention comprises a low molecular weight lipopolysaccharide having a molecular weight of 2000 to 20,000 as measured by the SDS-PAGE method, and a high molecular weight lipopolysaccharide having a molecular weight of more than 20,000 and not more than 100,000 as measured by the SDS-PAGE method, and it is preferable that the content of the low molecular weight lipopolysaccharide is 80% or more relative to the total amount of the low molecular weight lipopolysaccharide and the high molecular weight lipopolysaccharide. In addition, the lipopolysaccharide of the present invention can be used in an ELISA method. It reacts with the primary antibody 4E-11, and The value (E / L ratio) obtained by dividing the lipopolysaccharide quantitative value (E) by the lipopolysaccharide quantitative value (L) obtained by the Limulus test (endpoint colorimetric method) is preferably 1.0 or less. In addition, in the lipopolysaccharide of the present invention, the gram-negative bacterium is preferably the genus Pantoea. The present invention will now be described in detail. In the following description, percentages are by weight unless otherwise specified. Effect of the Invention

[0018] The method for producing lipopolysaccharide of the present invention contributes to occupational hygiene and environmental protection, and provides a method for producing lipopolysaccharide suitable for large-scale production. Specifically, the contribution to the above-mentioned occupational hygiene and environmental protection means that lipopolysaccharide can be extracted without using organic solvents that have a high environmental impact, thereby improving environmental, health, and safety risks. In addition, in the production of lipopolysaccharide, the risks of environmental protection can be reduced in addition to the risks of occupational hygiene management in the work environment management, work management, and health management, so that the above-mentioned risk control equipment can be reduced in the LPS production facility, and the overall production cost can be reduced. Furthermore, according to the production method of the present invention, lipopolysaccharide having high purity and a high content of low-molecular-weight lipopolysaccharide can be obtained, making it possible to provide lipopolysaccharide that is highly safe and physiologically active. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is an SDS-PAGE (silver staining) electrophoretic diagram. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In this specification, the above-mentioned solvent with a high environmental impact refers to an organic solvent used in the first step of obtaining an extract containing LPS, and further refers to a substance that is designated as a Class 1 designated chemical substance under the Act on Reporting, etc. of Releases of Chemical Substances to the Environment and Promoting Improvements in Their Management and that can be used as an organic solvent in all manufacturing processes except for the first step.

[0021] In the present specification, high-purity LPS means LPS in which LPS is the main component compared to proteins and nucleic acids, that is, LPS in which the LPS content (the percentage remaining after subtracting the protein and nucleic acid contents (weight) from the weight of the obtained LPS) is 60% by weight or more. In the present specification, "purity" means the purity of LPS and is expressed in weight %. The purity of LPS is preferably 70% by weight or more, more preferably 80% by weight or more, and even more preferably 90% by weight or more.

[0022] In addition, in this specification, low molecular weight lipopolysaccharide (hereinafter also referred to as low molecular weight LPS) is LPS whose molecular weight is determined by separating its constituent components by SDS-PAGE (silver staining method) and using the mobility of a protein size marker as an indicator, and high molecular weight lipopolysaccharide (hereinafter also referred to as high molecular weight LPS) is LPS whose molecular weight is determined by the above measurement, and is greater than 20,000 and less than 100,000. Furthermore, LPS containing a high content of low molecular weight lipopolysaccharide (low molecular weight LPS) means anything from one that is substantially free of high molecular weight lipopolysaccharide (high molecular weight LPS) to one that contains high molecular weight LPS, but in which the low molecular weight LPS content is 80% or more of the total of high molecular weight LPS and low molecular weight LPS. LPS that contains 80% or more of low molecular weight LPS of the total of high molecular weight LPS and low molecular weight LPS has a higher low molecular weight LPS content than LPS purified by conventional methods. More specifically, when LPS is separated into its constituent components by SDS-PAGE (silver staining method), the molecular weight is determined using the mobility of a protein size marker as an indicator, and the content of the constituent components is determined by the total image brightness of the SDS-PAGE image (after silver staining), for LPS containing low molecular weight lipopolysaccharides with a molecular weight of 2000 to 20,000 and high molecular weight lipopolysaccharides with a molecular weight of more than 20,000 and not more than 100,000, the content of the low molecular weight lipopolysaccharides relative to the total amount of the low molecular weight lipopolysaccharides and the high molecular weight lipopolysaccharides is 80% or more by the silver staining method of SDS-PAGE. In the following description, LPS with a low molecular weight LPS content of 80% or more is referred to as LPS rich in low molecular weight LPS.

[0023] In this specification, the yield of lipopolysaccharide applicable to large-scale production means that the yield of lipopolysaccharide produced from 1 kg of bacterial cell pellets is 4 g or more. The yield of lipopolysaccharide produced from 1 kg of bacterial cell pellets can be measured by HPLC.

[0024] In this specification, "A to B" means "A or more and B or less."

[0025] One embodiment of the present invention relates to a method for producing lipopolysaccharide, which comprises extracting and purifying lipopolysaccharide from gram-negative bacteria, and includes a first step of obtaining an extract containing the lipopolysaccharide by extracting the gram-negative bacteria with hot water, and a second step of obtaining lipopolysaccharide by purifying the extract or a solution containing LPS in the extract using reversed-phase liquid chromatography, wherein the reversed-phase column in the reversed-phase liquid chromatography has a packing material composed of a material having a functional group having 1 to 8 carbon atoms.

[0026] Generally, LPS may be an extract from the cell wall of a gram-negative bacterium or a modified product thereof, or may be a synthetic product. However, the LPS obtained by the method for producing LPS of the present invention is a purified extract from the cell wall of a gram-negative bacterium.

[0027] The above-mentioned gram-negative bacteria include, for example, Escherichia, Shigella, Salmonella, Yersinia, Viblio, Haemophilus, Pseudomonas, Legionella, Bordetella, Brucella, Francisella, Bacteroides, Neisseria, Chlamydia, Plesiomonas, Prophyromonas, Pantoea, Agrobacterium, Stenortophomonas, Serratia, Leclercia, Rahnella, Acidicaldus, Acidiphilium, Acidisphaera, Acidocella, Acidomonas, Asaia, Belnapia, Craurococcus , Gluconacetobacter, Gluconobacter, Kozakia, Leahibacter, Muricoccus, Neoasaia, Oleomonas, Paracraurococcus, Rhodopila, Roseococcus, Rubritepida, Saccharibacter, Stella, Swaminathania, Teichococcus, Zavarzinia, Achromobacter, Flavobacterium, Enterobacter, Acetobacter, Xanthomonas, Zymomonas, Roseomonas, Rhodobactor, Proteus, Klebsiella, Citrobacter, Acinetobacter, etc.

[0028] Among them, the Gram-negative bacteria is preferably at least one selected from the group consisting of Escherichia, Salmonella, Pantoea, Acetobacter, Zymomonas, Xanthomonas, Enterobacter, Roseomonas and Rhodobactor. These are contained in many foods and herbal medicines since ancient times, and their safety to the living body is guaranteed. In particular, Pantoea is currently used as a health food, and LPS extracted and purified from the Pantoea genus is said to be safer and more effective for its use. The Gram-negative bacteria is more preferably Pantoea. The production method of the present invention can provide a production method that contributes to occupational health and environmental conservation in the production of Pantoea-derived LPS, the demand of which is expected to increase, and a Pantoea-derived LPS with high purity and a high content of low molecular weight LPS.

