Agents for reducing endotoxin in proteoglycans derived from animal tissues and methods for producing proteoglycans

Abstract

Provided are an agent that is capable of reducing endotoxin and is used to reduce endotoxin in proteoglycans derived from animal tissues, and a method for producing a proteoglycan.　The agent used to reduce endotoxin in proteoglycans derived from animal tissues of the present disclosure contains a nonionic surfactant.

Description

Technical Field

[0001] The present disclosure relates to an agent for reducing endotoxin of proteoglycan derived from animal tissues and a method for producing proteoglycan.

Background Art

[0002] In the regenerative medicine market, regenerative medicine tissues with a sheet structure are mainstream. However, it is difficult to regenerate tissues having a three-dimensional structure with the tissues having the sheet structure. For this reason, induction of the three-dimensional structure tissues has been attempted. When constructing the three-dimensional structure tissues, there is a problem that it is difficult to reproduce cell-cell crosstalk and the structure of the extracellular matrix similar to that in vivo. Therefore, development of a material capable of reproducing the structure of the extracellular matrix is desired (Non-Patent Document 1).

[0003] In addition, for regenerative medicine tissues, in order to be used in animals such as humans, reduction of endotoxin (endotoxin) and approval based on biological raw material standards are required (Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Since the inventors found that proteoglycans are abundantly present in the extracellular matrix, they conceived the idea that proteoglycans could be used as the said material. Then, in order to use proteoglycans for medical purposes, the inventors removed endotoxins in proteoglycans by an endotoxin removal method using a basic organic solvent. However, in the said endotoxin removal method, for example, there occurred a problem that not only endotoxins were removed but also the molecular weight of a given proteoglycan could not be maintained.

[0007] Therefore, a first object of the present disclosure is to provide, for example, a method for producing proteoglycans that can reduce endotoxins and maintain the molecular weight of a given proteoglycan.

[0008] Another object of the present disclosure is to provide, for example, an agent for use in reducing endotoxins in proteoglycans derived from animal tissues that can reduce endotoxins, a method for producing proteoglycans, and the like.

Means for Solving the Problems

[0009] In order to achieve the first object, the method for producing proteoglycans of the present disclosure includes a reducing step of reducing endotoxins from proteoglycans derived from animal tissues using a nonionic surfactant.

[0010] The composition of the present disclosure is a composition containing proteoglycans derived from animal tissues, the composition contains proteoglycans and endotoxins, and the endotoxin content ratio with respect to the composition is 4 EU / mg or less.

[0011] In order to achieve the second object, the agent for use in reducing endotoxins in proteoglycans derived from animal tissues of the present disclosure contains a nonionic surfactant.

[0012] The method for producing a proteoglycan of the present disclosure includes a first reduction step of reducing endotoxin from a proteoglycan derived from an animal tissue using a nonionic surfactant, and a second reduction step of reducing endotoxin from the obtained proteoglycan by treating the proteoglycan with a chromatography carrier.

[0013] According to the present disclosure, for example, it is possible to provide a method for producing a proteoglycan that can reduce endotoxin and maintain the molecular weight of a predetermined proteoglycan. Further, according to the present disclosure, for example, it is possible to provide an agent for reducing endotoxin of a proteoglycan derived from an animal tissue that can reduce endotoxin, a method for producing a proteoglycan, and the like.

Brief Description of the Drawings

[0014]

Figure 1

Embodiments for Carrying Out the Invention

[0015] Hereinafter, the present disclosure will be specifically described with examples. Hereinafter, unless otherwise specified, each disclosure can incorporate the description of other disclosures.

[0016] <Definition> In the present specification, "proteoglycan" means a molecule (glycoprotein) in which a protein (core protein) and a glycosaminoglycan (GAG, also referred to as "polysaccharide" or "sugar chain") are covalently bonded. The proteoglycan exists, for example, as an extracellular matrix such as skin, organs, and cartilage. The glycosaminoglycan is usually known as a sugar chain having a long-chain structure without a branched structure. Examples of the proteoglycan include the following. · Aggrecan family (also referred to as lectican family or hyalectan family): Aggrecan, Versican, Neurocan, Brevican, etc. · Small Leucine Rich Proteoglycans (SLRPs) family: Biglycan, Decorin, Fibromodulin, Lumican, PG-Lb (Epiphycan), Keratocan, Mimecan, etc. · Proteoglycans of basement membrane: Perlecan, Agrin, Vermican, etc. · Other proteoglycans: Testican, Biglycan, Serglycin, Syndecan, Dystroglycan, Claustrin, Glypican, Keratocan, etc. The proteoglycans can be classified into chondroitin sulfate proteoglycans, dermatan sulfate proteoglycans, heparan sulfate proteoglycans, or keratan sulfate proteoglycans, for example, according to the types of GAGs bound to the proteins.

[0017] The GAGs include, for example, chondroitin, chondroitin sulfate (CS), dermatan sulfate (DS, chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. The chondroitin includes an O-type sugar chain having a disaccharide structure of glucuronic acid and N-acetylgalactosamine as a main disaccharide structure, and an iO-type sugar chain having a disaccharide structure of iduronic acid and N-acetylgalactosamine as a main structure (hereinafter, also referred to as "chondroitin sulfate O" and "chondroitin sulfate iO", respectively). The chondroitin sulfate (CS) has a structure in which a sulfate group is added to a sugar chain in which a disaccharide of glucuronic acid and N-acetylgalactosamine repeats. The chondroitin sulfate (CS) includes, for example, chondroitin sulfate A (type A) having a disaccharide structure of glucuronic acid and N-acetylgalactosamine 4-sulfate as a main disaccharide structure, chondroitin sulfate C (type C) having a disaccharide structure of glucuronic acid and N-acetylgalactosamine 6-sulfate as a main disaccharide structure, and the like. The dermatan sulfate (DS) has a structure in which a sulfate group is added to a sugar chain in which a disaccharide of iduronic acid and N-acetylgalactosamine repeats. The dermatan sulfate includes, for example, chondroitin sulfate iA (iA type) having a disaccharide structure of iduronic acid and N-acetylgalactosamine 4-sulfate as a main disaccharide structure, chondroitin sulfate iC (iC type) having a disaccharide structure of iduronic acid and N-acetylgalactosamine 6-sulfate as a main disaccharide structure, and the like. Each chondroitin sulfate has, for example, a disaccharide structure shown in FIG. 1 as a main disaccharide structure. In FIG. 1, the sulfate group (sulfonyl group) is bonded to a hydrogen atom, but the present disclosure is not limited thereto, and the sulfate group of the GAG may be, for example, ionized by desorption of a hydrogen atom or may form a salt.

[0018] As used herein, "endotoxin" refers to a toxic component derived from lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria. The endotoxin is generated, for example, when the cell wall of the Gram-negative bacteria is disrupted and released after the death of the Gram-negative bacteria. It is known that when the endotoxin enters the blood of humans or the like, it causes biological reactions such as fever, septic shock, and multiple organ failure. The endotoxin is composed of O-antigen polysaccharide, core polysaccharide, and lipid A. Since the endotoxin has a hydrophilic portion (the sugar portions of O-antigen polysaccharide and core polysaccharide) and a hydrophobic portion (lipid A), it can associate in an aqueous solution to form micelles.

[0019] As used herein, "chromatography carrier" refers to any type of stationary phase that separates a specific molecule from other molecules present in a mixture. Examples of the stationary phase of the chromatography carrier include resins, porous bodies such as monoliths (e.g., silica monoliths), and membranes. When the stationary layer is a resin, the chromatography carrier can be referred to as a chromatography resin.

[0020] As used herein, "micelle" means a spherical structure in which amphiphilic molecules having a hydrophobic portion and a hydrophilic portion aggregate in an aqueous solvent such as water, with the hydrophilic group portion facing the aqueous solvent side and the hydrophobic portion facing the inside. The concentration at which micelles are formed from molecular dispersion is called the critical micelle concentration (CMC) of that substance.

[0021] As used herein, "surfactant" means a compound having a hydrophilic group and a hydrophobic group (lipophilic group). The surfactant forms micelles at a concentration equal to or higher than the critical micelle concentration. Examples of the surfactant include anionic surfactants, cationic surfactants, zwitterionic surfactants, and nonionic surfactants. The anionic surfactant means a surfactant that dissociates into anions in an aqueous solution. The cationic surfactant means a surfactant that dissociates into cations in an aqueous solution. The zwitterionic surfactant means a surfactant that dissociates into cations or anions depending on the pH of the aqueous solution in the aqueous solution. The nonionic surfactant means a surfactant that does not dissociate into ions in an aqueous solution.

[0022] As used herein, "cloud point" means the temperature (phase transition temperature) at which cloudiness begins to occur when a solution containing a nonionic surfactant is heated. The cloudiness is caused by a sharp decrease in the water solubility of the nonionic surfactant due to the heating, resulting in the inability of the nonionic surfactant to form micelles. The sharp decrease in the water solubility is caused by the inability of the polyether chain of the nonionic surfactant to form hydrogen bonds with water. The cloud point can be measured, for example, by visual observation, measurement in accordance with the measurement standard for the cloud point of nonionic surfactants (ISO 1065:1991), and the method described in JP-A-2006-257395. The method described in JP-A-2006-257395 is as follows. · The method described on page 95 of "New Introduction to Surfactants" by Takehiko Fujimoto (Sanyo Chemical Industries, Ltd.). In this method, a nonionic compound diluted to a 1% by mass aqueous solution is placed in a test tube, a thermometer and a stirring rod are placed therein, and the temperature is slowly raised while gently stirring with the stirring rod. When the temperature reaches a certain temperature or higher, the originally transparent aqueous solution becomes cloudy. The temperature at which the cloudiness occurs is measured and defined as the cloud point. Note that the definition of the cloud point in surfactants is also mentioned in, for example, JIS K3211.

[0023] As used herein, the "ion exchange carrier" (ion exchanger) means a solid phase having free anions or free cations for exchange with anions or cations in a liquid passing through the solid phase or in the solid phase, which is positively or negatively charged. When the ion exchange carrier is positively charged and capable of exchanging anions, the ion exchange carrier may also be referred to as an anion exchange carrier or an anion exchanger. The anion exchange carrier has a positively charged ligand such as a primary, secondary, tertiary or quaternary amino group, for example. Also, when the ion exchange carrier is negatively charged and capable of exchanging the cations, the ion exchange carrier may also be referred to as a cation exchange carrier or a cation exchanger.

[0024] As used herein, the "hydrophobic interaction carrier" means a solid phase having a hydrophobic group and adsorbing the molecules according to the hydrophobicity of the molecules contained in a liquid passing through the solid phase or in the solid phase. Examples of the hydrophobic group include the following functional groups. · Alkyl groups such as octyl group and octadecyl group · Functional groups containing aryl groups such as phenyl group and benzyl group · Polyamine-based functional groups such as polylysine (e.g., ε-polylysine)

[0025] As used herein, the "flow-through mode" (F / T mode) means a technique of binding or adsorbing at least one target substance to be removed contained in a target to a stationary phase such as a chromatography resin and allowing at least one target substance to flow through for separation. The F / T mode is also referred to as a negative mode, for example.

[0026] As used herein, the "binding / elution mode" (B / E mode) means a technique of binding or adsorbing at least one target substance contained in a target to a stationary phase such as a chromatography resin and then eluting the target substance for separation.

