WO2025122009A1

WO2025122009A1 - A method for purifying a gelatin

Info

Publication number: WO2025122009A1
Application number: PCT/NL2024/050646
Authority: WO
Inventors: Susanna PILUSO; Bas Jeroen Oosterman
Original assignee: Sentryx BV
Current assignee: Sentryx BV
Priority date: 2023-12-04
Filing date: 2024-12-04
Publication date: 2025-06-12
Anticipated expiration: 2026-06-04

Abstract

The invention is directed to a method for reducing the endotoxin content of a gelatin, comprising the steps of providing a gelatin gel comprising the gelatin and endotoxins; 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 endotoxins to dissolve in the extraction solvent; followed by separating at least part of the extraction solvent with dissolved endotoxins from the gelatin gel to obtain a purified gelatin gel comprising a purified 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.

Description

Title: A method for purifying a gelatin

TECHNICAL FIELD

The present invention is in the field of gelatin. In particular, the present invention relates to a method for reducing the endotoxin content of gelatin, thereby producing a low or ultralow endotoxin gelatin.

BACKGROUND

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 describes 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).

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

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.

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. For applications of gelatin in pharmacy, biomedicine, food and the like, the level of endotoxins in the gelatin must be below certain limits. Endotoxins, also referred to as lipopolysaccharides (LPS), are components of the outer membrane of the cell wall of gram-negative bacteria. Even in very low amounts, endotoxins negatively impact the health of humans and animals by its abilities to potently activate and modulate the immune system, and i.a. induce inflammation responses. Severe cases such as sepsis characterized by a whole-body inflammatory state may be caused by endotoxins.

Accordingly, to be accepted onto the market, gelatins must comply to regulatory standards and endotoxins levels must be sufficiently low. Preferably, gelatins have endotoxin levels of less than 200 endotoxin units (EU)/g, more preferably less than 100 EU/g. Gelatins with an endotoxin content of less than 10 EU/g are most preferred. For instance, the US Food and Drug Administration (FDA) prescribes endotoxin limits for medical devices of 0.5 EU/mL or 20 EU/device for products that directly or indirectly contact the cardiovascular system and lymphatic system. For devices in contact with cerebrospinal fluid, the limit is 0.06 EU/mL or 2.15 EU/device. For devices that are in direct or indirect contact with the intraocular environment, a lower endotoxins limit may apply. As recommended by the FDA, endotoxin levels 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).

During the production and processing of gelatin, the gelatin may become microbially contaminated and/or degraded, leading to an increase of endotoxin level, which is highly undesirable. In such case, it is required to purify the gelatin and reduce the endotoxin levels.

Conventional procedures to reduce the endotoxin level of gelatins are based on ultrafiltration, adsorption techniques (e.g. activated carbon or other adsorber materials), anion-exchange chromatography, affinity chromatography (Petsch et al, Journal of Biotechnology 2000, 76, 97-119.) and size exclusion chromatography (Lee et al, Process Biochemistry 2003, 38, 1091-1098), or rely on the use of surfactants, for instance micelleforming surfactant such as Triton X-100 and X-102. Examples thereof are described in WO 2016/085345. The drawback of using micelles is the requirement of surfactants such as Triton X-100 and X-102, which are considered environmentally unfriendly. Further, the method is elaborate.

Ultrafiltration can be used to remove endotoxins from product solutions if the products are of low molecular weight. Usually, membranes with molecular weight cut-off (MWCO) of 10 kDa are used: the product permeates through the membrane and the endotoxins are retained. If product and endotoxins have similar molecular weights, this method is not efficient in decreasing the endotoxin levels.

Non-selective adsorption (e.g. activated carbon or other adsorber materials) is also used but not very effective in reducing high levels of endotoxins.

Anion-exchange chromatography can be used since endotoxins are negatively charged. However, protein co-adsorption might be a problem if solutions with acidic proteins are to be decontaminated. This process works better with basic protein that have a net positive charge, as described in Petsch et al., Journal of Biotechnology 2000, 76, 97-119.

Size exclusion chromatography is efficient only when product and contaminant have large difference in sizes (see Lee et al., Process Biochemistry 2003, 38, 1091-1098).

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for reducing the endotoxin content of a gelatin, which method overcomes one or more of the above-mentioned drawbacks of known methods to purify gelatin. The present invention accordingly provides a method for reducing the endotoxin content of a gelatin, comprising the steps of: a) providing a gelatin gel comprising the gelatin and endotoxins; b) contacting the gelatin gel with an extraction solvent having a temperature of at most 10 °C, allowing at least a part of the endotoxins to dissolve in the extraction solvent; c) separating at least part of the extraction solvent with dissolved endotoxins to obtain a purified gelatin gel comprising a purified 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.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors surprisingly found that the capacity of gelatin to swell, as well as the difference in solubility of endotoxins compared to the gelatin allows ready removal of the endotoxins by employing an extraction solvent. More particularly, by allowing the gelatin to swell, the endotoxins that are entrapped in the gelatin become accessible by the extraction solvent, allowing these endotoxins to be effectively and efficiently removed from the gelatin. In addition, most endotoxins have a different solubility in the extraction solvent, in particular in organic solvents, than the gelatin.

Accordingly, the present invention is directed to a method comprising the steps of: a) providing a gelatin gel comprising the gelatin and endotoxins; b) contacting the gelatin gel with an extraction solvent having a temperature of at most 10 °C, allowing at least a part of the endotoxins to dissolve in the extraction solvent; c) separating at least part of the extraction solvent with dissolved endotoxins to obtain a purified gelatin gel comprising a purified 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.

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.

