Agent for reducing endotoxin in proteoglycans derived from animal tissues, and method for producing proteoglycan

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

Agent for use in reducing endotoxins in proteoglycans derived from animal tissues and method for producing proteoglycans

[0001] The present disclosure relates to an agent for use in reducing endotoxins in proteoglycans derived from animal tissues and a method for producing proteoglycans.

[0002] In the regenerative medicine market, sheet-structured tissues for regenerative medicine are mainstream. However, it is difficult to regenerate tissues with a three-dimensional structure using sheet-structured tissues. For this reason, attempts have been made to induce tissues with such three-dimensional structures. When constructing such three-dimensional tissues, there is a problem in that it is difficult to reproduce the intercellular crosstalk and extracellular matrix structure that are similar to those found in vivo. Therefore, there is a need for the development of materials that can reproduce the structure of the extracellular matrix (Non-Patent Document 1).

[0003] Furthermore, tissues for regenerative medicine must be low in endotoxin and must meet the standards for biologically derived raw materials in order to be used in animals such as humans (Patent Document 1).

[0004] Takami Akagi, Mitsuru Akashi. "Construction of three-dimensional tissues by cell manipulation and its application to drug discovery research and regenerative medicine." Pharmaceutics 76.5 (2016): 294-300.

[0005] International Publication No. 2017 / 018524

[0006] The present inventors conceived the idea that proteoglycans could be used as the material because they are abundant in the extracellular matrix. To use proteoglycans for medical purposes, the present inventors then attempted to remove endotoxins from proteoglycans using an endotoxin removal method that uses a basic organic solvent. However, this endotoxin removal method not only removed endotoxins, but also failed to maintain the desired molecular weight of the proteoglycans.

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

[0008] A second object of the present disclosure is to provide, for example, an agent capable of reducing endotoxins, for use in reducing endotoxins in proteoglycans derived from animal tissues, and a method for producing proteoglycans.

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

[0010] The composition of the present disclosure is a composition containing proteoglycan derived from animal tissue, the composition containing proteoglycan and endotoxin, and the endotoxin content of 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 according to the present disclosure comprises a nonionic surfactant.

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

[0013] According to the present disclosure, it is possible to provide, for example, a method for producing proteoglycan that can reduce endotoxins and maintain a predetermined molecular weight of proteoglycan, etc. Furthermore, according to the present disclosure, it is possible to provide, for example, an agent for use in reducing endotoxins in proteoglycans derived from animal tissues, and a method for producing proteoglycans that can reduce endotoxins, etc.

[0014] FIG. 1 shows the main disaccharide structures of each chondroitin sulfate.

[0015] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0016] <Definition> As used herein, "proteoglycan" refers to a molecule (glycoprotein) in which a protein (core protein) and a glycosaminoglycan (GAG, also called "polysaccharide" or "sugar chain") are covalently bonded. The proteoglycan exists as an extracellular matrix in, for example, skin, organs, and cartilage. The glycosaminoglycan is generally known as a sugar chain having a long chain structure without a branched structure. Examples of the proteoglycan include the following: Aggrecan family (also called the 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. - Basement membrane proteoglycans: perlecan, agrin, vermacan, etc. - Other proteoglycans: testican, biglycan, serglycin, syndecan, dystroglycan, claustrin, glypican, keratocan, etc. The proteoglycans can be classified into, for example, chondroitin sulfate proteoglycans, dermatan sulfate proteoglycans, heparan sulfate proteoglycans, and keratan sulfate proteoglycans depending on the type of GAG bound to the protein.

[0017] Examples of the GAG ​​include chondroitin, chondroitin sulfate (CS), dermatan sulfate (DS, chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. Examples of the chondroitin include an O-type sugar chain whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine, and an iO-type sugar chain whose main structure is a disaccharide structure of iduronic acid and acetylgalactosamine (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 the disaccharide structure of glucuronic acid and acetylgalactosamine is repeated. Examples of the chondroitin sulfate (CS) include chondroitin sulfate A (type A), whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine tetrasulfate, and chondroitin sulfate C (type C), whose main disaccharide structure is a disaccharide structure of glucuronic acid and acetylgalactosamine hexasulfate. The dermatan sulfate (DS) has a structure in which a sulfate group is added to a sugar chain in which the disaccharide structure of iduronic acid and acetylgalactosamine is repeated. Examples of the dermatan sulfate include chondroitin sulfate iA (type iA), whose main disaccharide structure is a disaccharide structure of iduronic acid and acetylgalactosamine tetrasulfate, and chondroitin sulfate iC (type iC), whose main disaccharide structure is a disaccharide structure of iduronic acid and acetylgalactosamine hexasulfate. Each chondroitin sulfate has, for example, the disaccharide structure shown in FIG. 1 as its main disaccharide structure. In FIG. 1, the sulfate group (sulfo group) is bonded to a hydrogen atom, but the present disclosure is not limited to this. For example, the sulfate group of the GAG ​​may be ionized by eliminating the 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 produced, for example, when the cell wall of the gram-negative bacteria is destroyed and released after the death of the gram-negative bacteria. It is known that, when the endotoxin enters the bloodstream of humans, it can cause biological reactions such as fever, septic shock, and multiple organ failure. The endotoxin is composed of an O-antigen polysaccharide, a core polysaccharide, and lipid A. Because the endotoxin has a hydrophilic portion (the sugar moiety of the O-antigen polysaccharide and the core polysaccharide) and a hydrophobic portion (lipid A), it can associate in an aqueous solution to form micelles.

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

[0020] As used herein, "micelle" refers to a spherical structure formed by amphiphilic molecules having hydrophobic and hydrophilic moieties in an aqueous solvent such as water, with the hydrophilic moieties facing the aqueous solvent and the hydrophobic moieties facing inward. The concentration at which micelles are formed from molecular dispersion is called the critical micelle concentration (CMC) of the substance.

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

[0022] As used herein, the term "cloud point" refers to the temperature (phase transition temperature) at which a solution containing a nonionic surfactant begins to become cloudy when heated. The cloudiness occurs when the heating causes a rapid decrease in the water solubility of the nonionic surfactant, rendering the nonionic surfactant unable to form micelles. The rapid decrease in water solubility occurs when the polyether chains of the nonionic surfactant are unable to form hydrogen bonds with water. The cloud point can be measured, for example, visually, by measurement in accordance with the measurement standard for the cloud point of nonionic surfactants (ISO 1065:1991), or by the method described in JP 2006-257395 A. The method described in JP 2006-257395 A is as follows. - "New Introduction to Surfactants" by Takehiko Fujimoto (Sanyo Chemical Industries), page 95, etc., in which 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 in the tube, and the temperature is slowly raised while gently stirring with the stirring rod, and when the temperature reaches a certain level or higher, the transparent aqueous solution becomes cloudy, and the temperature at which this cloudiness occurs is measured and this temperature is taken as the cloud point. The definition of the cloud point of a surfactant is also given in, for example, JIS K3211.

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

[0024] In this specification, the term "hydrophobic interaction carrier" refers to a solid phase that has hydrophobic groups and adsorbs molecules contained on the solid phase or in a liquid that has passed through the solid phase depending on the hydrophobicity of the molecules. Examples of the hydrophobic groups include the following functional groups: - Alkyl groups such as octyl groups and octadecyl groups - Functional groups containing aryl groups such as phenyl groups and benzyl groups - Polyamine-based functional groups such as polylysine (for example, ε-polylysine)

[0025] As used herein, "flow-through mode" (F / T mode) refers to a technique in which at least one substance to be removed contained in a target is bound or adsorbed to a stationary phase such as a chromatography resin, and at least one target substance is allowed to flow through and be separated. The F / T mode is also referred to as, for example, negative mode.

[0026] As used herein, "bind / elute mode" (B / E mode) refers to a technique in which at least one target substance contained in a sample is bound or adsorbed to a stationary phase such as a chromatography resin, and then the target substance is eluted and separated.