[0029] In the first step, the gram-negative bacteria are extracted with hot water, thereby denaturing and removing the cell walls of the gram-negative bacteria, extracting LPS from the cell walls and proteins and nucleic acids within the cells of the gram-negative bacteria, and obtaining an extract containing LPS.

[0030] The temperature of the hot water in the first step is preferably 50 to 150° C. If the temperature of the hot water is higher than 150° C., the high temperature may cause thermal denaturation of the extracted LPS, whereas if the temperature is lower than 50° C., the cell walls of the gram-negative bacteria may not be denatured, and LPS may not be sufficiently extracted. The temperature of the hot water is more preferably 50 to 130° C., even more preferably 50 to 99° C., even more preferably 70 to 99° C., and most preferably 85 to 95° C. This is because LPS can be extracted efficiently and also because extraction at normal pressure is possible.

[0031] The time for extraction with hot water is preferably 10 to 120 minutes. If the extraction time exceeds 120 minutes, the LPS may be thermally decomposed, and if it is less than 10 minutes, the LPS may not be sufficiently extracted.

[0032] The temperature and time of the hot water extraction in the first step can be appropriately selected from the above-mentioned hot water temperatures and extraction times so that the optimum conditions depend on the type of Gram-negative bacteria used. When the gram-negative bacterium is of the genus Pantoea, the hot water temperature and extraction time for LPS extraction can be appropriately selected within the above-mentioned ranges, but hot water extraction is preferably performed at 70 to 99°C for 10 to 120 minutes, and more preferably at 85 to 95°C for 20 to 30 minutes.

[0033] The hot water extraction in the first step may be performed only once, or may be performed multiple times at the same or different temperatures. The water used in the hot water extraction is not particularly limited, and water commonly used in this field may be used.

[0034] In addition, the hot water extraction can be carried out using a method and apparatus commonly used in this technical field, and can be carried out under normal pressure, reduced pressure or increased pressure. In addition, a surfactant, a chelating agent, an organic acid salt, an inorganic salt, etc. may be added to the water used in the hot water extraction.

[0035] In the method for producing LPS according to one embodiment of the present invention, the first step and the second step may be carried out consecutively, or another step may be included between the first step and the second step. When the first step and the second step are carried out consecutively, the extract obtained in the first step is purified in the second step. When another step is included between the first step and the second step, the solution containing LPS in the extract obtained in the first step is purified in the second step.

[0036] Examples of other steps carried out between the first and second steps include ultrafiltration, enzyme treatment, ethanol precipitation, dialysis, solid-phase extraction, etc., and it is preferable to carry out ultrafiltration on the extract obtained in the first step. By carrying out ultrafiltration, the LPS in the extract is concentrated and at the same time, low-molecular-weight impurities (those that pass through an ultrafiltration membrane of 200,000 Daltons) are removed, which allows efficient purification using reverse-phase liquid chromatography in the second step. It is preferable that the extract obtained in the first step is ultrafiltered once or multiple times. Since LPS forms micelles and its apparent molecular weight increases, its molecular weight does not directly correspond to the molecular weight of the cutoff value of the ultrafiltration membrane.

[0037] In the production method of the present invention, when ultrafiltration is carried out between the first step and the second step, the ultrafiltration membrane used in the ultrafiltration has a cut-off molecular weight of 5,000 to 200,000 Daltons, preferably 8,000 to 100,000 Daltons, and more preferably 10,000 to 50,000 Daltons. The molecular weight cutoff is a term that indicates membrane properties defined in JIS K 3802 (membrane terminology).

[0038] The reversed-phase column of the reversed-phase liquid chromatography used in the second step has a packing material composed of a material having a functional group with 1 to 8 carbon atoms. By using a reversed-phase column having a packing material composed of a material having a functional group with a specific number of carbon atoms, impurities other than LPS remaining in the extract obtained in the first step or in the solution containing LPS in the extract can be removed, and LPS with a higher purity can be purified. In addition, LPS with a high content of low-molecular-weight LPS can be purified.

[0039] The filler is preferably composed of a material having a functional group having 2 to 6 carbon atoms, more preferably composed of a material having a functional group having 2 to 4 carbon atoms, and even more preferably composed of a material having a functional group having 4 carbon atoms.

[0040] The functional group is preferably an alkyl group. This is because impurities other than LPS remaining in the extract obtained in the first step can be removed, and LPS with a higher purity can be purified. In addition, this is because LPS can be sufficiently purified while suppressing the cost of the equipment used in the production method of the present invention. The packing material is preferably composed of a material having an alkyl group with 1 to 8 carbon atoms, more preferably composed of a material having an alkyl group with 2 to 6 carbon atoms, even more preferably composed of a material having an alkyl group with 2 to 4 carbon atoms, and most preferably composed of a material having an alkyl group with 4 carbon atoms. In other words, the packing material is most preferably composed of a material having a butyl group.

[0041] The material (base material) constituting the packing material is preferably silica gel. This is because impurities other than LPS remaining in the extract obtained in the first step can be removed to purify LPS with a higher purity. In addition, this is because LPS can be sufficiently purified while suppressing the cost of the equipment used in the production method of the present invention. The packing material is preferably silica gel having a functional group with 1 to 8 carbon atoms, more preferably silica gel having a functional group with 2 to 6 carbon atoms, even more preferably silica gel having a functional group with 2 to 4 carbon atoms, and most preferably silica gel having a functional group with 4 carbon atoms. The material (base material) constituting the packing material is preferably silica gel having an alkyl group. This is because impurities other than LPS remaining in the extract obtained in the first step can be removed, and LPS with a higher purity can be purified. In addition, this is because LPS can be sufficiently purified while suppressing the cost of the equipment used in the production method of the present invention. The packing material is preferably silica gel having an alkyl group with 1 to 8 carbon atoms, more preferably silica gel having an alkyl group with 2 to 6 carbon atoms, even more preferably silica gel having an alkyl group with 2 to 4 carbon atoms, and most preferably silica gel having an alkyl group with 4 carbon atoms. In other words, the packing material is most preferably silica gel having a butyl group.

[0042] The packing material preferably has an alkyl functional group and is made of silica gel, and the above-mentioned configurations may be appropriately combined.

[0043] The second step may also include a pretreatment of the extract obtained in the first step or a solution containing LPS in the extract, which is to be treated by reversed-phase liquid chromatography. The pretreatment may be an operation that is commonly used in this field and is suitable for separation by reversed-phase liquid chromatography. Specifically, a surfactant may be added to the extract obtained in the first step or a solution containing LPS in the extract, to solubilize LPS, which may then be purified by reversed-phase liquid chromatography. This is because the efficiency of separation by reversed-phase liquid chromatography can be improved. The second step preferably includes the above-mentioned solubilization treatment as a pretreatment for reversed-phase liquid chromatography.

