Use of fatty alcohol polyglycol ethers for reducing endotoxin activity and / or endotoxin in collagen-containing substances and / or collagen-derived substances

Abstract

The present invention relates to a method for reducing lipopolysaccharide activity and / or lipopolysaccharides in a collagen-containing substance and / or a collagen-derived substance, and to a product obtained by said method. The method comprises the steps of providing a collagen-containing substance and / or a collagen-derived substance containing lipopolysaccharides, contacting the collagen-containing substance and / or the collagen-derived substance containing lipopolysaccharides with a polyglycol ether of a fatty alcohol to obtain a mixture, and recovering the collagen-containing substance and / or the collagen-derived substance.

Description

Technical Field

[0001] The present invention relates to reducing lipopolysaccharide (LPS) activity and / or LPS in collagen-containing substances and / or collagen-derived substances, and to products obtained by this method.

Background Art

[0002] Collagen, collagen-containing substances or collagen-derived substances, such as the extracellular matrix (ECM), collagen peptides, gelatin, and (chemically) modified gelatin, are used in various fields ranging from the food industry to the medical and pharmaceutical fields. Interest in this type of substance stems from desirable properties such as biocompatibility and biodegradability. Collagen, as well as collagen-containing substances and collagen-derived substances, are generally considered safe, but the presence of endotoxins such as LPS poses problems for biomedical use.

[0003] LPS is located in the outer membrane of Gram-negative bacteria and functions as a permeability barrier, providing structural integrity and protection. LPS is composed of various polysaccharide chains and lipid A, which is a lipid moiety. Although the LPS molecule is about 10 kDa in size, it may form large aggregates called "micelles" in aqueous media. LPS is not secreted by bacteria but is released when the bacterial cell membrane is disrupted, so it is called an "endotoxin". LPS is highly immunogenic to humans, and even exposure to a small amount of LPS can cause serious side effects. Due to its toxicity, the amount of endotoxin permitted in medical devices is strictly regulated. For example, the US Food and Drug Administration (FDA) permits a maximum of 0.5 endotoxin units (EU) / g or 20 EU / device for products that come into direct or indirect contact with the cardiovascular and lymphatic systems. In the case of devices that come into contact with cerebrospinal fluid, this limit is even lower.

[0004] In the conventional manufacturing processes of collagen-containing substances and / or collagen-derived substances, it is difficult to avoid endotoxin contamination. Furthermore, once endotoxin is present, it is very difficult to remove it, especially from delicate substances containing proteins.

[0005] In the art, a method for removing LPS from a protein solution using a surfactant is described, for example, in International Publication No. WO 2016 / 085345. Although the above approach shows great potential, it relies on Triton X-100, which is controversial in terms of its use.

[0006] Therefore, there remains an unmet need for an efficient and safe method for reducing and / or removing LPS from collagen-containing substances and collagen-derived substances.

[0007] An object of the present invention is to solve one or more of the above problems. For this purpose, the present invention provides a method for reducing LPS activity and / or LPS from collagen-containing substances and / or collagen-derived substances. SUMMARY OF THE INVENTION

[0008] The inventors have identified an effective method for reducing LPS activity and / or LPS in collagen-containing substances and / or collagen-derived substances based on the use of fatty alcohol polyglycol ethers.

[0009] More specifically, the inventors have discovered that Laureth-9, a non-ionic and non-toxic micelle-forming surfactant, is effective in reducing LPS activity in collagen-containing substances and / or collagen-derived substances. Surprisingly, it was found that the concentration of Laureth-9 required to show effectiveness is significantly lower than that of Triton X-100.

[0010] Therefore, the inventors propose an improved method for reducing LPS activity.

[0011] In one aspect, the present invention relates to a method for reducing the LPS activity in a collagen-containing substance and / or a collagen-derived substance.

[0012] In one aspect, the present invention relates to the use of a polyglycol ether of a fatty alcohol for reducing the LPS activity in a collagen-containing substance and / or a collagen-derived substance.

[0013] In one aspect, the present invention relates to a collagen-containing substance and / or a collagen-derived substance having an LPS content of less than 3000 EU / g, preferably less than 1000 EU / g, more preferably less than 100 EU / g, still more preferably less than 10 EU / g, and most preferably less than 1 EU / g.

Brief Description of the Drawings

[0014]

Figure 1

Modes for Carrying Out the Invention

[0015] The present invention relates to a method for reducing the LPS activity in a collagen-containing substance and / or a collagen-derived substance, the method comprising one or more, preferably all, of the following steps: Preparing a collagen-containing substance and / or a collagen-derived substance containing LPS; Contacting the collagen-containing substance and / or the collagen-derived substance containing LPS with a polyglycol ether of a fatty alcohol to obtain a mixture; A step of recovering a collagen-containing substance and / or a collagen-derived substance, preferably having an LPS activity of less than 3000 EU / g, more preferably having an LPS activity of less than 1000 EU / g, still more preferably having an LPS activity of less than 100 EU / g, even more preferably having an LPS activity of less than 10 EU / g, and most preferably having an LPS activity of less than 1 EU / g.

[0016] As used herein, the term "reduction of LPS activity" means that the LPS level of the recovered collagen-containing substance and / or collagen-derived substance is lower than the LPS level of the starting material, i.e., the collagen-containing substance and / or collagen-derived substance before contact with the fatty alcohol polyglycol ether. In the present invention, "reduction of LPS activity" includes both reduction and / or removal of LPS in and / or from the starting material. The reduction and / or removal of LPS in and / or from the starting material can be determined using the Limulus Amebocyte Lysate (LAL) assay, a well-known method for the detection of LPS. LAL is an aqueous extract of the blood cells (amebocytes) of the horseshoe crab Limulus polyphemus. The reaction between LAL and LPS is the basis of the LAL assay and is used to qualitatively (e.g., visual inspection) or quantitatively (e.g., colorimetric or turbidimetric methods) determine the LPS content.