[0027] The present disclosure will be described below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, the descriptions in this disclosure and each embodiment can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean including the numerical or physical values ​​before and after it. In addition, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

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

[0029] As a result of extensive research, the inventors came up with the idea that endotoxins could be reduced from compositions containing proteoglycans and endotoxins by utilizing the difference in hydrophobicity and hydrophilicity between proteoglycans, which are glycoproteins, and endotoxins, which are glycolipids. Further research led the inventors to discover that endotoxins can be extracted from mixtures of proteoglycans and endotoxins using a nonionic surfactant, thereby establishing the present disclosure. It is presumed that the use of the nonionic surfactant reduces endotoxins through the mechanism described below. However, the present disclosure is not limited to this mechanism. Proteoglycans are composed of sugars and proteins and are hydrophilic components. On the other hand, endotoxins, as described above, have hydrophilic and hydrophobic portions. Therefore, when the proteoglycan, the endotoxin, and the nonionic surfactant are present in an aqueous solvent at a temperature where the nonionic surfactant can be dispersed in the aqueous solvent, the proteoglycan, the endotoxin, and the nonionic surfactant behave differently due to their differences in hydrophilicity and hydrophobicity. Specifically, the proteoglycan can be dispersed alone in the aqueous solvent. On the other hand, the endotoxin and the nonionic surfactant contain hydrophobic moieties, making them difficult to disperse alone in the aqueous solvent, and instead form a complex. Therefore, the endotoxin and the nonionic surfactant exist in the aqueous solvent in the form of a complex. If the aqueous solvent is heated in this state to a temperature above the cloud point of the nonionic surfactant, the complex containing the nonionic surfactant cannot maintain its dispersion 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 nonionic surfactant phase during the phase formation (phase separation).Therefore, in the present disclosure, it is presumed that the amount of endotoxin in a composition containing the proteoglycan can be reduced by extracting the endotoxin into a different phase from the composition containing the proteoglycan and 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-octylphenyl ether (formula (1) below). In formula (1), n ​​represents the degree of polymerization of ethylene glycol. For example, n is preferably 1 to 10, and more preferably 7 to 8. For example, the nonionic surfactant is a nonionic surfactant that undergoes a temperature-dependent phase transition. For example, one type of surfactant may be used alone, or multiple types may be used in combination. When multiple types of surfactants are used, for example, multiple types of surfactants may be mixed in advance.

[0031]

[0032] The animal is not particularly limited, and examples thereof include mammals (mammals) such as pigs; avian animals (birds) such as chickens; flatfish such as Japanese flounder, salmonids such as chum salmon and Atlantic salmon, and fish such as rays (including, for example, skate); etc. Preferred examples of the animal include pigs, poultry, flatfish, salmon, and rays.

[0033] The animal tissue is, for example, a tissue containing proteoglycan, and examples thereof include epithelial tissue such as skin, cartilaginous tissue such as cartilage, digestive organs, circulatory organs, respiratory organs, and placenta, etc. Specific examples of the animal tissue include cartilage, fins, digestive organs, circulatory organs, respiratory organs, and ears.

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

[0035] In the manufacturing method of the present disclosure, as described above, in the first reduction step, endotoxins are reduced from proteoglycans derived from animal tissues using the nonionic surfactant. Specifically, in the first reduction step, for example, as described above, endotoxins that have formed a complex with the nonionic surfactant are concentrated in the nonionic surfactant phase by forming a nonionic surfactant phase from the complex. Therefore, the manufacturing method of the present disclosure may include, prior to the first reduction step, a formation step of contacting the nonionic surfactant with the proteoglycans derived from animal tissues 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), for example, because this allows for efficient formation of the complex. The liquid system can also be referred to as being in the presence of a solvent such as an aqueous solvent. The liquid system preferably does not substantially contain an organic solvent, for example, a basic organic solvent. The term "substantially free" means, for example, that the organic solvent content is below the detection limit. Examples of the aqueous solvent include phosphate buffer, buffer solutions such as calcium (Ca) and / or magnesium (Mg)-free phosphate buffered saline, phosphate buffered saline, and PBS (Phosphate Buffered Saline), saline, sodium chloride solution, and mixtures thereof. The aqueous solvent may contain alcohol, such as ethanol, for example, because this allows for more efficient removal of the endotoxin. When the aqueous solvent contains alcohol, the alcohol is preferably present in an amount that does not inhibit the formation of a complex between the nonionic surfactant and the endotoxin. When the aqueous solvent contains an alcohol, the concentration of the alcohol is, for example, preferably more than 0 (v / v) % and less than 2 (v / v) %, and more preferably about 1 (v / v) %.

[0037] In the formation process, the contact between the nonionic surfactant and the proteoglycan can be carried out appropriately depending on the state of the nonionic surfactant and the proteoglycan. The nonionic surfactant and the proteoglycan to be contacted may be, for example, solid or liquid. When the nonionic surfactant and the proteoglycan are solid, the nonionic surfactant and the proteoglycan are preferably dispersed in a solvent before 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 blending (hereinafter collectively referred to as "mixing") the nonionic surfactant and the animal tissue-derived proteoglycan. 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. Thus, in the formation step, a coexistence system (mixed 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, the complex of the nonionic surfactant and the endotoxin preferably forms, for example, micelles. In this case, the coexistence system contains micelles of the nonionic surfactant, and the micelles can be said to contain the endotoxin.

[0038] In the forming step, the coexistence system is preferably further mixed, for example, after the contact. This can promote the formation of a complex between the endotoxin and the nonionic surfactant in the coexistence system in the forming step. The mixing may be performed using a stirring device such as a vortex mixer, by inversion mixing, or by ultrasonic treatment, but is preferably ultrasonic treatment.

[0039] In the formation step, the animal tissue-derived proteoglycan may be proteoglycan that has been pretreated, such as crudely purified or purified, from the animal tissue, or may be animal tissue containing the proteoglycan. Examples of the pretreatment include pulverizing, degreasing, and purifying the animal tissue. The pulverization can be performed, for example, by cutting, crushing, and sonication. The purification (extraction) can be performed, for example, by contacting the animal tissue or a pretreated product thereof with an extract. Examples of the extract include a guanidine hydrochloride aqueous solution, an acetic acid aqueous solution, a urea aqueous solution, and a magnesium chloride aqueous solution.

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

[0041] In the forming step, the temperature of the coexistence system is, for example, a temperature below the cloud point of the nonionic surfactant. The temperature can be set, for example, depending on 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, greater than -10°C and not greater than 40°C (more preferably, greater than -10°C and less than 22°C).

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

[0043] The proteoglycan may be pretreated prior to the formation step to reduce endotoxins by precipitating calcium salts. Specifically, the pretreatment can be carried out by, for example, contacting or mixing the proteoglycan with a phosphate buffer and separating or removing precipitates containing calcium phosphate salts and endotoxins formed after the contact or mixing. The calcium phosphate salts adsorb the endotoxins. Therefore, the production method of the present disclosure, for example, can more efficiently remove endotoxins by including the pretreatment. Furthermore, the supernatant obtained after removing the precipitates contains proteoglycans with reduced endotoxins. Therefore, for example, the supernatant may be recovered during the pretreatment, and the recovered supernatant may be used as the proteoglycan during the formation step. The pretreatment time is, for example, preferably 2 hours or more, more preferably 4 to 36 hours, and even more preferably 8 to 24 hours. The pretreatment temperature is, for example, preferably 18 to 25°C.

[0044] Next, in the first reduction step, the coexistence system containing the nonionic surfactant and the animal tissue-derived proteoglycan is heated from a temperature below the cloud point of the nonionic surfactant to a temperature above the cloud point. As a result, in the first reduction step, a surfactant phase containing the nonionic surfactant is formed, and the 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, the endotoxin in the complex is also concentrated in the surfactant phase when the surfactant phase is formed, 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 animal tissue-derived proteoglycan 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 referred to as, for example, an aqueous phase.

[0045] The cloud point varies depending on the type of nonionic surfactant. Therefore, in the concentration step, the temperature below the cloud point and the temperature above the cloud point can be appropriately adjusted depending on the type of nonionic surfactant. The cloud point can 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 achieved and measuring the aqueous solution using the cloud point measurement method described above, or by determining 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 determined by referring to, for example, the cloud point described in the accompanying instructions. Specifically, when the nonionic surfactant is polyethylene glycol tert-octylphenyl ether (degree of polymerization 7 to 8), the cloud point is, for example, approximately 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, for example, above -10°C and below 22°C, preferably approximately 0°C. 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 can be increased by heating the coexistence system, for example, using a temperature control device such as a heat block or an incubator.

