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

Abstract

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

Description

TECHNICAL FIELD

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

[0002] Collagen, products containing collagen or collagen-derived, such as extracellular matrix (ECM), collagen peptides, gelatin and (chemically) modified gelatin, have found applications in a variety of different fields, ranging from the food industry to the medical and pharmaceutical fields. Interest in these types of substances stems from their desirable characteristics, including their biocompatibility and biodegradability. Although collagen and substances containing collagen and collagen-derived are generally considered safe, their biomedical use is challenged by the presence of endotoxins, such as LPS.

[0003] LPS is located on the outer cell membrane of Gram-negative bacteria, where it provides structural integrity and protection by acting as a permeability barrier. LPS is composed of a variable polysaccharide chain and a lipid portion (lipid A). The size of the LPS molecule is about 10 kDa, but can form large aggregates in aqueous media, also known as “micelles”. LPS is not secreted by the bacteria, but is released upon disruption of the bacterial cell membrane, hence the term “endotoxin”. LPS is highly immunogenic to humans and can have severe adverse effects even upon exposure to small amounts of LPS. Due to the toxicity, the amount of endotoxin allowed on medical devices is strictly regulated. For example, the Food and Drug Administration (FDA) allows a maximum of 0.5 endotoxin units (EU) / g or 20 EU / device for products that come into direct / indirect contact with the cardiovascular and lymphatic system. For devices that come into contact with cerebrospinal fluid, the limit is even lower.

[0004] Avoiding endotoxin contamination is challenging in the traditional manufacturing process of products containing collagen and / or collagen-derived. Moreover, once present, endotoxins are extremely difficult to remove, especially from sensitive substances comprising proteins.

[0005] In the art, methods for removing LPS from protein solutions using surfactants have been described, for example in WO2016085345. While showing great promise, the above-mentioned method relies on Triton X-100, whose use is under discussion.

[0006] Therefore, there is still an unmet need to reduce and / or remove LPS from products containing collagen and collagen-derived in an effective and safe manner.

[0007] It is an object of the present invention to overcome one or more of the above-mentioned problems. To this end, the present invention provides a method for reducing LPS activity and / or LPS from a collagen-containing and / or collagen-derived product. SUMMARY

[0008] The present inventors identified an effective method for reducing LPS activity and / or LPS from a collagen-containing and / or collagen-derived product, which method is based on the use of a fatty alcohol polyglycol ether.

[0009] More specifically, the present inventors found that laureth-9, which is a non-ionic, non-toxic micelle-forming surfactant, effectively reduces LPS activity in a collagen-containing and / or collagen-derived product. Surprisingly, the concentration of laureth-9 required for effectiveness was found to be significantly lower than that of Triton X-100.

[0010] Accordingly, the present inventors provide an improved method for reducing LPS activity.

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

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

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

[0014] The present invention relates to a method for reducing LPS activity in a collagen-containing and / or collagen-derived product, the method comprising one or more of the following steps, preferably all of the following steps: - providing a collagen-containing and / or collagen-derived product comprising LPS,

[0015] - contacting the collagen-containing and / or collagen-derived product comprising LPS with a fatty alcohol polyglycol ether to provide a mixture,

[0016] - recovering a collagen-containing and / or collagen-derived product, preferably a collagen-containing and / or collagen-derived product having an LPS content of less than 3000 EU / g, more preferably a collagen-containing and / or collagen-derived product having an LPS content of less than 1000 EU / g, even more preferably a collagen-containing and / or collagen-derived product having an LPS content of less than 100 EU / g, yet even more preferably a collagen-containing and / or collagen-derived product having an LPS content of less than 10 EU / g, most preferably a collagen-containing and / or collagen-derived product having an LPS content of less than 1 EU / g.

[0017] The term "reducing LPS activity" as used herein means that the LPS level of the recovered collagen-containing and / or collagen-derived product is lower than the LPS level of the starting material (i.e. the collagen-containing and / or collagen-derived product prior to contact with the polyethylene glycol ether of a fatty alcohol). In the present application, "reducing LPS activity" includes both reducing and / or removing 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 a Limulus Amebocyte Lysate (LAL) assay, which is a well-known method for detecting LPS. LAL is an aqueous extract of blood cells (amebocytes) from the horseshoe crab Limulus polyphemus. The reaction between LAL and LPS is the basis of the LAL assay and is used to determine the LPS content qualitatively (e.g. visual inspection) or quantitatively (e.g. chromogenic or turbidimetric).

[0018] In one embodiment, the LPS activity of the recovered collagen-containing and / or collagen-derived product is preferably at least 10% lower than the LPS activity of the starting material. More preferably, the LPS activity of the recovered collagen-containing and / or collagen-derived product is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% lower than the LPS activity of the starting material. Preferably, the LPS activity of the starting material is less than 20000 EU / g (per gram of dry weight of the collagen-containing and / or collagen-derived product). More preferably, the LPS activity of the starting material is less than 15000 EU / g (per gram of dry weight of the collagen-containing and / or collagen-derived product), even more preferably less than 5000 EU / g (per gram of dry weight of the collagen-containing and / or collagen-derived product). The starting material can also have a higher or lower LPS activity, and if the LPS content is higher, the material can be pre-treated, for example with an ion exchange chromatography step.