[0027] Hereinafter, the present disclosure will be described with examples. However, the present disclosure is not limited to the following examples and can be arbitrarily modified and implemented. In addition, each description in the present disclosure and each embodiment can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used in the meaning including the numerical values or physical values before and after it. In this specification, the expression "A and / or B" includes "only A", "only B", and "both A and B".

[0028] <Method for producing proteoglycan> In one aspect, the present disclosure provides a method for producing proteoglycan. The production method of the present disclosure includes a first reduction step of reducing endotoxin from proteoglycan derived from animal tissues using a nonionic surfactant. According to the production method of the present disclosure, endotoxin can be reduced while maintaining the molecular weight of a predetermined proteoglycan.

[0029] As a result of intensive research, the inventors of the present invention have conceived the idea of reducing endotoxin from a composition containing proteoglycan, which is a glycoprotein, and endotoxin, which is a glycolipid, by taking advantage of the difference in the degree of hydrophobicity and hydrophilicity between them. Then, as a result of further research, the inventors of the present invention have found that endotoxin can be extracted from a mixture of the proteoglycan and the endotoxin by using a nonionic surfactant, and have thus established the present disclosure. It should be noted that the present disclosure is presumed to be able to reduce the endotoxin by the mechanism as described below by using the nonionic surfactant. However, the present disclosure is not limited to the following mechanism. The proteoglycan is composed of sugar and protein and is a hydrophilic component. On the other hand, as described above, the endotoxin has a hydrophilic portion and a hydrophobic portion. Therefore, when the proteoglycan, the endotoxin, and the nonionic surfactant coexist in an aqueous solvent under temperature conditions where the nonionic surfactant can be dispersed in the aqueous solvent, the proteoglycan, the endotoxin, and the nonionic surfactant exhibit different behaviors due to the difference in their hydrophilicity and hydrophobicity. Specifically, the proteoglycan can be dispersed alone in the aqueous solvent. On the other hand, since the endotoxin and the nonionic surfactant contain hydrophobic portions, it is difficult for them to be dispersed alone in the aqueous solvent, and the two gather to form a complex. Therefore, in the aqueous solvent, the endotoxin and the nonionic surfactant exist in the form of a complex. In this state, when the aqueous solvent is heated to a temperature exceeding the cloud point of the nonionic surfactant, the complex containing the nonionic surfactant cannot maintain its dispersed state in the aqueous solvent and forms a phase of the nonionic surfactant (surfactant phase). Since the endotoxin forms a complex with the nonionic surfactant, it is concentrated (extracted) in the phase of the nonionic surfactant during the formation of the phase (phase separation).Therefore, in the present disclosure, it is presumed that the amount of endotoxin in the composition containing the proteoglycan can be reduced by extracting the endotoxin into a different phase from the composition containing the proteoglycan and the endotoxin.

[0030] Examples of the nonionic surfactant include surfactants having a polyether chain such as polyoxyethylene alkyl phenyl ether (POEAE). Examples of the polyoxyethylene alkyl phenyl ether include polyethylene glycol tert-octyl phenyl ether (the following formula (1)). In the following formula (1), n represents the degree of polymerization of ethylene glycol. The n is preferably, for example, 1 to 10, and more preferably 7 to 8. The nonionic surfactant is, for example, a nonionic surfactant that undergoes a phase transition depending on temperature. The surfactant may be used, for example, alone or in combination of a plurality of types. When a plurality of types of surfactants are used, for example, a plurality of types of surfactants may be mixed and used in advance.

[0031]

Chemical formula

[0032] The animal is not particularly limited, and examples include mammalian animals (mammals) such as pigs; avian animals (birds) such as chickens; fish of the family Rajidae such as little skate, salmonids such as rainbow trout and Atlantic salmon, and fish such as rays (including, for example, thornback ray); etc. The animals are preferably pigs, birds, rays, salmon, and rays, etc.

[0033] The animal tissue is, for example, a tissue containing proteoglycan. As an example, epithelial tissue such as skin; cartilage tissue such as cartilage; digestive organs; circulatory organs; respiratory organs; and placenta, etc. can be mentioned. As a specific example, the animal tissue is, for example, cartilage, fin, digestive organ, circulatory organ, respiratory organ, and ear, etc.

[0034] The sugar chains of the proteoglycan include, for example, chondroitin, chondroitin sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. The sugar chain is preferably chondroitin sulfate.

[0035] In the production method of the present disclosure, as described above, in the first reduction step, the nonionic surfactant is used to reduce endotoxin from the proteoglycan derived from animal tissue. Specifically, in the first reduction step, for example, as described above, for the endotoxin forming a complex with the nonionic surfactant, the phase of the nonionic surfactant is formed from the complex to concentrate it in the phase of the nonionic surfactant. Therefore, the production method of the present disclosure may include a forming step of bringing the nonionic surfactant into contact with the proteoglycan derived from animal tissue prior to the first reduction step to form a complex of the nonionic surfactant containing the endotoxin.

[0036] In the formation step, the contact between the nonionic surfactant and the proteoglycan is preferably carried out in a liquid system (liquid phase) because, for example, the complex can be efficiently formed. The liquid system can also mean, for example, in the presence of a solvent such as an aqueous solvent. The liquid system preferably substantially does not contain an organic solvent such as a basic organic solvent. The term "substantially does not contain" means, for example, a content at which the content of the organic solvent is below the detection limit. Examples of the aqueous solvent include buffer solutions such as phosphate buffer, phosphate buffered saline without calcium (Ca) and / or magnesium (Mg), phosphate buffered saline, PBS (Phosphate Buffered Saline), saline, sodium chloride solution, or a mixed solution thereof. The aqueous solvent may contain an alcohol such as ethanol because, for example, the endotoxin can be more efficiently removed. When the aqueous solvent contains an alcohol, the alcohol is preferably contained within a range that does not inhibit the formation of the complex between the nonionic surfactant and the endotoxin. When the aqueous solvent contains an alcohol, the concentration of the alcohol is preferably more than 0 (v / v)% and less than 2 (v / v)%, and more preferably about 1 (v / v)%.

[0037] In the formation step, the contact between the nonionic surfactant and the proteoglycan can be appropriately carried out, for example, according to the states of the nonionic surfactant and the proteoglycan. The nonionic surfactant and the proteoglycan to be subjected to the contact may be, for example, solid or liquid. When the nonionic surfactant and the proteoglycan are solid, it is preferable that the nonionic surfactant and the proteoglycan are dispersed in a solvent in advance and subjected to the contact. When one of the proteoglycan and the nonionic surfactant is solid and the other is liquid, the contact can be carried out, for example, by a known solid-liquid contact method. Specifically, the contact can be carried out by mixing or admixing (hereinafter collectively referred to as "mixing") the nonionic surfactant and the proteoglycan derived from animal tissue. When the nonionic surfactant and the proteoglycan are solid, the contact can be carried out, for example, by adding the nonionic surfactant and the proteoglycan to a solvent. Thereby, in the formation step, a coexistence system (mixing system or mixed solution) of the nonionic surfactant and the proteoglycan containing a complex of the endotoxin and the nonionic surfactant can be prepared. In the coexistence system, it is preferable that the complex of the nonionic surfactant and the endotoxin forms micelles, for example. In this case, it can also be said that the coexistence system contains micelles of the nonionic surfactant and the micelles contain endotoxin.

[0038] In the formation step, it is preferable that the coexistence system is further mixed, for example, after the contact. Thereby, in the formation step, for example, the formation of the complex of the endotoxin and the nonionic surfactant in the coexistence system can be promoted. The mixing may be carried out, for example, using a stirring device such as a vortex mixer, by inversion mixing, or by ultrasonic treatment, but preferably by ultrasonic treatment.

[0039] In the formation step, the proteoglycan derived from the animal tissue may be a proteoglycan that has been pretreated such as roughly purified or purified from the animal tissue, or may be an animal tissue containing the proteoglycan. Examples of the pretreatment include treatments such as mincing, defatting, and purification of the animal tissue. The mincing can be carried out by treatments such as cutting, crushing, and ultrasonic treatment. The purification (extraction) can be carried out, for example, by bringing the animal tissue or its pretreated product into contact with an extraction solution. Examples of the extraction solution include an aqueous guanidine hydrochloride solution, an aqueous acetic acid solution, an aqueous urea solution, and an aqueous magnesium chloride solution.

[0040] The pH of the coexistence system is not particularly limited. For example, it is preferably pH 5 to pH 7.4, preferably pH 5.5 or higher (for example, pH 5.5 to pH 7.4), or pH 7 or lower (for example, pH 5 to pH 7, or pH 5.5 to pH 7). More preferably, it is pH 6 or higher (for example, pH 6 to pH 7.4 or pH 6 to pH 7), or pH 6.8 or lower (for example, pH 5 to pH 6.8, pH 5.5 to pH 6.8, or pH 6 to pH 6.8).

[0041] In the formation step, the temperature of the coexistence system (temperature) is, for example, a temperature below the cloud point of the nonionic surfactant. The temperature can be set according to, for example, the type of the nonionic surfactant and the concentration of the nonionic surfactant in the coexistence system. When the nonionic surfactant is a POEAE-based nonionic surfactant, the temperature is, for example, above -10°C and 40°C or lower (more preferably, above -10°C and less than 22°C).

[0042] In the forming step, the contents of the nonionic surfactant and the proteoglycan in the coexisting system can be set according to the content of endotoxin assumed to be contained in the proteoglycan. Specifically, when the content of endotoxin assumed to be contained in the proteoglycan is relatively high, the content of the nonionic surfactant in the coexisting system can be set relatively high, for example. On the other hand, when the content of endotoxin assumed to be contained in the proteoglycan is relatively low, the content of the nonionic surfactant in the coexisting system can be set relatively low, for example. The contents of the nonionic surfactant and the proteoglycan in the coexisting system may be set according to the content of the proteoglycan, for example. In this case, in the coexisting system, per 1 g of the proteoglycan, the nonionic surfactant can be, for example, 700 μg to 1400 μg (for example, 711 μg, 736 μg).

[0043] The proteoglycan may be subjected to a pretreatment for reducing endotoxin, for example, by precipitating a calcium salt prior to the forming step. Specifically, the pretreatment can be carried out, for example, by contacting or mixing the proteoglycan and a phosphate buffer solution and separating or removing the precipitate containing calcium phosphate salt and the endotoxin formed after the contacting or mixing. The calcium phosphate salt adsorbs the endotoxin, for example. Therefore, the production method of the present disclosure can remove the endotoxin more efficiently, for example, by including the pretreatment. Further, the supernatant after removing the precipitate contains, for example, the proteoglycan with reduced endotoxin. Therefore, in the pretreatment, for example, the supernatant may be recovered, and in the forming step, the recovered supernatant may be used as the proteoglycan. The time of the pretreatment is, for example, preferably 2 hours or more, more preferably 4 to 36 hours, and even more preferably 8 to 24 hours. The temperature of the pretreatment is, for example, preferably 18 to 25°C.