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

[0048] In the concentration step, for example, prior to the temperature increase, it is preferable to mix the coexistence system at a temperature below the cloud point. The mixing may be performed using a stirring device such as a vortex mixer, or by end-over-end mixing, preferably end-over-end mixing. The mixing time at a temperature below the cloud point can be set, for example, within a range in which the temperature of the coexistence system or the solvent of the coexistence 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 the components at a temperature equal to or higher than the cloud point. The mixing may be performed using, for example, a vortex mixer or by inversion mixing, preferably by inversion mixing. The mixing time at a temperature equal to or higher than the cloud point can be set, for example, within a range in which the temperature of the solvent becomes approximately uniform. The mixing time at a temperature equal to or higher than 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 from a phase other than the surfactant phase. As a result, the first reduction step can separate the surfactant phase into a layer structure including, for example, a layer formed from the surfactant phase and a layer formed from a phase other than the surfactant phase. By including the separation step in the first reduction step, recovery or removal of at least one of the surfactant phase and the phase other than the surfactant phase in the coexistence system can be easily performed. Therefore, the manufacturing method of the present disclosure can efficiently reduce endotoxins in the proteoglycan, for example. 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, specifically, by centrifugation. The phase other than the surfactant phase can be, for example, an aqueous phase. When the separation step is performed by centrifugation, the centrifugation conditions are, for example, about 9500 × g.

[0051] The time (separation time) in the separation step can be appropriately set within a range in which the surfactant phase can be separated from a phase other than the surfactant phase, for example, 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, specifically, a temperature equal to or higher than the cloud point. When the nonionic surfactant is polyethylene glycol tert-octylphenyl ether (degree of polymerization 7 to 8), the separation temperature is, for example, 22°C to 40°C.

[0053] The manufacturing method of the present disclosure may include a first recovery step in which a phase other than the surfactant phase after the separation is recovered in the first reduction step to recover the proteoglycan with reduced endotoxins. 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 of the supernatant. The recovery method may be performed using, for example, a dropper or the like.

[0054] In the production method of the present disclosure, for example, the first reduction step may be repeated one or more times using the recovered proteoglycan as the animal tissue-derived proteoglycan. 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 repeatedly carried out one or more times, the surfactant used in each first reduction step may be, for example, the same surfactant or different surfactants.

[0056] In the production method of the present disclosure, when the first reduction step is repeated one or more times, the formation step may be performed, for example, prior to the repeated first reduction step. In this case, it is preferable that the coexistence system in the first formation step contains, for example, the aqueous solvent, and that in the second or subsequent formation steps, the coexistence system contains, for example, an aqueous solvent containing the alcohol. This allows the production method of the present disclosure to remove the endotoxin more efficiently, for example.

[0057] Next, the production 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 "first proteoglycan"). Specifically, in the second reduction step, the first proteoglycan is treated with a chromatography carrier to reduce endotoxins from the proteoglycan. In the production method of the present disclosure, for example, by performing the second reduction step, endotoxins that are difficult to associate with the nonionic surfactant can be removed, thereby further reducing the amount of endotoxins.

[0058] It is presumed that the second reduction step of the present disclosure can further reduce the endotoxin by using the chromatography carrier through the mechanism described below. However, the present disclosure is not limited to the following mechanism. Gram-negative bacteria contain endotoxins having glycolipids of various types or structures. The glycolipids have different hydrophobicity and charge levels depending on their composition, and therefore the endotoxins have different hydrophobicity and charge levels depending on the Gram-negative bacteria from which they are derived. Furthermore, even within the same Gram-negative bacteria, the glycolipids produced are not uniform in composition but contain different compositions, resulting in different hydrophobicity and charge levels of the endotoxins. On the other hand, it is believed that the endotoxins that can be removed by the nonionic surfactant and the endotoxins that can be removed by the chromatography carrier have different hydrophobicity and charge levels. Therefore, in the present disclosure, it is estimated that after reducing endotoxins by treatment with the nonionic surfactant, endotoxins in the proteoglycans can be further reduced by further treatment with the chromatography carrier, taking advantage of the differences in the degree of hydrophobicity and / or the degree of charge between the endotoxins that can be removed.

[0059] In the second reduction step, the contact between the first proteoglycan and the chromatography carrier can be carried out appropriately, for example, depending on the state of the first proteoglycan. The first proteoglycan may be, for example, proteoglycan obtained after the first recovery step, or proteoglycan obtained after the solidification step described below for the proteoglycan obtained after the first recovery step. In the case of proteoglycan obtained after the solidification step, the proteoglycan is preferably dispersed in a solvent beforehand and then subjected to the contact. The solvent can be selected, for example, depending on the type of 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, an amine solvent to which a salt has been added, Tris-HCl buffer, HEPES, a potassium chloride aqueous solution, a sodium chloride aqueous solution, or a magnesium chloride aqueous solution. When a hydrophobic interaction resin is used as the chromatography support, the solvent may be, for example, an amine solvent to which a salt has been added, a Tris-HCl buffer solution, HEPES, an aqueous potassium chloride solution, an aqueous sodium chloride solution, or an aqueous magnesium chloride solution.

[0060] Examples of the chromatography resin include an ion exchange carrier such as an ion exchange resin, and a hydrophobic interaction carrier such as a hydrophobic interaction resin. Examples of the ion exchange carrier include an anion exchange resin and an anion exchange monolith. By using the hydrophobic interaction carrier or the anion exchange carrier as the chromatography carrier, in the second reduction step, for example, endotoxins in the proteoglycans obtained in the first reduction step can be adsorbed onto the chromatography carrier. Furthermore, by using the anion exchange carrier as the chromatography carrier, proteoglycans in the proteoglycans obtained in the first reduction step can be adsorbed onto the carrier. Examples of the anion exchange carrier include solid phases having, as functional groups, primary to tertiary amino groups such as diethylaminoethyl (DEAE) groups and polyamine-based functional groups such as polylysine (e.g., ε-polylysine); quaternary ammonium groups such as trimethylammonium groups and dimethylethanolammonium groups; etc. The hydrophobic interaction carrier may be, for example, a solid phase having, as a functional group, a polyamine functional group such as polylysine (e.g., ε-polylysine); or an alkyl group such as a butyl group, an octyl group, or an octadecyl group. The chromatography carrier may have one type of functional group or multiple functional groups. One type of chromatography carrier may be used alone, or multiple types may be used in combination. The shape of the chromatography carrier can be designed, for example, depending on the type of 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 a diethylaminoethyl (DEAE) group include TOYOPEARL (trademark) DEAE-650M (Tosoh Corporation). Examples of the chromatography resin containing polylysine include Cellufine ET Clean S (JNC Corporation) and Cellufine ET Clean L (JNC Corporation).Examples of the chromatography resin having an octyl group include Nucleosil 100-C8 (GL-Science). Examples of the chromatography resin having an octadecyl group include Nucleosil 100-C18 (GL-Science). Examples of the monolith having a quaternary ammonium group include CIMmultus (registered trademark) QA (Sartorius AG). Examples of the monolith having a diethylaminoethyl (DEAE) group include CIMmultus (registered trademark) DEAE (Sartorius AG). Examples of the monolith having a butyl group include CIMmultus (registered trademark) C4-HLD and CIMmultus (registered trademark) C4-A (Sartorius AG).

[0061] The second reduction step can be performed, for example, depending on the type of the chromatography carrier. When the chromatography carrier adsorbs the endotoxin but not the proteoglycan, the second reduction step includes, for example, an adsorption step of contacting the first proteoglycan with the chromatography carrier to adsorb the endotoxin onto 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 performed, for example, in 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 contacting the first proteoglycan with the chromatography carrier to adsorb the proteoglycan onto the chromatography carrier, and a recovery step (second recovery step) of recovering the adsorbed proteoglycan. That is, the second reduction step can be performed, for example, in B / E mode. When the second reduction step is in B / E mode, the recovery can also be referred to as elution.