[0017] In one embodiment, it is preferable that the LPS activity of the recovered collagen-containing substance and / or collagen-derived substance is 10% or more lower than that of the starting material. More preferably, the LPS activity of the recovered collagen-containing substance and / or collagen-derived substance is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more lower than that of the starting material. Preferably, the LPS activity of the starting material is less than 20,000 EU / g per 1 g of the dry weight of the collagen-containing substance and / or collagen-derived substance. More preferably, the LPS activity of the starting material is less than 15,000 EU / g per 1 g of the dry weight of the collagen-containing substance and / or collagen-derived substance, and even more preferably, less than 5,000 EU / g per 1 g of the dry weight of the collagen-containing substance and / or collagen-derived substance. The starting material may have a higher or lower LPS activity. When the LPS content is higher, the substance may be pretreated by an ion exchange chromatography step or the like.

[0018] In one embodiment, the collagen-containing substance and / or collagen-derived substance does not contain endotoxin (i.e., does not contain LPS). The term "does not contain endotoxin" may mean that endotoxin is absent and / or may mean "essentially free of endotoxin" (i.e., essentially free of LPS).

[0019] The term "free of" may be synonymous with "substantially free of". The term "substantially free of" includes that the amount of a substance or compound cannot be measured according to the standard techniques in the art and / or is below a specific threshold. Additionally or alternatively, the term "substantially free of" may mean that the endotoxin level is less than 10, preferably less than 1, more preferably less than 0.1, even more preferably less than 0.01, and most preferably less than 0.001 (all in units of EU / g or EU / ml). The expressions "free of" or "substantially free of" include that a substance or compound is completely absent (e.g., 0% by weight or 0 EU / g). The terms "absent", "free of", and "substantially free of" can be used interchangeably in the context of the present invention.

[0020] When the amount of LPS (activity) in the starting material is high, as described herein, it is particularly advantageous to use a polyglycol ether of a fatty alcohol for the removal of LPS (activity). For example, when the LPS content in the starting material is relatively high, such as 1000 EU / g or more, preferably 2500 EU / g or more, more preferably 5000 EU / g or more, even more preferably 10000 EU / g or more, and most preferably 50000 EU / g or more, the polyglycol ether of a fatty alcohol has been found to be more effective for the removal of LPS (activity) than other known methods such as Triton X-100. In one embodiment, the LPS content in the collagen-containing substance and / or the collagen-derived substance is 500 EU / g or more, or 1000 EU / g or more, or 2500 EU / g or more, or 5000 EU / g or more, or 10000 EU / g or more, or 50000 EU / g or more, and / or 50000 EU / g or less, or 10000 EU / g or less, or 5000 EU / g or less, or 2500 EU / g or less, or 1000 EU / g or less, or 500 EU / g or less. Additionally or alternatively, the LPS content in the collagen-containing substance and / or the collagen-derived substance is preferably 500 to 50000 EU / g, 1000 to 50000 EU / g, and most preferably 1000 to 10000 EU / g.

[0021] The present disclosure involves contacting a collagen-containing substance and / or a collagen-derived substance with a polyglycol ether of a fatty alcohol. The purpose may be to obtain a mixture or formulation in which an interaction between the collagen-containing substance and / or the collagen-derived substance and the polyglycol ether of the fatty alcohol is provided.

[0022] In one embodiment, the collagen-containing substance and / or the collagen-derived substance can be an extracellular matrix, natural collagen, gelatin, (chemically) modified gelatin, gelatin hydrolysate, and mixtures thereof. Natural collagen may include acid-extracted collagen and enzyme-soluble collagen, including telocollagen, atelocollagen, and fibrillar collagen.

[0023] The term "collagen" in the context of the present invention means an amino acid sequence containing a repeating (Gly-X-Y) sequence, preferably containing 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 200 or more, 300 or more, or 400 or more sequences of Gly-X-Y, where X and Y are amino acid residues independently selected from each other, provided that X and / or Y are more preferably proline. "Collagen" preferably has a sequence found in natural collagen of one or more animal species. Additionally or alternatively, "collagen" can mean the full-length sequence of (natural) collagen or a fragment or subunit thereof, preferably one or more of types I to XXVII collagen, more preferably one or more of types I, II, III, V, or X collagen, and even more preferably one or more of types I, II, or III collagen. For example, the term "collagen" may refer to an α1(I), α2(I), α1(II), or α1(III) chain, or a fragment thereof. The term "collagen" encompasses a triple helix structure formed by three subunits as present in natural collagen.

[0024] In the context of the present invention, "collagen" includes "gelatin", "collagen hydrolysate", and "hydrolyzed gelatin".

[0025] In the context of the present invention, the term "collagen hydrolysate" means a mixture of short chains of amino acids (dipeptides, tripeptides, oligopeptides, polypeptides) derived from the (partial) hydrolysis of native (full-length) collagen by enzymatic hydrolysis or the like. The degree of hydrolysis affects the average molecular weight (expressed in daltons, Da) of the final product. The term "collagen hydrolysate" may be used interchangeably or synonymously with the terms "hydrolyzed collagen" or "collagen peptide". In the context of the present invention, "collagen hydrolysate" includes collagen that has been hydrolyzed or partially hydrolyzed. "Collagen hydrolysate" in the context of the present invention can be produced from collagen-containing materials in a one-step process or via an intermediate gelatin step (i.e., whereby "hydrolyzed gelatin" is obtained). Thus, the term "collagen hydrolysate" includes hydrolyzed gelatin (i.e., hydrolyzed gelatin).

[0026] The collagen-containing substances and / or collagen-derived substances used herein can be obtained from one or more types of collagen known in the art. More specifically, the collagen-containing substances and / or collagen-derived substances used herein can be selected from the group including type I, II, and III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII collagen. Additionally or alternatively, the collagen-containing substances and / or collagen-derived substances used herein may be a mixture of one or more types of collagen.

