A method for purifying a modified gelatin

Abstract

The invention is directed to a method for purifying a modified gelatin, comprising the steps of providing a gelatin gel comprising the modified gelatin and contaminants; followed by contacting the gelatin gel with an extraction solvent having a temperature of at most 10 °C, allowing at least a part of the contaminants to dissolve in the extraction solvent; followed by separating at least part of the extraction solvent with dissolved contaminants from the gelatin gel to obtain a purified gelatin gel comprising a purified modified gelatin; followed by contacting the purified gelatin gel with water causing the purified gelatin gel to swell; optionally followed by repeating steps b) to d) to further purify the purified gelatin gel. The invention is further directed to products obtainable by the method, which products have an amount of contaminants of at most 200 ppm.

Description

[0001] Title: A method for purifying a modified gelatin

[0002] TECHNICAL FIELD

[0003] The present invention is in the field of modified gelatins. In particular, the present invention relates to a method for purifying a modified gelatin and relates to the purified modified gelatin obtainable by said method.

[0004] BACKGROUND

[0005] Biopolymers represent a great resource for the development and utilization of new functional materials due to their particular advantages such as biocompatibility, biodegradability and non-toxicity. A review by Ilic- Stojanovic et al. in Gels 9 (2023) 556 discloses hydrogels based on biopolymers such as natural proteins (e.g. fibrin, silk fibroin, collagen, keratin, gelatin) and natural polysaccharides (e.g. pectin, chitosan, hyaluronic acid, cellulose, carrageenan, alginate).

[0006] Of particular interest are modified gelatins, e.g. those that are modified by reacting the carboxylic groups and / or amino groups of the gelatin with compound that provide an additional functionality to the gelatin, e.g. the capacity to cross-link.

[0007] Gelatin is a mixture of water-soluble proteins derived from collagen. It dissolves in hot water and forms a physical gel on cooling.

[0008] Gelatin is obtainable by partial hydrolysis of collagen, obtained by aqueous extraction of skin, tendons, ligaments, bones etc., from e.g. bovine, porcine, poultry or fish, in acid or alkali conditions, or by enzymatic hydrolysis, as known in the art. Gelatin obtained by acid treatment is called “type A gelatin”, whereas “type B gelatin” is derived from alkali-based process. Due to a more extensive deamination of asparagine and glutamine in type B gelatin, the isoelectric points (IEP) of type A gelatin and type B gelatin are at pH 7.0-9.0 and pH 4.9- 5.1, respectively, which enables them to be positively and negatively charged at neutral physiological pH.

[0009] Gelatins present a class of materials made naturally and hence typically do not constitute a uniform molecule but comprises a variable amount of molecules of variable length.

[0010] The most extensively used and studied modification of gelatin is methacryloylation, resulting in methacryloyl-modified gelatin - also referred to as gelMA. An alternative to the well-established methacryloyl- gelatin is acryloyl- gelatin (see Billiet et al. Macromolecular Bioscience 13 (2013) 1531-45). Other modified gelatins are for instance tyramine-gelatins, as disclosed in Wang et al., Biomaterials 31 (2010) 1148-57, which also discloses the functionalization of gelatin through modification with a combination of 3-(4-hydroxyphenyl)-propionic acid (HP A), an excess of N- hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC). W02006 / 010066 discloses modification of gelatin using a carbodiimide-mediated coupling of tyramine to gelatin. W02020 / 050779 discloses hydrogels with tunable properties based on tyramine or hydroxyphenyl propionic acid based crosslinking agents. The compound 3-(4-hydroxyphenyl)propanoic acid ((HOCeH4CH2)2CO2H) is sometimes abbreviated as HP A, and is also known as phloretic acid or desaminotyrosine (sometimes abbreviated as DAT). These indications may be used interchangeably.

[0011] In Roch et al. Macromolecular Symposia 309-310 (2011) 182-189, a gelatin is disclosed that is modified with a carboxylic acid moiety R- (CH2)n-COOH. Roch et al. recommend the use of a certified low endotoxin containing gelatin (Type A, 200 Bloom, porcine). Gelatin was functionalized with DAT and desaminotyrosyl tyrosine (also abbreviated at DATT) respectively.

[0012] Gelatin can also be modified using the so-called Bolton-Hunter reagent, as described in Elvin et al. Biomaterials 31 (2010) 8323e8331. Herein, it is described that gelatin was derivatized to increase its Tyr content, using the Bolton-Hunter reagent (2V-succinimidyl-3-[4- hydroxyphenyl]propionate). After the reaction, the obtained derivatized gelatin was dialyzed against a phosphate-buffered saline (PBS) solution at 37 °C and subsequently freeze-dried.

[0013] During the modification of a gelatin (also referred to as functionalization or derivatization of gelatin), a number of reagents are typically used. These reagents, residues thereof and reaction products can remain present in the modified gelatin. For instance, upon reacting gelatin with 2V-succinimidyl-3-[4-hydroxyphenyl]propionate such as described by Elvin et al. (see above), hydrolysis products such as 3-[4-hydroxy]propionic acid (HP A, phloretic acid), N-hydroxysuccinimide (NHS) and unreacted reagents such as 2V-succinimidyl-3-[4-hydroxyphenyl]propionate can remain present in the modified gelatin composition. These remnants are considered undesirable, since they may interfere with the crosslinking and may impact the biocompatibility of the hydrogel (depending on the type of remnant).

[0014] Conventional procedures to reduce the impurity level of modified gelatins by removing the remnants rely on purifying the reaction mixture by dialysis against distilled water.

[0015] US 2023 / 0133041 disclose a method for purifying methacryloyl- gelatin and acryloyl-gelatin. This method does not lead to consistently low (meth)acrylic acid content. US8815277 disclose purification of gelatin by dialysis.

[0016] Typically, dialysis is performed for longer than two days. This procedure is time-consuming, it requires large volumes of water and come with the risk of other contamination. Moreover, dialysis can have an effect on the molecular weight distribution of the gelatin, and it can reduce the yield of the modified gelatin, especially if dialysis if performed for longer than 2 to 3 days. Further, the removal of remnants depends on their solubility in water as dialysis is usually performed in water because most of the membranes used for dialysis have a limited compatibility to organic solvents. Dialysis is therefore not efficient for large-scale production. Hence there is a need for a more efficient process allowing production at industrial scale.

