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

Abstract

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

Description

Technical Field

[0001] The present invention relates to reducing the activity of lipopolysaccharides (LPS) and / or LPS in products containing collagen and / or a source of collagen, and products obtained by this method. Background Art

[0002] Collagen, products containing collagen or collagen sources (e.g., extracellular matrix (ECM), collagen peptides, gelatin and (chemically) modified gelatin) have been used in a variety of different fields from the food industry to the medical and pharmaceutical fields. The interest in these types of substances stems from their desired characteristics, including their biocompatibility and biodegradability. Although collagen and substances containing collagen and collagen sources are generally considered safe, their biomedical use is challenged by the presence of endotoxins (e.g., LPS).

[0003] LPS is located on the outer cell membrane of Gram-negative bacteria, where LPS provides structural integrity and protection by acting as a permeation barrier. LPS is composed of variable polysaccharide chains and lipid moieties (lipid A). The size of LPS molecules is about 10kDa, but large aggregates can be formed in aqueous media, also referred to as "micelles". LPS is not secreted by bacteria, but released when the bacterial cell membrane is destroyed, so it is called "endotoxin". LPS has high immunogenicity to people, and even if exposed to a small amount of LPS, there will be violent adverse effects. Due to toxicity, the amount of endotoxin allowed on medical devices is strictly regulated. For example, the Food and Drug Administration (FDA) allows products that directly / indirectly contact the cardiovascular and lymphatic systems to be a maximum of 0.5 endotoxin units (endotoxin unit, EU) / g or 20EU / device. For devices in contact with cerebrospinal fluid, this limit is even lower.

[0004] Avoiding endotoxin contamination is challenging during conventional manufacturing of products containing collagen and / or collagen-derived products. Furthermore, once endotoxins are present, they are extremely difficult to remove, especially from sensitive materials including proteins.

[0005] In the art, methods for removing LPS from protein solutions using surfactants have been described, for example, in WO 2016085345. Although showing great promise, the above methods rely on Triton X-100, the use of which is under discussion.

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

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

[0008] The present inventors have identified an effective method for reducing LPS activity and / or LPS in products containing collagen and / or collagen sources, said method being based on the use of fatty alcohol polyglycol ethers.

[0009] More specifically, the inventors have found that laureth-9, which is a nonionic, non-toxic micelle-forming surfactant, is effective in reducing LPS activity in products containing collagen and / or collagen sources. Unexpectedly, the concentration of laureth-9 required for effectiveness was found to be significantly lower than that of Triton X-100.

[0010] The present inventors have therefore provided improved methods for reducing LPS activity.

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

[0012] In one aspect, the present invention relates to the use of polyethylene glycol ethers of fatty alcohols for reducing LPS activity in products containing collagen and / or a source of collagen.

[0013] In one aspect, the present invention relates to products containing collagen and / or collagen sources having an 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 product containing collagen and / or a source of collagen, said method comprising one or more of the following steps, preferably all of the following steps: - providing a product containing collagen and / or a source of collagen comprising LPS,

[0015] - contacting a product containing collagen and / or a collagen source comprising LPS with a polyethylene glycol ether of a fatty alcohol to provide a mixture,

[0016] - Recovering a product containing collagen and / or a source of collagen, preferably a product containing collagen and / or a source of collagen wherein the LPS content is less than 3000EU / g, more preferably a product containing collagen and / or a source of collagen wherein the LPS content is less than 1000EU / g, even more preferably a product containing collagen and / or a source of collagen wherein the LPS content is less than 100EU / g, even more preferably a product containing collagen and / or a source of collagen wherein the LPS content is less than 10EU / g, most preferably a product containing collagen and / or a source of collagen wherein the LPS content is less than 1EU / g.

[0017] The term "reducing LPS activity" as used herein means that the LPS level of the recovered product containing collagen and / or a source of collagen is lower than the LPS level of the starting material (i.e., the product containing collagen and / or a source of collagen before contact with the polyethylene glycol ether of fatty alcohol). In the present invention, "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., colorimetric or turbidimetric).

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

[0019] In one embodiment, the product containing collagen and / or a collagen source is endotoxin-free (ie, LPS-free).The term "endotoxin-free" may mean the absence of endotoxins and / or may mean "substantially free of endotoxins" (ie, substantially free of LPS).