[0044] Next, in the first reduction step, for the coexistence system containing the nonionic surfactant and the proteoglycan derived from animal tissue, the temperature is raised from a temperature below the cloud point of the nonionic surfactant to a temperature equal to or higher than the cloud point. Thereby, in the first reduction step, a surfactant phase containing the nonionic surfactant of the nonionic surfactant is formed, and endotoxin can be concentrated in the surfactant phase (concentration step). As described above, since the endotoxin and the nonionic surfactant form a complex, in the concentration step, when the surfactant phase is formed, the endotoxin in the complex is also concentrated in the surfactant phase, and the proteoglycan is contained in a phase other than the surfactant phase. Therefore, in the first reduction step, for example, the endotoxin in the proteoglycan derived from animal tissue can be separated or extracted into the surfactant phase, and the proteoglycan can be separated or extracted into a phase other than the surfactant phase. When an aqueous solvent is used as the coexistence system, the phase other than the surfactant phase can also be, for example, an aqueous phase.

[0045] The cloud point varies depending on the type of the nonionic surfactant. Therefore, in the concentration step, the temperature below the cloud point and the temperature above the cloud point can be appropriately adjusted according to the type of the nonionic surfactant. The cloud point may be determined, for example, by preparing an aqueous solution containing the nonionic surfactant so that the concentration of the nonionic surfactant in the coexistence system to be used is obtained, and measuring the aqueous solution by the above-described method for measuring the cloud point, or may be determined based on the known cloud point of the nonionic surfactant. When the nonionic surfactant is a commercially available product, the cloud point of the nonionic surfactant can be referred to, for example, the cloud point described in the attached instruction manual. As a specific example, when the nonionic surfactant is polyethylene glycol tert-octylphenyl ether (degree of polymerization: 7 to 8), the cloud point is, for example, about 22°C (preferably, 22°C to 25°C). Therefore, when polyethylene glycol tert-octylphenyl ether (degree of polymerization: 7 to 8) is used as the nonionic surfactant, the temperature below the cloud point can be set to, for example, exceeding -10°C and less than 22°C, preferably about 0°C. Further, the temperature above the cloud point is, for example, 22°C to 50°C, preferably about 40°C. When the nonionic surfactant is polyethylene glycol tert-octylphenyl ether (degree of polymerization: 1 to 5), the cloud point is, for example, about 38°C. When the nonionic surfactant is polyethylene glycol tert-octylphenyl ether (degree of polymerization: 9 to 10), the cloud point is, for example, about 64°C to 66°C.

[0046] In the concentration step, the temperature increase can be carried out by heating the coexistence system. The heating of the coexistence system can be carried out, for example, using a temperature adjustment device such as a heat block or a thermostat.

[0047] The rate of temperature increase (heating rate) from the temperature below the cloud point to the temperature above the cloud point in the coexistence system is not particularly limited. The temperature increase (heating) may be carried out, for example, continuously or stepwise.

[0048] In the concentration step, for example, prior to the temperature increase, it is preferable to mix the co-existing system at a temperature below the cloud point. The mixing may be performed, for example, using a stirring device such as a vortex mixer, or by inversion mixing, and preferably, it is inversion mixing. The mixing time at a temperature below the cloud point can be set, for example, within a range where the temperature of the co-existing system or the solvent of the co-existing system becomes approximately uniform. The mixing time at a temperature below the cloud point is, for example, 1 minute to 10 minutes.

[0049] In the concentration step, for example, after the temperature increase, it is preferable to mix at a temperature above the cloud point. The mixing may be performed, for example, using a vortex mixer or the like, or by inversion mixing, and preferably, it is inversion mixing. The mixing time at a temperature above the cloud point can be set, for example, within a range where the temperature of the solvent becomes approximately uniform. The mixing time at a temperature above the cloud point is, for example, 1 minute to 10 minutes.

[0050] The first reduction step may include a separation step of separating the surfactant phase and the phase other than the surfactant phase. Thereby, in the first reduction step, for example, it can be separated into a layer structure including a layer formed from the surfactant phase and a layer formed from the phase other than the surfactant phase. By including the separation step in the first reduction step, it is possible to easily perform the recovery or removal of at least one of the surfactant phase and the phase other than the surfactant phase in the co-existing system. Therefore, the production method of the present disclosure can efficiently reduce, for example, endotoxin in the proteoglycan. The separation step is preferably performed after the concentration step. The separation method can be performed, for example, by a separation method capable of separating two liquid phases, and as a specific example, it can be performed by centrifugation or the like. The phase other than the surfactant phase is, for example, an aqueous phase. When the separation step is performed by the centrifugation, the conditions of the centrifugation are, for example, about 9500×g.

[0051] The time in the separation step (separation time) can be appropriately set, for example, within a range where the surfactant phase and the phase other than the surfactant phase can be separated. The separation time is, for example, from 1 second to 60 seconds.

[0052] The temperature in the separation step (separation temperature) is, for example, a temperature at which the surfactant phase can be maintained, and as a specific example, it is a temperature equal to or higher than the cloud point. When the nonionic surfactant uses polyethylene glycol tert-octylphenyl ether (degree of polymerization 7 to 8), the separation temperature is, for example, from 22°C to 40°C.

[0053] The production method of the present disclosure may include, in the first reduction step, a first recovery step of recovering the proteoglycan with reduced endotoxin by recovering the phase other than the surfactant phase after the separation. The recovery method is not particularly limited as long as it can recover the aqueous phase, which is the phase other than the surfactant phase in the supernatant. The recovery method may be carried out, for example, using a dropper or the like.

[0054] The production method of the present disclosure may, for example, repeat the first reduction step one or more times with the recovered proteoglycan as the proteoglycan derived from the animal tissue. In the production method of the present disclosure, for example, by repeating the first reduction step one or more times, the amount of endotoxin in the proteoglycan can be further reduced.

[0055] When the first reduction step is repeated one or more times, the surfactants used in each first reduction step may be, for example, the same surfactant or different surfactants.

[0056] When the manufacturing method of the present disclosure repeatedly performs the first reduction step one or more times, for example, the forming step may be performed prior to the repeated first reduction step. In this case, in the first forming step, the coexisting system preferably contains, for example, the aqueous solvent, and in the forming steps after the second time, the coexisting system preferably contains, for example, an aqueous solvent containing the alcohol. Thereby, the manufacturing method of the present disclosure can, for example, remove the endotoxin more efficiently.

[0057] Next, the manufacturing method of the present disclosure may perform a second reduction step on the proteoglycan obtained in the first reduction step (hereinafter, also referred to as "the first proteoglycan"). Specifically, in the second reduction step, endotoxin is reduced from the proteoglycan by treating the first proteoglycan with a chromatography carrier. In the manufacturing method of the present disclosure, for example, by performing the second reduction step, endotoxin that is difficult to associate with the nonionic surfactant can be removed, so that the amount of endotoxin can be further reduced.

[0058] The second reduction step of the present disclosure is estimated to be able to further reduce the endotoxin by a mechanism as described below by using the chromatography carrier. However, the present disclosure is not limited to the following mechanism. Gram-negative bacteria have endotoxins with various types or structures of glycolipids. Since the hydrophobicity and charge of the glycolipids vary depending on their composition, the endotoxins have different degrees of hydrophobicity and charge depending on the Gram-negative bacteria from which they are derived. Also, even in the same Gram-negative bacteria, the composition of the glycolipids produced is not single and includes different compositions, so the degrees of hydrophobicity and charge of the endotoxins are different. On the other hand, it is considered that the endotoxins that can be removed by the nonionic surfactant and the endotoxins that can be removed by the chromatography carrier have different degrees of hydrophobicity and charge. Therefore, in the present disclosure, after reducing the endotoxin by treatment with the nonionic surfactant, by utilizing the difference in the degree of hydrophobicity and / or charge between the removable endotoxins, and further performing treatment using the chromatography carrier, it is estimated that the endotoxin in the proteoglycan can be further reduced.

[0059] In the second reduction step, the contact between the first proteoglycan and the chromatography carrier can be appropriately carried out, for example, according to the state of the first proteoglycan. The first proteoglycan may be, for example, the proteoglycan after the first recovery step, or may be the proteoglycan obtained after performing the solidification step described below on the proteoglycan after the first recovery step. When it is the proteoglycan after the solidification step, it is preferable that the proteoglycan is dispersed in a solvent in advance and subjected to the contact. The solvent can be selected, for example, according to the type of the chromatography carrier described below. As a specific example, when an anion exchange resin is used as the chromatography carrier, the solvent can be, for example, a solvent obtained by adding a salt to an amine-based solvent, Tris hydrochloride buffer solution, HEPES, aqueous potassium chloride solution, aqueous sodium chloride solution, aqueous magnesium chloride solution, etc. When a hydrophobic interaction resin is used as the chromatography carrier, the solvent can be, for example, a solvent obtained by adding a salt to an amine-based solvent, Tris hydrochloride buffer solution, HEPES, aqueous potassium chloride solution, aqueous sodium chloride solution, aqueous magnesium chloride solution, etc.

[0060] Examples of the chromatography resin include ion exchange carriers such as ion exchange resins, and hydrophobic interaction carriers such as hydrophobic interaction resins. Examples of the ion exchange carrier include anion exchange resins, anion exchange monoliths, and the like. By using the hydrophobic interaction carrier or the anion exchange carrier as the chromatography carrier, in the second reduction step, for example, endotoxin in the proteoglycan obtained in the first reduction step can be adsorbed onto the chromatography carrier. Further, by using the anion exchange carrier as the chromatography carrier, the proteoglycan in the proteoglycan obtained in the first reduction step can be adsorbed onto the carrier. Examples of the anion exchange carrier include a solid phase having, as a functional group, primary to tertiary amino groups such as diethylaminoethyl (DEAE) group, polyamine-based functional groups such as polylysine (for example, ε-polylysine); quaternary ammonium groups such as trimethylammonium group, dimethylethanolammonium group; and the like. Examples of the hydrophobic interaction carrier include a solid phase having, as a functional group, polyamine-based functional groups such as polylysine (for example, ε-polylysine); alkyl groups such as butyl group, octyl group, and octadecyl group; and the like. The chromatography carrier may have one type of functional group or a plurality of functional groups. The chromatography carrier may be used alone or in combination of a plurality of types. The shape of the chromatography carrier can be designed according to, for example, the type of the carrier. When the chromatography carrier is a chromatography resin, the shape of the chromatography resin is, for example, spherical or bead-like. When the chromatography carrier is a monolith, the shape of the monolith is, for example, cylindrical. The chromatography carrier may be, for example, a commercially available product. Examples of the chromatography resin having the diethylaminoethyl (DEAE) group include TOYOPEARL (trademark) DEAE-650M (Tosoh Corporation) and the like. Examples of the chromatography resin having polylysine include Selfine ET Clean S (JNC Corporation), Selfine ET Clean L (JNC Corporation), and the like.Examples of the chromatography resin having the octyl group include Nucleosil 100-C8 (GL-Science). Examples of the chromatography resin having the octadecyl group include Nucleosil 100-C18 (GL-Science). Examples of the monolith having the quaternary ammonium group include CIMmultus (registered trademark) QA (Sartorius AG). Examples of the monolith having the diethylaminoethyl (DEAE) group include CIMmultus (registered trademark) DEAE (Sartorius AG). Examples of the monolith having the butyl group include, for example, CIMmultus (registered trademark) C4-HLD, CIMmultus (registered trademark) C4-A (Sartorius AG).