[0044] The surfactant is preferably an ionic surfactant, since it has a high solubilizing effect on LPS. Examples of ionic surfactants include, but are not limited to, alkyl ammonium salts, bile acids, fusidic acid, amino acids, oligopeptide or polypeptide fatty acid conjugates, amino acids, glyceride esters of oligopeptides, glyceride esters of polypeptides, acyl lactylates, monoacetylated tartaric acid esters of monoglycerides, diacetylated tartaric acid esters of monoglycerides, monoacetylated tartaric acid esters of diglycerides, diacetylated tartaric acid esters of diglycerides, succinylated monoglycerides, citric acid esters of monoglycerides, citric acid esters of diglycerides, alginic acid, propylene glycol alginic acid esters, lecithin, hydrogenated lecithin, lysolecithin, hydrogenated lysolecithin, lysophospholipids, phospholipids, salts of alkyl sulfates, fatty acids, and pharmacologically acceptable salts thereof, but bile acids and / or their pharmacologically acceptable salts are preferred, since they have a particularly high solubilizing effect on LPS.

[0045] In addition, examples of the bile acids and pharmacologically acceptable salts thereof include chenodeoxycholic acid (CDCA), ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidic acid, glycodihydrofusidic acid, and pharmacologically acceptable salts thereof (e.g., sodium salts).

[0046] Among them, the surfactant is preferably deoxycholic acid and / or a pharmacologically acceptable salt thereof. The surfactant is also preferably deoxycholic acid and / or a salt thereof, since this has a particularly high solubilizing effect on LPS.

[0047] In the second step of the method for producing LPS of the present invention, the extract after the first step or the solution containing LPS in the extract may be subjected to various pretreatments, such as concentration adjustment, pH adjustment, ionic strength adjustment, addition of organic solvent, filtration, dialysis, etc., which are generally performed on sample solutions for reversed-phase liquid chromatography, in addition to the above-mentioned solubilization treatment, in order to improve the efficiency of separation by reversed-phase liquid chromatography.

[0048] The second step may also include a post-treatment of the separation liquid containing LPS obtained by using reversed-phase liquid chromatography. As the post-treatment, an operation suitable for the separation liquid of reversed-phase liquid chromatography can be performed, and as the operation suitable for the separation liquid, a suitable combination of separation liquid treatments usually used in the technical field to which the present invention belongs can be performed. Specifically, for example, methods utilizing differences in solubility such as distillation, salting out, and solvent precipitation; methods utilizing differences in molecular weight such as dialysis, ultrafiltration, gel filtration, PAGE, and SDS-PAGE; and methods utilizing differences in isoelectric point such as isoelectric focusing can be mentioned. Among them, distillation and / or ultrafiltration are preferably performed as the post-treatment, and distillation and ultrafiltration are more preferably performed. This is because impurities contained in the mobile phase of the reversed-phase liquid chromatography in the second step can be removed.

[0049] Furthermore, the method for producing LPS according to one embodiment of the present invention may include a step of concentrating or washing the LPS purified in the above-mentioned second step. The concentrating and washing steps can be carried out by appropriately combining treatments that are commonly used in the technical field to which the present invention pertains.

[0050] Furthermore, the method for producing LPS in one embodiment of the present invention may include a step of drying the LPS purified in the second step (drying step). The LPS purified in the second step often contains solvents such as volatile organic solvents and water contained in the mobile phase of the reversed-phase liquid chromatography in the second step. Therefore, the drying step preferably includes a step of removing the volatile organic solvent contained in the mobile phase of the reversed-phase liquid chromatography in the second step by dry distillation (evaporation) (organic solvent removing step) and / or a step of completely removing solvents such as water by freeze-drying (solvent removing step). By including such a drying step, the handleability of LPS can be improved.

[0051] The above-mentioned method for producing LPS of the present invention can be suitably used for large-scale production. The yield of LPS obtained by the method for producing LPS of the present invention is preferably 4 g or more of lipopolysaccharide produced from 1 kg of gram-negative bacterial cell pellets. The LPS obtained by the method for producing LPS of the present invention can be quantified by HPLC.

[0052] In another embodiment, the present invention relates to a lipopolysaccharide produced by the above-mentioned production method of the present invention. The lipopolysaccharide produced by the production method of the present invention is a high-purity LPS having a purity of 60% by weight or more, preferably 70% by weight or more, more preferably 80% by weight or more, and even more preferably 90% by weight or more.

[0053] The lipopolysaccharide according to another embodiment of the present invention is preferably produced from the above-mentioned gram-negative bacteria. The above-mentioned gram-negative bacteria is more preferably at least one selected from the group consisting of Escherichia, Salmonella, Pantoea, Acetobacter, Zymomonas, Xanthomonas, Enterobacter, Roseomonas and Rhodobactor, and more preferably Pantoea. These bacteria have been contained in many foods and herbal medicines since ancient times, and their safety to the living body is guaranteed. In particular, Pantoea is currently used as a health food, and LPS extracted and purified from the Pantoea genus is said to be safer and more effective for such use.

[0054] Another embodiment of the present invention, the lipopolysaccharide, comprises a low molecular weight lipopolysaccharide having a molecular weight of 2000-20000 as measured by SDS-PAGE method, and a high molecular weight lipopolysaccharide having a molecular weight of more than 20000 and not more than 100000 as measured by SDS-PAGE method, and the content of the low molecular weight lipopolysaccharide is preferably 80% or more relative to the total amount of the low molecular weight lipopolysaccharide and the high molecular weight lipopolysaccharide. LPS with a low molecular weight LPS content of 80% or more is extremely safe and has excellent biological activity, and is particularly useful.

[0055] In the present invention, the molecular weight of LPS is a value obtained by dividing the components by SDS-PAGE (silver staining) and determining the molecular weight using the mobility of a protein size marker as an index. The content ratio of low molecular weight LPS to high molecular weight LPS is a value obtained by determining the content of the components from the total image brightness of the SDS-PAGE image (after silver staining).

[0056] In another embodiment of the present invention, the lipopolysaccharide has a lipopolysaccharide quantitative value (E) by ELISA divided by a lipopolysaccharide quantitative value (L) by Limulus test (endpoint colorimetric method) (E / L ratio) of 1.0 or less. The E / L ratio is a value greater than 0.

[0057] The lipopolysaccharide quantitative value (E) by the ELISA method and the lipopolysaccharide quantitative value (L) by the Limulus test (endpoint colorimetric method) are measured, for example, by the following method.

[0058] (Quantitative method for LPS using ELISA) The quantification of LPS by ELISA is carried out, for example, by measuring the LPS content (E) (μg / mg) in a sample by ELISA using a glycan-specific antibody of the LPS to be measured (e.g., IP-PA1 glycan-specific antibody, manufactured by Natural Immunity Applied Technology Research Institute, Inc.). Specifically, ELISA measurement can be performed according to the following procedure.