[0017] Moreover, as indicated, the risk for microbial contamination and gelatin degradation increases during dialysis. This may cause the formation of endotoxins in the modified gelatin. Of particular importance for applications such as in pharmacy, biomedicine and food applications is the level of endotoxins. Even if a gelatin is used with a low level of endotoxins, the aforementioned microbial contamination and gelatin degradation may result in an increase of endotoxin level, which is highly undesirable. To be accepted onto the market, modified gelatins must comply to regulatory standards and lipopolysaccharide (LPS) endotoxins and process contaminants must be sufficiently low. Preferably, modified gelatins have endotoxin levels of less than 200 endotoxin units (EU) / g, more preferably less than 100 EU / g. Modified gelatins with an endotoxin content of less than 10 EU / g are most preferred.

[0018] WO 2022 / 258763A1 discloses a method for the modification of particular modified gelatins and subsequent purification using a two-step purification based on micelle formation. The drawback of using micelles is the requirement of surfactants such as Triton X-100 and X-102. Further, the method is elaborate and does not enable extensive removal of remnants.

[0019] SUMMARY OF THE INVENTION

[0020] An object of the present invention is to provide a method for purifying gelatin that overcomes one or more of the above-mentioned drawbacks of known methods to purify modified gelatin.

[0021] The present invention accordingly provides a method for purifying a modified gelatin, comprising the steps of: a) providing a gelatin gel comprising the modified gelatin with contaminants; b) contacting the gelatin gel with an extraction solvent having a temperature of at most 10 °C, allowing at least a part of the contaminants to dissolve in the extraction solvent; c) separating at least part of the extraction solvent with dissolved contaminants to obtain a purified gelatin gel comprising a purified modified gelatin; d) contacting the purified gelatin gel with water causing the purified gelatin gel to swell; e) optionally repeating steps b) to d) to further purify the purified gelatin gel, f) optionally contacting the purified gelatin gel obtained from either one of steps d) and e) with the extraction solvent at a temperature of less than 10 °C, and g) optionally drying the purified gelatin gel obtained from either one of steps d), e) and f) to obtain a purified gelatin powder.

[0022] In another aspect, the invention is directed to purified modified gelatin having an amount of contaminants of at most 200 ppm, preferably at most 150 ppm, more preferably at most 100 ppm, even more preferably at most 70 ppm, most preferably at more 50 ppm.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] The present inventors surprisingly found that the capacity of gelatin to swell, as well as the difference in solubility of typical contaminants compared to the gelatin allows ready removal of the contaminants by employing an extraction solvent. More particularly, by allowing the gelatin to swell, the contaminants that are entrapped in the gelatin become accessible by the extraction solvent, allowing these contaminants to be effectively and efficiently removed from the gelatin. In addition, most contaminants are remnants of the modification of the gelatin (see above), and more hydrophobic than gelatin. This leads to a different solubility in the extraction solvent, in particular in organic solvents, of the contaminants vis-a-vis the gelatin.

[0025] Accordingly, the present invention is directed to a method comprising the steps of: a) providing a gelatin gel comprising the modified gelatin and contaminants; b) contacting the gelatin gel with an extraction solvent having a temperature of at most 10 °C, allowing at least a part of the contaminants to dissolve in the extraction solvent; c) separating at least part of the extraction solvent with dissolved contaminants to obtain a purified gelatin gel comprising a purified modified gelatin; d) contacting the purified gelatin gel with water causing the purified gelatin gel to swell; e) optionally repeating steps b) to d) to further purify the purified gelatin gel, f) optionally contacting the purified gelatin gel obtained from either one of steps d) and e) with the extraction solvent at a temperature of less than 10 °C, and g) optionally drying the purified gelatin gel obtained from either one of steps d), e) and f) to obtain a purified gelatin powder.

[0026] The process of the invention may be performed in batch, semibatch or continuously. It may be performed on laboratory scale, but also on industrial scale.

[0027] Modified gelatin

[0028] In principle, the method of the present invention is applicable to any type of modified gelatin. Modified gelatin herein preferably means that the gelatin is functionalized by reacting its amine and / or carboxylic acid groups with compounds that provide an additional functionality to the gelatin.

[0029] Examples of modified gelatins that can be purified with the present process are for instance methacryloyl-gelatines and acryloyl- gelatins, as described in Billiet et al. Macromolecular Bioscience 13 (2013) 1531-45). Other modified gelatins are for instance tyramine-gelatins, as disclosed in Wang et al., Biomaterials 31 (2010) 1148-57, which also discloses the functionalization of gelatin through modification with 3-(4- hydroxyphenyl)-propionic acid. W02006 / 010066 discloses modification of gelatin using a carbodiimide-mediated coupling of tyramine to gelatin. Yet other modified gelatins include gelatin acrylamide, gelatin-PEG, thiolated gelatin, gelatin-furfuryl amine, gelatin-norbornene, gelatin-nitrocinnamate, as for instance those described in Van Hoorick et al, Acta Biomaterialia 2019, 97, 46-73. Further modified gelatins are known from Huang et al. Trends in Food Science & Technology 86 (2019) 260-269.

[0030] In particular embodiments, the gelatin is functionalized with an (meth)acrylate moiety, an acetyl moiety, a phenol moiety, a catechol moiety, a thiol moiety, a norbornene moiety, a tetrazine moiety, an azide moiety, a furan moiety, an allyl moiety, a maleimide moiety or any combination thereof. For instance, the modified gelatin may be functionalized with a 3- (4-hydroxyphenyl)-propionic acid, 3-mercaptopropionic acid, and 2-(5- norbornenyl)-acetic acid, preferably 3-(4-hydroxyphenyl)-propionic acid.

[0031] In preferred embodiments, the modified gelatin comprises gelatin- methacryloyl (GelMA), gelatin-desaminotyrosine (GelDAT), gelatin- desaminotyrosyl tyrosine (GelDATT), gelatin-tyramine (GelTyr), gelatin- caffeic acid (GelCA), gelatin- dihydrocaffeic acid (GelDHC), gelatin- gallic acid, gelatin-tannic acid, gelatin -p-coumaric acid, gelatin cinnamic acid, gelatin-sinapic acid, gelatin-rosmarinic acid, gelatin-dihydroxybenzoic acids (of which 3,4-dihydroxybenzoic acid is particularly preferred), gelatin-ferulic acid, gelatin -dopamine, gelatin-norbornene, gelatin- alkyne, gelatin- maleimide, gelatin-tetrazine, gelatin-azide, gelatin-divinyl sulfone, gelatin- thiol (GelSH) or a combination thereof, preferably GelDAT.

[0032] In preferred embodiments, the invention relates to type A gelatin. However, the modified gelatin may also be based on type B gelatin.

[0033] Moreover, in preferred embodiments, low-endotoxin modified gelatin is used, preferably having endotoxin levels of less than 200, more preferably less than 100 endotoxin units EU / g. Use of low-endotoxin gelatin with an endotoxin level of less than 10 EU / g is most preferred.