[0019] In one embodiment, the collagen-containing and / or collagen-derived product is endotoxin-free (i.e. LPS-free). The term "endotoxin-free" can mean that no endotoxin is present and / or can mean "substantially free of endotoxin" (i.e. substantially free of LPS).

[0020] The term "free of" can mean the same as "substantially free of". The term "substantially free of" encompasses an amount of a substance or compound that is not measurable and / or is below some threshold value according to standard techniques in the art. In addition or alternatively, the term "substantially free of" can mean an endotoxin level of less than 10, preferably less than 1, more preferably less than 0.1, even more preferably less than 0.01, most preferably less than 0.001 (all in EU / g or EU / ml). The phrase "free of" or "substantially free of" encompasses that the substance or compound is completely absent (e.g. 0 wt.% or 0 EU / g). The terms "absence", "free of" and "substantially free of" are used interchangeably in the context of the present application.

[0021] As described herein, the use of a polyethylene glycol ether of a fatty alcohol to remove LPS (activity) is particularly advantageous if the starting material has a high amount of LPS (activity). For example, it was found that the polyethylene glycol ether of a fatty alcohol is more effective in removing LPS (activity) than other known methods (e.g. Triton X-100) if the LPS content in the starting material is relatively high, e.g. at least 1000 EU / g, preferably at least 2500 EU / g, more preferably at least 5000 EU / g, even more preferably at least 10000 EU / g, most preferably at least 50000 EU / g. In one embodiment, the LPS content in a product containing collagen and / or collagen source is at least 500 EU / g, or at least 1000 EU / g, or at least 2500 EU / g, or at least 5000 EU / g, or at least 10000 EU / g, or at least 50000 EU / g, and / or not more than 50000 EU / g, or not more than 10000 EU / g, or not more than 5000 EU / g, or not more than 2500 EU / g, or not more than 1000 EU / g or not more than 500 EU / g. In addition or alternatively, the LPS content in a product containing collagen and / or collagen source is preferably 500 to 50000 EU / g, 1000 to 50000 EU / g, most preferably 1000 to 10000 EU / g.

[0022] The present disclosure comprises contacting a product containing collagen and / or collagen source with a polyethylene glycol ether of a fatty alcohol. The aim can be to provide a mixture or blend such that an interaction between the product containing collagen and / or collagen source and the polyethylene glycol ether of a fatty alcohol is provided.

[0023] In one embodiment, the product containing collagen and / or collagen source can be an extracellular matrix, native collagen, gelatin, (chemically) modified gelatin, gelatin hydrolysate and mixtures thereof. Native collagen can comprise acid-extracted and enzyme-soluble collagen, which includes telocollagen, atelocollagen and fibrillar collagen.

[0024] The term "collagen" in the context of the present application means an amino acid sequence comprising the repeating (Gly-X-Y) sequence, preferably comprising at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 300 or 400 sequences containing the sequence Gly-X-Y, wherein X and Y are independently selected amino acid residues from each other, but X and / or Y are more preferably proline. The "collagen" preferably has the sequence of a natural collagen present in one or more animal species. Additionally or alternatively, the "collagen" can mean the full-length sequence of a (natural) collagen or a fragment or subunit thereof, the (natural) collagen preferably being one or more of collagen types I to XXVII, more preferably one or even more of collagen types I, II, III, V or X, even more preferably one or more of collagen types I, II or III. For example, the term "collagen" can refer to the alpha-1 (I), alpha-2 (I), alpha-1 (II) or alpha-1 (III) chain, or fragments thereof. The term "collagen" encompasses a triple helix structure as formed by three subunits present in natural collagen.

[0025] "Collagen" in the context of the present application encompasses "gelatin", "collagen hydrolysate" and "hydrolyzed gelatin".

[0026] The term "collagen hydrolysate" in the context of the present application means a mixture of amino acid short chains (dipeptides, tripeptides, oligopeptides, polypeptides) derived from (partial) hydrolysis (e.g. by enzymatic hydrolysis) of natural (full-length) collagen. The degree of hydrolysis has an influence on the average molecular weight (expressed in Dalton, Da) of the final product. The term "collagen hydrolysate" can be used interchangeably with and synonymously to the term "hydrolyzed collagen" or "collagen peptide". "Collagen hydrolysate" in the context of the present application encompasses collagen subjected to hydrolysis or partial hydrolysis. "Collagen hydrolysate" in the context of the present application can be produced from collagen-containing material in a one-step process or via an intermediate gelatin stage (i.e. thus obtaining "hydrolyzed gelatin"). Thus, the term "collagen hydrolysate" encompasses hydrolyzed gelatin (i.e. gelatin subjected to hydrolysis).

[0027] The collagen- and / or collagen-derived containing product used herein can be derived from one or more types of collagen known in the art. More particularly, the collagen- and / or collagen-derived containing product used herein can be selected from the group comprising collagen types I, II and III, IV, V, VI, VII, VIII, IX, X, XI, XII, XII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXVIII. Additionally or alternatively, the collagen- and / or collagen-derived containing product used herein can be a mixture of one or more types of collagen.