[0062] When the second reduction step is performed in F / T mode, the chromatography carrier can be, for example, a hydrophobic interaction carrier such as the hydrophobic interaction resin. In the adsorption step, the contact between the first proteoglycan and the chromatography carrier can be performed, for example, by contacting a dispersion containing the first proteoglycan (hereinafter also referred to as "first dispersion") with the chromatography carrier. Specifically, the contact can be performed, for example, by mixing the first dispersion with the chromatography carrier. In the adsorption step, for example, contact between the first proteoglycan and the chromatography carrier causes an interaction between the endotoxin contained in the first proteoglycan and the chromatography carrier, depending on the properties of the chromatography carrier. As a result, in the adsorption step, the endotoxin is adsorbed to the chromatography carrier. On the other hand, in the adsorption step, for example, there is substantially no interaction between the proteoglycan contained in the first proteoglycan and the chromatography carrier, or the interaction is sufficiently low compared to the interaction between the endotoxin and the chromatography carrier. As a result, in the adsorption step, for example, the proteoglycan is not substantially adsorbed to the chromatography carrier, or the amount of adsorption is sufficiently low compared to the adsorption of the endotoxin, and the proteoglycan is maintained in the dispersion liquid.

[0063] For example, the description of the solvent in the second reduction step can be used for the solvent of the first dispersion.

[0064] In the adsorption step, the amount of the proteoglycan to be contacted with the chromatography carrier can be set, for example, depending on 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, when Cellufine ET Clean S (JNC Corporation) or Cellufine ET Clean L (JNC Corporation) (hydrophobic interaction resin) is used as the chromatography carrier slurry, 350 to 550 μg of the proteoglycan is contacted with 1 ml of the chromatography carrier slurry.

[0065] When the chromatography support is a hydrophobic interaction resin, the conditions of the first dispersion liquid to be brought into contact with the chromatography support can be set depending on the type of the hydrophobic interaction resin. Specific examples of the conditions of the first dispersion liquid include the following: 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 liquid fraction is recovered from a mixture of the first proteoglycan dispersion and the chromatography carrier, thereby recovering a dispersion containing the proteoglycan with reduced endotoxin (second dispersion). The second dispersion can be recovered using, for example, 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 washing solution to wash the chromatography carrier. In this case, the second recovery step may involve recovering a liquid fraction from a mixture of the washing solution and the chromatography carrier after the washing, thereby recovering a dispersion containing the endotoxin-reduced proteoglycan. 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 washing the chromatography carrier with the washing solution. Therefore, in the second recovery step, the recovery rate of the proteoglycan can be improved, for example, by washing the chromatography carrier with the washing solution and recovering the washing solution.

[0068] The solvent of the cleaning liquid may be, for example, the same solvent as that of the first dispersion liquid. The conditions of the cleaning liquid may be, for example, the same as those of the first dispersion liquid.

[0069] When the second reduction step is performed in B / E mode, the chromatography carrier can be an anion exchange carrier such as an anion exchange resin. In the adsorption step, the contact between the first proteoglycan and the chromatography carrier can be performed, for example, in the same manner as in the F / T mode. In the adsorption step, for example, the negatively charged first proteoglycan and endotoxin come into contact with the chromatography carrier, and the first proteoglycan and endotoxin bind to the chromatography carrier depending on 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 contacted with the chromatography carrier can be set, for example, depending on 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, when TOYOPEARL (trademark) DEAE-650M (Tosoh Corporation) (anion exchange resin) is used as the chromatography carrier slurry, 350 to 550 μg of the proteoglycan is contacted with 1 ml of the chromatography carrier slurry.

[0071] When the chromatography carrier is an anion exchange resin, the conditions of the first dispersion liquid to be brought into contact with the chromatography carrier can be set depending on the type of the anion exchange resin. Specific examples of the conditions of the first dispersion liquid include the following: 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 anionic charge and hydrophobicity. Therefore, in the second reduction step, after the adsorption step, the proteoglycan can be preferentially eluted from the chromatography carrier by, for example, passing a solvent having a higher salt concentration than the solvent used in the adsorption step through the chromatography carrier after adsorption. Therefore, the second reduction step preferably includes, for example, an elution step of eluting the proteoglycan adsorbed to 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 preferably has a higher salt concentration than the solvent used in the adsorption step. Specific examples of the elution solvent conditions include the following: 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, the eluate eluted in the elution step is recovered, thereby recovering a dispersion liquid (second dispersion liquid) containing the proteoglycan with reduced endotoxin.

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

[0076] The chromatography support may be used, for example, in a form packed in a column. In this case, the adsorption step can be carried out by loading the equilibration solution, the first dispersion liquid, a washing solution, an elution solution, etc. into the column packed with the chromatography support.

[0077] In the production method of the present disclosure, for example, the second reduction step may be repeated 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 performed multiple times. In the production method of the present disclosure, for example, the amount of endotoxin in the proteoglycan can be further reduced by performing the second reduction step multiple times. In this case, the explanation of the second reduction step can be used by replacing "proteoglycan obtained in the first recovery step" with "proteoglycan obtained in the second recovery step."

[0078] When the second reduction step is performed multiple times, the second reduction step may include, for example, performing the following steps (A) and (B) on the proteoglycan obtained in the first reduction step: step (A): an adsorption step of contacting the proteoglycan 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 contacting the proteoglycan with the chromatography carrier to adsorb proteoglycan onto the chromatography carrier, and a recovery step of recovering the adsorbed proteoglycan.

[0079] In the step (A), the chromatography support is preferably an ion exchange resin or a hydrophobic interaction resin, and in the step (B), the chromatography support is preferably, for example, an anion exchange support having a DEAE group.

[0080] When the second reduction step is performed multiple times, the second reduction step may include a first treatment step in which the proteoglycan obtained in the first reduction step is treated with the chromatography carrier, and a second treatment step in which the treated proteoglycan is treated with the chromatography carrier. Preferably, the chromatography carrier in the first treatment step and the chromatography carrier in the second treatment step are different chromatography carriers. The first treatment step and the second treatment step can be performed in the same manner as the second reduction step.

[0081] In the manufacturing method of the present disclosure, the endotoxin concentration of the proteoglycan contained in the recovered aqueous phase may be measured after the first reduction step or the second reduction step. The endotoxin concentration can be measured by a gel clot method (LAL test) or an optical quantification method (colorimetric method) using a lysate reagent made from a horseshoe crab hemocyte extract.

[0082] The endotoxin concentration in the first reduction step is, for example, 10 EU / mg or less, 7 EU / mg or less. The endotoxin concentration 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. The lower limit of the endotoxin concentration is preferably, for example, 0 EU / mg, but in reality, it is preferably above 0 EU / mg (approximately the detection limit).

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

[0084] (Conditions for measuring peak top molecular weight (Mp)) HPLC system: LC-20AD (Shimadzu Corporation) Column: TSKgel G5000-PWXL φ7.8 mm × 300 mm (Tosoh Corporation) Eluent: pH 6.8 phosphate buffer solution Flow rate: 0.5 mL / min Column temperature: 40°C Detector: Differential refractive index detector (RID-20A, 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, or 400,000 to 1,200,000.

[0086] The manufacturing method of the present disclosure may include a fractionation step of fractionating proteoglycans satisfying a predetermined peak top molecular weight from the recovered phase other than the 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 the proteoglycans while maintaining their predetermined molecular weight. The fractionation method can be carried out, for example, by a known method capable of fractionating components based on molecular weight. Specifically, the fractionation method may be carried out using an ultrafiltration membrane (molecular weight cutoff membrane) or a dialysis membrane, or may be carried out by, for example, silica gel column chromatography, gel filtration chromatography, ion exchange column chromatography, etc. The molecular weight cutoff of the molecular weight cutoff membrane can be set, for example, depending on the target molecular weight.

[0087] The time for the fractionation step (fractionation time) is not particularly limited as long as it is within a range in which the proteoglycans can be fractionated while maintaining their predetermined molecular weights, and the fractionation time is, for example, 1 day to 2 weeks.

[0088] The manufacturing 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 the proteoglycan while maintaining the predetermined molecular weight. The solidification step can be carried out, for example, by a known method capable of solidifying proteoglycan from a liquid containing the fractionated proteoglycan, specifically, by a drying process such as freeze-drying or spray-drying.

[0089] <Agent> In another aspect, the present disclosure provides an agent for use in reducing endotoxins in proteoglycans derived from animal tissues. The agent of the present disclosure includes a nonionic surfactant. The agent of the present disclosure can reduce endotoxins in proteoglycans derived from animal tissues, and is therefore suitable for use in producing the proteoglycans with reduced endotoxins.