[0020] The term "free of" may have the same meaning as "substantially free of". The term "substantially free of" encompasses that the amount of a substance or compound is not measurable according to standard techniques in the art and / or is below certain thresholds. In addition or as an alternative, 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 EU / g or EU / ml). The wording "free of" or "substantially free of" encompasses that a substance or compound is completely absent (e.g., 0 wt.% or 0 EU / g). The terms "absent", "free of" and "substantially free of" are used interchangeably in the context of the present invention.

[0021] As described herein, the use of polyethylene glycol ethers of fatty alcohols to remove LPS (activity) is particularly advantageous if the starting material has a high amount of LPS (activity). For example, it has been found that if the LPS content in the starting material is relatively high, such as 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, and most preferably at least 50000 EU / g, the polyethylene glycol ethers of fatty alcohols are more effective than other known methods (e.g., Triton X-100) in removing LPS (activity). In one embodiment, the LPS content in the product containing collagen and / or a source of collagen is at least 500EU / g, or at least 1000EU / g, or at least 2500EU / g, or at least 5000EU / g, or at least 10000EU / g, or at least 50000EU / g, and / or no more than 50000EU / g, or no more than 10000EU / g, or no more than 5000EU / g, or no more than 2500EU / g, or no more than 1000EU / g or no more than 500EU / g. In addition or alternatively, the LPS content in the product containing collagen and / or a source of collagen is preferably 500 to 50000EU / g, 1000 to 50000EU / g, and most preferably 1000 to 10000EU / g.

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

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

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

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

[0026] The term "collagen hydrolysate" in the context of the present invention means a mixture of short chains of amino acids (dipeptides, tripeptides, oligopeptides, polypeptides) derived from (partial) hydrolysis (e.g., by enzymatic hydrolysis) of native (full-length) collagen. The degree of hydrolysis has an influence on the average molecular weight (expressed in Daltons, Da) of the final product. The term "collagen hydrolysate" can be used interchangeably and synonymously with the term "hydrolyzed collagen" or "collagen peptides." "Collagen hydrolysate" in the context of the present invention encompasses collagen that has undergone hydrolysis or partial hydrolysis. "Collagen hydrolysate" in the context of the present invention can be produced from a collagen-containing substance in a one-step process or via an intermediate gelatin stage (i.e., thereby obtaining "hydrolyzed gelatin"). Therefore, the term "collagen hydrolysate" encompasses hydrolyzed gelatin (i.e., hydrolyzed gelatin).

[0027] The product containing collagen and / or collagen source used herein can be derived from one or more types of collagen known in the art. More particularly, the product containing collagen and / or collagen source used herein can be selected from comprising 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, the group of XXVIII type collagen. In addition or as a substitute, the product containing collagen and / or collagen source used herein can be a mixture of one or more types of collagen.

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

[0029] As known in the art, the term "collagen-derived product" as used herein refers to proteins and peptides that are part of the collagen matrix in the raw material and 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 a combination thereof. Gelatin is not composed of uniform protein molecules, but contains variable amounts of protein molecules of variable length. Preferably, the average molecular weight of the gelatin used herein is in the following range: 1500Da to 300kDa, preferably 2000D a Up to 300kD a , 4000D a Up to 300kD a , 5000D a Up to 300kD a , 10kD aTo 300kD, or 20kDa to 300kDa, more preferably 50kDa to 300kDa, most preferably 100kDa to 300kDa, for example 100kDa to 275kDa or 100kDa to 250kDa. The term "(chemically) modified gelatin" includes gelatin having one or more functional groups on the gelatin backbone, such as acrylamide, ferulic acid, methacryloyl (e.g. GelMA), desaminotyrosine (e.g. GelDAT), furfurylamine and norbornene (Klotz, BJ, Gawlitta, D., Rosenberg, AJ, Malda, J., & Melchels, FP (2016). Gelatin-methacryloyl hydrogels: toward 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 the hydrolysis of gelatin into peptide molecules with an average molecular weight of 70 kDa or less, typically 20 kDa or less, typically 100 to 15000 Da.

[0030] In one embodiment, the product containing collagen and / or a source of collagen 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 product containing collagen and / or a source of collagen 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 invention encompasses solutions comprising one or more co-solvents in addition to water, which may depend on the polyethylene glycol ether of the selected fatty alcohol. 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 invention are one or more selected from 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 a (dry) product containing collagen and / or a source of collagen with a solvent. The product containing collagen and / or a source of collagen is preferably dissolved at room temperature or 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, because elevated temperatures can increase the undesirable hydrolysis of the product containing collagen and / or a source of collagen. The previous steps can be performed at different temperatures, however preferably the highest of which is 68°C. Preferably, the mixing time includes a time of at least 10 minutes. Shorter or longer mixing times can also be used, however, for effectiveness and efficiency, the mixing time is preferably 10 to 60 minutes. Alternatively, the product containing collagen and / or a source of collagen can be directly contacted with a pure solution of Laureth-9.