[0059] 1) 34-G2 (immobilized antibody: Natural Immunity Applied Technology Research Institute, Lot. 201107-7, antibody against O antigen polysaccharide of IP-PA1) is diluted 800-fold with PBS(-) that has been sterilized by filtration at the time of use. Dispense 50 μL into a 96-well immunoplate, seal with parafilm to prevent evaporation, and store at 4°C. 2) Remove the 96-well immunoplate containing 34-G2 from 4°C, discard the liquid, and then add 200 μL / well of blocking solution (PBS(-) to which BSA has been added to make a final concentration of 3% (w / v)). Leave at 25°C for at least 30 minutes to prevent nonspecific adsorption of antibodies and other proteins. 3) After washing three times with 200 μL of washing solution (equivalent to 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% (v / v) Tween 20), add 50 μL of serially diluted LPS standard solution (LPS standard substance (e.g., Pantoea agglomerans-derived LPS standard, purity 95% or higher, manufactured by Natural Immunity Applied Technology Research Co., Ltd.)) to the wells. Add 50 μL of the sample solution to be measured to another well of the same 96-well immunoplate. Leave in a constant temperature bath at 25°C for 1 hour. 4) Wash the wells three times with 200 μL of washing solution, add 50 μL / well of 1000-fold diluted 4E-11 (primary antibody: Natural Immunity Applied Technology Research Institute, Lot. 20170702, antibody against O-antigen polysaccharide of IP-PA1) and leave in a 25°C incubator for 1 hour. 5) Wash the wells three times with 200 μL of washing solution, add 50 μL / well of 1000-fold diluted alkaline phosphatase-conjugated anti-mouse IgG immunoglobulin antibody (Sigma, Cat. 1418), and leave in a 25° C. incubator for 1 hour. 6) Wash the wells five times with 200 μL of washing solution, add 100 μL / well of color developing solution (disodium p-nitrophenyl phosphate hexahydrate (p-NPP): Nacalai Tesque, Cat. 25019-52, Lot. MOP2259), and leave at room temperature for 1 hour. 7) Then, measure the absorbance at 415 nm using a microplate reader (Molecular Devices SpectraMax, Plus384). Create a calibration curve using a quadratic curve. 8) Using the calibration curve, calculate the LPS concentration (μg / mL) in the sample solution from the absorbance of the sample solution. Divide the LPS concentration in the sample solution by the sample concentration (mg / mL) in the sample solution (calculated from the sample weight and solution volume at the time of preparation) to obtain the LPS content (E) (μg / mg) in the sample. The detection limit of the ELISA method used in this measurement is 1.6 ng / mL.

[0060] (Quantitative method for LPS using the Limulus test) The quantification of LPS by the Limulus test can be determined by measuring the LPS content (L) (μg / mg) in a sample using, for example, a Limulus measurement kit capable of measuring LPS specifically. Specifically, the Limulus test can be performed according to the following procedure.

[0061] (LPS quantification method using the Limulus test (endpoint colorimetric method)) Using a Limulus measurement kit capable of specifically measuring LPS (e.g., Endospecy ES-50M Set, manufactured by Seikagaku Corporation), the LPS content (L) (μg / mg) in the sample LPS to be measured is measured by endpoint colorimetric method using a microplate according to the attached instructions. For the measurement, CSE (10 ng / vial) (Seikagaku Corporation) is used as an endotoxin standard.

[0062] Based on the LPS contents (E and L) obtained by the above two methods, the ratio (E / L ratio) can be calculated. Here, first, the relationship between the structure and molecular weight of LPS is explained. LPS is structurally composed of O-antigen polysaccharide, core polysaccharide, and lipid A portion. Of these, the molecular weight of the lipid A portion in one LPS molecule does not differ greatly between LPS types. The molecular weight of the core polysaccharide portion in one LPS molecule also does not differ greatly between LPS types. On the other hand, the O-antigen polysaccharide portion in the LPS molecule has a repeating structure, and the degree of repeating varies greatly depending on the LPS type. As a result, the molecular weight of the O-antigen polysaccharide portion differs depending on the type of LPS, and the molecular weight of LPS differs. If the number of repeats of the O-antigen polysaccharide portion is high, the molecular weight of LPS is high, and if the number of repeats of the O-antigen polysaccharide portion is low, the molecular weight of LPS is low. In addition, LPS extracted from gram-negative bacteria is usually a mixture of LPS with different molecular weights. Next, the relationship between the structure of LPS and the LPS content (E and L) is explained. The amount of LPS (E) measured by ELISA is the amount of LPS calculated based on the amount of antibodies that recognize and bind to the O-antigen polysaccharide in the LPS structure. If the number of repeating O-antigen polysaccharides in the LPS structure is small or there is no O-antigen polysaccharide (i.e., if the molecular weight of LPS is small), there will not be a sufficient size of antibody binding site in the LPS molecule, and the antibody will not be able to bind to the LPS, resulting in a low measured value of the amount of LPS measured by ELISA. On the other hand, the amount of LPS (L) measured by the Limulus test is the amount of LPS calculated based on the reaction activity of the lipid A structure in the LPS structure, so that a measured value corresponding to the number of moles of LPS in the measurement sample can be obtained, regardless of the proportion or presence of O-antigen polysaccharide in the LPS structure. Therefore, when a fixed mass of LPS sample (a mixture of LPS with different molecular weights) is measured using the ELISA and endotoxin methods to determine the LPS content (E and L), a large E / L ratio means that the measured sample contains a large amount of LPS with a large molecular weight, and a small E / L ratio means that the measured sample contains a large amount of LPS with a small molecular weight.

[0063] The LPS obtained by the production method of the present invention preferably has an E / L ratio greater than 0 and equal to or less than 1.0. Such an LPS will ultimately have a higher content of low molecular weight LPS than LPS (IP-PA1) produced by a conventional production method. In the production method of the present invention, reverse phase chromatography is used in the purification step 2, so it is highly likely that highly lipophilic LPS (LPS with fewer hydrophilic glycans and a higher proportion of lipophilic Lipid A moiety) is selectively recovered. That is, by including a purification step using reverse phase chromatography, it is possible to produce LPS with a high content of low molecular weight LPS. As a result, the LPS obtained is safe and has high biological activity (cytokine induction ability) due to its high content of low molecular weight LPS. EXAMPLES

[0064] The present invention will be described in more detail below with reference to examples and comparative examples, but the scope of the present invention is not limited to the examples.

[0065] <Preparation of Gram-negative bacteria (Pantoea) used in the Examples and Comparative Examples> (preculture) 100 mL of pre-culture medium (LB medium: 1% tryptone (Nacalai Tesque, special reagent for microbial culture), 0.5% yeast extract (Nacalai Tesque, for microbial culture), 1% sodium chloride (Fujifilm Wako Pure Chemical Industries, special grade)) was dispensed into 250 mL baffled flasks (10 flasks), and sterilized at 121 ° C for 15 minutes in an autoclave (HIRAYAMA HV-50). In a clean bench, Pantoea agglomerans (Natural Immunity Applied Technology Research Institute, Inc.) was inoculated from an agar medium aseptically into the flask, which was then set in a shaking incubator (Sanki Seiki, RMS-20R) and shake-cultured. The shaking culture conditions were 35 ° C, 150 rpm, and the culture was performed for 22 hours.

[0066] (Main culture) 10 L of the main culture medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 0.2% glycerin (FUJIFILM Wako Pure Chemical Industries, Ltd.), 0.04% citric acid monohydrate (FUJIFILM Wako Pure Chemical Industries, Ltd., special grade), 0.2% dipotassium hydrogen phosphate (FUJIFILM Wako Pure Chemical Industries, Ltd., special grade), 0.07% ammonium hydrogen phosphate tetrahydrate (FUJIFILM Wako Pure Chemical Industries, Ltd., first grade), 0.01% Antifoam 204 (Sigma)) was placed in a 20 L jar fermenter (Marubishi Bioengine Co., Ltd.) and sterilized by autoclaving at 121°C for 30 minutes.

[0067] To the main culture medium, 10% magnesium sulfate heptahydrate (FUJIFILM Wako Pure Chemical Industries, Ltd., special grade) was aseptically added to make the concentration 0.04%. Furthermore, 500 mL of the preculture solution was aseptically poured in and inoculated, and main culture was performed. The main culture conditions were culture temperature: 30°C, stirring speed: 300 rpm, aeration amount: 18-20 L / min, and pH: 6.8-7.2. The culture solution was collected 16 hours after the start of culture. The above-mentioned main culture operation on a 10 L medium scale was carried out in two sets in parallel, and the sets were combined, homogenized (mixed), and then subjected to the subsequent cell recovery operation.