[0090] For the agent of the present disclosure, for example, the explanation of the method for producing proteoglycan of the present disclosure can be used.

[0091] <Composition> In another aspect, the present disclosure provides a composition comprising a proteoglycan derived from animal tissue. The composition of the present disclosure comprises a proteoglycan and an endotoxin, and the endotoxin content of 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 is preferably, for example, 0 EU / mg, but in reality, it is preferably above 0 EU / mg (approximately the detection limit).

[0092] The composition of the present disclosure includes, for example, a proteoglycan obtained by the method for producing a proteoglycan of the present disclosure. For example, the description of the method for producing a proteoglycan of the present disclosure can be used for the composition of the present disclosure.

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

[0094] Example 1 According to the manufacturing method of the present disclosure, it was possible to reduce endotoxins from proteoglycans derived from animal tissues, and also possible to maintain the predetermined molecular weight of the proteoglycans (molecular weight of about 450,000).

[0095] 7.8 mg of salmon-derived proteoglycan was added to 0.2 mol / L NaCl-containing phosphate buffer (pH 6). After the addition, the mixture was stirred and sonicated at approximately 23°C. 5 μl of Triton™-X114 was then added. After the addition, the mixture was mixed by end-over-end at 0°C for 6 minutes. After the end-over-end mixing, the mixture was mixed by end-over-end at 37°C for 5 minutes. After the end-over-end mixing, the mixture was centrifuged at 23°C for 30 seconds at 10,000 rpm. After the centrifugation, separation into two layers was confirmed. After confirmation, the upper layer containing the proteoglycan was collected and dialyzed for a molecular weight cutoff (MWCO: 12,000-16,000). After the dialysis, the proteoglycan was freeze-dried to obtain a proteoglycan solid. Thereafter, the endotoxin concentrations of the proteoglycan before treatment and the solid proteoglycan were measured by the LAL method using a kit (ToxinSensor Chromogenic LAL Endotoxin Assay Kit, manufactured by GenScript Inc.) Furthermore, the proteoglycan before treatment and the solid proteoglycan were subjected to HPLC analysis under the following measurement conditions.

[0096] (HPLC measurement conditions) HPLC system: LC-20AD (Shimadzu Corporation) Column: TSKgel G5000-PWXL Φ7.8 mm × 300 mm (Tosoh Corporation) Eluent: pH 6.8 phosphate buffer Flow rate: 0.5 mL / min Column temperature: 40°C Detector: differential refractive index detector (RID-10A, 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, the endotoxin concentration in the proteoglycan before treatment was 400 EU / mg, while the endotoxin concentration in the proteoglycan solid was 4 EU / mg. Furthermore, the peak top molecular weight of the proteoglycan before treatment was approximately 450,000, while the peak top molecular weight of the proteoglycan solid was approximately 450,000. These results demonstrate that the method of the present disclosure (Example 1) can reduce endotoxins from proteoglycans derived from animal tissues while maintaining the predetermined molecular weight of the proteoglycans.

[0098] The following Comparative Examples 1A to 1C were also carried out.

[0099] [Comparative Example 1A] Except for using ethanol containing 0.001 N sodium hydroxide instead of 0.2 mol / L NaCl-containing phosphate buffer (pH 6), the preparation of a sample of Comparative Example 1A, measurement of endotoxin concentration, and HPLC analysis were carried out in the same manner as in Example 1. As a result, it was confirmed that the predetermined molecular weight (approximately 450,000) of proteoglycan was maintained in the sample of Comparative Example 1A, but no reduction in endotoxin was confirmed.

[0100] [Comparative Example 1B] Preparation of a sample of Comparative Example 2, measurement of endotoxin concentration, and HPLC analysis were performed in the same manner as in Example 1, except that ethanol containing 0.01 N sodium hydroxide was used instead of 0.2 mol / L NaCl-containing phosphate buffer (pH 6). As a result, the endotoxin concentration in the sample of Comparative Example 1B was 167.8 EU / mg. From these results, it was found that the endotoxin concentration in the sample of Comparative Example 1B was not reduced as compared to Example 1.

[0101] [Comparative Example 1C] The sample of Comparative Example 1C was prepared, the endotoxin concentration was measured, and HPLC analysis was performed in the same manner as in Example 1, except that ethanol containing 0.025N sodium hydroxide was used instead of 0.2 mol / L NaCl-containing phosphate buffer (pH 6). 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 specified molecular weight (approximately 450,000). From these results, it was found that while the endotoxin concentration was reduced in the sample of Comparative Example 1C, the specified molecular weight of the proteoglycan was not maintained.

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

[0103] To a 0.05 mol / l phosphate buffer solution (pH 6.0) was added 7.8 mg of salmon-derived proteoglycan.

[0104] After the addition, 5 μl of Triton™-X114 was added. After the addition, the mixture was stirred and 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 × g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was collected, and a three-fold amount of saturated ethanol was added to cause crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 23°C for 3 minutes at 9060 × g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum-dried to obtain powdered proteoglycan. The endotoxin concentrations of the proteoglycan before treatment and the powdered proteoglycan were then measured by the LAL method. 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, whereas the endotoxin concentration in the powdered proteoglycan was 9.2 EU / mg. These results demonstrate that the method of the present disclosure (Example 2A) can reduce endotoxins from proteoglycans derived from animal tissues.

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

[0107] 7.8 mg of salmon-derived proteoglycan 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 standing, the precipitate was removed to obtain a supernatant. 5 μl of Triton™-X114 was then added. After the addition, the mixture was stirred and allowed to stand on ice for 10 minutes. After standing, the mixture was allowed to stand at 40°C for 10 minutes. After standing, the mixture was centrifuged at 23°C for 3 minutes at 9060×g. After centrifugation, separation into two layers was confirmed. After confirmation, the upper layer containing the proteoglycan was recovered, and a three-fold amount of saturated ethanol was added to cause crystallization. After 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 centrifugation, the saturated ethanol was removed, and the mixture was vacuum-dried to obtain powdered proteoglycan. The endotoxin concentration of the powdered proteoglycan from Example 2B was then measured using the same method as in Example 2A.

[0109] As a result, the endotoxin concentration in the powdered proteoglycan of Example 2B was 9.2 EU / mg. These results demonstrate that the method of the present disclosure (Example 2B) can also reduce endotoxins from proteoglycans derived from animal tissues.

[0110] The following Comparative Example 2 was also carried out.

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

[0112] After standing, the precipitate was removed to obtain a supernatant. Three times the amount of saturated ethanol was added to the supernatant to cause crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, centrifugation was performed under conditions of 23°C, 3 minutes, and 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum dried to obtain powdered proteoglycan. Thereafter, the endotoxin concentration of the powdered proteoglycan of Comparative Example 2 was measured using the same method as in Example 2A.

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

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

[0115] Powdered proteoglycan was obtained in the same manner as in Example 2A, except that PBS (pH 7.2, Fujifilm Wako Pure Chemical Industries, Ltd., 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 in the proteoglycan before treatment was 304.2 EU / mg, whereas the endotoxin concentration in the powdered proteoglycan was 21.4 EU / mg. These results demonstrate that the method of the present disclosure (Example 3A) can reduce endotoxins from proteoglycans derived from animal tissues.

[0117] [Example 3B] It was confirmed that endotoxins in proteoglycans derived from animal tissues can be reduced by repeatedly carrying out the reduction step in the production method of the present disclosure.

[0118] Salmon-derived proteoglycan was added to PBS (pH 7.2, Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No.: 164-28713). After the addition, the mixture was stirred and sonicated at 23°C to obtain a 5 mg / ml proteoglycan solution. Then, 1 (v / v)% 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was collected, avoiding the solution at the boundary between the lower and upper layers. After the collection, 1 (v / v)% Triton™-X114 was added. After the addition, the mixture was allowed to stand on ice for 10 minutes. After the incubation, the mixture was allowed to stand at 40°C for 10 minutes. After the incubation, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, it was confirmed that the mixture had separated into two layers. After the confirmation, the upper layer containing the proteoglycan was collected, and a three-fold amount of saturated ethanol was added to cause crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum dried to obtain powdered proteoglycan. Thereafter, the endotoxin concentration of the powdered proteoglycan of Example 3B was measured using the same method as in Example 2A.