[0032] In one embodiment, the aqueous solution may contain any concentration of products containing collagen and / or collagen sources. In a preferred embodiment, the aqueous solution contains at least 1 w / w% of products containing collagen and / or collagen sources, preferably at least 4 w / w%, more preferably at least 6 w / w%, even more preferably at least 8 w / w% of products containing collagen and / or collagen sources, still even more preferably at least 10 w / w% of products containing collagen and / or collagen sources, still even more preferably at least 15 w / w% of products containing collagen and / or collagen sources, and most preferably at least 20 w / w%.

[0033] In another preferred embodiment, the aqueous solution comprises 1 to 50 w / w% of a product containing collagen and / or a source of collagen, preferably 2 to 30 w / w%, more preferably 4 to 15 w / w% of a product containing collagen and / or a source of collagen, and even more preferably 6 to 10 w / w% of a product containing collagen and / or a source of collagen. The aqueous medium may comprise as much as possible a product containing collagen and / or a source of collagen 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% of a product containing collagen and / or a source of collagen. When using a product containing collagen and / or a source of collagen (e.g., gelatin hydrolyzate) having a relative molecular weight, a higher concentration of a product containing collagen and / or a source of collagen, such as at least 60 w / w%, 70 w / w%, 80 w / w%, and 90 w / w% is possible. Although the viscosity of aqueous solutions containing collagen and / or a source of collagen can be reduced by increasing the temperature, the elevated temperature may result in undesirable hydrolysis of products containing collagen and / or a source of collagen.

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

[0035] In the present disclosure, fatty alcohols (or long chain alcohols) are typically straight chain primary alcohols and can be derived from natural fats and oils in general. 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. Like fatty acids, they are typically referred to by the number of carbon atoms in the molecule, e.g., "C12 alcohols," i.e., alcohols with 12 carbons, such as dodecanol.

[0036] In one embodiment, the polyethylene glycol ether of a fatty alcohol comprises and / or comprises on average 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 polyglycol ether of a fatty alcohol comprises and / or comprises on average 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 polyglycol ether of a fatty alcohol comprises and / or comprises on average at least 1, 2, 3, 4, 5, 6, 7, 8, 9 oxyethylene groups. In yet another preferred embodiment, the polyglycol ether of a fatty alcohol comprises and / or comprises on average at most

[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 ethylene oxide groups. In a further preferred embodiment, the polyglycol ether of a fatty alcohol is preferably selected from one or more polyglycol ethers of a fatty alcohol comprising C12 and (on average) 9 ethylene oxide groups (e.g. Laureth-9), C12 and (on average) 10 ethylene oxide groups (e.g. Genapol X-100), C12 and (on average) 23 ethylene oxide groups (e.g. Brij 35), C16 and (on average) 10 ethylene oxide groups (e.g. Brij C10), C18 and (on average) 100 ethylene oxide groups (e.g. Brij S100), C18 and (on average) 10 ethylene oxide groups (e.g. Eco Brij S10).

[0039] In one embodiment, the polyglycol ether of a fatty alcohol comprises laureth-9. Laureth-9 itself is 3,6,9,12,15,18,21,24,27-nonaoxatriacontacan-1-ol. In a practical sense, it is a mixture of lauryl alcohol etherified with an average of 9 oxyethylene groups, dispersed by 1 to 20 oxyethylene groups.

[0040] Laureth-9 is a micelle-forming surfactant, which means that it is able to form micelles in solution. For this reason, the so-called critical micelle concentration (CMC) of a surfactant is defined as the concentration of the surfactant above which micelles begin to form. At a certain temperature, the so-called cloud point, the surfactant forms insoluble aggregates in an aqueous medium and phase separates.