[0028] The collagen-containing and / or collagen-derived product can be from synthetic natural (i.e. recombinant collagen), but is preferably derived from any animal raw material from which a collagen-containing and / or collagen-derived product can be prepared. In a preferred embodiment, the animal raw material is from a bovine, porcine, poultry or fish source. In another preferred embodiment, the collagen-containing and / or collagen-derived product is derived from skin and / or skin connective tissue. In yet another preferred embodiment, the collagen-containing and / or collagen-derived product is derived from cartilage. In yet another preferred embodiment, the collagen-containing and / or collagen-derived product is derived from bone.

[0029] As known in the art, the term "collagen-derived product" as used herein refers to proteins and peptides which are part of the collagen matrix in the raw material and which are processed to prepare a collagen-derived product, such as gelatin. Gelatin and / or (chemically) modified gelatin can be produced by (partial) alkaline hydrolysis and / or acid hydrolysis, by enzymatic hydrolysis, by thermal hydrolysis and / or combinations thereof. Gelatin does not consist of uniform protein molecules, but comprises variable amounts of protein molecules of variable length. Preferably, the average molecular weight of the gelatin as used herein is in the range of 1500 Da to 300 kDa, preferably 2000 Da to 250 kDa, more preferably 3000 Da to 200 kDa, even more preferably 4000 Da to 150 kDa, and most preferably 5000 Da to 100 kDa. a to 300 kD a , 4000 D a to 300 kD a , 5000 D a to 300 kD a , 10 kD aup to 300 kD, 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 on the gelatin backbone, such as acrylamide, ferulic acid, methacryloyl (e.g. GelMA), desaminotyrosine (e.g. GelDAT), furfurylamine and norbornene (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” products also include products obtainable after further processing of gelatin, such as gelatin hydrolysates, which are peptide preparations derived from hydrolysis of gelatin into peptide molecules having an average molecular weight of 70 kDa or less, typically 20 kDa or less, typically 100 to 15000 Da.

[0030] In one embodiment, the collagen and / or collagen-derived product containing can be in the form of a solution, wherein the solvent can comprise an acid (e.g. acetic acid, formic acid) or an organic solvent (e.g. supercritical carbon dioxide, acetone, preferably up to 30%). The collagen and / or collagen-derived product containing can also be in the form of an aqueous solution, which can comprise 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 application encompasses solutions comprising one or more co-solvents in addition to water, which can depend on the polyethylene glycol ether of the fatty alcohol chosen. The presence of a co-solvent can for example improve the solubility of the polyethylene glycol ether of the fatty alcohol. Some examples of co-solvents or surfactants known to the skilled person and suitable for the present application are selected from one or more of the following: ethanol, isopropanol, 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, preferably ethanol.

[0031] The solution can be prepared by dissolving (dry) the collagen and / or collagen- derived product containing product with the solvent. The collagen and / or collagen-derived product containing product is preferably dissolved at room temperature or at an elevated temperature, however preferably not higher than 68°C, more preferably not higher than 65°C, even more preferably not higher than 60°C, as an elevated temperature can increase the undesired hydrolysis of the collagen and / or collagen-derived product containing product. The previous steps can be performed at different temperatures, however preferably wherein the maximum is 68°C. Preferably, the mixing time comprises a period of at least 10 minutes. Also shorter or longer mixing times can be used, however, for effectiveness and efficiency the mixing time is preferably between 10 and 60 minutes. Alternatively, the collagen and / or collagen-derived product containing product can be contacted directly with a neat solution of lauryl alcohol polyether-9.

[0032] In one embodiment, the aqueous solution can comprise any concentration of collagen and / or collagen-derived product containing product. In a preferred embodiment, the aqueous solution comprises at least 1 w / w% collagen and / or collagen-derived product containing product, preferably at least 4 w / w%, more preferably at least 6 w / w%, even more preferably at least 8 w / w% collagen and / or collagen-derived product containing product, still even more preferably at least 10 w / w% collagen and / or collagen-derived product containing product, still even more preferably at least 15 w / w% collagen and / or collagen-derived product containing product, most preferably at least 20 w / w%.

[0033] In another preferred embodiment, the aqueous solution comprises between 1 and 50 w / w% collagen and / or collagen-derived product containing product, preferably between 2 and 30 w / w%, more preferably between 4 and 15 w / w% collagen and / or collagen-derived product containing product, even more preferably between 6 and 10 w / w% collagen and / or collagen-derived product containing product. The aqueous medium can comprise as much collagen and / or collagen-derived product containing product as possible until the aqueous solution becomes too viscous for processing according to the method. The aqueous solution of the present disclosure preferably comprises at most 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 w / w% collagen and / or collagen-derived product containing product. When collagen and / or collagen-derived product containing products are used having a relative molecular weight, such as gelatin hydrolysates, higher concentrations of collagen and / or collagen-derived product containing product are possible, such as at least 60, 70, 80 and 90 w / w%. Although the viscosity of the collagen and / or collagen-derived aqueous solution can be reduced by increasing the temperature, an elevated temperature can result in undesired hydrolysis of the collagen and / or collagen-derived product containing product.