[0034] The modified gelatin used herein may have a number-average molecular weight within the range of 1500 Da to 300 kDa. The molecular weight distribution of gelatin is usually measured by size exclusion chromatography (SEC) techniques, and eluted fractions are detected by UV adsorption and the measured data are evaluated by suitable software (see e.g. Olijve et al. Journal of Colloid and Interface Science 243 (2000) 476- 482).

[0035] The extend of the modification of the gelatin may be expressed in terms of the “degree of functionalization” (DoF). The DoF of gelatin generally refers to the percentage of functionalized primary amine groups with respect to the total primary amine groups present in the gelatin before functionalization. The gelatin before functionalization is herein referred to as the corresponding unmodified gelatin. The expressions “functionalized” and “modified” are interchangeable.

[0036] In modified gelatin that is functionalized with carboxylic acid compounds, these carboxylic acid compounds have preferably been reacted with the free amine groups of the corresponding unmodified gelatin. Such modified gelatine may, for instance, have a degree of carboxylic acid condensation, and hence DoF, of between 1% and 100%, preferably between 5% and 70%, more preferably between 10% and 60%, such as between 15% and 50%, more preferably between 20% and 40%, based on the total number of free primary amine groups of the corresponding unmodified gelatin. It should be realized that with a high DoF the risk of residual reagents and remnants of the reagents increases. Greater amount of contaminants require longer duration times in conventional dialysis. Accordingly, there is an increased risk that the endotoxin levels go up. The current method is therefore of particular interest to modified gelatins that have a DoF of more than 20%.

[0037] Similarly, the modified gelatin may also be functionalized by reacting the carboxylic acid groups of the corresponding unmodified gelatin. These carboxylic acid groups may for instance be reacted with amine compounds such as tyramine, leading to GelTyr.

[0038] Accordingly, more generally, the modified gelatin has a DoF in the range of 15-50%, preferably 20-40% more preferably about 30%, based on the number of free carboxylic groups and / or free primary amino groups in a corresponding unmodified gelatin.

[0039] The modified gelatin may be modified in more than one way. For instance, the carboxylic acid-gelatin may be further modified with a combination with two or more groups selected from the group consisting of (meth)acryloyl group, an acetyl group, a phenol group, a thiol group, a norbornene group, a tetrazine group, an azide group, a catechol group, an alkyne group, a polyphenolic group, a furan group, an allyl group, a maleimide group or any combination thereof. Double chemically functionalized gelatin may be produced as described in Hoch et al. Journal of Material Chemistry B (2012) :5675). In a preferred embodiment, the modified gelatin is modified with at least and an acetyl group and a phenol group.

[0040] In step a), the gelatin gel comprising the modified gelatin and contaminants is provided. This provision may comprise the following steps: al) forming a solution, preferably an aqueous solution, of the modified gelatin; a2) optionally treating the solution with charcoal; a3) cooling the, optionally treated, solution to form the gelatin gel, preferably to a temperature in the range of 1 to 10 °C, more preferably 2 to 8 °C, and preferably at a cooling rate of 1 °C / min; a4) optionally forming particle of the gelatin gel.

[0041] Step al) comprises forming a solution of the modified gelatin. This can be done in an adequate amount of water, which may be in the form of an aqueous buffer solution. Other solvents and / or co-solvents may be used as well. The amount of water and / or (co)solvent depends on the gelatin, e.g., its nature, the molecular weight, DoF, and its hydrophilicity. Typically, the solution of the modified gelatin has a concentration in the range of 3 to 20 wt%, more preferably 4 to 15 wt%. This also limits the amount of water that is used to form the solution in step la). Step al) may be performed at ambient or elevated temperatures, such as at least 37 °C or preferably 40 °C or even more, provided that the modified gelatin is not adversely affected by the temperature. Conveniently, the solution can be filtered. Still more conveniently, the solution can be combined with carbon black and filtered. This may help to remove contaminants and / or impurities in the modified gelatin, e.g., remaining reagents and remnants thereof caused by modifying the gelatin.

[0042] For instance, in step al), modified gelatin may be dissolved in an aqueous buffer solution, e.g. a solution comprising 2-(N- morpholino)ethanesulfonic acid (MES buffer) with a pH of about 6, or solution comprising phosphate-buffered saline (PBS buffer) with a pH of about 7.4, or in dimethyl sulfoxide (DMSO). Preferably the MES buffer is used. The modified gelatin is preferably dissolved in a concentration in the range of 5 to 10 wt%. Higher concentrations are not recommended, to avoid issues with the optional filtration step. If step a2) is carried out by the addition of carbon black, then this may be done immediately after the modification reaction when the solution comprising dissolved gelatin (or similar biopolymer) is still warm. At concentrations below 5 wt% it may become difficult to obtain a physical gel in step a3).

[0043] By reducing the temperature in step a3), the modified gelatin will solidify and form a physical gel. The temperature at which a gel is formed depends on the modified gelatin and its concentration. The temperature at which the gel solidifies can be easily found out by the person skilled in the art. For instance, in order to solidify modified gelatin, it is preferably cooled to a temperature in the range of 1 to 10 °C, preferably 2 to 8 °C. Cooling may be performed by any ordinary method. Preferably the modified gelatin is cooled slowly, e.g. at 1 °C / min in order to achieve a complete sol to gel transition and / or to avoid that the modified gelatin is only partially gelled.

[0044] Once the gel has solidified, particles may be formed thereof by any suitable means (step a4). The smaller the size of the particles, the easier the removal of contaminants from the particles upon extraction in steps b)-e). It has been found that gel particles with an average (D50) size in the range of 0.1 to 10, preferably in the range of 1 to 5 mm can be conveniently used in the extraction step b)-e). This can be achieved with conventional equipment, including cutters, choppers, dicers, slicers, shredders, or a blender or mixer or the like. On industrial scale, a static mixer may be used.

[0045] Contaminants

[0046] The method of the present invention is particularly suitable to remove contaminants that are remnants of the modification reaction to prepare the modified gelatin. However, in principle, any contaminant can be removed, also those not originating from the modification reaction. For instance, endotoxins can also favorably be removed with the present invention. It may accordingly be appreciated that the contaminants may depend on the type of modified gelatin that is to be purified. For instance, GelDAT (i.e. gelatin functionalized with DAT) may contain free DAT, as well as reagents, moieties or derivatives thereof that were used to couple DAT to the gelatin (for instance NHS). As will be elaborated in more detail below, using the present disclosure, the skilled person can readily determine which contaminant is to be removed and can accordingly select the appropriate extraction solvent.