[0027] The collagen-containing substance and / or the collagen-derived substance may be obtained from a synthetic composition (i.e., recombinant collagen), but it is preferably derived from any animal raw material from which the collagen-containing substance and / or the collagen-derived substance can be prepared. In a preferred embodiment, the animal raw material is derived from cattle, pigs, chickens, or fish. In another preferred embodiment, the collagen-containing substance and / or the collagen-derived substance is derived from skin and / or skin connective tissue. In yet another preferred embodiment, the collagen-containing substance and / or the collagen-derived substance is derived from cartilage. In yet another preferred embodiment, the collagen-containing substance and / or the collagen-derived substance is derived from bone.

[0028] As used herein, the term "collagen-derived substance" refers to proteins and peptides that are part of the collagen matrix in the raw material and are processed to prepare collagen-derived substances such as gelatin, for example. Gelatin and / or (chemically) modified gelatin may be produced by (partial) alkali and / or acid hydrolysis, enzymatic hydrolysis, thermal hydrolysis, and / or combinations thereof. Gelatin does not consist of uniform protein molecules but contains various amounts of protein molecules of various lengths. Preferably, the gelatin used herein has an average molecular weight in the range of 1500 Da to 300 kDa, preferably 2000 Da to 300 kDa, 4000 Da to 300 kDa, 5000 Da to 300 kDa, 10 kDa to 300 kDa, or 20 kDa to 300 kDa, more preferably 50 kDa to 300 kDa, most preferably 100 kDa to 300 kDa, for example 100 kDa to 275 kDa, or 100 kDa to 250 kDa. The term "(chemically) modified gelatin" encompasses gelatin having one or more functionalized groups, such as acrylamide, ferulic acid, methacryloyl (e.g., GelMA), deaminotyrosine (e.g., GelDAT), furfurylamine, norbornene, etc., on the gelatin backbone (Klotz, B. J., Gawlitta, D., Rosenberg, A. J., Malda, J., & Melchels, F. P. (2016). Gelatin-methacryloyl hydrogels: towards biofabrication-based tissue repair. Trends in biotechnology, 34(5), 394-407). "Collagen-derived" substances also include substances obtained by further processing gelatin, such as gelatin hydrolysates, which are peptide preparations obtained by hydrolyzing gelatin into peptide molecules with an average molecular weight of 70 kDa or less, usually 20 kDa or less, and usually 100 to 15000 Da.

[0029] In one embodiment, the collagen-containing substance and / or collagen-derived substance may be in the form of a solution, where the solvent may contain an acid (e.g., acetic acid, formic acid) or an organic solvent (e.g., supercritical carbon dioxide, acetone, preferably 30% or less). The collagen-containing substance and / or collagen-derived substance may be in the form of an aqueous solution, and the aqueous solution may contain any type of water such as tap water, distilled water, deionized water, and / or ultrapure water. The term "aqueous solution" in the context of the present invention encompasses a solution containing one or more co-solvents in addition to water, and the co-solvent may depend on the polyglycol ether of the selected fatty alcohol. The presence of the co-solvent can, for example, improve the solubility of the polyglycol ether of the fatty alcohol. As is recognized by those skilled in the art, co-solvents or surfactants suitable for the present invention include ethanol, isopropyl alcohol, propylene glycol, polyethylene glycol (PEG), sodium lauryl sulfate, polysorbate surfactants (e.g., polysorbate 20, polysorbate 80), sorbitan esters (e.g., sorbitan monolaurate, sorbitan monooleate), ethoxylated alcohols, ethoxylated fatty acids, ethoxylated fatty amines, and one or more selected from the group consisting of, preferably ethanol.

[0030] The solution can be prepared by dissolving the (dry) collagen-containing substance and / or collagen-derived substance in a solvent. The collagen-containing substance and / or collagen-derived substance is preferably dissolved at room temperature or a higher temperature, but since the undesirable hydrolysis of the collagen-containing substance and / or collagen-derived substance may increase at a higher temperature, it is preferably 68°C or less, more preferably 65°C or less, and even more preferably 60°C or less. It is possible to perform the previous step at a different temperature, but it is preferably 68°C or less. Preferably, the mixing time is 10 minutes or more. It is also possible to use a shorter or longer mixing time, but for effectiveness and efficiency, the mixing time is preferably 10 - 60 minutes. Alternatively, the collagen-containing substance and / or collagen-derived substance can also be brought into direct contact with a pure solution of laureth-9.

[0031] In one embodiment, the aqueous solution may contain a collagen-containing substance and / or a collagen-derived substance at any concentration. In a preferred embodiment, the aqueous solution contains a collagen-containing substance and / or a collagen-derived substance of 1 w / w% or more, preferably 4 w / w% or more, more preferably 6 w / w% or more, still more preferably 8 w / w% or more, even more preferably 10 w / w% or more, and most preferably 20 w / w% or more.

[0032] In another preferred embodiment, the aqueous solution contains a collagen-containing substance and / or a collagen-derived substance of 1 to 50 w / w%, preferably 2 to 30 w / w%, more preferably 4 to 15 w / w%, and still more preferably 6 to 10 w / w%. The aqueous medium can contain a collagen-containing substance and / or a collagen-derived substance in an amount that is not so high that the aqueous solution becomes too viscous to be processed according to the method. The aqueous solution of the present disclosure preferably contains a collagen-containing substance and / or a collagen-derived substance of 55 w / w% or less, 50 w / w% or less, 45 w / w% or less, 40 w / w% or less, 35 w / w% or less, 30 w / w% or less, 25 w / w% or less, 20 w / w% or less, 15 w / w% or less, 10 w / w% or less. When using a relatively low molecular weight collagen-containing substance and / or a collagen-derived substance such as a hydrolyzed gelatin, higher concentrations of a collagen-containing substance and / or a collagen-derived substance such as 60 w / w% or more, 70 w / w% or more, 80 w / w% or more, and 90 w / w% or more may be possible. By increasing the temperature, it is possible to lower the viscosity of the collagen-containing and / or collagen-derived aqueous solution, but an undesirable hydrolysis of the collagen-containing substance and / or the collagen-derived substance may occur due to the increase in temperature.