[0068] (Bacteria collection) The homogenized main culture was transferred to 1 L centrifuge tubes (20 tubes) and centrifuged at 4°C, 8000 rpm, and 20 minutes (high-speed refrigerated centrifuge: KUBOTA 7780). The supernatant was removed and the bacterial pellet was collected as a precipitate (total amount of bacterial pellet: 260 g). The obtained bacterial pellet was frozen and stored at -30°C.

[0069] The Pantoea agglomerans cell pellet (frozen preserved product) obtained by the above method was subjected to extraction and purification of LPS by the method of the Examples or Comparative Examples.

[0070] (Example 1) (Hot water extraction (first step) + ultrafiltration + C4 reverse phase HPLC (second step)) [1]Hot water extraction 78 g of Pantoea agglomerans cell pellets obtained by the above-mentioned preparation of gram-negative bacteria (Pantoea bacteria) were added and suspended in 400 mL of water for injection (Otsuka Pharmaceutical Factory). The pH of the suspension was measured with a pH meter (LAQUA, Horiba, Ltd.) to find that it was 6.0, so sodium hydroxide (FUJIFILM Wako Pure Chemical Industries, Ltd.) (10N) was added to neutralize it. Then, the mixture was heated at 90°C for 20 minutes using an autoclave (HIRAYAMA HV-50). After cooling, the mixture was transferred to a 1 L centrifuge tube and centrifuged at 8000 rpm for 20 minutes, and the supernatant (hot water extract) was collected.

[0071] [2] Ultrafiltration treatment 15 mL of the above hot water extract was added to an ultrafiltration tube (Amicon Ultra Centrifugal Filter Units) with a molecular weight cutoff of 30 kda and centrifuged (7000 rpm x 30 min) to obtain a concentrate containing LPS on the non-permeated side of the ultrafiltration membrane. 15 mL of water for injection was added to the concentrate, and the concentrate was washed by similar centrifugation. The same washing was repeated five more times, and the concentrate after washing was collected and diluted with water for injection to obtain 15 mL of ultrafiltered suspension.

[0072] [3] Pretreatment for chromatographic purification (solubilization treatment) To 1 mL of the ultrafiltered suspension, 3 mL of solubilization buffer (0.25% sodium deoxycholate, 200 mM sodium chloride, 10 mM Tris-HCl (pH 9.5)) was added, mixed well, and diluted 4-fold. Unsolubilized precipitates were removed using a syringe filter with a pore size of 0.45 μm to obtain approximately 3.5 mL of solubilized solution (a solution containing the LPS in the extract).

[0073] [4] Chromatographic purification A portion (1500 μL) of the above-mentioned solubilized solution was injected into a high-performance liquid chromatography (HPLC) apparatus, and the LPS corresponding component (sample fraction) was collected. As the column, a reversed-phase silica gel column with a butyl group (alkyl group having 4 carbon atoms) (C4 reversed-phase column) was used. That is, HPLC equipped with a C4 reversed-phase column (C4 reversed-phase HPLC) was used. As the mobile phase, an aqueous solution / methanol-based gradient separation with a small environmental load was adopted. Formic acid and triethylamine were added to the aqueous solution. The gradient was set to a gradient condition in which the concentration of the B solution was increased after flowing under the condition rich in the A solution. The specific separation conditions are as follows.

[0074] <HPLC Separation Conditions> · Apparatus: HPLC ("Prominence" manufactured by Shimadzu Corporation) · C4 reversed-phase column: XBridge Protein BEH C4 Column, 300 Å, 3.5 μm, 4.6 mm X 100 mm manufactured by Waters · Eluent: A solution (aqueous solution of 50 mM formic acid · 50 mM triethylamine) B solution: Methanol · Gradient conditions (volume %): 0 - 10 min: Constant concentration condition (95% of A solution / 5% of B solution), 10 - 35 min: Linear gradient condition (95% of A solution / 5% of B solution (10 min) → (5% of A solution / 95% of B solution (35 min)), 35 - 50 min: Constant concentration (5% of A solution / 95% of B solution) · Flow rate: 4.5 mL / min · Column temperature: 40 °C · Sample injection volume: 1500 μL · Detector (DAD): 210 nm

[0075] [5] Post-treatment of chromatographic purification (removal of organic solvent and concentration) The sample fraction separated by the chromatographic purification in [4] above was evaporated at 35°C (rotary evaporator) to remove methanol, and the unvolatilized aqueous solution was collected and water for injection was added to make up to 15 mL. The solution was added to an ultrafiltration tube (Amicon Ultra Centrifugal Filter Units) with a molecular weight cutoff of 10 kda and centrifuged (7000 rpm x 30 min) to obtain a concentrate containing LPS on the non-permeated side of the ultrafiltration membrane. 15 mL of water for injection was added to the concentrate, and the concentrate was washed by similar centrifugation. The same washing was repeated five more times, and the concentrate after washing was collected and diluted with water for injection to obtain 4 mL of aqueous solution.

[0076] [6] Freeze drying The resulting aqueous solution was freeze-dried using a vacuum freeze dryer (FD-2BU-SQ manufactured by Nippon Techno Service Co., Ltd.) (conditions: -20°C, 5 Pa, 15 hours) to obtain the final product, LPS according to Example 1.

[0077] (Example 2) (Hot water extraction + C4 reverse phase HPLC) The hot water extract obtained through step [1] of Example 1 was used. The ultrafiltration treatment of step [2] performed in Example 1 was not performed.

[0078] The same procedure as in step [3] of Example 1 was carried out, except that 1 mL of the suspension after hot water extraction was used instead of 1 mL of the ultrafiltered suspension used in the pretreatment (solubilization treatment) of the chromatographic purification in step [3] of Example 1, to obtain approximately 3.5 mL of a solubilized solution.

[0079] The solubilized solution obtained was subjected to the same operations as in steps [4] chromatographic purification, [5] post-chromatographic purification (removal of organic solvents and concentration), and [6] lyophilization in Example 1 to obtain the final product, LPS according to Example 2.

[0080] (Comparative Example 1) (TCA extraction + PhOH extraction + ultrafiltration)

[11] TCA extraction 130 g of Pantoea agglomerans cell pellets obtained by the above-mentioned procedure for preparing gram-negative bacteria (Pantoea bacteria) and distilled water were added to a 3 L beaker and mixed by stirring to prepare a 300 mg / mL suspension (433 mL). The suspension was mixed with an equal amount of 0.5M trichloroacetic acid (TCA) solution and stirred at 15-20°C for 3 hours (100 rpm). The mixture was then transferred to a centrifuge tube and centrifuged (8,000G x 15 minutes) to obtain a supernatant. This was added to a 3L beaker, and 10N NaOH (20μL / mL) was added to adjust the pH to 7.0 to obtain a TCA extract. To 300 mL of the obtained TCA extract, 600 mL of ethanol at -20°C was added and mixed by stirring, after which the above TCA extract was left to stand overnight at -20°C and centrifuged the next day (7,000G x 15 minutes) to obtain a precipitate. To the obtained precipitate, 40 mL of distilled water was added to prepare a suspension, which was then freeze-dried to obtain 1.7 g of dry TCA extract.

