Method for endotoxin reduction in water-soluble biopolymers
Patent Information
- Application Number
- PCT/EP2025/088018
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-03-10
- Filing Date
- 2025-12-18
- Publication Date
- 2026-09-17
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Abstract
Description
[0001] A 501 286 p Applicant: GELITA AG December 18, 2025 Uferstrasse 7
[0002] p-260 69412 Eberbach Method for endotoxin reduction in water-soluble biopolymers
[0003] The present invention relates to a method for reducing the content of endotoxins in an aqueous solution containing one or more water-soluble biopolymers.
[0004] The invention further relates to a water-soluble biopolymer obtainable according to this method.
[0005] Biopolymers such as gelatin or carrageenan are used in various applications, particularly in the medical field, in addition to food technology. This includes their use in parenteral pharmaceutical formulations, in the production of tissue implants, and as substrates for cell cultures. For such products, especially those with potential contact with the human bloodstream, the highest possible purity of the biopolymers used must be ensured, particularly with regard to potentially toxic components or impurities.
[0006] Many biopolymers, due to their derivation from animal or plant raw materials, can be contaminated with bacterial endotoxins, which even at extremely low concentrations have a pyrogenic effect, meaning they can trigger fever and inflammation in humans. The problem of potential endotoxin contamination affects not only biopolymers used in their natural, unmodified form, but also chemically or enzymatically modified biopolymers such as modified gelatins, modified starches, and modified celluloses.
[0007] By far the most relevant group of endotoxins are lipopolysaccharides (LPS), which are produced in the cell membrane of gram-negative bacteria and are released in A 501 286 p
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[0010] - 2 - are released during the lysis of bacterial cells. The terms endotoxin and lipopolysaccharide are often used synonymously. When endotoxins are mentioned in this description, this always includes lipopolysaccharides.
[0011] The endotoxin content can be determined using the LAL test and is expressed in the unit EU endotoxin units') per gram or milliliter.
[0012] Several methods are known to reduce the endotoxin content in medical products, particularly in biopolymers or compositions containing biopolymers. These methods include extraction, adsorption, and, if necessary, ultrafiltration. Thermal deactivation of endotoxins is also possible in principle, but is generally not feasible with biopolymers and pharmaceutical compositions due to their lack of thermal stability.
[0013] EP 3 223 869 Bl discloses a process for removing lipopolysaccharides from aqueous solutions of gelatin using micelle-forming surfactants, in particular alkylphenol ethoxylates. These form a complex with lipopolysaccharides, which is removed from the solution by an adsorbent. However, the surfactants used in EP 3 223 869 Bl, such as the product Triton X-100, are themselves toxicologically problematic and have been included by the EU in the list of substances of very high concern. The use of these surfactants in medical devices or in their manufacture should therefore be avoided wherever possible.
[0014] The present invention is based on the objective of proposing an improved method for reducing the content of endotoxins in an aqueous solution containing one or more water-soluble biopolymers.
[0015] This problem is solved according to the invention by a method comprising the steps: A 501 286 p
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[0018] - 3 -a) Providing one or more water-soluble biopolymers containing endotoxins;
[0019] b) Producing an aqueous solution of the endotoxin-containing biopolymer(s), wherein the aqueous solution further contains at least one phospholipid;
[0020] c) Incubate the aqueous solution for a period of approximately 1 minute or more;
[0021] d) Contacting the aqueous solution with a solid adsorbent; and
[0022] e) Separation of the aqueous solution from the solid adsorbent.
[0023] In the process according to the invention, phospholipids are used to extract the endotoxins from the aqueous solution. Due to their polar molecular structure, phospholipids are able to form complexes with the endotoxins, particularly in the form of micelles, which can then be separated from the aqueous solution of the biopolymer(s) using a solid adsorbent. In contrast to synthetic nonionic surfactants such as Triton X-100, phospholipids are natural substances or their derivatives, and any residues present in medical products are largely harmless.
[0024] At the beginning of the process, in step a), an aqueous solution of the endotoxin-containing biopolymer(s) can already be provided, e.g., an aqueous solution obtained from the manufacturing process of a biopolymer. In step b), the at least one phospholipid is then added to this solution to produce the solution containing the biopolymer and phospholipid.