[0033] The polyglycol ether of fatty alcohol can be a polyglycol ether of C8-C20 fatty alcohol, preferably a polyglycol ether of C8-C18 fatty alcohol, more preferably a polyglycol ether of C8-C16 fatty alcohol, and most preferably a polyglycol ether of C10-C14 fatty alcohol. In a preferred embodiment, the polyglycol ether of fatty alcohol is preferably selected from the list including polyglycol ether of 1-decanol, polyglycol ether of 1-dodecanol, polyglycol ether of 1-tetradecanol, polyglycol ether of 1-cetyl alcohol, polyglycol ether of 1-palmitoleyl alcohol, polyglycol ether of 1-octadecenol alcohol, and / or polyglycol ether of 1-stearyl alcohol. Most preferably, the polyglycol ether of fatty alcohol is the polyglycol ether of 1-dodecanol.

[0034] In the present disclosure, the fatty alcohol (or long-chain alcohol) is typically a straight-chain primary alcohol and is typically obtained from natural fats and oils. Related fatty alcohols include lauryl alcohol (C12), myristyl alcohol (C14), stearyl alcohol (C18), and oleyl alcohol (C16). Fatty alcohols typically have an even number of carbon atoms and one alcohol group (-OH) bonded to the terminal carbon. Some are unsaturated and some are branched. Similar to fatty acids, they are often generically named by the number of carbon atoms in the molecule, such as "C12 alcohol" which is an alcohol having 12 carbon atoms (such as dodecanol).

[0035] In one embodiment, the polyglycol ether of fatty alcohol contains and / or on average contains 1 to 20 oxyethylene groups, preferably 5 to 18 oxyethylene groups, more preferably 8 to 16 oxyethylene groups, and most preferably 10 to 14 oxyethylene groups.

[0036] In a more preferred embodiment, the polyglycol ether of the fatty alcohol contains, and / or on average contains, from 1 to 100 oxyethylene groups, more preferably from 2 to 75 oxyethylene groups, still more preferably from 3 to 50 oxyethylene groups, even more preferably from 4 to 40 oxyethylene groups, even more preferably from 5 to 30 oxyethylene groups, even more preferably from 6 to 20 oxyethylene groups, and most preferably from 7 to 10 oxyethylene groups. In another preferred embodiment, the polyglycol ether of the fatty alcohol contains, on average, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more oxyethylene groups. In yet another preferred embodiment, the polyglycol ether of the fatty alcohol contains, and / or on average contains, 200 or fewer, 175 or fewer, 150 or fewer, 125 or fewer, 100 or fewer, 75 or fewer, 50 or fewer, 45 or fewer, 40 or fewer, 39 or fewer, 38 or fewer, 37 or fewer, 36 or fewer, 35 or fewer, 34 or fewer, 33 or fewer, 32 or fewer, 31 or fewer, 30 or fewer, 29 or fewer, 28 or fewer, 27 or fewer, 26 or fewer, 25 or fewer, 24 or fewer, 23 or fewer, 22 or fewer, 21 or fewer, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer oxyethylene groups. In yet another preferred embodiment, the polyglycol ether of the fatty alcohol is preferably selected from one or more polyglycol ethers of fatty alcohols containing C12 and (on average) 9 oxyethylene groups (e.g., laureth-9), C12 and (on average) 10 oxyethylene groups (e.g., Genapol X-100), C12 and (on average) 23 oxyethylene groups (e.g., Brij 35), C16 and (on average) 10 oxyethylene groups (e.g., Brij C10), C18 and (on average) 100 oxyethylene groups (e.g., Brij S100), C18 and (on average) 10 oxyethylene groups (e.g., Eco Brij S10).

[0037] In one embodiment, the polyglycol ether of the fatty alcohol contains laureth-9. Laureth-9 itself is 3,6,9,12,15,18,21,24,27-nonaoxanonatriacontan-1-ol. Practically, this is lauryl alcohol etherified with an average of 9 oxyethylene groups (a mixture of oxyethylene groups ranging from 1 to 20).

[0038] Laureth-9 is a surfactant that forms micelles and can form micelles in solution. Therefore, the so-called critical micelle concentration (CMC) of the surfactant is defined as the concentration of the surfactant at which micelles begin to form. At a specific temperature called the so-called cloud point, the surfactant forms insoluble aggregates in the aqueous medium and phase-separates.

[0039] In one embodiment, the polyglycol ether of the fatty alcohol is preferably present in the mixture at a concentration above the CMC. Preferably, the polyglycol ether of the fatty alcohol is present at a concentration of at least 3 times the CMC. More preferably, the polyglycol ether of the fatty alcohol is present at a concentration of at least 5 times the CMC. Even more preferably, the polyglycol ether of the fatty alcohol is present at a concentration of at least 10 or 20 times the CMC. Most preferably, the polyglycol ether of the fatty alcohol is present at a concentration of at least 30 times the CMC. Preferably, the aqueous solution contains at least 0.001 w / w%, more preferably at least 0.002 w / w%, even more preferably at least 0.004 w / w%, even more preferably at least 0.006 w / w%, even more preferably at least 0.008 w / w%, even more preferably at least 0.01 w / w%, even more preferably at least 0.03 w / w%, and most preferably at least 0.05 w / w% of the polyglycol ether of the fatty alcohol, where the polyglycol ether of the fatty alcohol is preferably laureth-9.