[0041] In one embodiment, the polyglycol ether of fatty alcohol is preferably present in the mixture at a concentration equal to or higher than CMC. Preferably, the polyglycol ether of fatty alcohol is present at a concentration of at least 3 times CMC. More preferably, the polyglycol ether of fatty alcohol is present at a concentration of at least 5 times CMC. Even more preferably, the polyglycol ether of fatty alcohol is present at a concentration of at least 10 or 20 times CMC. Most preferably, the polyglycol ether of fatty alcohol is present at a concentration of at least 30 times CMC. Preferably, the aqueous solution comprises at least 0.001w / w%, more preferably at least 0.002w / w%, even more preferably at least 0.004w / w%, also even more preferably at least 0.006w / w%, also even more preferably at least 0.008w / w%, also even more preferably at least 0.01w / w%, also even more preferably at least 0.03w / w%, most preferably at least 0.05w / w% polyglycol ether of fatty alcohol, wherein the polyglycol ether of 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%, and even more preferably 0.004 to 0.06 w / w% polyglycol ether of a fatty alcohol, wherein the polyglycol ether of a fatty alcohol is preferably laureth-9. Even more preferably, the aqueous solution comprises 0.001 to 0.05 w / w% polyglycol ether of a fatty alcohol, wherein the polyglycol 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% polyglycol ether of a fatty alcohol, wherein the polyglycol ether of a fatty alcohol is preferably laureth-9. The exact concentration of the polyglycol ether of a fatty alcohol may also be adjusted depending on the LPS content in the product containing collagen and / or a source of collagen. For example, if the starting material has a low LPS content, a low concentration of polyethylene glycol ether of fatty alcohol (i.e. not exceeding the CMC value to a large extent) may be sufficient. The concentration of polyethylene glycol ether of fatty alcohol can be expressed relative to the amount of product containing collagen and / or a source of collagen present in the aqueous solution. Preferably, the polyethylene glycol ether of fatty alcohol is present in a concentration of 0.1 to 10 mg, even more preferably 0.2 to 8 mg, and even more preferably 0.9 to 8 mg per 1 gram of product containing collagen and / or a source of collagen present in the aqueous solution. In addition or as an alternative, the polyethylene glycol ether of fatty alcohol is preferably present in a concentration of at least 0.1, 0.2, 0.4, 0.6, 0.8, 1 mg of polyethylene glycol ether of fatty alcohol per 1 gram of product containing collagen and / or a source of collagen. Additionally or alternatively, the polyethylene glycol ether of a fatty alcohol is preferably present in a concentration of up to 10, 8, 6, 5, 4, 2 mg polyethylene glycol ether of a fatty alcohol per 1 g of the product containing collagen and / or a collagen source.

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

[0044] In certain embodiments, for example, when the polyethylene glycol ether of a fatty alcohol contains and / or contains on average 1 to 8, preferably 1 to 5, more preferably 1 to 3 oxyethylene groups, an ethanol solution or a solution containing another cosolvent as disclosed herein can be used. In addition or alternatively, when the polyethylene glycol ether of a fatty alcohol contains and / or contains on average less than 5, 4, 3, 2, 1 oxyethylene groups, an ethanol solution or a solution containing 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 can, for example, contain 10 to 70 (v / v)%, 20 to 60 (v / v)%, or 30% to 50% ethanol.

[0045] The contact time period between the product containing collagen and / or collagen source and the polyglycol ether of fatty alcohol is long enough to allow mixing. In addition or in lieu thereof, the contact time period is selected so as to reach the expected endotoxin activity. Preferably, the contact time period between the product containing collagen and / or collagen source and the polyglycol ether of fatty alcohol is at least 1 minute. More preferably, the contact time period is at least 5 minutes, even more preferably at least 10 minutes, also even more preferably at least 20 minutes, also even more preferably at least 30 minutes, also even more preferably at least 60 minutes. More preferably, the product containing collagen and / or collagen source and the polyglycol ether of fatty alcohol are hatched for 1 to 60 minutes, more preferably 5 to 30 minutes, most preferably 20 to 30 minutes.

[0046] In one embodiment, the product containing collagen and / or a source of collagen can be recovered by contacting the mixture with an adsorbent. The adsorbent can be any suitable adsorbent that is capable of binding a surfactant and preferably also binding LPS. Advantageously, the aqueous medium is free-flowing when the surfactant is added and when in contact with the adsorbent. Although the viscosity and gelation temperature vary between different products containing collagen and / or a source of collagen, the solution may 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 the adsorbent to be properly separated.

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

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

[0049] In one embodiment, the solid adsorbent comprises activated carbon, such as Norit SX Plus or Norit ROX0.8 (Cabot, the Netherlands).

[0050] In one embodiment, the adsorbent is disposed in a filter. Additionally or alternatively, the adsorbent may be stacked in a column. The contacting step between the mixture and the solid adsorbent is performed for a sufficient amount of time that allows for proper adsorption of the polyglycol ether of 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 may be required to achieve the desired recovered product compared to incubation over longer time periods. Longer time periods are also possible, but less desirable from a process efficiency perspective. Preferably, the adsorption step can be performed once.