[0025] Similarly, in step b), the endotoxin-containing biopolymer(s) can be added to an aqueous solution of the at least one phospholipid. Previously insoluble components can be separated from such an aqueous solution of the at least one phospholipid. A 501 286 p
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[0028] - 4 - The group of substances known as phospholipids, which can be considered natural ionic surfactants, includes both phosphoglycerides and sphingomyelins, whose structure is based on glycerol or sphingosine as a polyhydric alcohol, respectively. In phospholipids, the hydroxyl groups of glycerol or sphingosine are esterified with a (usually substituted) phosphoric acid and at least one fatty acid.
[0029] Particularly preferred in the process according to the invention is the at least one phospholipid selected from the group of lysophospholipids, especially lysophosphoglycerides and lysosphingomyelins. In these lyso compounds, the glycerol or sphingosine each has a free, unesterified hydroxyl group adjacent to the phosphoric acid-esterified hydroxyl group. Lysophospholipids can be obtained from the corresponding fully esterified phospholipids by enzymatic cleavage of a fatty acid using phospholipase A2.
[0030] The lysophospholipids used in step b) preferably comprise one or more La-lysophosphoglycerides with the structural formula (I):
[0031]
[0032] wherein R is selected from saturated and unsaturated acyl groups, preferably having a chain length of 6 to 30 carbon atoms, more preferably of 10 to 18 carbon atoms. The latter include in particular the acyl groups of capric acid (C10), lauric acid (C12), myristic acid (CM), palmitic acid (Cie) and stearic acid (Cis).
[0033] X in formula (I) is selected from hydrogen, a choline residue, an ethanolamine residue, an inositol residue, a serine residue and an A 501 286 p
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[0036] - 5 - Glycerol residue. Accordingly, the at least one phospholipid preferably comprises one or more lysophosphatidic acids, lysophosphatidylcholines, lysophosphatidylethanolamines, lysophosphatidylinositols, lysophosphatidylglycerols or mixtures thereof.
[0037] In a particularly preferred embodiment, the at least one phospholipid comprises a lysophosphatidylcholine. Lysophosphatidylcholines have been shown to be particularly effective in reducing the endotoxin content in aqueous solutions of biopolymers within the framework of the process according to the invention.
[0038] In step b) of the process according to the invention, the lysophosphatidylcholine can be used in the form of a composition that further comprises one or more additional phospholipids. These phospholipids can include either further lysophosphoglycerides described above or fully esterified phosphoglycerides. The composition preferably comprises approximately 60 wt.% lysophosphatidylcholine or more, more preferably approximately 80 wt.% or more.
[0039] The lysophosphatidylcholine used in the process according to the invention is advantageously obtained from a vegetable oil, preferably soybean oil or sunflower oil, or from egg yolk. Phosphatidylcholine, also known as lecithin, can be obtained from such plant or animal raw materials in a first step. The compositions described above, which, in addition to lysophosphatidylcholine as the main component, comprise one or more phospholipids, can then be obtained from this by enzymatic reaction with phospholipase A2.
[0040] The at least one phospholipid is typically present in the aqueous solution prepared in step b) in an amount of approximately 0.001 to 0.5 wt.%, preferably approximately 0.02 to approximately 0.4 wt.%, and more preferably 0.05 to approximately 0.3 wt.%, based on the aqueous solution. The [A 501 286 p]
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[0043] - 6 - The amount of phospholipids required for effective endotoxin reduction may depend in particular on the original content of endotoxins in the aqueous solution, as well as on the type of biopolymer(s).
[0044] The incubation of the aqueous solution with the at least one phospholipid in step c) of the process according to the invention preferably takes place at a temperature of approximately 30 to approximately 70 °C, more preferably at approximately 40 to approximately 70 °C.
[0045] 65 °C. The efficiency of the inventive method tends to be improved by higher incubation temperatures, whereby the maximum usable temperature may be limited by the thermal stability of the type of biopolymer(s).