[0040] In another embodiment, the aqueous solution preferably contains 0.001 to 0.5 w / w% of a polyglycol ether of a fatty alcohol, more preferably 0.004 to 0.5 w / w%, still more preferably 0.004 to 0.1 w / w%, and even more preferably 0.004 to 0.06 w / w%, where the polyglycol ether of the fatty alcohol is preferably laureth-9. Even more preferably, the aqueous solution contains 0.001 to 0.05 w / w% of a polyglycol ether of a fatty alcohol, where the polyglycol ether of the fatty alcohol is preferably laureth-9. Even more preferably, the aqueous solution contains 0.002 to 0.02 w / w%, still more preferably 0.004 to 0.01 w / w% of a polyglycol ether of a fatty alcohol, where the polyglycol ether of the fatty alcohol is preferably laureth-9. The exact concentration of the polyglycol ether of the fatty alcohol may be adjusted according to the LPS content in the collagen-containing substance and / or the collagen-derived substance. For example, when the LPS content of the starting material is low, a lower concentration of the polyglycol ether of the fatty alcohol (i.e., not significantly exceeding the CMC value) may be sufficient. The concentration of the polyglycol ether of the fatty alcohol may also be expressed in relation to the amount of the collagen-containing substance and / or the collagen-derived substance present in the aqueous solution. Preferably, the polyglycol ether of the fatty alcohol is present at a concentration of 0.1 to 10 mg, still more preferably 0.2 to 8 mg, and even more preferably 0.9 to 8 mg per 1 g of the collagen-containing substance and / or the collagen-derived substance present in the aqueous solution. Additionally or alternatively, the polyglycol ether of the fatty alcohol is preferably present at a concentration of 0.1 mg or more, 0.2 mg or more, 0.4 mg or more, 0.6 mg or more, 0.8 mg or more, 1 mg or more of the polyglycol ether of the fatty alcohol per 1 g of the collagen-containing substance and / or the collagen-derived substance. Additionally or alternatively, the polyglycol ether of the fatty alcohol is preferably present at a concentration of 10 mg or less, 8 mg or less, 6 mg or less, 5 mg or less, 4 mg or less, 2 mg or less of the polyglycol ether of the fatty alcohol per 1 g of the collagen-containing substance and / or the collagen-derived substance.

[0041] In a preferred embodiment, the aqueous solution contains 6 to 10 w / w% of a collagen-containing substance and / or a collagen-derived substance, and 0.004 to 0.06 w / w% of a polyglycol ether of a fatty alcohol, where the polyglycol ether of the fatty alcohol is preferably laureth-9. Since the proportion of CMC is also known to depend on the composition of the aqueous solution, it may vary more widely.

[0042] In certain embodiments, for example, when the polyglycol ether of the fatty alcohol contains and / or on average contains 1 to 8, preferably 1 to 5, more preferably 1 to 3 oxyethylene groups, an ethanol solution, or a solution containing another co-solvent disclosed herein can be used. Additionally or alternatively, when the polyglycol ether of the fatty alcohol contains and / or on average contains less than 5, less than 4, less than 3, less than 2, less than 1 oxyethylene group, an ethanol solution, or a solution containing another co-solvent disclosed herein can be used, and results equivalent to or even better than those obtained when using an aqueous solution may be obtained. The ethanol solution can contain, for example, 10 to 70 (v / v)%, 20 to 60 (v / v)%, or 30 to 50% ethanol.

[0043] The contact time between the collagen-containing substance and / or the collagen-derived substance and the polyglycol ether of the fatty alcohol is long enough for mixing. Additionally or alternatively, the contact time is selected such that the expected endotoxin activity is achieved. Preferably, the contact time between the collagen-containing substance and / or the collagen-derived substance and the polyglycol ether of the fatty alcohol is 1 minute or more. More preferably, the contact time is 5 minutes or more, even more preferably 10 minutes or more, still more preferably 20 minutes or more, still more preferably 30 minutes or more, still more preferably 60 minutes or more. More preferably, the collagen-containing substance and / or the collagen-derived substance and the polyglycol ether of the fatty alcohol are incubated for 1 to 60 minutes, more preferably 5 to 30 minutes, and most preferably 20 to 30 minutes.

[0044] In one embodiment, the collagen-containing substance and / or the collagen-derived substance may be recovered by contacting the mixture with an adsorbent. The adsorbent can be a surfactant and any suitable adsorbent that can preferably also bind LPS. When adding the surfactant and when contacting with the adsorbent, it is advantageous for the aqueous medium to be in a free flowing state. The viscosity and the gelation temperature vary depending on the collagen-containing substance and / or the collagen-derived substance, but the solution is likely to be free flowing at a temperature of at least 30°C and is likely to be sufficiently processable. The free flowing solution not only enables optimal contact between the medium and the absorbent, but also enables proper separation of the adsorbent.

[0045] In one embodiment, the adsorbent is solid.

[0046] In one embodiment, the solid adsorbent is hydrophobic.

[0047] In one embodiment, the solid adsorbent includes activated carbon, such as Norit SX Plus or Norit ROX 0.8 (Cabot, Netherlands).

[0048] In one embodiment, the absorbent is provided in a filter. Alternatively or additionally, the adsorbent may be deposited in a column. The contacting step of the mixture with the solid adsorbent is carried out for a sufficient time so that the fatty alcohol polyglycol ether and / or LPS can be properly adsorbed.

[0049] In one embodiment, the mixture may be recovered, for example, by filtration, sedimentation, or centrifugation. Preferably, the mixture is contacted with the solid adsorbent for 5 minutes to 1 hour, more preferably 10 to 30 minutes. A shorter time may also be possible, but more adsorbent may be required compared to incubating for a longer time to obtain the desired recovered product. A longer time may also be possible, but it is not very desirable from the perspective of process efficiency. Preferably, the adsorption step is carried out once.