[0034] The polyethylene glycol ether of a fatty alcohol can be a polyethylene glycol ether of a C8-C20 fatty alcohol, preferably a C8-C18 fatty alcohol, more preferably a C8-C16 fatty alcohol, most preferably a C10-C14 fatty alcohol. In a preferred embodiment, the polyethylene glycol ether of a fatty alcohol is preferably selected from the list of polyethylene glycol ethers comprising 1-decanol, 1-dodecanol, 1-tetradecanol, 1-cetyl alcohol, 1-palmrotyl alcohol, 1-octadecenol and / or 1-stearyl alcohol. Most preferably, the polyethylene glycol ether of a fatty alcohol is a polyethylene glycol ether of 1-dodecanol.

[0035] In the present disclosure, a fatty alcohol (or long-chain alcohol) is typically a straight-chain primary alcohol and can typically be derived from natural fats and oils. Some related fatty alcohols are lauryl alcohol (C12), myristyl alcohol (C14), stearyl alcohol (C18) and oleyl alcohol (C16). Fatty alcohols typically have an even number of carbon atoms and a single alcohol group (-OH) attached to the terminal carbon. Some are unsaturated and some are branched. As with fatty acids, they are typically referred to by the number of carbon atoms in the molecule, e.g. "C12 alcohol", i.e. an alcohol with 12 carbons, such as dodecanol.

[0036] In one embodiment, the polyethylene glycol ether of a fatty alcohol comprises and / or on average comprises 1 to 20 oxyethylene groups, preferably 5 to 18 oxyethylene groups, more preferably 8 to 16 oxyethylene groups, most preferably 10 to 14 oxyethylene groups.

[0037] In a more preferred embodiment, the polyethylene glycol ether of a fatty alcohol comprises and / or on average comprises 1 to 100 oxyethylene groups, more preferably 2 to 75 oxyethylene groups, even more preferably 3 to 50 oxyethylene groups, still even more preferably 4 to 40 oxyethylene groups, still even more preferably 5 to 30 oxyethylene groups, still even more preferably 6 to 20 oxyethylene groups, most preferably 7 to 10 oxyethylene groups. In another preferred embodiment, the polyethylene glycol ether of a fatty alcohol comprises and / or on average comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 oxyethylene groups. In yet another preferred embodiment, the polyethylene glycol ether of a fatty alcohol comprises and / or on average comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 oxyethylene groups.

[0038] 200, 175, 150, 125, 100, 75, 50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 225, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9 oxyethylene groups. In yet another preferred embodiment, the polyethylene glycol ether of a fatty alcohol is preferably selected from one or more polyethylene glycol ethers of a fatty alcohol comprising C12and (on average) 9 oxyethylene groups (e.g. Lauryl ether-9), C12and (on average) 10 oxyethylene groups (e.g. Genapol X-100), C12and (on average) 23 oxyethylene groups (e.g. Brij 35), C16and (on average) 10 oxyethylene groups (e.g. Brij C10), C18and (on average) 100 oxyethylene groups (e.g. Brij S100), C18and (on average) 10 oxyethylene groups (e.g. Eco Brij S10).

[0039] In one embodiment, the polyethylene glycol ether of a fatty alcohol comprises Lauryl ether-9. Lauryl ether-9 is 3,6,9,12,15,18,21,24,27-nonaoxanonadecan-1-ol in itself. In practical terms, it is lauryl alcohol etherified with an average of 9 oxyethylene groups, a mixture dispersed with 1 to 20 oxyethylene groups.

[0040] Lauryl ether-9 is a micelle-forming surfactant, which means that it is able to form micelles in solution. To this end, the so-called critical micelle concentration (CMC) of a surfactant is defined as the concentration of the surfactant above which micelles start to form. At certain temperatures, the so-called cloud point, surfactants form insoluble aggregates in aqueous media and phase separate.

[0041] In one embodiment, the polyethylene glycol ether of a fatty alcohol is preferably present in the mixture in a concentration equal to or higher than the CMC. Preferably, the polyethylene glycol ether of a fatty alcohol is present in a concentration of at least 3 times the CMC. More preferably, the polyethylene glycol ether of a fatty alcohol is present in a concentration of at least 5 times the CMC. Even more preferably, the polyethylene glycol ether of a fatty alcohol is present in a concentration of at least 10 or 20 times the CMC. Most preferably, the polyethylene glycol ether of a fatty alcohol is present in a concentration of at least 30 times the CMC. Preferably, the aqueous solution comprises at least 0.001 w / w %, more preferably at least 0.002 w / w %, even more preferably at least 0.004 w / w %, yet even more preferably at least 0.006 w / w %, yet even more preferably at least 0.008 w / w %, yet even more preferably at least 0.01 w / w %, yet even more preferably at least 0.03 w / w %, most preferably at least 0.05 w / w % of the polyethylene glycol ether of a fatty alcohol, wherein the polyethylene glycol ether of a fatty alcohol is preferably laureth-9.