[0046] The incubation in step c) is preferably carried out for a period of approximately
[0047] 1 to approximately 120 min, preferably from approximately 5 to approximately 50 min, more preferably from approximately 20 to approximately 40 min. In particular, there may be a correlation with the incubation temperature, such that at a lower temperature a longer incubation time is generally required to ensure sufficient endotoxin reduction in the aqueous solution.
[0048] In step d) of the process according to the invention, the aqueous solution is brought into contact with a solid adsorbent to adsorb the complexes formed from the endotoxins and phospholipids. A variety of adsorbents known to those skilled in the art can be used for this purpose. Preferably, the solid adsorbent is selected from activated carbon, amorphous silicas, and aromatic polymeric adsorbents. The adsorbent is preferably added to the aqueous solution in powder or particle form, and the separation of the aqueous solution from the solid adsorbent in the subsequent step e) is advantageously carried out by filtration. Before separating the adsorbent, a further incubation is advantageously carried out for a period of approximately 1 minute or more, preferably from approximately 5 to approximately 50 minutes, and more preferably from approximately 20 to approximately 10 minutes.
[0049] 40 min. A 501 286 p
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[0052] - 7 - The amount of solid adsorbent used in step d) depends primarily on the amount of the at least one phospholipid added in step b). Preferably, the solid adsorbent is added to the aqueous solution in a weight ratio of 2:1 to 10:1, more preferably from 4:1 to 8:1, in each case based on the at least one phospholipid.
[0053] Instead of adding the solid adsorbent to the aqueous solution, a continuous adsorption process can also be carried out in step d), in which the adsorbent is present, for example, in the form of a column onto which the aqueous solution is applied.
[0054] The process according to the invention can be used to reduce the endotoxin content of various types of water-soluble biopolymers. The biopolymer(s) in the solution provided in step a) are preferably selected from proteins, in particular from gelatins, collagen hydrolysates, albumins and enzymes, and from polysaccharides, in particular from pectins, alginates, carrageenan, modified starches, modified celluloses and mixtures thereof.
[0055] The process according to the invention is particularly suitable for the endotoxin reduction of gelatin, which may be contaminated with endotoxins due to its manufacturing process. The aqueous solution in step a) of the process can, in particular, contain one or more gelatins obtained from collagen-containing animal starting materials, such as, in particular, bovine bone, bovine hide, or pigskin. The gelatin can be produced by means of an acidic digestion process (Type A) or by means of an alkaline digestion process (Type B). However, the process according to the invention is also applicable to gelatins produced by recombinant gene expression.
[0056] In addition to conventional, unmodified gelatin, the process according to the invention can also be advantageously used to reduce the endotoxin content of chemically or enzymatically modified gelatins. In this case, A 501 286 p
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[0059] - 8 - In this case, the gelatin is preferably modified with one or more chemical groups selected from acrylic, methacrylic, carboxyl, acyl, hydroxy, amino, phenyl, allyl, thiol, norbornene, azide, furan, tetrazine, maleimide, organophosphorus, desaminothyrosine and organometallic groups, as well as their derivatives.
[0060] Gelatins modified with methacrylic or desaminothyrosine groups are of particular importance in the production of tissue implants or tissue models using bioprinting. In these applications, the greatest possible reduction of endotoxins is of paramount importance.
[0061] The concentration of the biopolymer(s) in the aqueous solution can vary widely depending on the biopolymer and application, particularly on the degree of water solubility of the biopolymer(s). Preferably, the aqueous solution in step a) contains an amount of approximately 0.1 to approximately 60 wt.% of the biopolymer(s), more preferably approximately 0.3 to approximately 30 wt.%, and even more preferably approximately 0.4 to approximately 100 wt.%.
[0062] 20% by weight.
[0063] Accordingly, in the process according to the invention, the amount of the at least one phospholipid used in relation to the amount of the biopolymer(s) in the aqueous solution in step b) can vary over a wide range. Advantageously, this ratio is in the range of 1:500 to 1:1, more preferably in the range of 1:50 to 1:10.
[0064] The inventive method preferably further comprises the step:
[0065] f) Obtain the water-soluble biopolymer(s) from the aqueous solution according to step e).A 501 286 p
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[0068] - 9 - The biopolymer(s) can be obtained, in particular, by concentrating and / or drying the aqueous solution. Corresponding processes, which may differ depending on the type of biopolymer(s), are known to those skilled in the art.