[0047] In typical embodiments, the contaminants are selected from the group consisting of 3-mercaptopropionic acid (3-MPA), 2-(5-norbornenyl)- acetic acid, methyl methacrylate (MMA), methacrylic acid (MAA), methacrylic anhydride (MA), poly(methyl methacrylate) (PMMA), glycidyl methacrylate, desaminotyrosine (DAT), desaminotyrosyl tyrosine (DATT), tyramine (Tyr), caffeic acid (CA), dihydrocaffeic acid (DHC), 4- hydroxycinnamic acid (p-coumaric acid), gallic acid, tannic acid, dopamine, ferulic acid, sinapic acid, rosmarinic acid, dihydroxybenzoic acids, norbornene, A-hydroxysuccinimide (NHS), carbodiimides such as N,N'- dicyclohexylcarbodiimide (DCC), A,A'-ethyl(dimethyl aminopropyl) and carbodiimide (EDC) and l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), ureas such a A,A'-dicyclohexylurea, A,A'-ethyl(dimethyl aminopropyl)urea and l-ethyl-3-(3-dimethylaminopropyl)urea, alkynes such as propiolic acid, 3-butynoic acid, 4-pentynoic acid, 5-hexynoic acid, 3- maleidopropionic acid, methyltetrazine-amine, azide-PEG4-NHS ester (full name: 3-(2-{2-[2-(2-azido-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid 2, 5- dioxo-pyrrolidin-l-yl ester, CAS nr. 944251-24-5), divinyl sulfone, 2- iminothiolane (SH), thiolactic acid, endotoxins, and combinations thereof, preferably wherein the contaminants are selected from the group consisting of DAT, Tyr, CA, 4-hydroxycinnamic acid, rosmarinic acid, and DHC, and combinations thereof, most preferably DAT.

[0048] This group is i.a. based on typically known functionalization reactants (also referred to as functionalization agents) and reagents. For instance, as non-limitative examples, the carboxylic acids methyl methacrylate (MMA), methacrylic acid (MA), poly(methyl methacrylate) (PMMA), desaminotyrosine (DAT), desaminotyrosyl tyrosine (DATT), tyramine (Tyr), caffeic acid (CA), dihydrocaffeic acid (DHC), 4- hydroxycinnamic acid, gallic acid, tannic acid, jo-coumaric acid, cinnamic acid, sinapic acid, rosmarinic acid, dihydroxybenzoic acids (such as 3,4- dihydroxybenzoic acid), ferulic acid, dopamine and norbornene are commonly used reactants used to functionalized gelatin with. For the functionalization reaction, carbodiimides can be used as reagents, which may also remain as remnants in the gelatin. These reagents can also undergo a reaction, for instance with water, to form for instance ureas such as A,A'-dicyclohexylurea, A,A'-ethyl(dimethyl aminopropyl)urea, l-ethyl-3- (3-dimethylaminopropyl)urea and the like. Similarly, NHS may be the result of using the so-called Bolton-Hunter reagent or other activated ester to functionalize the gelatin.

[0049] The relevance or importance of reducing the amount of contaminants to a certain level depends on the type of contaminants and the intended use of the purified modified gelatin. For instance, the biocompatibility of MA is worse than that of DAT. Hence, the tolerable level of residual MA in the purified modified gelatin is typically lower than for that of DAT.

[0050] Endotoxins as used herein mean LPS (see Wang, X., Quinn, P.J. (2010). Endotoxins: Lipopolysaccharides of Gram-Negative Bacteria. In: Wang, X., Quinn, P. (eds) Endotoxins: Structure, Function and Recognition. Subcellular Biochemistry, vol 53. Springer, Dordrecht). The levels of LPS are referred herein, are levels determined in accordance with the LAL-test (see K. L. Williams, “Endotoxins: pyrogens, LAL-testing and depyrogenation”, Informa Healthcare. New York 2007).

[0051] The relevance or importance of reducing the amount of endotoxins to a certain level depends on the type of endotoxins and the intended use of the purified gelatin. For instance, for applications of gelatin in pharmacy, biomedicine, food and the like, the level of endotoxins in the gelatin must be below certain limits. Extraction procedure and extraction solvents

[0052] In step b) of the present process, the gelatin gel, or the particles thereof, are contacted with an extraction solvent. This may also be a combination of solvents. Similarly, in each step that the extraction solvent is used (steps b), each of steps e) and step f)), the extraction solvent may be the same of different. The same extraction solvent may be the same for all steps b), e) and f). However, in embodiments wherein mixtures of contaminants are to be removed, it may be preferable to use a mixture of solvents and apply different ratios of the solvents for the various steps and cycles (vide infra).

[0053] The extraction solvent is selected based on the solubility of the contaminants to be removed from the modified gelatin. In other words, the solvent is selected for its ability to dissolve the functionalization agents and their residues used in modifying the gelatin. On the other hand, the extraction solvent should not dissolve the gel. Hence, in a preferred embodiment, the extraction solvent exhibits a higher dissolution capacity for the contaminants than for the modified gelatin. Preferably, the extraction solvent exhibits a dissolution capacity for the contaminants of at least 15 mg / ml at 10 °C. For this reason, an organic solvent is typically used for extraction. However, also preferably, the extraction solvent is preferably miscible with water to avoid different liquid phases when the gel is contacted with the extraction solvent. Miscible with water herein means that a 50 / 50 volume ratio of water and the extraction solvent at 20 °C gives a single liquid phase.

[0054] Contacting the gelatin gel with the extraction solvent preferably leads to a shrinkage of the gel, i.e. a reduction in volume. Without wishing to be bound by theory, the inventors believe that this advantageously leads to an active expulsion of the solvent with contaminants from the gelatin gel. When in the next step water is again added causing the gel to swell (e.g. in step d) or e)), the gel is behaving as a sponge that is repeatedly taking up and releasing solvent, allowing it to be rinsed.

[0055] The inventors found that in particular embodiments, it is preferred to use a mixture of water and the organic solvent that is miscible with water as the extraction solvent. Without wishing to be bound by theory, the inventors believe that the shrinkage of the organic solvents may actively repulse the contaminants of the gel and while this may be beneficial to repulse the contaminants from the gel, this principle may also lead to a reduced accessibility of the contaminants in the gel to the extraction solvent. Accordingly, to limit the shrinkage, in all or some of the extraction steps (or cycles), some water may be mixed with the organic solvent to form the extraction solvent. The optimal ratio of water to organic solvent can be found by determining the amount of contaminant that is extracted for different ratios, at a given contaminant concentration, temperature, and incubation period. Accordingly, in particular preferred embodiments, the extraction solvent used in all or some of the extraction cycles comprises water and organic solvent that is miscible with water, preferably in a volume ratio of the organic solvent to water of 10:1 to 10:1, more preferably 5:1 to 1:5, most preferably 3:1 to 1:3 such as about 1:1.