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

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

[0121] Powdered proteoglycan was obtained in the same manner as in Example 2A, except that PBS (pH 7.2, Fujifilm Wako Pure Chemical Industries, Ltd., 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 in the proteoglycan before treatment was 403.7 EU / mg, while the endotoxin concentration in the powdered proteoglycan was 14.0 EU / mg. These results demonstrate that the proteoglycan method of the present disclosure (Example 4A) can reduce endotoxins from proteoglycans derived from animal tissues.

[0123] [Example 4B] It was confirmed that the use of an aqueous solvent containing 1 (v / v) % ethanol as a coexistence system in the formation step of the production method of the present disclosure can reduce endotoxins in proteoglycans derived from animal tissues.

[0124] Salmon-derived proteoglycan was added to PBS (pH 7.2, Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No.: 164-28713). After the addition, the mixture was stirred and sonicated at approximately 23°C to obtain a 5 mg / ml proteoglycan solution. Then, 1 (v / v)% ethanol and 1 (v / v)% Triton™-X114 were 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was collected, and a three-fold volume of saturated ethanol was added to crystallize the mixture. 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 x g. After centrifugation, the saturated ethanol was removed, and the proteoglycan was dried in vacuo to obtain a powder. Thereafter, the endotoxin concentration of the powdered proteoglycan of Example 4B was measured in the same manner as in Example 2A.

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

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

[0127] Powdered proteoglycan was obtained in the same manner as in Example 3B, except that PBS (pH 7.2, Fujifilm Wako Pure Chemical Industries, Ltd., 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. These results demonstrate that the endotoxin concentration in 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 endotoxins in proteoglycans derived from animal tissues can be reduced by repeatedly performing the reduction step in the manufacturing method of the present disclosure and using an aqueous solvent containing 1 (v / v)% ethanol as the coexistence system in the second formation step.

[0130] Salmon-derived proteoglycan was added to PBS (pH 7.2, Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No.: 164-28713). After the addition, the mixture was stirred and sonicated at approximately 23°C to obtain a 5 mg / ml proteoglycan solution. Then, 1 (v / v)% 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was recovered so as not to contain the solution at the boundary between the lower and upper layers. After the recovery, 1% ethanol and 1 (v / v)% Triton™-X114 were 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was collected, and a three-fold amount of saturated ethanol was added to cause crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum dried to obtain powdered proteoglycan. The endotoxin concentration of the powdered proteoglycan of Example 4D was then measured by the LAL method using the same method as in Example 2A.

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

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

[0133] After the preparation, 1% Triton™-X114 was added. After the addition, the mixture was stirred and 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After confirmation, the upper layer containing the proteoglycan was collected, and a three-fold amount of saturated ethanol was added to cause crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum-dried to obtain powdered proteoglycan. Thereafter, the endotoxin concentrations of the untreated proteoglycan and the powdered proteoglycan of Example 5A were measured using the same method as in Example 2A.

[0134] As a result, the endotoxin concentration in the proteoglycan before treatment was 362.6 EU / mg, whereas the endotoxin concentration in the powdered proteoglycan of Example 5A was 10.5 EU / mg. These results demonstrate that the manufacturing method of the present disclosure can reduce the endotoxin concentration in proteoglycan.

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

[0136] After the preparation, 1% Triton™-X114 was added. After the addition, the mixture was stirred and 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was recovered so as not to contain the solution at the boundary between the lower and upper layers. After the recovery, 1 (v / v)% 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was collected, and a three-fold amount of saturated ethanol was added to crystallize it. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum dried to obtain powdered proteoglycan. Then, the endotoxin concentration of the powdered proteoglycan of Example 5B was measured using the same method as in Example 2A.

[0137] As a result, the endotoxin concentration in the powdered proteoglycan of Example 5B was 9.7 EU / mg. These results demonstrate that the endotoxin concentration in 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, the mixture was stirred and 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, the mixture was centrifuged at 37°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was collected, and a three-fold amount of saturated ethanol was added to cause crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 37°C for 3 minutes at 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum-dried to obtain powdered proteoglycan. The endotoxin concentration of the powdered proteoglycan of Example 5C was then measured using the same method as in Example 2A.

[0140] As a result, the endotoxin concentration in the powdered proteoglycan of Example 5C was 6.9 EU / mg. These results demonstrate that the endotoxin concentration in 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 preparation, 1% Triton™-X114 was added. After the addition, the mixture was stirred and 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was collected so as not to contain the solution at the boundary between the lower and upper layers. After the collection, a 1 (v / v)% mixture of Triton™-X45 and Triton™-X100 in a 3:10 ratio was added. After the addition, 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, the mixture was centrifuged at 37°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was collected, and a three-fold volume of saturated ethanol was added to cause crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 37°C for 3 minutes at 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum dried to obtain powdered proteoglycan. Thereafter, the endotoxin concentration of the powdered proteoglycan of Example 5D was measured using the same method as in Example 2A.

[0143] As a result, the endotoxin concentration in the powdered proteoglycan of Example 5D was 9.2 EU / mg. These results demonstrate that the endotoxin concentration in 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% Triton™-X114 was added. After the addition, the mixture was stirred and 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After confirmation, the upper layer containing the proteoglycan was collected so as not to contain the solution at the boundary between the lower and upper layers. After the collection, a 1 (v / v)% mixture of Triton™-X45 and Triton™-X100 in a 3:20 ratio was added. After the addition, 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, the mixture was centrifuged at 37°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After the confirmation, the upper layer containing the proteoglycan was collected, and a three-fold volume of saturated ethanol was added to crystallize it. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 37°C for 3 minutes at 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum dried to obtain powdered proteoglycan. Then, the endotoxin concentration of the powdered proteoglycan of Example 5E was measured using the same method as in Example 2A.

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

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

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

[0149] After the preparation, 1% Triton™-X114 was added. After the addition, the mixture was stirred and 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After confirmation, the upper layer containing the proteoglycan was collected, and a three-fold amount of saturated ethanol was added to cause crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum-dried to obtain powdered proteoglycan. The powdered proteoglycan was then subjected to chromatography using an anion exchange resin. Specifically, the powdered proteoglycan was dissolved in 0.2 mol / L NaCl. After dissolution, the sample was charged onto a DEAE M column (manufactured by TOSOH Corporation), and the NaCl solution containing the proteoglycan was passed through. After passing through, the column was washed with 0.2 mol / L NaCl. After washing, 0.5 mol / L NaCl was passed through to elute the proteoglycan. The eluate containing the eluted proteoglycan was then desalted as specified, and lyophilized as specified to obtain powdered proteoglycan. The endotoxin concentration of the powdered proteoglycan of Example 6A was then measured using the same method as in Example 2A.

[0150] As a result, the endotoxin concentration in the proteoglycan treated with Triton™-X114 and DEAE chromatography was 0.83 EU / mg. These results demonstrate that the manufacturing method of the present disclosure can reduce the endotoxin concentration in proteoglycan. While proteoglycan was eluted using 0.5 mol / L NaCl in Example 6A, it was confirmed that proteoglycan could also be eluted using 1 mol / L and 2 mol / L NaCl.

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

[0153] After the preparation, 1% Triton™-X114 was added. After the addition, the mixture was stirred and 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After confirmation, the upper layer containing the proteoglycan was collected, and a three-fold amount of saturated ethanol was added to cause crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum-dried to obtain powdered proteoglycan. The powdered proteoglycan was then subjected to chromatography using an anion exchange resin. Specifically, the powdered proteoglycan was dissolved in 0.2 mol / L NaCl. After dissolution, the sample was charged to a Cellufine™ ET Clean S (manufactured by JNC Corporation), and the NaCl solution containing the proteoglycan was passed through, and the flow-through was collected. After passing through, the column was washed with 0.2 mol / L NaCl, and the flow-through was collected. The flow-through containing the proteoglycan was then desalted as specified and freeze-dried as specified to obtain powdered proteoglycan. The endotoxin concentration of the powdered proteoglycan of Example 6B was then measured using the same method as in Example 2A.