[0069] The present invention further relates to a water-soluble biopolymer obtainable according to the inventive process, wherein the water-soluble biopolymer has an endotoxin content, in particular of lipo-polysaccharides, of approximately 300 EU / g or less. Preferably, the biopolymer has an endotoxin content of approximately 100 EU / g or less, more preferably of approximately 30 EU / g or less, and even more preferably of approximately 30 EU / g or less.
[0070] 10 EU / g or less, in particular approximately 1 EU / g or less. Correspondingly low limit values can be achieved with the method according to the invention.
[0071] In a preferred embodiment of the invention, the biopolymer comprises a gelatin, in particular an unmodified gelatin or a chemically or enzymatically modified gelatin.
[0072] The biopolymer according to the invention is preferably free of synthetic surfactants, in particular free of non-ionic surfactants.
[0073] Examples
[0074] Example 1: Endotoxin reduction in gelatin
[0075] In this example, the reduction of endotoxin levels was investigated using the inventive method with various compositions containing phospholipids.
[0076] A type A gelatin from pigskin, contaminated with endotoxins and with a Bloom strength of 311 and an A 501 286 p, was used as a water-soluble biopolymer.
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[0079] - 10 - A viscosity of 4.5 mPa-s was used. The original endotoxin content of this gelatin before reduction was 372 EU / g, determined using the LAL test.
[0080] The commercially available phospholipid compositions used in the various experiments are listed in Table 1 below:
[0081] Table 1
[0082]
[0083] To carry out the procedure, 900 g of a 10 wt% aqueous solution of gelatin were prepared and pre-tempered to 60 °C. After adding the respective phospholipid composition up to a proportion of 0.25 wt% or 0.15 wt% in the aqueous solution (see Table 2 below), this solution was shaken at 60 °C for 30 min on an incubation shaker at 200 rpm. In a comparison sample, no phospholipids were added.
[0084] After incubation, 9 g or 5.4 g of activated carbon (Norit SX, Norit Germany) were added to the aqueous solution as a solid adsorbent (see Table 2 below), i.e., in a weight ratio of 4:1 based on A 501 286 p
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[0087] - 11 -the phospholipid composition. The solution was shaken again for 30 min at 200 rpm and the activated carbon was removed via a paper filter (Schleicher & Schüll, 150 mm, 602 eh).
[0088] The gelatin was obtained from the aqueous solution in a known manner by gelling, drying and grinding, and its endotoxin content was determined using the LAL test.
[0089] The respective amounts of phospholipids and adsorbents, as well as the endotoxin content of the obtained gelatin, are given in the following Table 2:
[0090] Table 2
[0091] <
[0092] <
[0093] <
[0094] <
[0095] <
[0096] <
[0097]
[0098] With all the phospholipid compositions tested, the original endotoxin content of 372 EU / g could be reduced to less than 300 EU / g, in most cases to below 100 EU / g. In the comparative example without phospholipids, adsorption with activated carbon only led to a reduction to 346 EU / g.
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[0101] - 12 - However, the composition with 80% lysophos-phatidylcholine (along with 20% other phospholipids) is clearly the most effective, with which a reduction of the endotoxin content to below 1 EU / g could be achieved.
[0102] To determine the proportion of phospholipids removed by the solid adsorbent, the samples were each ashed, and the phosphate content of the original gelatin (cO), the phosphate content after addition of the corresponding amount of phospholipid composition (cl), and the phosphate content of the recovered gelatin (c2) were determined. The proportion (A) of removed phospholipids is then calculated using the formula
[0103] A = (cl - c2) / (cl - cO)
[0104] The results are shown in Table 3 below.
[0105] Table 3
[0106]
[0107] Even though phospholipids are harmless to health, many applications require the most complete possible removal of phospholipids from the purified gelatin. The results show that, with the method according to the invention, at least for some phospholipid compositions, extensive or complete removal by the solid adsorbent can be achieved. A 501 286 p
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[0110] - 13 - Example 2: Varying the amount of adsorbent
[0111] The experiment according to example 1.1.1 above was repeated with an amount of 0.45 g of the product LPC 80 (La-lysophosphatidylcholine), corresponding to a proportion of 0.05 wt% in the aqueous solution. Different amounts of activated carbon (Norit SX) were added as a solid adsorbent.