[0061] The second reduction step can be carried out, for example, according to the type of the chromatography carrier. When the chromatography carrier adsorbs the endotoxin and does not adsorb the proteoglycan, the second reduction step includes, for example, an adsorption step of bringing the first proteoglycan into contact with the chromatography carrier to adsorb the endotoxin to the chromatography carrier, and a recovery step (second recovery step) of recovering the proteoglycan with reduced endotoxin. That is, the second reduction step can be carried out, for example, in the F / T mode. On the other hand, when the chromatography carrier adsorbs the endotoxin and the proteoglycan, the second reduction step includes, for example, an adsorption step of bringing the first proteoglycan into contact with the chromatography carrier to adsorb the proteoglycan to the chromatography carrier, and a recovery step (second recovery step) of recovering the adsorbed proteoglycan. That is, the second reduction step can be carried out, for example, in the B / E mode. When the second reduction step is in the B / E mode, the recovery can also be referred to as elution.

[0062] When the second reduction step is carried out in the F / T mode, as the chromatography carrier, for example, a hydrophobic interaction carrier such as the above-mentioned hydrophobic interaction resin can be used. In the adsorption step, the contact between the first proteoglycan and the chromatography carrier can be carried out, for example, by bringing a dispersion liquid containing the first proteoglycan (hereinafter also referred to as "the first dispersion liquid") into contact with the chromatography carrier. Specifically, the contact can be carried out, for example, by mixing the first dispersion liquid and the chromatography carrier. In the adsorption step, for example, due to the contact between the first proteoglycan and the chromatography carrier, an interaction occurs between the endotoxin contained in the first proteoglycan and the chromatography carrier according to the properties of the chromatography carrier. As a result, in the adsorption step, the endotoxin is adsorbed onto the chromatography carrier. On the other hand, in the adsorption step, for example, substantially no interaction occurs between the proteoglycan contained in the first proteoglycan and the chromatography carrier, or the interaction is sufficiently lower compared with the interaction between the endotoxin and the chromatography carrier. As a result, in the adsorption step, for example, the proteoglycan is not substantially adsorbed onto the chromatography carrier, or the adsorption amount is sufficiently lower compared with the adsorption of the endotoxin, and the proteoglycan is maintained in the dispersion liquid.

[0063] As the solvent of the first dispersion liquid, for example, the description of the solvent in the description of the second reduction step can be incorporated.

[0064] In the adsorption step, the amount of the proteoglycan to be brought into contact with the chromatography carrier can be set according to, for example, the amount of endotoxin adsorbed by the chromatography carrier and the amount of endotoxin in the first proteoglycan. As a specific example, in the adsorption step, for example, when Selfine ET Clean S (JNC Corporation) or Selfine ET Clean L (JNC Corporation) (hydrophobic interaction resin) is used in the slurry of the chromatography carrier, 350 to 550 μg of the proteoglycan is brought into contact with 1 ml of the slurry of the chromatography carrier.

[0065] When the chromatography carrier is a hydrophobic interaction resin, the conditions of the first dispersion liquid to be used for contact with the chromatography carrier can be set according to the type of the hydrophobic interaction resin. As specific examples, the conditions of the first dispersion liquid are as follows. Salt concentration in the first dispersion liquid: (In the case of NaCl) 0.1 to 0.2 mol / l or 0.15 to 0.2 mol / l

[0066] Next, in the second recovery step, for example, a dispersion liquid (second dispersion liquid) containing the proteoglycan with reduced endotoxin can be recovered by recovering a liquid fraction from the mixed liquid of the dispersion liquid of the first proteoglycan and the chromatography carrier. The second dispersion liquid can be obtained, for example, by using a known solid-liquid separation method.

[0067] In the second recovery step, after recovering the second dispersion, the chromatography carrier may be brought into contact with a cleaning solution to clean the chromatography carrier. In this case, in the second recovery step, after the cleaning, a dispersion containing the proteoglycan with reduced endotoxin may be recovered by recovering a liquid fraction from the mixed solution of the cleaning solution and the chromatography carrier. The recovered dispersion may be recovered as the second dispersion. Even after the second recovery step is performed, the proteoglycan remains around the chromatography carrier. Therefore, in the second recovery step, the proteoglycan can be extracted and recovered by cleaning the chromatography carrier with the cleaning solution. For this reason, in the second recovery step, for example, the recovery rate of the proteoglycan can be improved by cleaning the chromatography carrier with the cleaning solution and recovering the cleaning solution.

[0068] The solvent of the cleaning solution can be, for example, the same solvent as the first dispersion. The conditions of the cleaning solution can be, for example, the same as those of the first dispersion.

[0069] When the second reduction step is performed in the B / E mode, an anion exchange carrier such as an anion exchange resin can be used as the chromatography carrier. In the adsorption step, the contact between the first proteoglycan and the chromatography carrier can be performed, for example, in the same manner as the F / T mode. In the adsorption step, for example, by contacting the first proteoglycan and endotoxin having a negative charge with the chromatography carrier, the first proteoglycan and the endotoxin bind to the chromatography carrier according to the properties of the chromatography carrier. As a result, in the adsorption step, the proteoglycan and the endotoxin are adsorbed to the chromatography carrier.

[0070] In the adsorption step, the amount of the proteoglycan to be brought into contact with the chromatography carrier can be set according to, for example, the amount of endotoxin adsorbed by the chromatography carrier and the amount of endotoxin in the first proteoglycan. As a specific example, in the adsorption step, for example, when TOYOPEARL (trademark) DEAE-650M (Tosoh Corporation) (anion exchange resin) is used in the slurry of the chromatography carrier, 350 to 550 μg of the proteoglycan is brought into contact with 1 ml of the slurry of the chromatography carrier.

[0071] When the chromatography carrier is an anion exchange resin, the conditions of the first dispersion liquid to be used for contact with the chromatography carrier can be set according to the type of the anion exchange resin. As a specific example, the conditions of the first dispersion liquid are as follows. Salt concentration in the first dispersion liquid: (In the case of NaCl) 0.1 to 0.2 mol / l or 0.15 to 0.2 mol / l

[0072] The proteoglycan has, for example, a sulfo group. On the other hand, the endotoxin has, for example, a phosphate group. Therefore, the proteoglycan and the endotoxin differ in the charge and hydrophobicity of anions. Thus, in the second reduction step, after the adsorption step, a solvent having a higher salt concentration than the solvent in the adsorption step is passed through the chromatography carrier after adsorption, so that the proteoglycan can be preferentially eluted from the chromatography carrier. For this reason, the second reduction step preferably includes, for example, an elution step of eluting the proteoglycan adsorbed on the chromatography carrier after the adsorption step.

[0073] In the elution step, examples of the elution solvent include an aqueous potassium chloride solution, an aqueous sodium chloride solution, and an aqueous magnesium chloride solution. The elution solvent is preferably a solvent having a higher salt concentration than the solvent used in the adsorption step, for example. As a specific example, the conditions of the elution solvent are as follows. Salt concentration of the elution solvent: (In the case of NaCl) 0.4 to 2 mol / l, 0.45 to 1 mol / l, or 0.5 to 1 mol / l

[0074] Next, in the second recovery step, for example, by recovering the eluate eluted in the elution step, a dispersion (second dispersion) containing the proteoglycan with reduced endotoxin can be recovered.

[0075] In the second reduction step, prior to the adsorption step, the chromatographic carrier may be equilibrated. When an ion exchange resin or a hydrophobic interaction resin is used for the chromatographic carrier, as the equilibration solution of the chromatographic resin, for example, the same solvent as the solvent used in the adsorption step can be used.

[0076] The chromatographic carrier may be used, for example, in a form filled in a column. In this case, the adsorption step can be carried out by loading the equilibration solution, the first dispersion, the washing solution, the elution solution, etc. onto the column filled with the chromatographic carrier.

[0077] In the production method of the present disclosure, for example, the second reduction step may be repeatedly carried out one or more times on the proteoglycan recovered in the second recovery step, that is, in the production method of the present disclosure, the second reduction step may be carried out a plurality of times. In the production method of the present disclosure, for example, by carrying out the second reduction step a plurality of times, the amount of endotoxin in the proteoglycan can be further reduced. In this case, the description in the second reduction step can be applied by substituting "the proteoglycan obtained in the first recovery step" with "the proteoglycan obtained in the second recovery step".

[0078] When the second reduction step is carried out a plurality of times, the second reduction step may be carried out, for example, the following steps (A) and (B) on the proteoglycan obtained in the first reduction step. Step (A): An adsorption step of bringing the proteoglycan into contact with the chromatography carrier to adsorb endotoxin onto the chromatography carrier, and A recovery step of recovering the proteoglycan with reduced endotoxin; (B) step: An adsorption step of bringing the proteoglycan into contact with the chromatography carrier to adsorb the proteoglycan onto the chromatography carrier, and A recovery step of recovering the adsorbed proteoglycan.

[0079] In the step (A), it is preferable to use an ion exchange resin or a hydrophobic interaction resin as the chromatography carrier. In the step (B), it is preferable to use, for example, an anion exchange carrier having a DEAE group as the chromatography carrier.

[0080] When the second reduction step is carried out a plurality of times, the second reduction step may include a first treatment step of treating the proteoglycan obtained in the first reduction step with the chromatography carrier, and a second treatment step of treating the treated proteoglycan with the chromatography carrier. The chromatography carrier in the first treatment step and the chromatography carrier in the second treatment step are preferably, for example, different chromatography carriers. The first treatment step and the second treatment step can be carried out in the same manner as the second reduction step.

[0081] In the production method of the present disclosure, after the first reduction step or after the second reduction step, the endotoxin concentration of the proteoglycan contained in the recovered aqueous phase may be measured. The measurement of the endotoxin concentration can be carried out by, for example, a gel clotting method (LAL test) and an optical quantification method (colorimetric method) using a lysate reagent made from hemocyte extracts of horseshoe crabs.

[0082] The concentration of endotoxin in the first reduction step is, for example, 10 EU / mg or less, 7 EU / mg or less. Also, the concentration of endotoxin after the second reduction step is, for example, 4 EU / mg or less or 2 EU / mg or less, preferably 1 EU / mg or less, more preferably 0.61 EU / mg or less or 0.4 EU / mg or less, and even more preferably 0.04 EU / mg or less. Note that the lower limit of the concentration of the endotoxin is preferably 0 EU / mg, for example, but in reality, it is preferably more than 0 EU / mg (about the detection limit).

[0083] In the production method of the present disclosure, after the first reduction step or the second reduction step, the molecular weight of the proteoglycan contained in the recovered aqueous phase may be measured. The molecular weight of the proteoglycan means the peak top molecular weight measured by the GPC (Gel Permeation Chromatography) method. The molecular weight of the proteoglycan can be measured by using the GPC method or the like. The GPC method is carried out under the following conditions, for example, and the standard sample (molecular weight marker, pullulan) is individually injected into the HPLC system to obtain a molecular weight calibration curve for calculation.

[0084] (Measurement conditions for peak top molecular weight (Mp)) HPLC system: LC-20AD (manufactured by Shimadzu Corporation) Column: TSKgel G5000-PWXL φ7.8 mm × 300 mm (manufactured by Tosoh Corporation) Eluent: Phosphate buffer solution with pH 6.8 Flow rate: 0.5 mL / min Column temperature: 40 °C Detector: Differential refractive index detector (RID-20A manufactured by Shimadzu Corporation) Injection volume: 50 μL Molecular weight marker: Shodex STANDARD P-82 (pullulan)

[0085] The peak top molecular weight of the proteoglycan after the first reduction step or the second reduction step is, for example, 300,000 to 1,300,000, 400,000 to 1,200,000.