[0154] As a result, the endotoxin concentration in the proteoglycan treated with Triton™-X114 and polylysine chromatography was 1.93 EU / mg. These results demonstrate that the production method of the present disclosure can reduce the endotoxin concentration in proteoglycan.

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

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

[0157] After the preparation, 1% Triton™-X114 was added. After the addition, the mixture was stirred and 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, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, separation into two layers was confirmed. After confirmation, the upper layer containing the proteoglycan was collected, and a three-fold amount of saturated ethanol was added to cause crystallization. After the crystallization, the saturated ethanol was removed, and saturated ethanol was added again. After the addition, the mixture was centrifuged at 23°C for 3 minutes at 9060 x g. After the centrifugation, the saturated ethanol was removed, and the mixture was vacuum-dried to obtain powdered proteoglycan. The powdered proteoglycan was then subjected to chromatography using an anion exchange resin. Specifically, the powdered proteoglycan was dissolved in 0.2 mol / L NaCl. After dissolution, the sample was charged to a Cellufine™ ET Clean L (manufactured by JNC Corporation), and the NaCl solution containing the proteoglycan was passed through, and the flow-through was collected. After passing through, the column was washed with 0.2 mol / L NaCl, and the flow-through was collected. The flow-through containing the proteoglycan was then desalted as specified and freeze-dried as specified to obtain powdered proteoglycan. The endotoxin concentration of the powdered proteoglycan of Example 6C was then measured using the same method as in Example 2A.

[0158] As a result, the endotoxin concentration in the proteoglycan treated with Triton™-X114 and polylysine chromatography was 1.93 EU / mg. These results demonstrate that the production method of the present disclosure can reduce the endotoxin concentration in proteoglycan.

[0159] [Comparative Example 6A] Salmon-derived proteoglycan was subjected to chromatography using an anion exchange resin. Specifically, the salmon-derived proteoglycan was dissolved in 0.2 mol / L NaCl. After dissolution, the sample was charged onto a DEAE M column (manufactured by TOSOH Corporation), and the NaCl solution containing the dissolved proteoglycan was passed through. After passing through, the column was washed with 0.2 mol / L NaCl. After washing, 0.5 mol / L NaCl was passed through to elute the proteoglycan. The eluate containing the eluted proteoglycan was then desalted as specified and freeze-dried as specified to obtain powdered proteoglycan. The endotoxin concentration of the powdered proteoglycan of Comparative Example 6A was then measured using the same method as in Example 2A.

[0160] As a result, the endotoxin concentration in the proteoglycan that had only been subjected to DEAE chromatography treatment was 90.6 EU / mg. These results demonstrate that the endotoxin concentration in proteoglycan can be reduced by the production method of the present disclosure.

[0161] [Comparative Example 6B] Salmon-derived proteoglycan was subjected to chromatography using an anion exchange resin. Specifically, the salmon-derived proteoglycan was dissolved in 0.2 mol / L NaCl. After dissolution, the sample was charged to a Cellufine™ ET Clean S (manufactured by JNC Corporation), and the NaCl solution containing the proteoglycan was passed through, and the flow-through was collected. After passing through, the column was washed with 0.2 mol / L NaCl, and the flow-through was collected. The flow-through containing the proteoglycan was then desalted as specified, and powdered proteoglycan was obtained by lyophilization as specified. The endotoxin concentration of the powdered proteoglycan of Comparative Example 6B was then measured using the same method as in Example 2A.

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

[0163] [Comparative Example 6C] Salmon-derived proteoglycan was subjected to chromatography using an anion exchange resin. Specifically, the proteoglycan was dissolved in 0.2 mol / L NaCl. After dissolution, the sample was charged to a Cellufine™ ET Clean L (manufactured by JNC Corporation), and the NaCl solution containing the proteoglycan was passed through, and the flow-through was collected. After passing through, the column was washed with 0.2 mol / L NaCl, and the flow-through was collected. The flow-through containing the proteoglycan was then desalted as specified, and powdered proteoglycan was obtained by lyophilization as specified. The endotoxin concentration of the powdered proteoglycan of Comparative Example 6C was then measured using the same method as in Example 2A.

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

[0165] Although the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications that can be understood by a person 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 publications cited herein are incorporated by reference into this specification in their entirety as if the contents 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, the disclosures of which are incorporated herein in their entireties.

[0168] <Appendix> Some or all of the above embodiments and examples can be described as, but are not limited to, the following appendices. <Agents> (Appendix A1) An agent for use in reducing endotoxins in proteoglycans derived from animal tissues, comprising a nonionic surfactant. (Appendix A2) The agent according to Appendix A1, wherein the nonionic surfactant is a polyoxyethylene alkyl ether (POEAE)-based nonionic surfactant. (Appendix A3) The agent according to Appendix A1 or A2, wherein the nonionic surfactant is polyethylene glycol tert-octylphenyl ether. (Appendix A4) The agent according to any one of Appendix A1 to A3, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray. <Method for producing proteoglycan> (Appendix A5) A method for producing proteoglycan, comprising: a first reduction step of reducing endotoxins from proteoglycans derived from animal tissue using a nonionic surfactant; and a second reduction step of reducing endotoxins from the proteoglycans by treating the resulting proteoglycans with a chromatography carrier. (Appendix A6) The method according to Appendix A5, wherein the second reduction step comprises: an adsorption step of contacting the proteoglycans obtained in the first reduction step with the chromatography carrier to adsorb endotoxins onto the chromatography carrier; and a recovery step of recovering the proteoglycans with reduced endotoxins. (Appendix A7) The method according to Appendix A5, wherein the second reduction step comprises: an adsorption step of contacting the proteoglycans obtained in the first reduction step with the chromatography carrier to adsorb proteoglycans onto the chromatography carrier; and a recovery step of recovering the adsorbed proteoglycans. (Appendix A8) The manufacturing method according to any one of Appendices A5 to A7, wherein the second reduction step is carried out multiple times.(Appendix A9) The manufacturing method according to any one of Appendices A5 to A8, in which the second reduction step involves carrying out the following steps (A) and (B) on the proteoglycan obtained in the first reduction step: Step (A): an adsorption step of contacting the proteoglycan 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 contacting the proteoglycan with the chromatography carrier to adsorb proteoglycan onto the chromatography carrier, and a recovery step of recovering the adsorbed proteoglycan. (Appendix A10) The manufacturing method according to Appendix A8 or A9, wherein the second reduction step includes: 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, wherein the chromatography carrier is an ion exchange carrier or a hydrophobic interaction carrier, and the chromatography carrier in the first treatment step and the chromatography carrier in the second treatment step are different chromatography carriers. (Appendix A11) The manufacturing method according to any of Appendixes A5 to A9, wherein the chromatography carrier is a chromatography resin. (Appendix A12) The manufacturing method according to Appendix A11, wherein the chromatography resin is an ion exchange resin and / or a hydrophobic interaction resin. (Appendix A13) The manufacturing method according to Appendix A12, wherein the ion exchange resin is an anion exchange resin. (Appendix A14) The manufacturing method according to Appendix A13, wherein the anion exchange resin is a resin having a diethylaminoethyl group as a functional group. (Appendix A15) The production method according to Appendix A12, wherein 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.(Appendix A16) The manufacturing method according to any one of Appendices A5 to A15, wherein the first reduction step comprises: a concentration step of raising the temperature of a coexistence system containing the nonionic surfactant and the animal tissue-derived proteoglycan from a temperature below the cloud point of the nonionic surfactant to a temperature equal to or higher than the cloud point of the nonionic surfactant to form a surfactant phase containing the nonionic surfactant and concentrate the endotoxin in the surfactant phase; and a recovery step of recovering the proteoglycan with reduced endotoxin by recovering a phase other than the surfactant phase. (Appendix A17) The manufacturing method according to Appendices A16, wherein the first reduction step comprises a separation step of separating the surfactant phase from a phase other than the surfactant phase, and the recovery step recovers the phase other than the surfactant phase after separation. (Appendix A18) The manufacturing method according to Appendices A16 or A17, wherein the phase other than the surfactant phase is an aqueous phase. (Appendix A19) The manufacturing method according to Appendices A17 or A18, wherein the separation is by centrifugation. (Appendix A20) The manufacturing method according to any one of Appendices A16 to A19, wherein the coexistence system contains micelles of the nonionic surfactant, and the micelles contain the endotoxin. (Appendix A21) The manufacturing method according to any one of Appendices A5 to A20, wherein prior to the first reduction step, the method includes a formation step of contacting the nonionic surfactant with the animal tissue-derived proteoglycan to form micelles of the nonionic surfactant containing the endotoxin, and in the concentration step, the surfactant phase is formed from the micelles and the endotoxin is concentrated in the surfactant phase. (Appendix A22) The manufacturing method according to any one of Appendices A5 to A21, wherein the nonionic surfactant is a polyoxyethylene alkyl ether (POEAE)-based nonionic surfactant. (Appendix A23) The manufacturing method according to any one of Appendices A5 to A22, wherein the nonionic surfactant is polyethylene glycol tert-octylphenyl ether. (Appendix A24) The method of any one of Appendices A5 to A23, wherein the peak top molecular weight of the proteoglycan after the second reduction step is 300,000 to 1,300,000. (Appendix A25) The method of any one of Appendices A5 to A24, wherein the sugar chain of the proteoglycan is chondroitin sulfate.(Appendix A26) The method according to any one of Appendices A5 to A25, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray.