[0050] In one embodiment, the method disclosed herein may include contacting a collagen-containing substance and / or a collagen-derived substance containing lipopolysaccharide with a polyglycol ether of a fatty alcohol, and subsequently obtaining a powder in a drying step. In one embodiment, the method disclosed herein includes contacting a mixture with an adsorbent, and subsequently obtaining a powder in a drying step. Preferably, the drying is spray drying. In one embodiment, the drying step may be part of a step of recovering an (optional) collagen-containing substance and / or a collagen-derived substance. When the drying step is part of a step of recovering an (optional) collagen-containing substance and / or a collagen-derived substance, a powder is obtained in the step of contacting an (optional) mixture with an adsorbent and subsequent (optional) recovery by, for example, filtration, sedimentation, or centrifugation, followed by the drying step.

[0051] Furthermore, the use of a polyglycol ether of a fatty alcohol for reducing LPS (activity) in a collagen-containing substance and / or a collagen-derived substance is also described.

[0052] Furthermore, the use of a polyglycol ether of lauryl alcohol for reducing LPS (activity) in a collagen-containing substance and / or a collagen-derived substance is also described.

[0053] In one aspect, the present invention relates to a collagen-containing substance and / or a collagen-derived substance, optionally obtained by the method disclosed herein. In a preferred embodiment, the collagen-containing substance and / or the collagen-derived substance is gelatin or hydrolyzed gelatin. In another preferred embodiment, the collagen-containing substance and / or the collagen-derived substance is a collagen hydrolysate.

[0054] In one embodiment, the collagen-containing substance and / or collagen-derived substance, optionally obtained by the method according to the preceding claims, preferably has an LPS content of less than 3000 EU / g, preferably less than 1000 EU / g, more preferably less than 100 EU / g, still more preferably less than 10 EU / g, and most preferably less than 1 EU / g.

[0055] In one embodiment, the collagen-containing substance and / or collagen-derived substance may contain a polyglycol ether of one or more fatty alcohols as disclosed herein, preferably laureth-9. In one embodiment, the residue of the polyglycol ether of the fatty alcohol in the collagen-containing substance and / or collagen-derived substance is less than 1000 ppm, optionally less than 750 ppm, or less than 500 ppm, or less than 250 ppm, or less than 100 ppm, or less than 75 ppm, or less than 50 ppm, or less than 25 ppm, or less than 10 ppm, or less than 5 ppm, or less than 2 ppm, or less than 1 ppm, or less than 0.1 ppm. In one embodiment, the amount of the polyglycol ether of the fatty alcohol in the collagen-containing substance and / or collagen-derived substance is preferably from 0.01 to 10 ppm. In one embodiment, the amount of the polyglycol ether of the fatty alcohol in the collagen-containing substance and / or collagen-derived substance is preferably from 0.05 to 5 ppm. In one embodiment, the amount of the polyglycol ether of the fatty alcohol in the collagen-containing substance and / or collagen-derived substance is preferably from 0.1 to 1 ppm. In one embodiment, the amount of the polyglycol ether of the fatty alcohol in the collagen-containing substance and / or collagen-derived substance is preferably from 0.2 to 0.5 ppm. In one embodiment, the amount of the polyglycol ether of the fatty alcohol in the collagen-containing substance and / or collagen-derived substance is preferably from 0.3 to 0.4 ppm.

[0056] Additionally or alternatively, the present invention also relates to a collagen-containing substance and / or a collagen-derived substance, optionally obtained by the method according to the preceding claims, having an LPS content of less than 3000 EU / g, preferably less than 1000 EU / g, more preferably less than 100 EU / g, still more preferably less than 10 EU / g, and most preferably less than 1 EU / g, and a residue of a polyglycol ether of a fatty alcohol being 0.001 to 100 ppm, 0.001 to 50 ppm, 0.001 to 25 ppm, 0.001 to 10 ppm, 0.001 to 5 ppm, 0.001 to 1 ppm, preferably 0.004 to 2 ppm, 0.004 to 1 ppm, 0.004 to 0.8 ppm, 0.004 to 0.6 ppm, 0.004 to 0.4 ppm, 0.004 to 0.2 ppm, where the polyglycol ether of the fatty alcohol is preferably laureth-9.

[0057] In one embodiment, a collagen-containing substance and / or a collagen-derived substance, optionally obtained by the method according to the preceding claims, may find uses in various industries such as the food, pharmaceutical, and cosmetic industries. From this perspective, a collagen-containing substance and / or a collagen-derived substance, optionally obtained by the method of the present invention, may be used for medical devices, pharmaceuticals, and / or cosmetic applications. The collagen-containing substance and / or the collagen-derived substance may be used as a gelling agent, a texturizer, and / or a hydrogel. From this perspective, the present invention further relates to the use of a collagen-containing substance and / or a collagen-derived substance, preferably the use of a collagen-containing substance and / or a collagen-derived substance in medical devices and / or pharmaceuticals and / or cosmetics (applications).

Examples

[0058] Example 1. Effect of Triton X-100 and laureth-9 on the reduction of LPS activity To examine the effect of reducing the LPS activity of Laureth-9, a porcine-derived gelatin solution of 8 w / w% (high bloom, high viscosity, initial LPS content 800 - 1200 EU / g) was prepared with ultrapure water and contacted with different concentrations of Laureth-9. Laureth-9 was added to the gelatin solution at concentrations of 0.0015 w / w%, 0.004 w / w%, or 0.015 w / w% and mixed for 30 minutes. Then, the gelatin solution (containing Laureth-9) was filtered and the gelatin solution was recovered. The unfiltered gelatin solution, and the first 25 mL and the last 100 mL of the filtered gelatin solution were sampled and stored at -20 °C until analysis. To compare the potential effect of Laureth-9 with the prior art, one example using 0.09 w / w% of Triton X-100 was included in the experiment.