[0086] The production method of the present disclosure may include a fractionation step of fractionating a proteoglycan that satisfies a predetermined peak top molecular weight from the phase other than the recovered surfactant phase after the first reduction step or the second reduction step. The fractionation method is not particularly limited as long as it can fractionate within a range that can maintain the molecular weight of a predetermined proteoglycan. The fractionation method can be carried out, for example, by a known method capable of fractionating components based on molecular weight. Specifically, it may be carried out using an ultrafiltration membrane (molecular weight fractionation membrane) or a dialysis membrane. For example, it may also be carried out by silica gel column chromatography, gel filtration chromatography, ion exchange column chromatography, etc. The fractionation molecular weight of the molecular weight fractionation membrane can be set according to the target molecular weight, for example.

[0087] The time (fractionation time) in the fractionation step is not particularly limited as long as it can fractionate within a range that can maintain the molecular weight of a predetermined proteoglycan. The fractionation time is, for example, 1 day to 2 weeks.

[0088] The production method of the present disclosure may include a solidification step of solidifying the fractionated proteoglycan after the fractionation step. The solidification method is not particularly limited as long as it can solidify within a range that can maintain the molecular weight of a predetermined proteoglycan. The solidification step can be carried out, for example, by a known method capable of solidifying proteoglycan from the liquid containing the proteoglycan after fractionation. Specifically, it can be carried out by a drying treatment such as freeze-drying or spray drying.

[0089] <Agent> In another aspect, the present disclosure provides an agent for use in reducing endotoxin of proteoglycan derived from animal tissues. The agent of the present disclosure contains a nonionic surfactant. According to the agent of the present disclosure, since it can reduce the endotoxin of proteoglycan derived from animal tissues, it can be suitably used for the production of the proteoglycan with reduced endotoxin.

[0090] The agent of the present disclosure can, for example, make use of the description of the method for producing the proteoglycan of the present disclosure.

[0091] <Composition> In another aspect, the present disclosure provides a composition comprising a proteoglycan derived from animal tissue. The composition of the present disclosure contains a proteoglycan and endotoxin, and the endotoxin content ratio with respect to the composition is 4 EU / mg or less, or 2 EU / mg or less, preferably 1 EU / mg or less, more preferably 0.61 EU / mg or less or 0.4 EU / mg or less, and even more preferably 0.04 EU / mg or less. The lower limit of the endotoxin content ratio is preferably 0 EU / mg, for example, but in reality, it is preferably more than 0 EU / mg (about the detection limit).

[0092] The composition of the present disclosure contains, for example, a proteoglycan obtained by the method for producing the proteoglycan of the present disclosure. The composition of the present disclosure can, for example, make use of the description of the method for producing the proteoglycan of the present disclosure.

Examples

[0093] Next, examples of the present disclosure will be described. However, the present disclosure is not limited by the following examples. Commercially available reagents were used based on their protocols unless otherwise indicated. Note that "mol / l" may also be denoted as "M".

[0094] [Example 1] By the production method of the present disclosure, endotoxin could be reduced from a proteoglycan derived from animal tissue, and a predetermined molecular weight of the proteoglycan (molecular weight of about 450,000) could be maintained.

[0095] 7.8 mg of salmon-derived proteoglycan was added to 0.2 mol / l NaCl containing phosphate buffer (pH 6). After the addition, stirring and ultrasonic treatment were performed at about 23°C. Then, 5 μl of Triton (trademark)-X114 was added. After the addition, inversion mixing was performed under the conditions of 0°C for 6 minutes. After the inversion mixing, inversion mixing was performed under the conditions of 37°C for 5 minutes. After the inversion mixing, centrifugation was performed under the conditions of 23°C for 30 seconds at 10,000 rpm. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered and dialysis was performed with a fractional molecular weight (MWCO: 12,000 - 16,000). After the dialysis, the proteoglycan was lyophilized to obtain a proteoglycan solid. Then, for the proteoglycan before treatment and the proteoglycan solid, the concentration of endotoxin was measured by the LAL method using a kit (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, manufactured by GenScript). Also, for the proteoglycan before treatment and the proteoglycan solid, HPLC analysis was performed under the following measurement conditions.

[0096] (Measurement conditions for HPLC) HPLC system: LC-20AD (manufactured by Shimadzu Corporation) Column: TSKgel G5000-PWXL Φ7.8 mm × 300 mm (manufactured by Tosoh Corporation) Eluent: Phosphate buffer pH 6.8 Flow rate: 0.5 mL / min Column temperature: 40°C Detector: Differential refractive index detector (RID-10A manufactured by Shimadzu Corporation) Injection volume: 50 μL Molecular weight marker: Shodex STANDARD P-82 (pullulan) The peak top molecular weight (Mp) and weight average molecular weight (Mw) / number average molecular weight (Mn) of the molecular weight marker are as follows. STD P-800: Mp: 739,000, Mw / Mn: 1.24 STD P-400: Mp: 348,000, Mw / Mn: 1.33 STD P-200: Mp: 216,000, Mw / Mn: 1.22 STD P-100: Mp: 107,000, Mw / Mn: 1.12 STD P-50: Mp: 49,400, Mw / Mn: 1.08 STD P-20: Mp: 22,000, Mw / Mn: 1.08 STD P-10: Mp: 9,800, Mw / Mn: 1.07 STD P-5: Mp: 6,300, Mw / Mn: 1.09

[0097] As a result, in the proteoglycan before treatment, the endotoxin concentration was 400 EU / mg, while in the proteoglycan solid, the endotoxin concentration was 4 EU / mg. Also, in the proteoglycan before treatment, the peak top molecular weight was about 450,000, and in the proteoglycan solid, the peak top molecular weight was about 450,000. From the above results, it was found that by the method of the present disclosure (Example 1), endotoxin can be reduced from the proteoglycan derived from animal tissues and the predetermined molecular weight of the proteoglycan can be maintained.

[0098] In addition, the following Comparative Examples 1A to 1C were also carried out.

[0099] [Comparative Example 1A] The sample of Comparative Example 1A was prepared, the endotoxin concentration was measured, and HPLC analysis were performed in the same manner as in Example 1, except that ethanol containing 0.001 N sodium hydroxide was used instead of the phosphate buffer (pH 6) containing 0.2 mol / l NaCl. As a result, in the sample of Comparative Example 1A, the maintenance of the predetermined molecular weight (about 450,000) of the proteoglycan was confirmed, but the reduction of endotoxin was not confirmed.

[0100] [Comparative Example 1B] Instead of using a phosphate buffer (pH 6) containing 0.2 mol / l NaCl, ethanol containing 0.01 N sodium hydroxide was used, and in the same manner as in Example 1, the sample of Comparative Example 2 was prepared, the endotoxin concentration was measured, and HPLC analysis was performed. As a result, in the sample of Comparative Example 1B, the endotoxin concentration was 167.8 EU / mg. From the above results, it was found that in the sample of Comparative Example 1B, the reduction of endotoxin was not achieved compared to Example 1.

[0101] [Comparative Example 1C] Instead of using a phosphate buffer (pH 6) containing 0.2 mol / l NaCl, ethanol containing 0.025 N sodium hydroxide was used, and in the same manner as in Example 1, the sample of Comparative Example 1C was prepared, the endotoxin concentration was measured, and HPLC analysis was performed. As a result, in the sample of Comparative Example 3, the endotoxin concentration was 33.7 EU / mg, and the molecular weight of the proteoglycan was the predetermined molecular weight (about 450,000). From the above results, it was found that in the sample of Comparative Example 1C, while the endotoxin concentration was reduced, the molecular weight of the predetermined proteoglycan was not maintained.

[0102] [Example 2A] It was confirmed that the endotoxin in the proteoglycan derived from animal tissues can be reduced using the production method of the present disclosure.

[0103] 7.8 mg of proteoglycan derived from salmon was added to 0.05 mol / l phosphate buffer (pH 6.0).

[0104] After the addition, 5 μl of Triton-X114 was added. After the addition, stirring was performed and the mixture was allowed to stand on ice for 10 minutes. After the standing, the mixture was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was performed at 23°C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that the mixture was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, three times the amount of saturated ethanol was added, and crystallization was performed. After the crystallization, the saturated ethanol was removed and saturated ethanol was added again. After the addition, centrifugation was performed at 23°C for 3 minutes at 9060×g. After the centrifugation, the saturated ethanol was removed and vacuum drying was performed to obtain powdered proteoglycan. Thereafter, the endotoxin concentration was measured by the LAL method for the proteoglycan before treatment and the powdered proteoglycan. For the LAL method, a kit (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, manufactured by GenScript) was used.

[0105] As a result, the endotoxin concentration in the proteoglycan before treatment was 250.7 EU / mg, while the endotoxin concentration in the powdered proteoglycan was 9.2 EU / mg. From the above results, it was found that endotoxin can be reduced from proteoglycans derived from animal tissues by the method of the present disclosure (Example 2A).

[0106] [Example 2B] It was confirmed that the endotoxin in proteoglycans derived from animal tissues can be reduced by using the production method of the present disclosure.

[0107] 7.8 mg of proteoglycan derived from salmon was added to 0.05 mol / l phosphate buffer (pH 6.0), and after the addition, the mixture was allowed to stand at 23°C for 24 hours.

[0108] After the standing, the precipitate was removed to obtain a supernatant. Then, 5 μl of Triton (trademark)-X114 was added. After the addition, stirring was performed and the mixture was allowed to stand on ice under the condition of 10 minutes. After the standing, the mixture was allowed to stand at 40 °C for 10 minutes. After the standing, centrifugation was performed at 23 °C for 3 minutes under the condition of 9060×g. After the centrifugation, it was confirmed that the mixture was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, three times the amount of saturated ethanol was added, and crystallization was performed. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 23 °C for 3 minutes under the condition of 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Then, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Example 2B was measured.

[0109] As a result, in the powdered proteoglycan of Example 2B, the endotoxin concentration was 9.2 EU / mg. From the above results, it was found that endotoxin can also be reduced from proteoglycan derived from animal tissues in the method (Example 2B) of the present disclosure.

[0110] In addition, the following Comparative Example 2 was also carried out.

[0111] [Comparative Example 2] 7.8 mg of proteoglycan derived from salmon was added to 0.05 mol / l phosphate buffer (pH 6.0). After the addition, the mixture was allowed to stand at 23 °C for 24 hours.

[0112] After the standing, the precipitate was removed to obtain a supernatant. Three times the amount of saturated ethanol was added to the supernatant for crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 23 °C for 3 minutes under the condition of 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Then, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Comparative Example 2 was measured.

[0113] As a result, in the powdered proteoglycan of Comparative Example 2, the endotoxin concentration was 170.1 EU / mg.

[0114] [Example 3A] It was confirmed that the endotoxin in the proteoglycan derived from animal tissue can be reduced by the production method of the present disclosure.

[0115] A powdered proteoglycan was obtained in the same manner as in Example 2A, except that PBS (pH 7.2, manufactured by Fujifilm Wako Pure Chemical Corporation, Cat No.: 164 - 28713) was used as the solvent for the proteoglycan solution. Thereafter, the endotoxin concentration of the powdered proteoglycan of Example 3A was measured in the same manner as in Example 2A.