[0042] In another embodiment, the aqueous solution preferably comprises 0.001 to 0.5 w / w %, more preferably 0.004 to 0.5 w / w %, even more preferably 0.004 to 0.1 w / w %, yet even more preferably 0.004 to 0.06 w / w % of the polyethylene glycol ether of a fatty alcohol, wherein the polyethylene glycol ether of a fatty alcohol is preferably laureth-9. Even more preferably, the aqueous solution comprises 0.001 to 0.05 w / w % of the polyethylene glycol ether of a fatty alcohol, wherein the polyethylene glycol ether of a fatty alcohol is preferably laureth-9. Even more preferably, the aqueous solution comprises 0.002 to 0.02 w / w %, even more preferably 0.004 to 0.01 w / w % of the polyethylene glycol ether of a fatty alcohol, wherein the polyethylene glycol ether of a fatty alcohol is preferably laureth-9. The exact concentration of the polyethylene glycol ether of a fatty alcohol can also be adjusted depending on the LPS content in the product containing collagen and / or collagen source. For example, if the starting material has a low LPS content, a low concentration of the polyethylene glycol ether of a fatty alcohol (i.e. not greatly exceeding the CMC value) can be sufficient. The concentration of the polyethylene glycol ether of a fatty alcohol can be expressed relative to the amount of the product containing collagen and / or collagen source present in the aqueous solution. Preferably, the polyethylene glycol ether of a fatty alcohol is present in a concentration of 0.1 to 10 milligrams, even more preferably 0.2 to 8 milligrams, yet even more preferably 0.9 to 8 milligrams per 1 gram of the product containing collagen and / or collagen source present in the aqueous solution. In addition or alternatively, the polyethylene glycol ether of a fatty alcohol is preferably present in a concentration of at least 0.1, 0.2, 0.4, 0.6, 0.8, 1 milligram of the polyethylene glycol ether of a fatty alcohol per 1 gram of the product containing collagen and / or collagen source. In addition or alternatively, the polyethylene glycol ether of a fatty alcohol is preferably present in a concentration of at most 10, 8, 6, 5, 4, 2 milligrams of the polyethylene glycol ether of a fatty alcohol per 1 gram of the product containing collagen and / or collagen source.

[0043] In a preferred embodiment, the aqueous solution comprises 6 to 10 w / w % of the product containing collagen and / or collagen origin and 0.004 to 0.06 w / w % of the polyethylene glycol ether of a fatty alcohol, wherein the polyethylene glycol ether of a fatty alcohol is preferably laureth-9. The percentage of CMC can even vary more broadly, as it is known to also depend on the composition of the aqueous solution.

[0044] In certain embodiments, for example when the polyethylene glycol ether of a fatty alcohol comprises and / or on average comprises 1 to 8, preferably 1 to 5, more preferably 1 to 3 oxyethylene groups, an ethanol solution or a solution comprising another cosolvent as disclosed herein can be used. In addition or alternatively, when the polyethylene glycol ether of a fatty alcohol comprises and / or on average comprises less than 5, 4, 3, 2, 1 oxyethylene groups, an ethanol solution or a solution comprising another cosolvent as disclosed herein can be used, which can give similar or even better results than when using an aqueous solution. The ethanol solution may, for example, comprise 10 to 70 (v / v) %, 20 to 60 (v / v) % or 30 % to 50 % ethanol.

[0045] The period of time of contact between the product containing collagen and / or collagen origin and the polyethylene glycol ether of a fatty alcohol is long enough to allow mixing. In addition or alternatively, the period of time of contact is chosen such that the intended endotoxin activity is reached. Preferably, the period of time of contact between the product containing collagen and / or collagen origin and the polyethylene glycol ether of a fatty alcohol is at least 1 minute. More preferably, the period of time of contact is at least 5 minutes, even more preferably at least 10 minutes, still even more preferably at least 20 minutes, still even more preferably at least 30 minutes, still even more preferably at least 60 minutes. More preferably, the product containing collagen and / or collagen origin is incubated with the polyethylene glycol ether of a fatty alcohol for 1 to 60 minutes, more preferably 5 to 30 minutes, most preferably 20 to 30 minutes.

[0046] In an embodiment, the product containing collagen and / or collagen origin can be recovered by contacting the mixture with an adsorbent. The adsorbent can be any suitable adsorbent which is capable of binding the surfactant, and preferably also the LPS. Advantageously, the aqueous medium is free-flowing at the time of addition of the surfactant and at the time of contact with the adsorbent. Although the viscosity and gelation temperature differ between different products containing collagen and / or collagen origin, the solution can be free-flowing and well processable at a temperature of at least 30 °C. A free-flowing solution not only allows optimal contact between the medium and the adsorbent, but also enables proper separation of the adsorbent.

[0047] In an embodiment, the adsorbent is a solid.

[0048] In an embodiment, the solid adsorbent is hydrophobic.

[0049] In one embodiment, the solid adsorbent comprises activated charcoal, such as Norit SX Plus or Norit ROX 0.8 (Cabot, the Netherlands).