[0059] The LPS concentration of the recovered gelatin samples was measured using the EndoZyme II LPS quantification rFC assay kit (BioMerieux, Marcy l'Etoile, France), a type of LAL assay. Recombinant factor C (rFC) is activated by endotoxin binding, and then in a series of reactions, the active enzyme cleaves the synthetic substrate to generate a fluorescent compound. Next, the fluorescent compound is measured, and the intensity of the compound is directly proportional to the amount of endotoxin present in the analyzed sample.

[0060] The analysis was performed using a Biotek Synergy MX microplate reader (Agilent Technologies, California, USA), and the fluorescence signal was measured at an excitation wavelength of 380 nm and an emission wavelength of 445 nm.

[0061] Table 1 shows that when using 0.004 w / w% and 0.015 w / w% of Laureth-9, the endotoxin levels are lower than when using Triton X-100. It is also shown that since acceptable LPS levels (i.e., less than 10 EU / g) have already been reached before filtration, an additional filtration step is not essential.

[0062]

Table 1

[0063] When other (higher) concentrations of laureth-9, such as 0.01 w / w%, 0.025 w / w%, and 0.1 w / w% laureth-9, were tested, similar results to those of the 0.015 w / w% laureth-9 group were obtained. Furthermore, when other collagen-containing substances and / or collagen-derived substances, such as high bloom high viscosity gelatin, low bloom low viscosity gelatin, and (chemical) modified gelatin having one or more functional groups in the gelatin backbone, such as methacryloyl (GelMA), deaminotyrosine (GelDAT), etc., were used, similar results were obtained.

[0064] Experiments were also conducted to evaluate the decrease in LPS activity of polyglycol ethers of several fatty alcohols with different lengths of fatty alcohol and different average numbers of oxyethylene groups (using high bloom, high viscosity porcine-derived gelatin) (Table 2). Table 2 shows that LPS was effectively decreased in the polyglycol ethers of different fatty alcohols tested.

[0065] [Table 2]

[0066] Example 2. Effect of Laureth-9 on the Decrease in LPS Activity in Porcine-Derived and Bovine-Derived Gelatin To determine the effect of laureth-9 on LPS removal in different types of gelatin, porcine-derived gelatin (Type A) and bovine-derived gelatin (Type B) with initial LPS concentrations of 14000 - 16000 EU / g and 4000 - 7000 EU / g, respectively, were evaluated. The same protocol as in Example 1 was followed.

[0067] Laureth-9 showed a sufficient decrease in LPS concentration in both Type A gelatin and Type B gelatin, and the LPS concentration reached around 3000 EU / g or less (Table 3).

[0068] [Table 3]

[0069] Example 3. Efficacy of LPS removal by Laureth-9 and Triton X-100 To compare the efficacy of Laureth-9 and Triton X-100, gelatin containing different initial concentrations of LPS (i.e., 1000 EU / g, 5000 EU / g, and 15000 EU / g) was incubated with Laureth-9 or Triton X-100. After incubation, the recovered gelatin samples were tested for LPS content as described in Example 1.

[0070] As shown in Figure 1, when using gelatin with a relatively high LPS activity (i.e., >5000 EU / g), the efficacy of LPS reduction by Laureth-9 is higher than that of Triton X-100. (Supplementary Note) The present disclosure includes the following aspects: Item 1: A step of preparing a collagen-containing substance and / or a collagen-derived substance containing a lipopolysaccharide, and a step of bringing the collagen-containing substance and / or the collagen-derived substance containing the lipopolysaccharide into contact with a polyglycol ether of a fatty alcohol to obtain a mixture, and a step of recovering the collagen-containing substance and / or the collagen-derived substance, wherein the lipopolysaccharide activity is preferably less than 3000 EU / g, more preferably less than 1000 EU / g, still more preferably less than 100 EU / g, even more preferably less than 10 EU / g, and most preferably less than 1 EU / g, the step of recovering; A method for reducing the lipopolysaccharide activity in a collagen-containing substance and / or a collagen-derived substance, comprising the above steps. Item 2: The method according to Item 1, wherein the collagen-containing substance and / or the collagen-derived substance is selected from the group consisting of an extracellular matrix, natural collagen, gelatin, (chemically) modified gelatin, gelatin hydrolyzate, and mixtures thereof. Item 3: The method according to Item 1 or 2, wherein the collagen-containing substance and / or the collagen-derived substance is in the form of a solution. Item 4: The method according to any one of Items 1 to 3, wherein the collagen-containing substance and / or the collagen-derived substance is in the form of an aqueous solution. Item 5: The method according to Item 4, wherein the aqueous solution contains 1 w / w% or more of the collagen-containing substance and / or the collagen-derived substance. Item 6: The method according to Item 4 or 5, wherein the aqueous solution contains 1 to 50 w / w% of the collagen-containing substance and / or the collagen-derived substance. Item 7: The method according to any one of Items 1 to 6, wherein the polyglycol ether of the fatty alcohol is a polyglycol ether of lauryl alcohol. Item 8: The method according to any one of Items 1 to 7, wherein the polyglycol ether of the fatty alcohol contains 1 to 20 oxyethylene groups. Item 9: The method according to any one of Items 1 to 8, wherein the polyglycol ether of the fatty alcohol contains laureth-9. Item 10: The method according to any one of Items 4 to 9, wherein the aqueous solution contains 0.001 w / w% or more of the polyglycol ether of the fatty alcohol. Item 11: The method according to any one of Items 4 to 10, wherein the aqueous solution contains 0.001 to 0.05 w / w% of the polyglycol ether of the fatty alcohol. Item 12: The method according to any one of Items 1 to 11, wherein the mixture is brought into contact with an adsorbent. Item 13: The method according to Item 12, wherein the adsorbent is a solid. Item 14: The method according to Item 13, wherein the solid adsorbent is hydrophobic. Item 15: The method according to Item 14, wherein the hydrophobic adsorbent contains activated carbon. Item 16: The method according to any one of Items 1 to 15, wherein the absorbent is provided in a filter. Item 17: The method according to any one of Items 1 to 16, wherein the collagen-containing substance and / or collagen-derived substance is recovered by filtration. Item 18: Use of a polyglycol ether of a fatty alcohol for reducing the lipopolysaccharide activity in a collagen-containing substance and / or collagen-derived substance. Item 19: Use of a polyglycol ether of lauryl alcohol for reducing the lipopolysaccharide activity in a collagen-containing substance and / or collagen-derived substance. Item 20: A collagen-containing substance and / or collagen-derived substance obtained by the method according to any one of Items 1 to 17. Item 21: The collagen-containing substance and / or collagen-derived substance according to Item 20, having a lipopolysaccharide content of less than 3000 EU / g. Item 22: The collagen-containing substance and / or collagen-derived substance according to Item 20 or 21, containing less than 10 ppm, preferably less than 2 ppm, of a polyglycol ether of a fatty alcohol. Item 23: The collagen-containing substance and / or collagen-derived substance according to Item 22, containing less than 1 ppm, preferably less than 0.1 ppm, of a polyglycol ether of a fatty alcohol. Item 24: The collagen-containing substance and / or collagen-derived substance according to any one of Items 20 to 23, containing a polyglycol ether of a fatty alcohol. Item 25: The collagen-containing substance and / or collagen-derived substance according to any one of Items 22 to 24, wherein the polyglycol ether of the fatty alcohol contains laureth-9. Item 26: The collagen-containing substance and / or collagen-derived substance according to any one of Items 20 to 25, wherein the collagen-containing substance and / or collagen-derived substance is one or more selected from the group consisting of gelatin, hydrolyzed gelatin, and collagen hydrolysate. Item 27: Use of the collagen-containing substance and / or collagen-derived substance according to any one of Items 20 to 26 in medical devices, pharmaceuticals, and / or cosmetics.