[0056] In some embodiments, it may be preferred that in at least one of the extraction cycles, steps b) and c) are carried out with essentially only water as the extraction solvent. This is particular preferred if one of the contaminants is particularly well soluble in water (e.g. NHS). In these embodiments however, it is preferred that the extraction solvent in at least the first extraction cycle comprises an organic solvent, more preferably is essentially free from water, to allow efficient removal of present water from the gelatin gel provided in step a) and thereby reduce the volume of the composition to be purified. In step f), it is preferred that the extraction solvent is essentially free from water, as this will facilitate the further processing such as optional drying step g).

[0057] The extraction solvent in steps b), e) and f) and the water in steps d) and e) are preferably used at a temperature of at most 10 °C, preferably at most 4 °C, to avoid redissolving the gel. In a most preferred embodiment, the temperature of the gelatin gel in steps c) to f) is maintained at a temperature of at most 10 °C.

[0058] It may thus be appreciated that depending on the chemical agent used for the modification a different extraction solvent may be used. For instance, the Bolton-Hunter reagent used in the preparation of GelDAT, i.e. 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, is sufficiently soluble in water, acetonitrile, chloroform, DMSO, alcohols and ethyl acetate. NHS is sufficiently soluble in water, dimethyl formamide, alcohols and ethyl acetate. DAT is sufficiently soluble in ethanol, ether, DMSO, and hot water. Accordingly, ethanol and water (up to 10 °C) are preferably used in the extraction process in the preparation of GelDAT. Similar selections may be used for other modified gelatins.

[0059] Suitable organic extraction solvents may be selected from the group consisting of dimethyl sulfoxide (DMSO), N,N- dim ethylformamide (DMF), Os-Os saturated and unsaturated hydrocarbons, chlorinated C1-C2 hydrocarbons such as dichloromethane and chloroform, C4-C8 ethers, C1-C4 esters of C2-C6 carboxylic acids, Ci-Ce aldehydes, C3-C6 ketones, Ci-Ce alcohols, and combinations thereof, preferably ethanol.

[0060] A non-limitative list of potentially suitable solvents is provided in the Table 1 hereafter, wherein in the first column the functionalization agents are disclosed for modifying a gelatin below the corresponding modified gelatin. In the subsequent rows, examples are provided of extractions solvents that do not redissolve the corresponding modified gelatin gel (first row), at least not at 10 °C or less. DMSO can be used as reaction solution when modifying the gelatin. As modified gelatin is soluble in DMSO at a temperature above 40 °C, it is preferably used as extraction solvent at 10 °C or less. Table 1

[0061] Whereas the above Table 1 focuses on the functionalization agent, it may be appreciated that the contaminants to be removed may be remnants thereof or other chemical agents used to facilitate the modification agent. In Wang et al., Biomaterials 31 (2010) 1148-57, functionalization of gelatin occurs through modification with a combination of 3-(4- hydroxyphenyl)-propionic acid (HP A), an excess of N-hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). It is therefore important that a solvent is selected that dissolves HP A, NHS and EDC, as well as derivatives thereof that may be form such as the corresponding (hydrolysis) urea products like l-ethyl-3-(3- dimethylaminopropyl)-urea.

[0062] The extraction procedure preferably starts with the extraction solvent (step b) and preferably ends with the extraction solvent (optional step 1), to remove water from within the purified gelatin gel, and causing the gel to shrink. After contact with the extraction solvent in step b), and removal of this in step c), incubation in water causes the gel to swell in step d). The gel preferably remains in contact with water for a duration long enough to cause an expansion of at least 10% by volume. The water used in step d) has a temperature of at most 10 °C, preferably at most 4 °C.

[0063] In step c), at least part of the extraction solvent, preferably the majority of the added extraction solvent (i.e. preferably more than 50 vol%, more preferably more than 80 vol%) with dissolved contaminants is separated from the gel to obtain a purified gel. This separation can be carried out by known techniques such as decantation, filtration, suction and the like.

[0064] The subsequent contact with extraction solvent causes the gel to collapse and loose water.

[0065] Preferably, this sequence of steps b) to d) (herein also referred to as extraction cycle, or simply cycle) is repeated once or twice or as often as needed to further reduce the level of impurities, as embodied by optional step e). This step e) may be repeated with different extraction solvents in each cycle. Preferably, the last step in purification procedure is performed with the extraction solvent that is essentially free from water (step f), followed by drying of the gel, e.g. in an oven (step g). This last step leads to a purified gelatin powder. Essentially free from water means that the organic solvent contains less than 5 wt% water, preferably less than 1 wt%, more preferably less than 0.5 wt%.

[0066] In embodiments wherein a mixture of contaminants is to be removed, it may be beneficial to use different solvents to extract each contaminant; depending on the solubility of the contaminants. For instance, one solvent may be preferred to extract one contaminant while another solvent may be preferred to extract another contaminant. Although a mixture of these solvents may be used to extract both contaminants and each cycle of steps b)-d) can be carried out with the same extraction solvent composition (i.e. the same combination of solvents, in the same ratio), it may also be feasible, and in fact in some embodiments preferred, to use a different composition of the extraction solvents for different extraction cycles of steps b)-d). Different composition herein refers to a different combination of solvents, as well as to a different ratio of the same combination of solvents. When different compositions of extraction solvents are used in the various solvents, it is preferred to gradually change from one composition to the other. For example, if both an organic solvent and water are used, it is preferred to start with only organic solvent as the extraction solvent and gradually (albeit stepwise) introduce water into the extraction solvent for the subsequent cycles, and finally gradually reduce the amount of water in the last cycles.

[0067] Purified modified gelatin

[0068] The method for purifying the modified gelatin leads to a purified modified gelatin. This purified modified gelatin is a further aspect of the present invention.

[0069] The purified modified gelatin may be used for a variety of applications. The purified modified gelatin may for instance be used for the preparation of hydrogels such as disclosed in EP3983018, and find use products for treating patents such as disclosed in EP3723641, NL2023208 and WO 2023 / 002017. A hydrogel obtainable from the purified modified gelatin is yet a further aspect of the present invention. The hydrogel may for instance be obtained by cross-linking the gelatin. The hydrogel may or may not incorporate living cells, drugs and / or growth factors, etc. Other applications include the manufacture or repair of tissue (e.g. cartilage, soft tissue) in a human or nonhuman animal, and the use as a bio-ink or bio-resin for the 3-dimensional bio -fabrication or 3- dimensional bioprinting of a biological construct. The biological construct may be any animal tissue or organ, or part thereof, that is able to be manufactured using a bio-fabrication or bioprinting technique, e.g. a scaffold containing cells which may be porous or non-porous.