[0169] <Method for Proteoglycan Production> (Appendix B1) A method for producing proteoglycan, comprising a reduction step of reducing endotoxins from animal tissue-derived proteoglycan using a nonionic surfactant. (Appendix B2) The reduction step comprises: a concentration step of raising the temperature of a coexistence system containing the nonionic surfactant and the animal tissue-derived proteoglycan from a temperature below the cloud point of the nonionic surfactant to a temperature above the cloud point of the nonionic surfactant to form a surfactant phase containing the nonionic surfactant and concentrate the endotoxins in the surfactant phase; and a recovery step of recovering the proteoglycan with reduced endotoxins by recovering the phase other than the surfactant phase. (Appendix B3) The reduction step comprises a separation step of separating the surfactant phase from the phase other than the surfactant phase, and the recovery step involves recovering the phase other than the surfactant phase after separation. (Appendix B4) The production method according to Appendix B3, wherein the phase other than the surfactant phase is an aqueous phase. (Appendix B5) The manufacturing method according to Appendices B3 or B4, wherein the separation is performed by centrifugation. (Appendix B6) The manufacturing method according to any one of Appendices B2 to B5, wherein the coexistence system contains micelles of the nonionic surfactant, and the micelles contain the endotoxin. (Appendix B7) The manufacturing method according to any one of Appendices B2 to B6, wherein prior to the reduction step, a formation step is included in which the nonionic surfactant is contacted with the animal tissue-derived proteoglycan to form micelles of the nonionic surfactant containing the endotoxin, and in the concentration step, a surfactant phase is formed from the micelles, and the endotoxin is concentrated in the surfactant phase. (Appendix B8) The manufacturing method according to any one of Appendices B1 to B7, wherein the nonionic surfactant is a polyoxyethylene alkyl ether (POEAE)-based nonionic surfactant. (Appendix B9) The manufacturing method according to any one of Appendices B1 to B8, wherein the nonionic surfactant is polyethylene glycol tert-octylphenyl ether. (Appendix B10) The production method according to any one of Appendices B1 to B9, wherein the peak top molecular weight of the proteoglycan after the reduction step is 300,000 to 1,300,000.(Appendix B11) The production method according to any one of Appendices B1 to B10, wherein the sugar chain of the proteoglycan is chondroitin sulfate. (Appendix B12) The production method according to any one of Appendices B1 to B11, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray. <Composition> (Appendix B13) A composition comprising a proteoglycan derived from animal tissue, wherein the composition comprises a proteoglycan and an endotoxin, and wherein the endotoxin content in the composition is 4 EU / mg or less. (Appendix B14) The composition according to Appendices B13, wherein the peak top molecular weight of the proteoglycan is 300,000 to 1,300,000. (Appendix B15) The composition according to Appendices B13 or B14, wherein the sugar chain of the proteoglycan is chondroitin sulfate. (Appendix B16) The composition of any of Appendices B13 to B15, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray.

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

Claims

1. a first reduction step of reducing endotoxins from proteoglycan derived from animal tissue using a coexistence system containing a nonionic surfactant and proteoglycan derived from animal tissue; a second reduction step of reducing endotoxins from the proteoglycan by treating the resulting proteoglycan with a chromatography carrier; The method for producing proteoglycan, wherein the pH of the coexistence system is 6.8 or less.

2. A first reduction step of reducing endotoxins from proteoglycan derived from animal tissue using a nonionic surfactant; a second reduction step of reducing endotoxins from the proteoglycan by treating the resulting proteoglycan with a chromatography carrier; The method for producing proteoglycan, wherein the chromatography carrier is an anion exchange carrier.

3. The second reduction step includes: an adsorption step of contacting the proteoglycan obtained in the first reduction step with the chromatography carrier to adsorb endotoxin onto the chromatography carrier; A recovery step of recovering the proteoglycan with reduced endotoxin; The method of claim 1 or 2, comprising:

4. The second reduction step includes: an adsorption step of contacting the proteoglycan obtained in the first reduction step with the chromatography carrier to adsorb the proteoglycan onto the chromatography carrier; A recovery step of recovering the adsorbed proteoglycan; The method of claim 1 or 2, comprising:

5. The method according to claim 1 or 2, wherein the second reduction step is carried out multiple times.

6. The method according to claim 1 or 2, wherein the second reduction step comprises carrying out the following steps (A) and (B) on the proteoglycan obtained in the first reduction step: (A) Process: an adsorption step of contacting the proteoglycan with the chromatography carrier to adsorb endotoxin onto the chromatography carrier; and a recovery step of recovering the proteoglycan with reduced endotoxin; (B) Process: an adsorption step of contacting the proteoglycan with the chromatography carrier to adsorb the proteoglycan onto the chromatography carrier; and A recovery step of recovering the adsorbed proteoglycan.

7. The second reduction step includes: a first treatment step of treating the proteoglycan obtained in the first reduction step with the chromatography carrier; a second treatment step of treating the treated proteoglycan with the chromatography carrier; The chromatography support is an ion exchange support or a hydrophobic interaction support, 6. The method of claim 5, wherein the chromatography carrier in the first process step and the chromatography carrier in the second process step are different chromatography carriers.

8. The method according to claim 1 or 2, wherein the chromatography support is a chromatography resin.

9. The process according to claim 8, wherein the chromatography resin is an ion exchange resin and / or a hydrophobic interaction resin.

10. The method according to claim 9 , wherein the ion exchange resin is an anion exchange resin.

11. The method according to claim 10, wherein the anion exchange resin is a resin having a diethylaminoethyl group as a functional group.

12. The method according to claim 9 , wherein 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.

13. The manufacturing method described in claim 1, wherein the pH in the coexistence system is pH 5 to pH 6.

8.

14. The first reduction step includes: a concentrating step of heating a coexistence system containing the nonionic surfactant and the proteoglycan derived from the animal tissue from a temperature lower than the cloud point of the nonionic surfactant to a temperature equal to or higher than the cloud point of the nonionic surfactant to form a surfactant phase containing the nonionic surfactant, and concentrating the endotoxin in the surfactant phase; The method for producing the present invention according to claim 1 or 2, further comprising a recovery step of recovering the proteoglycan having reduced endotoxins by recovering phases other than the surfactant phase.

15. The first reduction step includes a separation step of separating the surfactant phase from a phase other than the surfactant phase, The method according to claim 14 , wherein the recovery step recovers a phase other than the surfactant phase after the separation.

16. The process of claim 14, wherein the phase other than the surfactant phase is an aqueous phase.

17. The method of claim 15, wherein the separation is by centrifugation.

18. the coexistence system includes micelles of the nonionic surfactant, The micelle contains the endotoxin. The method of claim 14.

19. a formation step, prior to the first reduction step, of contacting the nonionic surfactant with the proteoglycan derived from the animal tissue to form micelles of the nonionic surfactant containing the endotoxin; The method according to claim 14, wherein in the concentrating step, the surfactant phase is formed from the micelles, and the endotoxin is concentrated in the surfactant phase.

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

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

22. The 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.

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

24. The method of claim 1 or 2, wherein the animal is selected from the group consisting of salmon, pig, poultry, flounder, and ray.