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

[0053] Also described is the use of polyethylene glycol ethers of fatty alcohols for reducing LPS (activity) in products containing collagen and / or a source of collagen.

[0054] Also described is the use of polyethylene glycol ethers of lauryl alcohol for reducing LPS (activity) in products containing collagen and / or a collagen source.

[0055] In one aspect, the present invention relates to products containing collagen and / or a source of collagen that are optionally obtainable by the methods disclosed herein. In a preferred embodiment, the product containing collagen and / or a source of collagen is gelatin or hydrolyzed gelatin. In another preferred embodiment, the product containing collagen and / or a source of collagen is a collagen hydrolyzate.

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

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

[0058] As a supplement or alternative, the present invention also relates to a product containing collagen and / or a collagen source, optionally obtainable by the method according to the preceding claims, comprising an 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 ether of fatty alcohol is preferably laureth-9.

[0059] In one embodiment, the product containing collagen and / or a source of collagen, which may be obtained by the method according to the preceding claims, may be used in a variety of industries, such as in the food, pharmaceutical and cosmetic industries. In view of this, the product containing collagen and / or a source of collagen, which may be obtained by the method of the present invention, may be used in medical devices, pharmaceuticals and / or cosmetic applications, and the product containing collagen and / or a source of collagen may be used as a gelling agent, a texturizing agent and / or a hydrogel. In view of this, the present invention also relates to the use of the product containing collagen and / or a source of collagen, preferably the use of the product containing collagen and / or a source of collagen in medical devices and / or pharmaceuticals and / or cosmetics (applications). BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 is a graph showing the effectiveness of laureth-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 laureth-9 or Triton X-100, and the y-axis shows the LPS content (EU / g) after exposure to laureth-9 or Triton X-100.

[0061] Example

[0062] Example 1. Effects of Triton X-100 and Laureth-9 in reducing LPS activity.

[0063] To determine the effect of laureth-9 on reducing LPS activity, 8 w / w% (high bloom, high viscosity, initial LPS content 800 to 1200 EU / g) porcine-derived gelatin solutions were prepared in ultrapure water and exposed to different concentrations of laureth-9. Laureth-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. Afterwards, the gelatin solution (containing laureth-9) was filtered and the gelatin solution was recovered. The unfiltered gelatin solution was sampled along with the initial 25 mL and the final 100 mL of the filtered gelatin solution and stored at -20°C until analysis. To compare the potential effects of laureth-9 with the prior art, an example using 0.09 w / w% Triton X-100 was included in the experiment.

[0064] EndoZyme II LPS quantitative rFC assay kit (Biomerieux, Marcy-l'Etoile, France) was used to determine the LPS concentration in the recovered gelatin samples, which is the type of LAL assay. Recombinant Factor C (rFC) is activated by endotoxin binding, and in a cascade reaction, the active enzyme then cuts the 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] Analysis was performed using a Biotek Synergy MX microplate reader (Agilent Technologies, California, United States) and the fluorescence signal was measured at an excitation wavelength of 380 nm and an emission wavelength of 445 nm.

[0066] Table 1 shows that the use of 0.004 w / w% and 0.015 w / w% laureth-9 resulted in lower endotoxin levels than when using Triton X-100. In addition, it is shown that an additional filtration step is not mandatory because acceptable LPS levels (i.e., below 10 EU / g) were already achieved 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] 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), similar results were obtained as in the 0.015 w / w% laureth-9 group. In addition, similar results were obtained when other products containing collagen and / or collagen sources were used, such as high bloom strength high viscosity gelatin, low bloom strength low viscosity gelatin, and (chemically) modified gelatin having one or more functional groups on the gelatin backbone, such as methacryloyl (GelMA), desaminotyrosine (GelDAT).

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

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

[0071]

[0072] Example 2. Effect of Laureth-9 in reducing LPS activity in gelatin of porcine and bovine origin.

[0073] 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) were evaluated at initial LPS concentrations of 14,000 to 16,000 EU / g and 4,000 to 7,000 EU / g, respectively. The same protocol as in Example 1 was followed.

[0074] Laureth-9 showed satisfactory reduction in LPS concentration of both type A and type B gelatin, reaching LPS concentrations of about 3000 EU / g or less (Table 3).