[0070] It may be appreciated that the purified modified gelatin has essentially the same functionalization (both in functionalization type as well as DoF) as the modified gelatin that was provided in step b) of the process. Hence, the purified modified gelatin is typically functionalized with an (meth)acrylate moiety, an acetyl moiety, a phenol moiety, a catechol moiety, a thiol moiety, a norbornene moiety, a tetrazine moiety, an azide moiety, a furan moiety, an allyl moiety, a maleimide moiety or any combination thereof. For instance, the purified modified gelatin is functionalized with one or more of 3- (4-hydroxyphenyl)-propionic acid, 3-(SH)-propionic acid, and 2-(5-norbornenyl)-acetic acid, preferably 3-(4-hydroxyphenyl)-propionic acid.

[0071] In particular embodiments, the purified modified gelatin can be cross-linked to form the hydrogel. It may thus be appreciated that before cross-linking, the purified modified gelatin is not yet cross-linked and can be dissolved in a solvent such as a PBS buffer or water at 40 °C. Dissolution may for instance be useful for analysis (e.g. GPC analysis) or further treatment of the gelatin.

[0072] In further preferred embodiments, the purified modified gelatin comprises gelatin-methacryloyl (GelMA), gelatin-desaminotyrosine (GelDAT), gelatin-desaminotyrosyl tyrosine (GelDATT), gelatin-tyramine (GelTyr), gelatin-caffeic acid (GelCA), Gelatin-dihydrocaffeic acid (GelDHC), gelatin-gallic acid, gelatin- / ? -coumaric acid, gelatin cinnamic acid, gelatin- dopamine, gelatin-norbornene, gelatin-alkyne, gelatin-maleimide, gelatin- tetrazine, gelatin-azide, gelatin-divinyl sulfone, gelatin-thiol (GelSH) or a combination thereof, most preferably GelDAT.

[0073] Advantageously, the method of the present invention does not substantially influence the weight average molecular weight (Mw) of modified gelatin. In contrast, conventional methods known to purify modified gelatin using ultrafiltration do substantially influence the weight average molecular weight (Mw) of modified gelatin.

[0074] The purified modified gelatin can be characterized by a low amount of each of the contaminants and / or impurities. Preferably, the purified modified gelatin may have an amount of at most 200 ppm, preferably at most 150 ppm, more preferably at most 100 ppm, even more preferably at most 70 ppm, most preferably at most 50 ppm for each individual contaminant. Notably, the amounts of contaminants detailed herein refer to the amount of each individual contaminant that may be present in the modified gelatin and thus not to the total amount of contaminants that may be present.

[0075] In view of the above disclosure, it may be appreciated that the contaminants is one or more selected from the group consisting of 3- mercaptopropionic acid (3-MPA), 2-(5-norbornenyl)-acetic acid, 3-(4- hydroxyphenyl)-propionic acid, methyl methacrylate (MMA), methacrylic acid (MA), poly(methyl methacrylate) (PMMA), desaminotyrosine (DAT), desaminotyrosyl tyrosine (DATT), tyramine (Tyr), caffeic acid (CA), dihydrocaffeic acid (DHC), gallic acid, tannic acid, dopamine, norbornene, N- hydroxysuccinimide (NHS), carbodiimides such as N,N’- dicyclohexylcarbodiimide (DOC), A,A’-ethyl(dimethyl aminopropyl) and carbodiimide (EDO) and l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDO), ureas such a A,A’-dicyclohexylurea, A,A’-ethyl(dimethyl aminopropyl)urea and l-ethyl-3-(3-dimethylaminopropyl)urea, alkynes such as propiolic acid, 3-butynoic acid, 4-pentynoic acid, and 5-hexynoic acid, 3- maleidopropionic acid, methyltetrazine-amine, azide-PEG4-NHS ester, divinyl sulfone, 2-iminothiolane (SH), thiolactic acid and combinations thereof, preferably wherein the contaminants are selected from the group consisting of DAT, Tyr, 4-hydroxycinnamic acid, caffeic acid, dihydrocaffeic acid, 3-(4-hydroxyphenyl)-propionic acid, and combinations thereof, most preferably DAT.

[0076] In particularly preferred embodiments, the purified modified gelatin comprises less than 100 ppm, more preferably less than 70 ppm of each of DAT, Tyr, 4-hydroxycinnamic acid, caffeic acid, dihydrocaffeic acid, 3 -(4-hydroxyphenyl) -propionic acid, rosmarinic acid, and combinations thereof. The purified modified gelatin even more preferably comprises less than 70 ppm DAT, yet even more preferably less than 50 ppm DAT.

[0077] Unless explicitly disclosed otherwise, the amount of a particular contaminant is herein expressed as concentration of the contaminant in part per million (ppm), based on the total weight of the modified purified gelatin including the contaminant.

[0078] An appropriate method to determine the amount of contaminants is to directly determine this amount, for instance by completely dissolving the purified modified gelatin and measuring the weight of contaminants in solution, with ultraviolet (UV) assessment vide infra), relative to the total weight of the purified modified gelatin that was dissolved. Alternative methods known in the art that indirectly measure the amount of contaminants by filtration of the gelatin and subsequent measurement of the filtrate are less accurate (see for instance WO 2022 / 258763A1), because a substantial amount of contaminants may be entrapped in the gelatin and thus remain in the residue that is not measured. Thus, these indirect measurements do not give the amount of contaminants based on the total mass of the purified modified gelatin, unlike the present disclosure, but rather based on the mass of the filtrate. Hence results of indirect measurements known in the prior art cannot be compared to results of direct measurement according to the present disclosure.

[0079] The following exemplary procedure can be followed to directly determine the amount of contaminants in the purified modified gelatin according to the present invention. Purified modified gelatin is dissolved in PBS buffer (pH 6.6) and supplemented with sodium chloride (0.5M) at 40 °C. After complete dissolution of the purified modified gelatin, the solution is analyzed by Gel Permeation Chromatography (GPC). Because in GPC molecules are separated based on their size, residual chemicals deriving from the functionalization process can be separated from the functionalized gelatin, and from each other, and their individual concentration can be determined using standard detection techniques such as those based on refractive index (RI) detection, ultraviolet (UV) detection, light scattering detection and the like. Masses for the contaminants are calculated by detector calibration of their signal and set in relation to the mass of the purified modified gelatin sample.

[0080] For example, for purified modified gelatin functionalized with DAT, DAT and NHS may remain in the purified modified gelatin. The concentration of both compounds can be determined by detector calibration of the UV signal at 280 nm.