[0116] As a result, the endotoxin concentration of the proteoglycan before treatment was 304.2 EU / mg, while the endotoxin concentration of the powdered proteoglycan was 21.4 EU / mg. From the above results, it was found that the endotoxin can be reduced from the proteoglycan derived from animal tissue by the method (Example 3A) of the present disclosure.

[0117] [Example 3B] It was confirmed that the endotoxin in the proteoglycan derived from animal tissue can be reduced by repeating the reduction step in the production method of the present disclosure.

[0118] To PBS (pH 7.2, manufactured by FUJIFILM Wako Pure Chemical Corporation, Cat No.: 164-28713), a proteoglycan derived from salmon was added. After the addition, stirring and ultrasonic treatment were carried out at 23°C to obtain a proteoglycan solution of 5 mg / ml. Then, 1 (v / v)% Triton™-X114 was added. After the addition, it was allowed to stand on ice for 10 minutes. After the standing, it was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was carried out at 23°C for 3 minutes under 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered so as not to contain the solution at the boundary between the lower layer and the upper layer. After the recovery, 1 (v / v)% Triton™-X114 was added. After the addition, it was allowed to stand on ice for 10 minutes. After the standing, it was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was carried out at 23°C for 3 minutes under 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, three times the amount of saturated ethanol was added, and crystallization was carried out. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was carried out at 23°C for 3 minutes under 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was carried out to obtain powdered proteoglycan. Then, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Example 3B was measured.

[0119] As a result, in the powdered proteoglycan of Example 3B, the endotoxin concentration was 7.8 EU / mg. Also, in Example 3B, compared with Example 3A, the endotoxin concentration was reduced to about 1 / 3. From the above results, it was found that by repeating the formation step and the reduction step in the production method of the present disclosure, the endotoxin concentration of the proteoglycan can be further reduced.

[0120] [Example 4A] It was confirmed that the endotoxin in the proteoglycan derived from animal tissue can be reduced by the production method of the present disclosure.

[0121] A powdered proteoglycan was obtained in the same manner as in Example 2A, except that PBS (pH 7.2, manufactured by Fujifilm Wako Pure Chemical Corporation, Cat No.: 164-28713) was used as the solvent for the proteoglycan solution. Thereafter, the endotoxin concentration of the powdered proteoglycan of Example 4A was measured in the same manner as in Example 2A.

[0122] As a result, the endotoxin concentration of the proteoglycan before treatment was 403.7 EU / mg, whereas the endotoxin concentration of the powdered proteoglycan was 14.0 EU / mg. From the above results, it was found that the method of the proteoglycan of the present disclosure (Example 4A) can reduce endotoxin from proteoglycans derived from animal tissues.

[0123] [Example 4B] It was confirmed that endotoxin in proteoglycans derived from animal tissues can be reduced by using an aqueous solvent containing 1 (v / v)% ethanol in the coexisting system of the forming step of the production method of the present disclosure.

[0124] To PBS (pH 7.2, manufactured by Fujifilm Wako Pure Chemical Corporation, Cat No.: 164 - 28713), a proteoglycan derived from salmon was added. After the addition, stirring and ultrasonic treatment were performed at about 23°C to obtain a 5 mg / ml proteoglycan solution. Then, 1 (v / v)% ethanol and 1 (v / v)% Triton (trademark)-X114 were added. After the addition, it was allowed to stand on ice for 10 minutes. After the standing, it was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was performed at 23°C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, three times the amount of saturated ethanol was added, and crystallization was carried out. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 23°C for 3 minutes at 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Then, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Example 4B was measured.

[0125] As a result, in the powdered proteoglycan of Example 4B, the endotoxin concentration was 13.7 EU / mg. From the above results, it was found that by using an aqueous solvent containing 1 (v / v)% ethanol in the coexistence system of the formation step of the production method of the present disclosure, endotoxin can be reduced from proteoglycans derived from animal tissues.

[0126] [Example 4C] It was confirmed that by repeatedly performing the reduction step in the production method of the present disclosure, endotoxin in proteoglycans derived from animal tissues can be reduced.

[0127] A powdered proteoglycan was obtained in the same manner as in Example 3B, except that PBS (pH 7.2, manufactured by Fujifilm Wako Pure Chemical Corporation, Cat No.: 164-28713) was used as the solvent for the proteoglycan solution. Thereafter, the endotoxin concentration of the powdered proteoglycan of Example 4C was measured by the LAL method in the same manner as in Example 2A.

[0128] As a result, the endotoxin concentration in the powdered proteoglycan of Example 4C was 12.6 EU / mg. From the above results, it was found that the endotoxin concentration of the proteoglycan can be further reduced by repeating the reduction step in the production method of the present disclosure.

[0129] [Example 4D] It was confirmed that the endotoxin in the proteoglycan derived from animal tissue can be reduced by repeating the reduction step in the production method of the present disclosure and using an aqueous solvent containing 1 (v / v)% ethanol in the coexisting system of the second forming step.

[0130] To PBS (pH 7.2, manufactured by FUJIFILM Wako Pure Chemical Corporation, Cat No.: 164-28713), a proteoglycan derived from salmon was added. After the addition, stirring and ultrasonic treatment were performed at about 23°C to obtain a proteoglycan solution of 5 mg / ml. Thereafter, 1 (v / v)% Triton™-X114 was added. After the addition, it was allowed to stand on ice for 10 minutes. After the standing, it was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was performed at 23°C for 3 minutes under 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered so as not to contain the solution at the boundary between the lower layer and the upper layer. After the recovery, 1% by volume of ethanol and 1 (v / v)% Triton™-X114 were added. After the addition, it was allowed to stand on ice for 10 minutes. After the standing, it was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was performed at 23°C for 3 minutes under 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, 3 times the volume of saturated ethanol was added, and crystallization was carried out. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 23°C for 3 minutes under 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Thereafter, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Example 4D was measured by the LAL method.

[0131] As a result, in the powdered proteoglycan of Example 4D, the endotoxin concentration was 9.7 EU / mg. From the above results, it was found that by repeating the reduction step in the production method of the present disclosure and using an alcohol-containing aqueous solvent in the coexistence system of the second forming step, the endotoxin concentration of the proteoglycan can be further reduced.

[0132] [Example 5A] A proteoglycan derived from salmon was added to 0.05 mol / l phosphate buffer (pH 6.0) to prepare a proteoglycan solution of 5 mg / ml.

[0133] After the preparation, 1% Triton-X114 was added. After the addition, stirring was performed, and the mixture was allowed to stand on ice under the condition of 10 minutes. After the standing, the mixture was allowed to stand under the conditions of 40 °C for 10 minutes. After the standing, centrifugation was performed under the conditions of 23 °C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that the mixture was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, three times the amount of saturated ethanol was added, and crystallization was performed. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugal separation was performed under the conditions of 23 °C for 3 minutes at 9060×g. After the centrifugal separation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Thereafter, in the same manner as in Example 2A, the endotoxin concentration was measured for the proteoglycan before treatment and the powdered proteoglycan of Example 5A.

[0134] As a result, in the proteoglycan before treatment, the endotoxin concentration was 362.6 EU / mg, whereas in the powdered proteoglycan of Example 5A, the endotoxin concentration was 10.5 EU / mg. From the above results, it was found that the endotoxin concentration of proteoglycan can be reduced by the production method of the present disclosure.

[0135] [Example 5B] Salmon-derived proteoglycan was added to 0.05 mol / l phosphate buffer (pH 6.0) to prepare a proteoglycan solution of 5 mg / ml.

[0136] After the preparation, 1% by volume of Triton-X114 was added. After the addition, stirring was performed, and the mixture was allowed to stand on ice for 10 minutes. After the standing, the mixture was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was performed at 23°C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that separation occurred into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered so as not to contain the solution at the interface between the lower layer and the upper layer. After the recovery, 1% (v / v) of Triton-X114 was added. After the addition, the mixture was allowed to stand on ice for 10 minutes. After the standing, the mixture was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was performed at 23°C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that separation occurred into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, three times the volume of saturated ethanol was added, and crystallization was performed. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 23°C for 3 minutes at 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Thereafter, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Example 5B was measured.

[0137] As a result, in the powdered proteoglycan of Example 5B, the endotoxin concentration was 9.7 EU / mg. From the above results, it was found that the endotoxin concentration of proteoglycan can be reduced by the production method of the present disclosure.

[0138] [Example 5C] Salmon-derived proteoglycan was added to 0.05 mol / l phosphate buffer (pH 6.0) to prepare a 5 mg / ml proteoglycan solution.

[0139] After the preparation, 1% Triton-X45 was added. After the addition, stirring was performed, and the mixture was allowed to stand at 4°C for 10 minutes. After the standing, the mixture was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was performed at 37°C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that the mixture was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, three times the amount of saturated ethanol was added, and crystallization was performed. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 37°C for 3 minutes at 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Thereafter, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Example 5C was measured.

[0140] As a result, in the powdered proteoglycan of Example 5C, the endotoxin concentration was 6.9 EU / mg. From the above results, it was found that the endotoxin concentration of proteoglycan can be reduced by the production method of the present disclosure.

[0141] [Example 5D] Salmon-derived proteoglycan was added to 0.05 mol / l phosphate buffer (pH 6.0) to prepare a 5 mg / ml proteoglycan solution.

[0142] After the above preparation, 1% Triton-X114 was added. After the addition, stirring was performed and the mixture was allowed to stand on ice for 10 minutes. After the standing, it was allowed to stand at 40 °C for 10 minutes. After the standing, centrifugation was performed at 23 °C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered so as not to contain the solution at the interface between the lower layer and the upper layer. After the recovery, a 1 (v / v)% mixture obtained by mixing Triton-X45 and Triton-X100 at a ratio of 3:10 was added. After the addition, it was allowed to stand at 4 °C for 10 minutes. After the standing, it was allowed to stand at 40 °C for 10 minutes. After the standing, centrifugation was performed at 37 °C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, three times the amount of saturated ethanol was added, and crystallization was performed. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 37 °C for 3 minutes at 9060×g. After the centrifugation, the saturated ethanol was removed, vacuum drying was performed, and a powdered proteoglycan was obtained. Thereafter, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Example 5D was measured.

[0143] As a result, in the powdered proteoglycan of Example 5D, the endotoxin concentration was 9.2 EU / mg. From the above results, it was found that the endotoxin concentration of the proteoglycan can be reduced by the production method of the present disclosure.

[0144] [Example 5E] Salmon-derived proteoglycan was added to 0.05 mol / l phosphate buffer (pH 6.0) to prepare a 5 mg / ml proteoglycan solution.

[0145] After the preparation, 1% by volume of Triton-X114 was added. After the addition, stirring was performed, and the mixture was allowed to stand on ice for 10 minutes. After the standing, it was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was performed at 23°C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered so as not to contain the solution at the interface between the lower layer and the upper layer. After the recovery, a 1 (v / v)% mixture prepared by mixing Triton-X45 and Triton-X100 at a ratio of 3:20 was added. After the addition, it was allowed to stand at 4°C for 10 minutes. After the standing, it was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was performed at 37°C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, three times the amount of saturated ethanol was added, and crystallization was performed. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 37°C for 3 minutes at 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Thereafter, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Example 5E was measured.

[0146] As a result, in the powdered proteoglycan of Example 5E, the endotoxin concentration was 6.9 EU / mg. From the above results, it was found that the endotoxin concentration of proteoglycan can be reduced by the production method of the present disclosure.