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

[0076]

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

[0078] To compare the effectiveness of laureth-9 and Triton X-100, gelatin with different starting 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.

[0079] like Figure 1 As can be seen in the Figure 2, when gelatin with relatively high LPS activity (ie, >5000 EU / g) was used, the LPS-reducing efficacy of laureth-9 was higher than that of Triton X-100.

Claims

1. A method for reducing the activity of lipopolysaccharides in a product containing collagen and / or a source of collagen, the method comprising the following steps: - providing a product containing collagen and / or a source of collagen comprising lipopolysaccharide, - contacting the product containing collagen and / or a collagen source comprising lipopolysaccharide with a polyethylene glycol ether of a fatty alcohol to provide a mixture, - Recovering a product containing collagen and / or collagen sources, wherein the lipopolysaccharide activity is preferably lower than 3000EU / g, more preferably lower than 1000EU / g, even more preferably lower than 100EU / g, yet even more preferably lower than 10EU / g, most preferably lower than 1EU / g.

2. The method according to claim 1, wherein the product containing collagen and / or a collagen source is selected from the group consisting of extracellular matrix, natural collagen, gelatin, (chemically) modified gelatin, gelatin hydrolysate and mixtures thereof.

3. A method according to any one of the preceding claims, wherein the product containing collagen and / or a source of collagen is in the form of a solution.

4. A method according to any one of the preceding claims, wherein the product containing collagen and / or a source of collagen is in the form of an aqueous solution.

5. The method according to claim 4, wherein the aqueous solution comprises at least 1 w / w% of a product containing collagen and / or a collagen source.

6. The method according to claim 4 or 5, wherein the aqueous solution comprises 1 to 50 w / w% of a product containing collagen and / or a collagen source.

7. The method according to any one of the preceding claims, wherein the polyethylene glycol ether of a fatty alcohol is a polyethylene glycol ether of lauryl alcohol.

8. The process according to any one of the preceding claims, wherein the polyethylene glycol ether of a fatty alcohol comprises from 1 to 20 oxyethylene groups.

9. The method according to any one of the preceding claims, wherein the polyglycol ether of a fatty alcohol comprises laureth-9.

10. The method according to any one of claims 4 to 9, wherein the aqueous solution comprises at least 0.001 w / w % of the polyethylene glycol ether of the fatty alcohol.

11. The method according to any one of claims 4 to 10, wherein the aqueous solution comprises 0.001 to 0.05 w / w % of polyethylene glycol ether of fatty alcohol.

12. The method according to any one of the preceding claims, wherein the mixture is contacted with an adsorbent.

13. The method of claim 12, wherein the adsorbent is a solid.

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

15. The method of claim 14, wherein the hydrophobic adsorbent comprises activated carbon.

16. A method according to any one of the preceding claims, wherein the adsorbent is provided in a filter.

17. A method according to any one of the preceding claims, wherein the product containing collagen and / or a source of collagen is recovered by filtration.

18. Use of polyethylene glycol ethers of fatty alcohols for reducing the activity of lipopolysaccharides in products containing collagen and / or a source of collagen.

19. Use of polyethylene glycol ethers of lauryl alcohol for reducing the activity of lipopolysaccharides in products containing collagen and / or collagen sources.

20. A product containing collagen and / or a source of collagen obtainable by the method according to any one of claims 1 to 17.

21. The product containing collagen and / or collagen source according to claim 20, wherein the lipopolysaccharide content is less than 3000 EU / g.

22. Product containing collagen and / or a collagen source according to claim 20 or 21, comprising polyethylene glycol ethers of fatty alcohols in an amount of less than 10 ppm, preferably less than 2 ppm.

23. The product containing collagen and / or a collagen source according to claim 22, comprising polyethylene glycol ethers of fatty alcohols in an amount of less than 1 ppm, preferably less than 0.1 ppm.

24. A product containing collagen and / or a source of collagen according to any one of claims 20 to 23, comprising a polyethylene glycol ether of a fatty alcohol.

25. A product containing collagen and / or a source of collagen according to any one of claims 22 to 24, wherein the polyethylene glycol ether of a fatty alcohol comprises laureth-9.

26. The product containing collagen and / or a source of collagen according to any one of claims 20 to 25, wherein the product containing collagen and / or a source of collagen is one or more selected from gelatin, hydrolyzed gelatin and collagen hydrolysate.

27. Use of a product containing collagen and / or a source of collagen according to any one of claims 20 to 26 in medical device, pharmaceutical and / or cosmetic applications.

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