[0050] In one embodiment, the adsorbent is provided in a filter. In addition or in the alternative, the adsorbent can be stacked in a column. The contacting step between the mixture and the solid adsorbent is performed for a sufficient amount of time which allows for proper adsorption of the polyethylene glycol ether of a fatty alcohol and / or LPS.

[0051] In one embodiment, the mixture can be recovered by, for example, 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. Shorter time periods are possible, but more adsorbent can be required to achieve the desired recovered product compared to longer time periods of incubation. Longer time periods are also possible, but are less desirable from a process efficiency point of view. Preferably, the adsorption step can be performed once.

[0052] In one embodiment, the method disclosed herein can comprise contacting a collagen and / or collagen-derived containing product comprising lipopolysaccharide with a polyethylene glycol ether of a fatty alcohol, followed by a drying step, thereby allowing to obtain a powder. In one embodiment, the method disclosed herein comprises a step of contacting the mixture with an adsorbent, followed by a drying step, thereby allowing to obtain a powder. Preferably, the drying is spray drying. In one embodiment, the drying step can be part of the (optional) step of recovering the collagen and / or collagen-derived containing product. When the drying step is part of the (optional) step of recovering the collagen and / or collagen-derived containing product, the (optional) step is contacting the mixture with an adsorbent and (optional) recovery by, for example, filtration, sedimentation or centrifugation (if followed by a drying step), thereby allowing to obtain a powder.

[0053] Also described is the use of a polyethylene glycol ether of a fatty alcohol for reducing LPS (activity) in a collagen and / or collagen-derived containing product.

[0054] Also described is the use of a polyethylene glycol ether of lauryl alcohol for reducing LPS (activity) in a collagen and / or collagen-derived containing product.

[0055] In one aspect, the present application relates to a collagen and / or collagen-derived containing product, which can optionally be obtained by the method disclosed herein. In a preferred embodiment, the collagen and / or collagen-derived containing product is gelatin or hydrolyzed gelatin. In another preferred embodiment, the collagen and / or collagen-derived containing product is a collagen hydrolysate.

[0056] In one embodiment, the collagen and / or collagen-derived containing product, optionally obtainable by the method of the preceding claims, preferably has a LPS content of less than 3000 EU / g, preferably less than 1000 EU / g, more preferably less than 100 EU / g, even more preferably less than 10 EU / g, most preferably less than 1 EU / g.

[0057] In one embodiment, the collagen and / or collagen-derived containing product can comprise one or more polyethylene glycol ethers of fatty alcohols as disclosed herein, preferably laureth-9. In one embodiment, the collagen and / or collagen-derived containing product can comprise less than 1000 ppm of residues of polyethylene glycol ethers of fatty alcohols, optionally even less than 750 ppm, or even less than 500 ppm, or even less than 250 ppm, or even less than 100 ppm, or even less than 75 ppm, or even less than 50 ppm, or even less than 25 ppm, or even less than 10 ppm, or even less than 5 ppm, or even less than 2 ppm, or even less than 1 ppm, or even less than 0.1 ppm. In one embodiment, the amount of polyethylene glycol ethers of fatty alcohols in the collagen and / or collagen-derived containing product is preferably 0.01 to 10 ppm. In one embodiment, the amount of polyethylene glycol ethers of fatty alcohols in the collagen and / or collagen-derived containing product is preferably 0.05 to 5 ppm. In one embodiment, the amount of polyethylene glycol ethers of fatty alcohols in the collagen and / or collagen-derived containing product is preferably 0.1 to 1 ppm. In one embodiment, the amount of polyethylene glycol ethers of fatty alcohols in the collagen and / or collagen-derived containing product is preferably 0.2 to 0.5 ppm. In one embodiment, the amount of polyethylene glycol ethers of fatty alcohols in the collagen and / or collagen-derived containing product is preferably 0.3 to 0.4 ppm.

[0058] Additionally or alternatively, the present application also relates to a collagen and / or collagen-derived containing product, optionally obtainable by the method of the preceding claims, comprising a LPS content of less than 3000 EU / g, preferably less than 1000 EU / g, more preferably less than 100 EU / g, even more preferably less than 10 EU / g, most preferably less than 1 EU / g, and 0.001 to 100, 0.001 to 50, 0.001 to 25, 0.001 to 10, 0.001 to 5, 0.001 to 1 ppm of residues of polyethylene glycol ethers of fatty alcohols, preferably 0.004 to 2, 0.004 to 1, 0.004 to 0.8, 0.004 to 0.6, 0.004 to 0.4, 0.004 to 0.2 ppm of residues of polyethylene glycol ethers of fatty alcohols, wherein the polyethylene glycol ethers of fatty alcohols are preferably laureth-9.