[0147] [Example 6A] It was confirmed that endotoxin can be reduced from proteoglycan derived from animal tissue by the production method of the present disclosure.

[0148] Proteoglycan derived from salmon was added to 0.05 mol / l phosphate buffer (pH 6.0) to prepare a proteoglycan solution at 5 mg / ml.

[0149] After the preparation, 1% by volume of Triton-X114 was added. After the addition, stirring was performed, and the mixture was allowed to stand on ice for 10 minutes. After the standing, the mixture was allowed to stand at 40°C for 10 minutes. After the standing, centrifugation was performed at 23°C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that the mixture was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, 3 volumes of saturated ethanol was added, and crystallization was performed. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 23°C for 3 minutes at 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Thereafter, chromatography was performed on the powdered proteoglycan using an anion exchange resin. Specifically, the powdered proteoglycan was dissolved in 0.2 mol / l NaCl. After the dissolution, the sample was charged into a DEAE M column (manufactured by TOSOH Corporation), and the NaCl in which the proteoglycan was dissolved was passed through. After the passage, the column was washed with 0.2 mol / l NaCl. After the washing, 0.5 mol / l NaCl was passed through to elute the proteoglycan. Thereafter, the eluate containing the eluted proteoglycan was subjected to predetermined desalting, and powdered proteoglycan was obtained by predetermined freeze-drying. Thereafter, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Example 6A was measured.

[0150] As a result, in the proteoglycan treated with Triton-X114 and subjected to chromatography using DEAE, the endotoxin concentration was 0.83 EU / mg. From the above results, it was found that the endotoxin concentration of proteoglycan can be reduced by the production method of the present disclosure. In Example 6A, the proteoglycan was eluted by passing 0.5 mol / l NaCl, but it was also confirmed that the proteoglycan could be eluted in the same manner when 1 mol / l and 2 mol / l NaCl were used.

[0151] [Example 6B] It was confirmed that endotoxin can be reduced from proteoglycans derived from animal tissues by the production method of the present disclosure.

[0152] Proteoglycan derived from salmon was added to 0.05 mol / l phosphate buffer (pH 6.0) to prepare a proteoglycan solution of 5 mg / ml.

[0153] After the preparation, 1% by volume of Triton (trademark)-X114 was added. After the addition, stirring was performed and the mixture was allowed to stand on ice for 10 minutes. After the standing, it was allowed to stand at 40 °C for 10 minutes. After the standing, centrifugation was performed at 23 °C for 3 minutes at 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, 3 times the volume of saturated ethanol was added, and crystallization was carried out. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 23 °C for 3 minutes at 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Thereafter, chromatography was performed on the powdered proteoglycan using an anion exchange resin. Specifically, the powdered proteoglycan was dissolved in 0.2 mol / l NaCl. After the dissolution, sample charging was performed on Selfine (trademark) ET Clean S (manufactured by JNC Corporation), the NaCl in which the proteoglycan was dissolved was passed through, and the flow-through was recovered. After the passage, the column was washed with 0.2 mol / l NaCl, and the flow-through was recovered. Thereafter, the flow-through containing proteoglycan was subjected to predetermined desalting, and powdered proteoglycan was obtained by predetermined freeze-drying. Thereafter, the endotoxin concentration of the powdered proteoglycan of Example 6B was measured in the same manner as in Example 2A.

[0154] As a result, in the proteoglycan that had been subjected to Triton (trademark)-X114 treatment and chromatographic treatment with polylysine, the endotoxin concentration was 1.93 EU / mg. From the above results, it was found that the endotoxin concentration of proteoglycan can be reduced by the production method of the present disclosure.

[0155] [Example 6C] It was confirmed that the production method of the present disclosure can reduce endotoxin from proteoglycans derived from animal tissues.

[0156] Proteoglycan derived from salmon was added to 0.05 mol / l phosphate buffer (pH 6.0) to prepare a proteoglycan solution at 5 mg / ml.

[0157] After the preparation, 1% Triton-X114 was added. After the addition, stirring was performed, and it was allowed to stand on ice for 10 minutes. After the standing, it was allowed to stand at 40 °C for 10 minutes. After the standing, centrifugation was performed at 23 °C for 3 minutes under the condition of 9060×g. After the centrifugation, it was confirmed that it was separated into two layers. After the confirmation, the upper layer containing the proteoglycan was recovered, three times the amount of saturated ethanol was added, and crystallization was carried out. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed at 23 °C for 3 minutes under the condition of 9060×g. After the centrifugation, the saturated ethanol was removed, and vacuum drying was performed to obtain powdered proteoglycan. Then, chromatography was performed on the powdered proteoglycan using an anion exchange resin. Specifically, the powdered proteoglycan was dissolved in 0.2 mol / l NaCl. After the dissolution, a sample charge was applied to Sephacryl (trademark) ET Clean L (manufactured by JNC), the NaCl in which the proteoglycan was dissolved was passed through, and the flow-through was recovered. After the passage, the column was washed with 0.2 mol / l NaCl, and the flow-through was recovered. Then, the flow-through containing the proteoglycan was subjected to predetermined desalting, and powdered proteoglycan was obtained by predetermined lyophilization. Then, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Example 6C was measured.

[0158] As a result, in the proteoglycan subjected to Triton-X114 treatment and chromatography treatment with polylysine, the endotoxin concentration was 1.93 EU / mg. From the above results, it was found that the endotoxin concentration of the proteoglycan can be reduced by the production method of the present disclosure.

[0159] [Comparative Example 6A] Regarding the proteoglycan derived from salmon, chromatography was performed using an anion exchange resin. Specifically, the proteoglycan derived from salmon was dissolved in 0.2 mol / l NaCl. After the dissolution, the sample was charged onto a DEAE M column (manufactured by TOSOH Corporation), and the NaCl in which the proteoglycan was dissolved was passed through. After the passage, the column was washed with 0.2 mol / l NaCl. After the washing, 0.5 mol / l NaCl was passed through to elute the proteoglycan. Thereafter, the eluate containing the eluted proteoglycan was subjected to predetermined desalting, and a powdered proteoglycan was obtained by predetermined freeze-drying. Thereafter, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Comparative Example 6A was measured.

[0160] As a result, in the proteoglycan subjected only to the chromatography treatment with DEAE, the endotoxin concentration was 90.6 EU / mg. From the above results, it was found that the endotoxin concentration of the proteoglycan can be reduced by the production method of the present disclosure.

[0161] [Comparative Example 6B] Regarding the proteoglycan derived from salmon, chromatography was performed using an anion exchange resin. Specifically, the proteoglycan derived from salmon was dissolved in 0.2 mol / l NaCl. After the dissolution, the sample was charged onto Selfine (trademark) ET Clean S (manufactured by JNC Corporation), the NaCl in which the proteoglycan was dissolved was passed through, and the flow-through was collected. After the passage, the column was washed with 0.2 mol / l NaCl, and the flow-through was collected. Thereafter, the flow-through containing the proteoglycan was subjected to predetermined desalting, and a powdered proteoglycan was obtained by predetermined freeze-drying. Thereafter, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Comparative Example 6B was measured.

[0162] As a result, in the proteoglycan that had undergone only the chromatography treatment with polylysine, the endotoxin concentration was 122.2 EU / mg.

[0163] [Comparative Example 6C] For the proteoglycan derived from salmon, chromatography was performed using an anion exchange resin. Specifically, the proteoglycan was dissolved in 0.2 mol / l NaCl. After the dissolution, the sample was charged into Selfine (trademark) ET Clean L (manufactured by JNC), and the NaCl in which the proteoglycan was dissolved was passed through, and the flow-through was collected. After the passage, the column was washed with 0.2 mol / l NaCl, and the flow-through was collected. Then, the flow-through containing the proteoglycan was subjected to predetermined desalting, and a powdered proteoglycan was obtained by predetermined lyophilization. Thereafter, in the same manner as in Example 2A, the endotoxin concentration of the powdered proteoglycan of Comparative Example 6C was measured.

[0164] As a result, in the proteoglycan that had undergone only the chromatography treatment with polylysine, the endotoxin concentration was 101.7 EU / mg.

[0165] As described above, the present disclosure has been described with reference to the embodiments and examples, but the present disclosure is not limited to the above embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0166] The patents, patent applications, and documents cited in this specification are incorporated herein by reference in their entirety as if the content thereof were specifically set forth herein.

[0167] This application claims priority based on Japanese Patent Application No. 2023-022139 filed on February 16, 2023, Japanese Patent Application No. 2023-127199 filed on August 3, 2023, and Japanese Patent Application No. 2023-203052 filed on November 30, 2023, and incorporates herein by reference all of their disclosures.