[0081] Advantageously, the present method gives purified modified gelatin having lower amounts of contaminants and / or endotoxins compared to methods known in the art such as dialysis. Interestingly, the present method does not substantially increase the endotoxin levels. Moreover, the method of the present invention may even reduce the endotoxin level, depending on the selection of the extraction solvent and number of cycles. This is therefore different from dialysis, wherein the endotoxin content of the modified gelatin may increase. Modified gelatins with exceptionally low endotoxin content may therefore be prepared, e.g. when starting with a low endotoxin gelatin. Some of the low endotoxin, ultra-pure modified gelatins are in fact new. The endotoxin level can be determined by the LAL test, which detects soluble LPS from Gram-negative bacteria (see K. L. Williams, “Endotoxins: pyrogens, LAL-testing and depyrogenation”, Informa Healthcare. New York 2007).

[0082] EXAMPLES

[0083] The invention can be illustrated by the following non-limiting examples.

[0084] Example 1 - preparation of GelDAT

[0085] In a typical experiment, low endotoxin gelatin type A (5 wt.%) (25 g) was added to MES buffer (490 mL; pH 5.2-5.8) in a 2.0 L round-bottom flask while stirring at 45 °C, until gelatin was completely dissolved. Bolton- Hunter reagent (1.3377 g, 2:1 ratio) was dissolved in 10 mL DMSO and added dropwise to the gelatin solution. The reaction was allowed to proceed for 2h at 45 °C. Accordingly a modified gelatin, GelDAT was prepared with a degree of functionalization of about 30%.

[0086] The gelatin was mixed with charcoal and filtered while the gelatin is still warm. After filtration, the reaction mixture is transferred to a beaker and placed overnight at 4 °C to induce physical gelation.

[0087] Example 2 - purification of GelDAT

[0088] The gelatin gel (about 25 g in about 490 g of MES buffer) as prepared in Example 1 was crushed into granules in the form of soft “jelly” particles having a size of 5 mm or less using a blender.

[0089] The granules were then purified in 6 cycles comprising incubating in an extraction solvent at 4 °C while mixing for an incubation time; whereupon the solvent was decanted and replaced with water at 4 °C. After thorough mixing, the water was removed by decantation and the next cycle was commenced. Details of the cycles including the extraction solvent and the incubation time are provided in Table 2.

[0090] Table 2

[0091] The ethanol in the extraction solvent facilitates the extraction of DAT while the water in the extraction solvent facilitates the extraction of NHS. Further, in contact with water, the gelatin gel swelled and then shrunk when exposed to the organic solvent. This improved the removal of more contaminants.

[0092] After cycle nr. 6, the extraction solvent was decanted and no further water was added. The gelatin granules were dried in an oven.

[0093] GPC analysis revealed a total amount of free DAT in the gelatin powder of 70 ppm.

[0094] Example 3 - influence of purification method on weight average molecular weight

[0095] A modified gelatin (GelDAT, weight average molecular weight 153000) was purified in a method according to Example 2.

[0096] In a comparative experiment, the same modified gelatin (GelDAT, weight average molecular weight 153000) was purified using ultrafiltration. The modified gelatin, immediately after synthesis, was purified using an ultrafiltration system in which impurities were separated from the modified gelatin through a membrane with molecular weight cut off (MWCO) of 10 kDa.

[0097] The resulting purified gelDAT samples were analyses to determine the average molecular weight by GPC-MALS (Multi angle Light Scattering). The results are provided in Table 3.

[0098] The results show that the weight average molecular weight of the GelDAT remains nearly unaffected when using the method of the present invention, whereas it is reduced when using ultrafiltration.

[0099] Table 3.

[0100] Comparative Example 1 - dialysis

[0101] The following experiment was carried out in triplicate. Modified gelatin powder (GelDAT) was purified in a procedure as described in US8287906. HPCL analysis revealed a total amount of free DAT in the gelatin powder of 103±6 ppm.

Claims

Claims1. A method for purifying a modified gelatin, comprising the steps of: a) providing a gelatin gel comprising the modified gelatin and contaminants; b) contacting the gelatin gel with an extraction solvent having a temperature of at most 10 °C, allowing at least a part of the contaminants to dissolve in the extraction solvent; c) separating at least part of the extraction solvent with dissolved contaminants from the gelatin gel to obtain a purified gelatin gel comprising a purified modified gelatin; d) contacting the purified gelatin gel with water causing the purified gelatin gel to swell; e) optionally repeating steps b) to d) to further purify the purified gelatin gel, f) optionally contacting the purified gelatin gel obtained from either one of steps d) and e) with the extraction solvent at a temperature of less than 10 °C, and g) optionally drying the purified gelatin gel obtained from either one of steps d), e) and f) to obtain a purified gelatin powder.

2. The method of claim 1, wherein the contaminants are selected from the group consisting of 3-mercaptopropionic acid (3-MPA), 2-(5- norbornenyl)-acetic acid, methyl methacrylate (MMA), methacrylic acid (MAA), methacrylic anhydride (MA), poly(methyl methacrylate) (PMMA), glycidyl methacrylate, desaminotyrosine (DAT), desaminotyrosyl tyrosine (DATT), tyramine (Tyr), caffeic acid (CA), 4-hydroxycinnamic acid (p- coumaric acid), cinnamic acid, dihydrocaffeic acid (DHC), gallic acid, tannic acid, dopamine, ferulic acid, sinapic acid, rosmarinic acid, dihydroxybenzoicacids, norbornene, iV-hydroxysuccinimide (NHS), carbodiimides such as 2V,2V’-dicyclohexylcarbodiimide (DCC), 2V,2V’-ethyl(dimethyl aminopropyl) and carbodiimide (EDC) and l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC), ureas such a 2V,2V’-dicyclohexylurea, N,N’- ethyl(dimethyl aminopropyl)urea and l-ethyl-3-(3- dimethylaminopropyl)urea, propiolic acid, alkynes such as 3-butynoic acid, 4-pentynoic acid, 5-hexynoic acid, 3-maleimidopropionic acid, methyltetrazine-amine, azide-PEG4-NHS ester, divinyl sulfone, 2- iminothiolane (SH), thiolactic acid, endotoxins, and combinations thereof, preferably wherein the contaminants are selected from the group consisting of DAT, Tyr, CA, 4-hydroxycinnamic acid, and DHC, and rosmarinic acid, and combinations thereof, most preferably DAT.

3. The method according to any of the previous claims, wherein the modified gelatin is functionalized with an (meth)acrylate moiety, an acetyl moiety, a phenol moiety, a catechol moiety, a thiol moiety, a norbornene moiety, a tetrazine moiety, an azide moiety, a furan moiety, an allyl moiety, a maleimide moiety, or any combination thereof.