[0081]

[12] Phenol extraction 1.6 g of the obtained dried TCA extract and 40 mL of distilled water were added to a beaker, and the mixture was stirred to prepare a 40 mg / mL suspension. Next, 40 mL of 90% aqueous phenol solution heated to 68°C was added to the suspension, and the mixture was stirred at 68°C for 20 minutes. After the suspension was cooled to 20°C or less, the suspension was transferred to a centrifuge tube and centrifuged at room temperature (7,000G x 30 minutes). After centrifugation, the upper layer (1st extract) of the centrifuge tube was collected, and the lower layer was returned to the beaker used for phenol extraction. 40 mL of distilled water was added to the beaker and stirred at 68°C for 20 minutes. After the suspension was cooled to 20°C or less, the resulting suspension was transferred to a centrifuge tube and centrifuged at room temperature (7,000G x 30 minutes). The upper layer (2nd extract) of the centrifuge tube after centrifugation was collected and combined with the 1st extract to obtain a phenol-extracted aqueous layer.

[0082]

[13] Ultrafiltration treatment The total amount of the phenol-extracted aqueous layer solution (about 80 mL) described above was added to an ultrafiltration tube with a molecular weight cut-off of 10 kDa (Amicon Ultra centrifugal filter units), and centrifuged (7000 rpm × 30 minutes) to obtain a concentrated solution containing LPS on the non-permeate side of the ultrafiltration membrane. Further, 15 mL of water for injection was added to the concentrated solution, and centrifugation was performed in the same manner to wash the concentrated solution. The same washing was repeated 11 more times, and the washed concentrated solution was collected, diluted with water for injection to the mark, and a 15 mL ultrafiltered suspension was obtained.

[14] Freeze-drying The obtained ultrafiltered suspension was freeze-dried using a vacuum freeze dryer (FD-2BU-SQ manufactured by Nippon Techno Service Co., Ltd.) (conditions: -20 °C, 5 Pa, 15 h) to obtain the LPS according to Comparative Example 1, which is the final product.

[0083] (Comparative Example 2) (Hot water extraction + ultrafiltration) Using 15 mL of the hot water extract obtained through the step [1] of Example 1, the same operations as in the step [2] of Example 1 were performed to carry out ultrafiltration treatment. The suspension obtained by the ultrafiltration treatment was 15 mL. Regarding the obtained ultrafiltered suspension, the same operations as in the freeze-drying in the step [6] of Example 1 were performed to obtain the LPS according to Comparative Example 2, which is the final product.

[0084] (Comparative Example 3) (Only hot water extraction) Regarding the hot water extract obtained through the step [1] of Example 1, the same operations as in the freeze-drying in the step [6] of Example 1 were performed to obtain the LPS according to Comparative Example 3, which is the final product.

[0085] <Quantification of LPS> The LPS obtained in each of the above-described examples and comparative examples (hereinafter referred to as samples) was dissolved in the following eluent, and the quantification of LPS was performed by the following HPLC method. Further, from the quantification results, the LPS yield per 1 kg of the cell pellet was calculated and described in Table 1 below.

[0086] <HPLC method> Each sample was appropriately diluted with the eluent and injected into the HPLC. Quantification was performed by the absolute calibration curve method.

[0087] <HPLC Separation Conditions> · Apparatus: HPLC (Agilent "HPLC 1260 Infinity") · Column: PL-aquagel-OH 30 manufactured by Agilent (particle size 8 μm, length 300 mm × inner diameter 7.5 mm) connected in series · Eluent: 0.25% sodium deoxycholate, 200 mM sodium chloride, 10 mM Tris-HCl aqueous solution (pH 9.5) · Flow rate: 1.0 mL / min · Column temperature: 40 °C · Sample injection volume: 50 μL · Detector: Differential refractive index detector · Standard substance: LPS standard from Pantoea agglomerans (purity 95% or higher)

[0088] <Measurement of Concentrations of Proteins and Nucleic Acids in Each Sample> The concentrations of proteins and nucleic acids remaining in each sample were measured by the following method. The results are shown in Table 1 below.

[0089] (Measurement of Protein Content) Each sample whose weight was measured was dissolved in water, and the protein content was measured by the BCA method (Thermo "Pierce BCA Protein Assay Kit"). Furthermore, the protein content was determined by multiplying the volume of the aqueous solution. Then, the protein content rate in each sample was determined by the following formula (1) and shown in Table 1. (Formula 1) Protein content rate (%) = (protein content (mg) / sample (mg)) × 100

[0090] (Measurement of Nucleic Acid Content) The weight of each sample was measured and dissolved in water to obtain an aqueous solution, and the absorbance of each sample aqueous solution at 260 nm and 320 nm was measured using a spectrophotometer (SpectraMaxPlus384, manufactured by Molecular Devices) with water as a control, and the nucleic acid concentration Ac in the aqueous solution was calculated using the following formula 2. (Formula 2) Ac(μg / mL)=OD260nm-OD320nm)×50 Furthermore, the nucleic acid content was calculated by multiplying the solution volume by the amount of the solution. The nucleic acid content in each sample was then calculated using the following formula 3, and is shown in Table 1. (Formula 3) Nucleic acid content (%) = (Nucleic acid content (mg) / sample (mg)) × 100

[0091] (Calculation of LPS purity) The purity of LPS in each sample was calculated using the following formula 4, and is shown in Table 1. (Formula 4) LPS purity (%) = 100 - protein content (%) - nucleic acid content (%)

[0092] (Calculation method for LPS yield per 1 kg of bacterial pellet) When WLPS (g) of LPS is obtained by purifying v (mL) of the extract (hot water or TCA) V (mL) obtained from the bacterial pellet W (g), the LPS yield per kg of bacterial pellet is WLPS × (V / v) × (1000 / W) (g). Based on this, the LPS yields in the Examples and Comparative Examples were calculated and are shown in Table 1.

[0093] <Maximum treatment concentration of Class 1 designated chemical substances> (Calculation method for maximum treatment concentration of Class 1 designated chemical substances) In each process, if a (mL) of solvent component A, b (mL) of component B, and c (mL) of component C are mixed, and B is a Class 1 designated chemical substance, the treatment concentration of the Class 1 designated chemical substance is {b / (a+b+c)}×100(%). The treatment concentration of the Class 1 designated chemical substance was calculated in each process employed in the examples or comparative examples, and the highest one was taken as the maximum treatment concentration of the Class 1 designated chemical substance, and is shown in Table 1.

[0094] The substances designated as Type 1 designated chemical substances (Act on Reporting, etc. of Releases of Chemical Substances and Promoting Improvements in Their Management) used in preparing the sample in Comparative Example 1 were trichloroacetic acid and phenol, and their maximum processing concentrations during the manufacturing process are shown in Table 1 below.

[0095] In addition, the substance designated as a Type 1 designated chemical substance (Act on Reporting, etc. of Releases of Chemical Substances and Promoting Improvements in Their Management) used in preparing the samples of Examples 1 and 2 was triethylamine, and the maximum processing concentration in the manufacturing process is shown in Table 1 below.