[0168] <Supplementary Note> Some or all of the above-described embodiments and examples may be described as follows, but are not limited thereto. <Agent> (Supplementary Note A1) An agent for reducing endotoxin of proteoglycan derived from animal tissue, which contains a nonionic surfactant. (Supplementary Note A2) The agent according to Supplementary Note A1, wherein the nonionic surfactant is a polyoxyethylene alkyl ether (POEAE)-type nonionic surfactant. (Supplementary Note A3) The agent according to Supplementary Note A1 or A2, wherein the nonionic surfactant is polyethylene glycol tert-octylphenyl ether. (Supplementary Note A4) The agent according to any one of Supplementary Notes A1 to A3, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, and skate. <Method for Producing Proteoglycan> (Supplementary Note A5) A first reduction step of reducing endotoxin from proteoglycan derived from animal tissue using a nonionic surfactant, and A second reduction step of reducing endotoxin from the obtained proteoglycan by treating the proteoglycan with a chromatography carrier, and A method for producing proteoglycan, comprising: (Supplementary Note A6) The second reduction step includes An adsorption step of bringing the proteoglycan obtained in the first reduction step into contact with the chromatography carrier to adsorb endotoxin onto the chromatography carrier, and A recovery step of recovering the proteoglycan with reduced endotoxin, and The production method according to Supplementary Note A5, including (Supplementary Note A7) The second reduction step is An adsorption step of bringing the proteoglycan obtained in the first reduction step into contact with the chromatography carrier to adsorb the proteoglycan on the chromatography carrier, and A recovery step of recovering the adsorbed proteoglycan, and The production method according to Supplementary Note A5, including (Supplementary Note A8) The production method according to any one of Supplementary Notes A5 to A7, wherein the second reduction step is carried out a plurality of times. (Supplementary Note A9) The production method according to any one of Supplementary Notes A5 to A8, wherein the second reduction step performs the following steps (A) and (B) on the proteoglycan obtained in the first reduction step: (A) step: An adsorption step of bringing the proteoglycan into contact with the chromatography carrier to adsorb endotoxin on the chromatography carrier, and A recovery step of recovering the proteoglycan with reduced endotoxin; (B) step: An adsorption step of bringing the proteoglycan into contact with the chromatography carrier to adsorb the proteoglycan on the chromatography carrier, and A recovery step of recovering the adsorbed proteoglycan. (Supplementary Note A10) The second reduction step is A first treatment step of treating the proteoglycan obtained in the first reduction step with the chromatography carrier, and A second treatment step of treating the treated proteoglycan with the chromatography carrier, including The chromatography carrier is an ion exchange carrier or a hydrophobic interaction carrier, The chromatographic carrier in the first treatment step and the chromatographic carrier in the second treatment step are different chromatographic carriers, and the production method described in Supplementary Note A8 or A9. (Supplementary Note A11) The chromatographic carrier is a chromatographic resin, and the production method described in any one of Supplementary Notes A5 to A9. (Supplementary Note A12) The chromatographic resin is an ion exchange resin and / or a hydrophobic interaction resin, and the production method described in Supplementary Note A11. (Supplementary Note A13) The ion exchange resin is an anion exchange resin, and the production method described in Supplementary Note A12. (Supplementary Note A14) The anion exchange resin is a resin having a diethylaminoethyl group as a functional group, and the production method described in Supplementary Note A13. (Supplementary Note A15) The hydrophobic interaction resin is a resin having a polyamine-based functional group, an octyl group, and / or an octadecyl group as a functional group, and the production method described in Supplementary Note A12. (Supplementary Note A16) The first reduction step is For the coexistence system containing the nonionic surfactant and the proteoglycan derived from animal tissue, the temperature is raised from a temperature below the cloud point of the nonionic surfactant to a temperature above the cloud point to form a surfactant phase containing the nonionic surfactant, and a concentration step of concentrating the endotoxin in the surfactant phase; A recovery step of recovering the proteoglycan with reduced endotoxin by recovering the phase other than the surfactant phase, and the production method described in any one of Supplementary Notes A5 to A15. (Supplementary Note A17) The first reduction step includes a separation step of separating the surfactant phase and the phase other than the surfactant phase, In the recovery step, the phase other than the surfactant phase after the separation is recovered, and the production method described in Supplementary Note A16. (Supplementary Note A18) The phase other than the surfactant phase is an aqueous phase, and the production method described in Supplementary Note A16 or A17. (Supplementary Note A19) The separation is separation by centrifugation, and the production method described in Supplementary Note A17 or A18. (Supplementary Note A20) The coexistence system contains micelles of the nonionic surfactant, The micelles contain the endotoxin, The production method described in any one of Supplementary Notes A16 to A19. (Supplementary Note A21) Prior to the first reduction step, a formation step of contacting the nonionic surfactant with the proteoglycan derived from animal tissue to form micelles of the nonionic surfactant containing the endotoxin is included. In the concentration step, a surfactant phase is formed from the micelles, and the endotoxin is concentrated in the surfactant phase. The production method described in any one of Supplementary Notes A5 to A20. (Supplementary Note A22) The nonionic surfactant is a polyoxyethylene alkyl ether (POEAE) - type nonionic surfactant. The production method described in any one of Supplementary Notes A5 to A21. (Supplementary Note A23) The nonionic surfactant is polyethylene glycol tert - octylphenyl ether. The production method described in any one of Claims 1 to 22. (Supplementary Note A24) The peak top molecular weight of the proteoglycan after the second reduction step is 300,000 to 1,300,000. The production method described in any one of Supplementary Notes A5 to A23. (Supplementary Note A25) The sugar chain of the proteoglycan is chondroitin sulfate. The production method described in any one of Supplementary Notes A5 to A24. (Supplementary Note A26) The animal is selected from the group consisting of salmon, pig, chicken, flounder, and skate. The production method described in any one of Supplementary Notes A5 to A25.

[0169] <Method for Producing Proteoglycan> (Appendix B1) A method for producing a proteoglycan, comprising a reduction step of reducing endotoxin from a proteoglycan derived from an animal tissue using a nonionic surfactant. (Appendix B2) The reduction step includes: For a coexistence system containing the nonionic surfactant and the proteoglycan derived from an animal tissue, raising the temperature from a temperature below the cloud point of the nonionic surfactant to a temperature above the cloud point to form a surfactant phase containing the nonionic surfactant, and concentrating the endotoxin in the surfactant phase, a concentration step; A recovery step of recovering the proteoglycan with reduced endotoxin by recovering a phase other than the surfactant phase, the production method according to Appendix B1. The production method according to Appendix B1, comprising: (Appendix B3) The reduction step includes a separation step of separating the surfactant phase and a phase other than the surfactant phase, In the recovery step, a phase other than the surfactant phase after the separation is recovered, the production method according to Appendix B2. (Appendix B4) The phase other than the surfactant phase is an aqueous phase, the production method according to Appendix B3. (Appendix B5) The separation is separation by centrifugation, the production method according to Appendix B3 or B4. (Appendix B6) The coexistence system contains micelles of the nonionic surfactant, The micelles contain the endotoxin, The production method according to any one of Appendices B2 to B5. (Appendix B7) Prior to the reduction step, a formation step of bringing the nonionic surfactant into contact with the proteoglycan derived from an animal tissue to form micelles of the nonionic surfactant containing the endotoxin is included, In the concentration step, the surfactant phase is formed from the micelles, and the endotoxin is concentrated in the surfactant phase, the production method according to any one of Appendices B2 to B6. (Supplementary Note B8) The non-ionic surfactant is a polyoxyethylene alkyl ether (POEAE)-type non-ionic surfactant, and the production method according to any one of Supplementary Notes B1 to B7. (Supplementary Note B9) The non-ionic surfactant is polyethylene glycol tert-octylphenyl ether, and the production method according to any one of Supplementary Notes B1 to B8. (Supplementary Note B10) The peak top molecular weight of the proteoglycan after the reduction step is 300,000 to 1,300,000, and the production method according to any one of Supplementary Notes B1 to B9. (Supplementary Note B11) The sugar chain of the proteoglycan is chondroitin sulfate, and the production method according to any one of Supplementary Notes B1 to B10. (Supplementary Note B12) The animal is selected from the group consisting of salmon, pig, chicken, flounder, and skate, and the production method according to any one of Supplementary Notes B1 to B11. <Composition> (Supplementary Note B13) A composition containing a proteoglycan derived from an animal tissue, The composition contains a proteoglycan and endotoxin, The endotoxin content ratio with respect to the composition is 4 EU / mg or less, the composition. (Supplementary Note B14) The peak top molecular weight of the proteoglycan is 300,000 to 1,300,000, the composition according to Supplementary Note B13. (Supplementary Note B15) The sugar chain of the proteoglycan is chondroitin sulfate, the composition according to Supplementary Note B13 or B14. (Supplementary Note B16) The animal is selected from the group consisting of salmon, pig, chicken, flounder, and skate, and the composition according to any one of Supplementary Notes B13 to B15.

Industrial Applicability

[0170] As described above, according to the present disclosure, it is possible to provide, for example, an agent for reducing endotoxin, which can reduce endotoxin, for use in reducing endotoxin of proteoglycan derived from animal tissues, a method for producing proteoglycan, and the like. Therefore, the present disclosure can be said to be extremely useful, for example, in the pharmaceutical field.

Claims

1. Using a coexistence system containing a nonionic surfactant and a proteoglycan derived from animal tissue, a first reduction step of reducing endotoxin from the proteoglycan derived from animal tissue, and a second reduction step of reducing endotoxin from the proteoglycan by treating the obtained proteoglycan with a chromatography carrier, wherein the pH in the coexistence system is 6.8 or less, the chromatography carrier is an anion exchange carrier, the first reduction step is For a coexistence system containing the nonionic surfactant and the proteoglycan derived from animal tissue, heating from a temperature below the cloud point of the nonionic surfactant to a temperature above the cloud point to form a surfactant phase containing the nonionic surfactant, and concentrating the endotoxin in the surfactant phase; a concentrating step, and a recovery step of recovering the proteoglycan with reduced endotoxin by recovering a phase other than the surfactant phase. A method for producing a proteoglycan.

2. The second reduction step is an adsorption step of bringing the proteoglycan obtained in the first reduction step into contact with the chromatography carrier to adsorb endotoxin onto the chromatography carrier, and a recovery step of recovering the proteoglycan with reduced endotoxin. The production method according to claim 1.

3. The second reduction step is an adsorption step of bringing the proteoglycan obtained in the first reduction step into contact with the chromatography carrier to adsorb the proteoglycan onto the chromatography carrier, and a recovery step of recovering the adsorbed proteoglycan. The production method according to claim 1 or 2.

4. The production method according to claim 1 or 2, wherein the second reduction step is carried out a plurality of times.

5. The second reduction step is carried out on the proteoglycan obtained in the first reduction step by performing the following steps (A) and (B). The production method according to claim 1 or 2: Step (A): an adsorption step of bringing the proteoglycan into contact with the chromatography carrier to adsorb endotoxin onto the chromatography carrier, and a recovery step of recovering the proteoglycan with reduced endotoxin; Step (B): an adsorption step of bringing the proteoglycan into contact with the chromatography carrier to adsorb the proteoglycan onto the chromatography carrier, and a recovery step of recovering the adsorbed proteoglycan. An adsorption step of bringing the proteoglycan into contact with the chromatography carrier to adsorb the proteoglycan onto the chromatography carrier, and A recovery step of recovering the adsorbed proteoglycan.

6. The second reduction step is A first treatment step of treating the proteoglycan obtained in the first reduction step with a chromatography carrier, and A second treatment step of treating the treated proteoglycan with a chromatography carrier, The chromatography carrier in the first treatment step and the chromatography carrier in the second treatment step are different chromatography carriers, and One of the chromatography carrier in the first treatment step and the chromatography carrier in the second treatment step is the anion exchange carrier, and the other is a hydrophobic interaction carrier. The production method according to claim 1 or 2.

7. The chromatography carrier is a chromatography resin. The production method according to claim 6.

8. The chromatography resin is an ion exchange resin and / or a hydrophobic interaction resin. The production method according to claim 7.

9. The ion exchange resin is an anion exchange resin. The production method according to claim 8.

10. The anion exchange resin is a resin having a diethylaminoethyl group as a functional group. The production method according to claim 9.

11. The hydrophobic interaction resin is a resin having a polyamine-based functional group, an octyl group and / or an octadecyl group as a functional group. The production method according to claim 8.

12. The pH in the coexistence system is pH 5 to pH 6.

8. The production method according to claim 1 or 2.

13. The first reduction step includes a separation step of separating the surfactant phase and the phase other than the surfactant phase, In the recovery step, the phase other than the surfactant phase after the separation is recovered. The production method according to claim 1 or 2.

14. The phase other than the surfactant phase is an aqueous phase. The production method according to claim 1 or 2.

15. The separation is separation by centrifugation. The production method according to claim 13.

16. The coexistence system includes micelles of the nonionic surfactant, The micelles contain the endotoxin, The production method according to claim 1 or 2.

17. Prior to the first reduction step, a formation step is included in which the nonionic surfactant is brought into contact with the proteoglycan derived from animal tissue to form micelles of the nonionic surfactant containing the endotoxin. The production method according to claim 1 or 2, wherein in the concentration step, a surfactant phase is formed from the micelles and the endotoxin is concentrated in the surfactant phase.

18. The production method according to claim 1 or 2, wherein the nonionic surfactant is a polyoxyethylene alkyl ether (POEAE) - based nonionic surfactant.

19. The production method according to claim 1 or 2, wherein the nonionic surfactant is polyethylene glycol tert - octylphenyl ether.

20. The production method according to claim 1 or 2, wherein the peak top molecular weight of the proteoglycan after the second reduction step is 300,000 to 1,300,000.

21. The production method according to claim 1 or 2, wherein the sugar chain of the proteoglycan is chondroitin sulfate.

22. The production method according to claim 1 or 2, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, and skate.

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