[0112] The respective quantities and results are given in the following Table 4:
[0113] Table 4
[0114] < <
[0115]
[0116] These experiments show that a higher proportion of adsorbent particularly improves the removal of phospholipids.
[0117] Example 3: Use of different adsorbents
[0118] The experiment according to the above example 1.1.1 was repeated with the same proportions using a different solid adsorbent (Ex. 3.1), namely a synthetic amorphous silica (Trisyl 300, WR Grace).
[0119] In a further experiment (Ex. 3.2), a continuous adsorption procedure was carried out. After incubation of the aqueous gelatin solution (900 g) with the phospholipid composition (2.25 g LPC 80), this A 501 286 p
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[0122] - 14 - add to a column with a volume of 200 ml of an aromatic polymeric adsorbent (AmberLite SD-2, DuPont).
[0123] The results are shown in Table 5 below:
[0124] Table 5
[0125] < <
[0126]
[0127] Example 4: Varying the amount of phospholipids
[0128] The experiment according to examples 1.1.1 and 1.1.2 above was repeated with different amounts of the product LPC 80 (La-lysophosphatidylcholine), corresponding to a proportion of 0.002 wt.% and 0.05 wt.% in the aqueous solution. The solid adsorbent was added in each case at a ratio of 8:1 with respect to the phospholipid.
[0129] The respective quantities and results are given in the following Table 6:
[0130] Table 6
[0131] < <
[0132]
[0133] A 501 286 p
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[0136] - 15 - The results show that even with an extremely small amount of phospholipids, an effective reduction of the endotoxin content is possible.
[0137] Example 5: Endotoxin reduction in different gelatins
[0138] The experiment according to the above example 2.2 with an amount of 0.45 g of the product LPC 80 (La-Lysophosphatidylcholine) and 2.7 g of activated charcoal (Norit SK), i.e. a ratio of adsorbent to phospholipid of 6:1, was repeated with different gelatins.
[0139] The gelatins in Examples 5.1 to 5.4 are derived from pigskin, and the gelatins in Examples 5.5 to 5.8 are derived from cattle bones. Further parameters of the gelatins and the results are given in Table 7 below.
[0140] Table 7
[0141] <
[0142]
[0143] In all gelatins, the process according to the invention resulted in a significant reduction of the endotoxin content, even at high initial values, to (with one exception) significantly below 100 EU / g.
Claims
A 501 286 p December 18, 2025 p-260 - 16 - Patent claims 1. Method for reducing the content of endotoxins in an aqueous solution containing one or more water-soluble biopolymers, comprising the steps of: a) Providing one or more water-soluble biopolymers containing endotoxins; b) Producing an aqueous solution of the endotoxin-containing biopolymer(s), wherein the aqueous solution further contains at least one phospholipid; c) Incubate the aqueous solution for a period of approximately 1 minute or more; d) Contacting the aqueous solution with a solid adsorbent; and e) Separation of the aqueous solution from the solid adsorbent.
2. The method of claim 1, wherein in step a) an aqueous solution of the endotoxin-containing biopolymer(s) is provided, and in step b) the at least one phospholipid is added to this solution.
3. The method of claim 1, wherein in step b) an aqueous solution of the at least one phospholipid is provided and the endotoxin-containing biopolymer(s) is added to this solution.
4. Method according to one of the preceding claims, wherein the at least one phospholipid is selected from the group of lysopholipids, in particular lysophosphoglycerides and lysosphingomyelins.
5. The method of claim 4, wherein the lysophospholipids comprise one or more La-lysophosphoglycerides having the structural formula (I): A 501 286 p December 18, 2025 p-260 - 17 - where X is selected from hydrogen, a choline residue, an ethanolamine residue, an inositol residue, a serine residue and a glycerol residue, and wherein R is selected from saturated and unsaturated acyl residues, preferably having a chain length of 6 to 30 carbon atoms, more preferably of 10 or 18 carbon atoms.