[0096] [Table 1]

[0097] From the results of Table 1 above, in Examples 1 and 2, by combining hot water extraction and C4 reverse phase column HPLC, regardless of whether ultrafiltration was performed or not, it was possible to produce LPS of sufficient purity without using organic solvents with high environmental impact as an extraction solvent, and by minimizing the amount of Class 1 designated chemicals used throughout the entire manufacturing process. It can be seen that the purity is as high as that of Comparative Example 1, which is a conventional LPS manufacturing method using TCA and phenol with high environmental impact, and the yield is comparable. It can also be seen that both the LPS yield and LPS purity are insufficient when using only hot water extraction, or only hot water extraction and ultrafiltration. From these results, it is clear that by combining hot water extraction and HPLC using a specific reverse phase column, it is possible to produce LPS with a satisfactory level of both LPS yield and LPS purity without using organic solvents with high environmental impact in the extraction process, and by minimizing the amount of Class 1 designated chemicals used throughout the entire manufacturing process.

[0098] Next, for the LPS (LPS purity 94.4%) prepared by the method of Example 2, the molecular weight distribution was evaluated by SDS-PAGE gel electrophoresis in order to clarify the structural characteristics of LPS. In addition, the LPS quantitative value (E) by ELISA and the LPS quantitative value (L) by Limulus test (endpoint colorimetric method) were obtained, and the ratio (E / L ratio) was evaluated. The measurement method and results are described below.

[0099] (Measurement sample) Samples prepared by the production method of Example 2 (LPS purity 94.4%) (Lot.2, Lot.3) IP-PA1 standard product (LPS derived from Pantoea agglomerans, LPS purity 97.3%, manufactured by Natural Immunity Application Technology Research Institute Co., Ltd., Lot.4) (measured as a comparison target)

[0100] Note that the IP-PA1 standard product is LPS derived from Pantoea agglomerans, but its production method is different from that of the present invention. IP-PA1 was obtained by extraction with hot phenol water and purification by anion exchange chromatography.

[0101] (Measurement of molecular weight distribution by SDS-PAGE gel electrophoresis) (Measurement method) LPS was fractionated by Tricine-SDS-PAGE and visualized by silver staining. Tricine-SDS-PAGE was referred to the paper by Schaegger H. et al. (1). A 15% polyacrylamide gel manufactured by ATTO Corporation was used, and Tricine was added only to the electrophoresis buffer. The silver staining method was referred to the paper by Tsai et al. (2), and stained using the Silver Staining II Kit for Electrophoresis Wako manufactured by Fujifilm Wako Pure Chemical Corporation.

[0102] Reference papers: (1) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Schaegger H, von Jagow G. Anal Biochem. 1987 Nov 1;166(2):368-79 (2)A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Tsai CM, Frasch CE. Anal Biochem. 1982 Jan 1;119(1):115-9.

[0103] (Preparation of sample solution for electrophoresis and electrophoresis) The IP-PA1 standard and the LPS of Example 2 (Lot. 2, Lot. 3) were subjected to electrophoresis. Distilled water was added to the IP-PA1 standard and LPS (Lot. 2, Lot. 3) in Example 2 to make the concentration 500 μg / mL. 12 μL of each was dispensed into three 1.5 mL tubes, and the same amount (12 μL) of sample buffer (Nacalai Tesque, sample buffer (for SDS-PAGE, 2-fold concentrated, containing 2-ME)) was added and mixed well, and the mixture was heated for 5 minutes in a heating block (aluminum block heater, Synix) at 100°C. After the treatment, the mixture was rapidly cooled in ice water, and the sample was collected at the bottom of the tube by centrifugation, and 20 μL was applied to the gel. The electrophoresis buffer was prepared according to the composition shown in Table 2 below.

[0104] [Table 2]

[0105] Size markers (Bio-Rad Laboratories, Precision Plus Protein Dual Extra Standard, molecular weight sizes: 250, 150, 100, 75, 50, 37, 25, 20, 15, 10, 5, 2 kDa) were added to 1 μL of sample buffer (Nacalai Tesque, sample buffer (for SDS-PAGE, 2x concentrated, containing 2-ME)) diluted 2x with distilled water (39 μL), and 20 μL was applied to the gel. Immediately after applying the size markers and samples, electrophoresis was started at a constant current of 20 mA, and electrophoresis was terminated when BPB (marker dye) had migrated to a position 1 cm from the bottom of the separation gel (about 60 minutes). After electrophoresis, the gel plate was removed from the device and the gel was placed in a fixing solution for silver staining.

[0106] (Silver staining method) Staining was performed using Wako Silver Staining Kit for Electrophoresis II manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. The staining procedure was performed according to the attached instructions.

[0107] The obtained electrophoretic diagram is shown in the SDS-PAGE (silver staining) diagram in Figure 1. The SDS-PAGE in Figure 1 is arranged in the following order from the left: IP-PA1 standard (Lot. 4) (N = 3), LPS (Lot. 2) (N = 3) of Example 2, LPS (Lot. 3) (N = 3) of Example 2, and a protein size marker. The vertical axis in Figure 1 indicates molecular weight. The molecular weights calculated based on the protein size markers are shown in Table 3 below.

[0108] (Analysis of the total brightness value of each band) Next, the image data was read using Image J software (free image processing software, http: / / imagej.nih.gov / ij / , ImageJ 1.52a), and the total brightness value (brightness value x area) of the high molecular weight bands and low molecular weight bands in each lane was calculated. The results of measuring the total brightness value of low molecular weight and high molecular weight components by SDS-PAGE are shown in Table 4 below.

[0109] [Table 3]

[0110]

Table 4

[0111] (Measurement results) As a result of electrophoresis, the molecular weights determined based on the protein size marker were 5,000 to 20,000 for the low molecular weight region bands in both the IP-PA1 standard product and the LPS of Example 2. For the high molecular weight region bands, the IP-PA1 standard product was 20,000 to 70,000, while the LPS of Example 2 had a narrower distribution of 25,000 to 60,000. Next, regarding the total luminance value of each band, in the IP-PA1 standard product, the ratio of low molecular weight LPS was 74.9% in terms of luminance value. In LPS Lot.2 of Example 2, it was 82.3%, and in Lot.3 it was 84.6%. It was shown that for both lots of the LPS of Example 2, the ratio of low molecular weight LPS was higher than that of the IP-PA1 standard product.

[0112] (Measurement of E / L ratio) (Quantification method of LPS by ELISA) The LPS content (E) (μg / mg) in the sample was measured by ELISA using an IP-PA1 sugar chain-specific antibody (manufactured by Natural Immunity Application Technology Research Co., Ltd.). Specifically, ELISA measurement was performed according to the following procedure.