[0059] In one embodiment, the collagen and / or collagen-derived containing product, optionally obtainable by the method of the preceding claims, can be applied in various industries, for example in the food, pharmaceutical and cosmetic industries. In view thereof, the collagen and / or collagen-derived containing product, optionally obtainable by the method of the present application, can be used in medical devices, pharmaceutical and / or cosmetic applications, which collagen and / or collagen-derived containing product can be used as a gelling agent, a texturizing agent and / or a hydrogel. In view thereof, the present application also relates to the use of a collagen and / or collagen-derived containing product, preferably the use of a collagen and / or collagen-derived containing product in medical devices and / or pharmaceutical and / or cosmetic (applications). BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 is a graph showing the effectiveness of lauryl alcohol polyether-9 and Triton X-100 in removing LPS from gelatin with different starting levels of LPS. The x-axis shows the LPS content (EU / g) before exposure to lauryl alcohol polyether-9 or Triton X-100, and the y-axis shows the LPS content (EU / g) after exposure to lauryl alcohol polyether-9 or Triton X-100.

[0061] EMBODIMENT

[0062] Example 1. Role of Triton X-100 and lauryl alcohol polyether-9 in reducing LPS activity.

[0063] To determine the role of lauryl alcohol polyether-9 in reducing LPS activity, a porcine-derived gelatin solution of 8 w / w% (high bloom, high viscosity, initial LPS content 800-1200 EU / g) was prepared in ultrapure water and exposed to different concentrations of lauryl alcohol polyether-9. Lauryl alcohol polyether-9 was added to the gelatin solution at concentrations of 0.0015 w / w%, 0.004 w / w% and 0.015 w / w% and mixed for 30 minutes. After that, the gelatin solution (containing lauryl alcohol polyether-9) was filtered and the gelatin solution was recovered. The unfiltered gelatin solution was sampled together with the first 25 mL and the last 100 mL of the filtered gelatin solution and stored at -20°C until analysis. To compare the potential role of lauryl alcohol polyether-9 with the state of the art, one example with 0.09 w / w% Triton X-100 was included in the experiment.

[0064] The LPS concentration in the recovered gelatin samples was determined using the EndoZyme II LPS Quantitative rFC assay kit (Biomerieux, Marcy-l’Etoile, France), which is a type of LAL assay. Recombinant Factor C (rFC) is activated by endotoxin binding and, in a cascade reaction, the active enzyme subsequently cleaves a synthetic substrate, resulting in the production of a fluorescent compound. The fluorescent compound is then measured and the intensity of the compound is directly proportional to the amount of endotoxin present in the analyzed sample.

[0065] The fluorescence signal was measured at an excitation wavelength of 380 nm and an emission wavelength of 445 nm using a Biotek Synergy MX microplate reader (Agilent Technologies, California, United States) for the analysis.

[0066] Table 1 shows that the use of 0.004 w / w% and 0.015 w / w% laureth-9 results in lower endotoxin levels than the use of Triton X-100. In addition, it is also shown that an additional filtration step is not mandatory, as acceptable LPS levels (i.e. below 10 EU / g) have already been reached before filtration. Table 1. Reduction of lipopolysaccharide (LPS) in porcine-derived gelatin solutions after exposure to various concentrations of laureth-9 and Triton X-100

[0067]

[0068] Similar results were obtained when other (higher) concentrations of laureth-9 were tested (e.g.: 0.01 w / w%, 0.025 w / w% and 0.1 w / w% laureth-9) as compared to the 0.015 w / w% laureth-9 group. In addition, similar results were obtained when other collagen- and / or collagen-derived containing products were used, such as high bloom strength high viscosity gelatin, low bloom strength low viscosity gelatin and (chemically) modified gelatins with one or more functionalized groups on the gelatin backbone, such as methacryloyl (GelMA), desamino tyrosine (GelDAT).

[0069] Experiments were also performed (also using porcine-derived gelatin of high bloom strength, high viscosity) to evaluate the reduction of LPS activity for several fatty alcohol polyglycol ethers of different fatty alcohols with different average number of oxyethylene groups (Table 2). Table 2 shows that LPS was effectively reduced for the different fatty alcohol polyglycol ethers tested.

[0070] Table 2. Reduction of lipopolysaccharide (LPS) in porcine-derived gelatin solutions after exposure to various surfactants.

[0071]

[0072] Example 2. Role of lauryl alcohol polyether-9 in reducing LPS activity in porcine- and bovine-derived gelatins.

[0073] To determine the role of lauryl alcohol polyether-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 to 16000 EU / g and 4000 to 7000 EU / g, respectively, were evaluated. The same protocol as in Example 1 was followed.

[0074] Lauryl alcohol polyether-9 showed a satisfactory reduction of LPS concentration for both type A and type B gelatin, reaching a LPS concentration of about 3000 EU / g or lower (Table 3).

[0075] Table 3. Reduction of lipopolysaccharide (LPS) in porcine-derived gelatin solutions and bovine-derived gelatin solutions after exposure to various concentrations of lauryl alcohol polyether-9.

[0076]

[0077] Example 3. Effectiveness of LPS removal by lauryl alcohol polyether-9 and Triton X-100

[0078] To compare the effectiveness of lauryl alcohol polyether-9 and Triton X-100, gelatin with different initial concentrations of LPS (i.e. 1000 EU / g, 5000 EU / g and 15000 EU / g) was incubated with lauryl alcohol polyether-9 or Triton X-100. After incubation, the LPS content of the recovered gelatin samples was tested according to that described in Example 1.