6. Method according to claim 5, wherein the at least one phospholipid comprises one or more lysophosphatidic acids, lysophosphatidylcholines, lysophosphatidylethanolamines, lysophosphatidylinositols, lysodiphosphatidylglycerols or mixtures thereof, preferably a lysophosphatidylcholine.
7. The method of claim 6, wherein the lysophosphatidylcholine in step b) is used in the form of a composition which further comprises one or more further phospholipids, wherein the composition preferably comprises approximately 60 wt.% lysophosphatidylcholine or more, more preferably approximately 80 wt.% or more.
8. Method according to claim 6 or 7, wherein the lysophosphatidylcholine is obtained from a vegetable oil, preferably from soybean oil or sunflower oil, or from egg yolk.
9. Method according to any one of the preceding claims, wherein the aqueous solution prepared in step b) contains the at least one phospholipid in an amount of approximately 0.001 to approximately 0.5 wt.%, preferably A 501 286 p December 18, 2025 p-260 - 18 - from approximately 0.02 to approximately 0.4 wt.%, preferably from approximately 0.05 to approximately 0.3 wt.%, each based on the aqueous solution.
10. A method according to any of the preceding claims, wherein in step c) the incubation takes place at a temperature of approximately 30 to approximately 70 °C, preferably at approximately 40 to approximately 65 °C; and / or wherein in step c) the incubation takes place for a period of approximately 1 to approximately 120 min, preferably from approximately 5 to approximately 50 min, more preferably from approximately 20 to approximately 100 min. 40 min.
11. Method according to one of the preceding claims, wherein in step d) the solid adsorbent is selected from activated carbon, amorphous silicas and aromatic polymeric adsorbents.
12. Method according to any of the preceding claims, wherein the solid adsorbent is added to the aqueous solution in a weight ratio of 2:1 to 10:1, preferably 4:1 to 8:1, in each case based on the at least one phospholipid.
13. Method according to any of the preceding claims, wherein the biopolymer(s) in the solution provided in step a) are selected from proteins, in particular from gelatins, collagen hydrolysates, albumins and enzymes, and from polysaccharides, in particular from pectins, alginates, carrageenan, modified starches, modified celluloses and mixtures thereof.
14. The method of claim 13, wherein the aqueous solution in step a) contains one or more gelatins obtained from collagen-containing animal starting materials or produced by recombinant gene expression. A 501 286 p December 18, 2025 p-260 - 19 - 15. Method according to claim 14, wherein the gelatin comprises a chemically modified gelatin, and wherein the gelatin is preferably modified with one or more chemical groups selected from acrylic, methacrylic, carboxyl, acyl, hydroxy, amino, phenyl, allyl, thiol, norbornene, azide, furan, tetrazine, maleimide, organophosphorus, desaminotyrosine and organometallic groups, as well as their derivatives.
16. Method according to one of the preceding claims, wherein the aqueous solution in step a) contains an amount of approximately 0.1 to approximately 60 wt.% of the biopolymer(s), preferably approximately 0.3 to approximately 30 wt.%, more preferably approximately 0.4 to approximately 20 wt.%.
17. Method according to one of the preceding claims, wherein in the aqueous solution in step b) the weight ratio of the at least one phospholipid to the water-soluble biopolymer(s) is in the range of 1:500 to 1:1, preferably in the range of 1:50 to 1:
10.
18. Method according to any of the preceding claims, further comprising the step of: f) Obtaining the water-soluble biopolymer(s) from the aqueous solution according to step e).
19. Water-soluble biopolymer obtainable according to the process of any one of the preceding claims, wherein the water-soluble biopolymer has an endotoxin content, in particular of lipopolysaccharides, of approximately 300 EU / g or less, preferably approximately 100 EU / g or less, more preferably approximately 30 EU / g or less, and even more preferably approximately 10 EU / g or less, and in particular approximately 1 EU / g or less. A 501 286 p December 18, 2025 p-260 - 20 - 20. Water-soluble biopolymer according to claim 19, wherein the biopolymer comprises a gelatin, in particular an unmodified gelatin or a chemically or enzymatically modified gelatin.
21. Water-soluble biopolymer according to claim 19 or 20, which is free of synthetic surfactants, in particular free of non-ionic surfactants. * * *