4. The method according to any of the previous claims, wherein the modified gelatin is functionalized with 4-hydroxycinnamic acid, caffeic acid, dihydrocaffeic acid, 3-(4-hydroxyphenyl)-propionic acid, rosmarinic acid, dihydroxybenzoic acids (DHBA), or combinations thereof, preferably a combination of caffeic acid and dihydrocaffeic acid or a combination of 3-(4- hydroxyphenyl)-propionic acid and dihydrocaffeic acid or a combination of 3- (4-hydroxyphenyl)-propionic acid and caffeic acid.

5. The method according to any of the previous claims, wherein the modified gelatin comprises gelatin-methacryloyl (GelMA), gelatin- desaminotyrosine (GelDAT), gelatin-desaminotyrosyl tyrosine (GelDATT), gelatin-tyramine (GelTyr), gelatin-caffeic acid (GelCA), Gelatin-dihydrocaffeic acid (GelDHC), gelatin- gallic acid, gelatin-tannic acid, gelatin-p-coumaric acid, gelatin -cinnamic acid, gelatin-sinapic acid, gelatin- rosmarinic acid, gelatin-dihydroxybenzoic acids, gelatin-ferulic acid, gelatin- dopamine, gelatin-norbornene, gelatin-alkyne, gelatin-maleimide, gelatin- tetrazine, gelatin-azide, gelatin-divinyl sulfone, gelatin-thiol (GelSH) or a combination thereof, preferably GelDAT.

6. The method according to any of the previous claims, wherein the extraction solvent comprises an organic solvent, preferably an organic solvent that is miscible with water.

7. The method according to any of the previous claims, wherein the extraction solvent exhibits a higher dissolution capacity for the contaminants than for the modified gelatin.

8. The method according to any of the previous claims, wherein the extraction solvent exhibits a dissolution capacity for the contaminants of at least 15 mg / ml at 10 °C.

9. The method according to any of the previous claims, wherein the extraction solvent comprises a solvent selected from the group consisting of dimethyl sulfoxide, 2V,2V-dimethylformamide, chlorinated C1-C2 hydrocarbons such as dichloromethane and chloroform, C5-C8 saturated and unsaturated hydrocarbons, C4-C8 ethers, C1-C4 esters of C2-C6 carboxylic acids, Ci-Ce aldehydes, C3-C6 ketones, Ci-Ce alcohols, and combinations thereof, preferably ethanol.

10. The method according to any of the previous claims, wherein the extraction solvent in steps b) and c) comprises a combination of water and an organic solvent that is miscible in water.

11. The method according to any of the previous claims, wherein the water used in step d) has a temperature of at most 10 °C, preferably at most 4 °C.

12. The method according to any of the previous claims, wherein the temperature of the gelatin gel in steps c) to f) is maintained at a temperature of at most 10 °C, preferably at most 4 °C.

13. The method according to any of the previous claims, wherein step e) is carried out at least one time, wherein each of sequential steps b) to d) are extraction cycles, and wherein the extraction solvent is different for at least two different extraction cycles.

14. The method according to the previous claim, comprising at least three extraction cycles, wherein the first extraction cycle is carried out with an organic solvent that is miscible with water as the extraction solvent, the middle extraction cycle is carried out with a combination of water and said organic solvent, and the last extraction cycle is carried out with said organic solvent.

15. The method according to any of the previous claims, wherein the gelatin gel is in the form of gel particles.

16. The method according to any of the previous claims, wherein the modified gelatin has a degree of functionalization (DoF) in the range of 15- 50%, preferably 20-40% more preferably about 30%, based on the number of free carboxylic groups and / or primary amino groups in a corresponding unmodified gelatin.

17. The method according to any of the previous claims, wherein the modified gelatin has a number-average molecular weight within the rangeof 1500 Da to 300 kDa, preferably between 2000 Da and 300 kDa, more preferably between 90 kDa and 150 kDa.

18. The method according to any of the previous claims, wherein the purified modified gelatin used has an endotoxin level of less than 200 endotoxin units (EU) / g, preferably less than 100 EU / g, more preferable less than 10 EU / g, as determined by the LAL-test.

19. The method according to any of the previous claims, wherein step a) of providing the gelatin gel comprising the modified gelatin, comprises the following steps: al) forming a solution, preferably an aqueous solution, of the modified gelatin; a2) optionally treating the solution with charcoal; a3) cooling the, optionally treated, solution to form the gelatin gel, preferably to a temperature in the range of 1 to 10 °C, more preferably 2 to 8 °C, and preferably at a cooling rate of 1 °C / min; a4) optionally forming particle of the gelatin gel.

20. Purified modified gelatin comprising one or more contaminants that each individually are present in an amount of at most 200 ppm, preferably at most 150 ppm, more preferably at most 100 ppm, even more preferably at most 70 ppm, most preferably at most 50 ppm, based on the total mass of the purified modified gelatin.

21. Purified modified gelatin according to claim 20, wherein the one or more contaminants is one or more selected from the group consisting of 3- mercaptopropionic acid (3-MPA), 2-(5-norbornenyl)-acetic acid, methyl methacrylate (MMA), methacrylic acid (MAA), methacrylic anhydride (MA), poly(methyl methacrylate) (PMMA), glycidyl methacrylate, desaminotyrosine (DAT), desaminotyrosyl tyrosine (DATT), tyramine (Tyr),caffeic acid (CA), 4-hydroxycinnamic acid (p-coumaric acid), cinnamic acid, dihydrocaffeic acid (DHC), gallic acid, tannic acid, dopamine, ferulic acid, sinapic acid, rosmarinic acid, dihydroxybenzoic acids, norbornene, N- hydroxysuccinimide (NHS), carbodiimides such as N,N’- dicyclohexylcarbodiimide (DCC), A,A’-ethyl(dimethyl aminopropyl) and carbodiimide (EDC) and l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC), ureas such a A,A’-dicyclohexylurea, A,A’-ethyl(dimethyl aminopropyl)urea and l-ethyl-3-(3-dimethylaminopropyl)urea, propiolic acid, alkynes such as 3-butynoic acid, 4-pentynoic acid, 5-hexynoic acid, 3- maleimidopropionic acid, methyltetrazine-amine, azide-PEG4-NHS ester, divinyl sulfone, 2 -iminothiolane (SH), thiolactic acid, endotoxins, and combinations thereof, preferably wherein the contaminants are selected from the group consisting of DAT, Tyr, CA, 4-hydroxycinnamic acid, DHC, and rosmarinic acid, and combinations thereof, most preferably DAT.

22. Purified modified gelatin according to any of claims 20-21, wherein the one or more contaminants comprises DAT and the purified modified gelatin has an amount of DAT of less than 70 ppm, preferably less than 50 ppm, based on the total mass of the purified modified gelatin.

23. Hydrogel obtainable from a purified modified gelatin as prepared by the method according to any of claims 1 to 19 or the modified gelatin according to any of claims 20-22.

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