[0079] As can be seen in Table 4, lauryl alcohol polyether-9 showed a higher LPS reduction potency than Triton X-100 when using gelatin with a relatively high LPS activity (i.e. > 5000 EU / g). Figure 1 As can be seen in Table 4, lauryl alcohol polyether-9 showed a higher LPS reduction potency than Triton X-100 when using gelatin with a relatively high LPS activity (i.e. > 5000 EU / g).

Claims

1. A method for reducing lipopolysaccharide activity in a collagen-containing and / or collagen-derived product, the method comprising the steps of: - providing a collagen-containing and / or collagen-derived product comprising lipopolysaccharide, - contacting the collagen-containing and / or collagen-derived product comprising lipopolysaccharide with a polyethylene glycol ether of a fatty alcohol comprising 9 to 10 oxyethylene groups to provide a mixture, - recovering a collagen-containing and / or collagen-derived product, wherein the lipopolysaccharide activity is below 3000 EU / g.

2. The method according to claim 1, wherein the collagen-containing and / or collagen-derived product is selected from the group consisting of extracellular matrix, native collagen, gelatin, modified gelatin, and mixtures thereof.

3. The method according to claim 1, wherein the collagen-containing and / or collagen-derived product is selected from chemically modified gelatin.

4. The method according to claim 1, wherein the collagen-containing and / or collagen-derived product is selected from gelatin hydrolysate.

5. The method according to any one of claims 1 to 4, wherein the collagen-containing and / or collagen-derived product is in the form of a solution.

6. The method according to any one of claims 1 to 4, wherein the collagen-containing and / or collagen-derived product is in the form of an aqueous solution.

7. The method according to claim 6, wherein the aqueous solution comprises at least 1 w / w% collagen-containing and / or collagen-derived product.

8. The method according to claim 6 or 7, wherein the aqueous solution comprises 1 to 50 w / w% collagen-containing and / or collagen-derived product.

9. The method according to any one of claims 1 to 4, wherein the polyethylene glycol ether of a fatty alcohol comprising 9 to 10 oxyethylene groups is a polyethylene glycol ether of lauryl alcohol.

10. The method according to any one of claims 1 to 4, wherein the polyethylene glycol ether of a fatty alcohol comprising 9 to 10 oxyethylene groups comprises laureth-9.

11. The method according to claim 6, wherein the aqueous solution comprises at least 0.001 w / w% of the polyethylene glycol ether of a fatty alcohol comprising 9 to 10 oxyethylene groups.

12. The method according to claim 6, wherein the aqueous solution comprises 0.001 to 0.05 w / w% of the polyethylene glycol ether of a fatty alcohol comprising 9 to 10 oxyethylene groups.

13. The method according to any one of claims 1 to 4, wherein the mixture is contacted with an adsorbent.

14. The method according to claim 13, wherein the adsorbent is a solid.

15. The method according to claim 14, wherein the solid adsorbent is hydrophobic.

16. The method according to claim 15, wherein the hydrophobic adsorbent comprises activated carbon.

17. The method according to any one of claims 1 to 4, wherein the adsorbent is provided in a filter.

18. The method according to any one of claims 1 to 4, wherein the collagen-containing and / or collagen-derived product is recovered by filtration.

19. Use of a polyethylene glycol ether of a fatty alcohol comprising 9 to 10 oxyethylene groups for reducing lipopolysaccharide activity in a product containing collagen and / or collagen derived.

20. Use of a polyethylene glycol ether of lauryl alcohol for reducing lipopolysaccharide activity in a product containing collagen and / or collagen derived.

21. A product containing collagen and / or collagen derived obtainable by the method of any one of claims 1 to 18.

22. The product containing collagen and / or collagen derived according to claim 21 having a lipopolysaccharide content of less than 3000 EU / g.

23. The product containing collagen and / or collagen derived according to claim 21 or 22 comprising polyethylene glycol ether of a fatty alcohol comprising 9 to 10 oxyethylene groups in an amount of less than 10 ppm.

24. The product containing collagen and / or collagen derived according to claim 23 comprising polyethylene glycol ether of a fatty alcohol comprising 9 to 10 oxyethylene groups in an amount of less than 1 ppm.

25. The product containing collagen and / or collagen derived according to claim 21 or 22 comprising polyethylene glycol ether of a fatty alcohol comprising 9 to 10 oxyethylene groups.

26. The product containing collagen and / or collagen derived according to claim 23, wherein the polyethylene glycol ether of a fatty alcohol comprising 9 to 10 oxyethylene groups comprises lauryl alcohol polyether-9.

27. The product containing collagen and / or collagen derived according to claim 21 or 22, wherein the product containing collagen and / or collagen derived is one or more selected from the group consisting of gelatin, hydrolyzed gelatin and collagen hydrolysate.

28. Use of the product containing collagen and / or collagen derived according to any one of claims 21 to 27 in medical devices, pharmaceuticals and / or cosmetic applications.

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