Claims

1. A step of preparing a collagen-containing substance and / or a collagen-derived substance containing a lipopolysaccharide; A step of contacting the collagen-containing substance and / or the collagen-derived substance containing the lipopolysaccharide with a polyglycol ether of a fatty alcohol; A step of recovering the collagen-containing substance and / or the collagen-derived substance; A method for reducing the lipopolysaccharide activity in a collagen-containing substance and / or a collagen-derived substance, comprising the above steps.

2. The method according to claim 1, wherein the lipopolysaccharide activity in the collagen-containing substance and / or the collagen-derived substance recovered in the step of recovering is less than 3000 EU / g.

3. The method according to claim 1, wherein the collagen-containing substance and / or the collagen-derived substance is selected from the group consisting of extracellular matrix, natural collagen, gelatin, (chemically) modified gelatin, gelatin hydrolyzate, and mixtures thereof.

4. The method according to claim 1, wherein the collagen-containing substance and / or the collagen-derived substance is in the form of an aqueous solution.

5. The method according to claim 4, wherein the aqueous solution contains 1 to 50 w / w% of the collagen-containing substance and / or the collagen-derived substance.

6. The method according to claim 1, wherein the polyglycol ether of the fatty alcohol is a polyglycol ether of lauryl alcohol and / or contains 1 to 20 oxyethylene groups.

7. The method according to claim 1, wherein the polyglycol ether of the fatty alcohol contains laureth-9.

8. The method according to claim 4, wherein the aqueous solution contains 0.001 w / w% or more of the polyglycol ether of the fatty alcohol.

9. The step of contacting the collagen-containing substance and / or the collagen-derived substance containing the lipopolysaccharide with the polyglycol ether of the fatty alcohol is for obtaining a mixture, and the mixture is contacted with an adsorbent. The method according to claim 1.

10. The method according to claim 1, wherein the collagen-containing substance and / or the collagen-derived substance is recovered by filtration.

11. Use of a polyglycol ether of a fatty alcohol for reducing the lipopolysaccharide activity in a collagen-containing substance and / or a collagen-derived substance.

12. The use according to claim 11, wherein the polyglycol ether of the fatty alcohol is a polyglycol ether of lauryl alcohol.

13. The lipopolysaccharide content is less than 3,000 EU / g, and contains a polyglycol ether of a fatty alcohol, wherein the content of the polyglycol ether of the fatty alcohol is less than 10 ppm, a collagen-containing substance and / or a collagen-derived substance.

14. The collagen-containing substance and / or collagen-derived substance according to claim 13, wherein the polyglycol ether of the fatty alcohol is laureth-9.

15. The collagen-containing substance and / or collagen-derived substance according to claim 13, wherein the collagen-containing substance and / or collagen-derived substance is one or more selected from the group consisting of gelatin, hydrolyzed gelatin, and collagen hydrolyzate.

16. A collagen-containing substance and / or a collagen-derived substance obtained by the method according to any one of claims 1 to 10.

17. The collagen-containing substance and / or collagen-derived substance according to claim 16, wherein the lipopolysaccharide content is less than 3,000 EU / g.

18. The collagen-containing substance and / or collagen-derived substance according to claim 16, wherein the content of the polyglycol ether of the fatty alcohol is less than 10 ppm.

19. The collagen-containing substance and / or collagen-derived substance according to claim 16, which contains a polyglycol ether of a fatty alcohol.

20. The collagen-containing substance and / or collagen-derived substance according to claim 19, wherein the polyglycol ether of the fatty alcohol is laureth-9.

21. The collagen-containing substance and / or collagen-derived substance according to claim 16, wherein the collagen-containing substance and / or collagen-derived substance is one or more selected from the group consisting of gelatin, hydrolyzed gelatin, and collagen hydrolyzate.

22. Use of the collagen-containing substance and / or collagen-derived substance according to claim 16 in medical devices, pharmaceuticals, and / or cosmetics.

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