[0113] 1) 34-G2 (immobilized antibody: manufactured by Natural Immunity Application Technology Research Co., Ltd., Lot. 201107-7, antibody against the O antigen polysaccharide of IP-PA1) was diluted 800-fold with PBS(-) sterilized by filter filtration at the time of use. 50 μL of it was dispensed into each well of a 96-well immunoplate, sealed with parafilm so that the liquid would not evaporate, and stored at 4°C. 2) The 96-well immunoplate with 34-G2 added was taken out from 4°C, the liquid was discarded, and then a blocking solution (a solution with BSA added to PBS(-) to a final concentration of 3% (w / v)) was added at a rate of 200 μL / well and left at 25°C for 30 minutes or more to prevent non-specific adsorption of the antibody and other proteins. 3) After washing three times with 200 μL of washing solution (equivalent to 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% (v / v) Tween 20), 50 μL of serially diluted IP-PA1 standard (IP-PA1 purity 97.2% or higher: Natural Immunity Applied Technology Research Co., Ltd., Lot. 4) was added to the wells. 50 μL of the sample solution to be measured was added to another well of the same 96-well immunoplate. The plate was left in a constant temperature bath at 25°C for 1 hour. 4) The wells were washed three times with 200 μL of washing solution, and 50 μL / well of 1000-fold diluted 4E-11 (primary antibody: Natural Immunity Applied Technology Research Institute, Inc., Lot. 20170702, antibody against O-antigen polysaccharide of IP-PA1) was added, and the wells were left in a 25°C incubator for 1 hour. 5) The wells were washed three times with 200 μL of washing solution, and 50 μL / well of 1000-fold diluted alkaline phosphatase-conjugated anti-mouse IgG immunoglobulin antibody (Sigma, Cat. 1418) was added and allowed to stand in a 25° C. incubator for 1 hour. 6) The wells were washed five times with 200 μL of washing solution, and 100 μL / well of color developing solution (disodium p-nitrophenyl phosphate hexahydrate (p-NPP): Nacalai Tesque, Cat. 25019-52, Lot. MOP2259) was added and allowed to stand at room temperature for 1 hour. 7) The absorbance at 415 nm was then measured using a microplate reader (Molecular Devices SpectraMax, Plus384). A calibration curve was created using a quadratic curve. 8) Using the calibration curve, the LPS concentration in the sample solution (μg / mL) was calculated from the absorbance of the sample solution. The LPS content (E) (μg / mg) in the sample was calculated by dividing the LPS concentration in the sample solution by the sample concentration in the sample solution (mg / mL) (calculated from the sample weight and solution volume at the time of preparation). The detection limit of this ELISA method is 1.6 ng / mL.

[0114] The IP-PA1 standard (Lot. 4) and the LPS (Lot. 2 and Lot. 3) of Example 2 were subjected to LPS quantification by ELISA. The measurement results are shown in Table 5 below.

[0115] (LPS quantification method using the Limulus test (endpoint colorimetric method)) The LPS content (L) (μg / mg) in the samples (LPS (Lot. 2 and Lot. 3) and IP-PA1 standard sample (Lot. 4) in Example 2) was measured by endpoint colorimetry using a microplate using a Limulus measurement kit (Endospecy ES-50M set, manufactured by Seikagaku Corporation) capable of specific measurement of LPS, in accordance with the attached instructions. For the measurements, CSE (10 ng / vial) (Seikagaku Corporation) was used as an endotoxin standard. The measurement results are shown in Table 5 below.

[0116] Based on the LPS contents (E and L) determined by the above two methods, the ratio (E / L ratio) was calculated. The results are shown in Table 5 below.

[0117] [Table 5]

[0118] (Measurement results) The E / L ratio of IP-PA1 produced by the conventional method was 1.64, whereas that of LPS in Example 2 was a low value of 0.71 to 0.72.

[0119] Since LPS is a substance derived from a living organism, it is considered to be a mixture of molecules with different structures and molecular weights. Therefore, it is difficult to clearly define the product of the present invention by a structural formula. Therefore, the characteristics of the manufactured product were clarified by evaluating the characteristic values ​​due to the structure. That is, the measurement results of SDS-PAGE gel electrophoresis confirmed that an LPS mixture was obtained by manufacturing the LPS according to the method of the embodiment in which the ratio of low molecular weight LPS to the total LPS was higher than that of LPS (IP-PA1) manufactured by the conventional manufacturing method. In addition, the fact that the E / L ratio value of the LPS manufactured by the method of the embodiment is smaller than that of LPS (IP-PA1) manufactured by the conventional manufacturing method indicates that the glycan portion, which contributes greatly to the molecular weight, is shorter. This result also confirmed that an LPS mixture was obtained by manufacturing the LPS according to the method of the embodiment in which the ratio of low molecular weight LPS to the total LPS was higher than that of LPS (IP-PA1) manufactured by the conventional manufacturing method.

[0120] In other words, since the production method of the present invention uses reverse phase chromatography in the purification process, it is possible that highly lipid-soluble LPS (LPS with few hydrophilic glycans and a high proportion of lipid-soluble Lipid A moiety) was selectively recovered.

[0121] As described above, the LPS of the present invention is produced by a specific production method, thereby obtaining a product (LPS) in which the proportion of low molecular weight LPS is high in the total LPS. Because of its molecular weight distribution, this LPS is highly safe and has high physiological activity (cytokine induction ability).

Claims

1. A method for producing lipopolysaccharide, comprising extracting and purifying lipopolysaccharide from gram-negative bacteria, a first step of extracting the gram-negative bacteria with hot water to obtain an extract containing the lipopolysaccharide; a second step of purifying the extract or a solution containing lipopolysaccharide in the extract using reverse phase liquid chromatography to obtain lipopolysaccharide; The reversed-phase column in the reversed-phase liquid chromatography has a packing material made of a material having a functional group having 1 to 8 carbon atoms. A method for producing lipopolysaccharide, comprising the steps of:

2. The method for producing lipopolysaccharide according to claim 1, wherein the filler is composed of a material having a functional group having 2 to 6 carbon atoms.

3. 3. The method for producing lipopolysaccharide according to claim 1, wherein the filler is composed of a material having a functional group having 2 to 4 carbon atoms.

4. The method for producing lipopolysaccharide according to any one of claims 1 to 3, wherein the filler is composed of a material having a functional group having four carbon atoms.

5. The method for producing lipopolysaccharide according to any one of claims 1 to 4, wherein the functional group is an alkyl group.

6. The method for producing lipopolysaccharide according to any one of claims 1 to 5, wherein the temperature of the hot water in the first step is 50 to 150°C.

7. The method for producing lipopolysaccharide according to any one of claims 1 to 6, wherein the temperature of the hot water in the first step is 50 to 99°C.

8. The method for producing lipopolysaccharide according to any one of claims 1 to 7, wherein the temperature of the hot water in the first step is 70 to 99°C.

9. The method for producing lipopolysaccharide according to any one of claims 1 to 8, wherein the temperature of the hot water in the first step is 85 to 95°C.

10. The method for producing lipopolysaccharide according to any one of claims 1 to 9, wherein the gram-negative bacterium is at least one selected from the group consisting of Escherichia, Salmonella, Pantoea, Acetobacter, Zymomonas, Xanthomonas, Enterobacter, Roseomonas and Rhodobacter.

11. The method for producing lipopolysaccharide according to any one of claims 1 to 10, wherein the gram-negative bacterium is Pantoea agglomerans.

12. Lipopolysaccharide produced by the manufacturing method described in claim 11, characterized in that it reacts with primary antibody 4E-11 by ELISA and has an E / L ratio (E=1.0 or less) obtained by dividing the lipopolysaccharide quantitative value (E) by the lipopolysaccharide quantitative value (L) by the Limulus test (endpoint colorimetric method).

13. The lipopolysaccharide according to claim 12, characterized in that it comprises a low molecular weight lipopolysaccharide having a molecular weight of 2,000 to 20,000 as measured by SDS-PAGE method, and a high molecular weight lipopolysaccharide having a molecular weight of more than 20,000 and not more than 100,000 as measured by SDS-PAGE method, and the content of the low molecular weight lipopolysaccharide relative to the total amount of the low molecular weight lipopolysaccharide and the high molecular weight lipopolysaccharide is 80% or more.

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