Gelatin

The gelatin for purification in accordance with the present invention comprises a certain level of endotoxins. It may be appreciated that the purified gelatin (either in gel or in powder form) comprises a lower level of endotoxins compared to the gelatin before purification. In principle the present invention is not limited with respect to the starting level of endotoxins in the gelatin to be purified. The process enables removal of both high and low amounts of endotoxins. In case the starting gelatin comprises high levels of endotoxins, it may be preferred to carry out step e) one, two, three or more times. The gelatin to be purified (herein also referred to as the starting gelatin) may have an endotoxin level of more than 200 EU/g. The starting gelatin may however also have a lower endotoxin level, for instance of less than 200 EU/g, or even less than 100 EU/g. Use of low-endotoxin starting gelatin with an endotoxin level of less than 10 EU/g is also possible.

In principle, the method of the present invention is applicable to any type of gelatin. In preferred embodiments, the invention relates to type A gelatin. However, the gelatin may also be based on type B gelatin.

Further, the gelatin may be modified or unmodified (also referred to as functionalized or unfunctionalized). Modified gelatin herein means that the gelatin is chemically functionalized by covalently reacting the amine and/or carboxylic acid groups with functional compounds. Unmodified (or unfunctionalized) gelatin herein means that the amine and/or carboxylic acid groups are not covalently reacted with functional compounds.

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.

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. In more specific 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.

The gelatin used herein may have a number-average molecular weight within the range of 1500 Da to 300 kDa, or more. 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). Advantageously, the method of the present invention does not significantly influence the weight average molecular weight (Mw) of the gelatin. In contrast, conventional methods known to purify gelatin using ultrafiltration do influence the weight average molecular weight (Mw) of gelatin.

In step a), the gelatin gel comprising the gelatin and endotoxins is provided. This provision may comprise the following steps: al) forming a solution, preferably an aqueous solution, of the 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.

Step al) comprises forming a solution of the 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, molecular weight, possible functionalization, and its hydrophilicity. In a preferred embodiment, solution of the gelatin comprises a chelating agent, for example ethylenediaminotetraacetic acid (EDTA), sodium citrate or another chelating agent for divalent cations. Advantageously, chelating agents may destabilize endotoxin aggregates and convert these to the oligomeric or preferably monomeric form with molecular weights of about 10 kDa. Endotoxins removal is easier in the monomeric form.

Typically, the solution of the gelatin has a concentration in the range of 3 to 20 wt%, more preferably 4 to 15 wt% of gelatin. 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 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 endotoxins and/or impurities in the gelatin, e.g., remaining reagents and remnants thereof caused by modifying the gelatin.

For instance, in step al), gelatin may be dissolved in an aqueous buffer solution, e.g. a solution comprising 2-(A-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 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).

By reducing the temperature in step a3), the gelatin will solidify and form a physical gel. The temperature at which a gel is formed depends on the 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 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 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 gelatin is only partially gelled.

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 endotoxins 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 mm, 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.

Endotoxins

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. Wilhams, “Endotoxins: pyrogens, LAL-testing and depyrogenation”, Informa Healthcare. New York 2007). In accordance with the present invention, LAL-testing can be carried out via a gel clot, turbidimetric or chromogenic. Chromogenic is preferred.

The method of the present invention is particularly suitable to remove endotoxins. Advantageously, the method may remove other contaminants and/or impurities as well.

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.

Extraction procedure and extraction solvents

In step b) of the present process, the gelatin gel, or the particles thereof, are contacted with an extraction solvent. This may 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 used for all steps b), e) and f)- However, in some embodiments 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).

The extraction solvent is selected based on the solubility of the endotoxins to be removed from 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 endotoxins than for the gelatin. Preferably, the extraction solvent exhibits a dissolution capacity for the endotoxins 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.

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 endotoxins 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.

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 endotoxins of the gel and while this may be beneficial to repulse the endotoxins from the gel, this principle may also lead to a reduced accessibility of the endotoxins 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 endotoxins that is extracted for different ratios, at a given endotoxin 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 1:10, more preferably 5:1 to 1:5, most preferably 3:1 to 1:3 such as about 1:1. Ethanol and water (up to 10 °C) are preferably used in the extraction process.

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. 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).

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.

Suitable organic extraction solvents may be selected from the group consisting of dimethyl sulfoxide (DMSO), N,N- dim ethylformamide (DMF), Cs-Cs 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, the organic extraction solvent is selected from the group consisting of ethanol, methanol, DMSO, DMF, ethyl acetate, chloroform, acetone, diethyl ether and combinations thereof. Most preferably, the extraction solvent comprises ethanol.

It may be appreciated that the extraction solvent should have an extremely low amount of endotoxins, for instance less than 1 EU/ml, before contacting the gelatin with the solvent in steps b), e) and f).

The extraction procedure preferably starts with the extraction solvent (step b) and preferably ends with the extraction solvent (optional step f), 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.

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 endotoxins 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.

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

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 endotoxins, 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%.

Although a mixture of solvents may be used 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.

Purified gelatin and products thereof

The method for purifying the gelatin leads to a purified gelatin without introducing surfactants.

Advantageously, the purified gelatin is essentially free from surfactants such as Triton X-100 and X-102 that are used for the micellebased purification methods as for example described in WO 2016/085345. Accordingly, a particular aspect of the present invention is the purified gelatin product obtainable from the method as described herein, wherein said purified gelatin product has an endotoxin level of less than 200 endotoxin units (EU)/g, preferably less than 100 EU/g, more preferably of less than 10 endotoxin units (EU)/g, as determined by the LAL-test, and are essentially free of surfactants. Essentially free herein means that the gelatin comprises less than 10 ppb, preferably less than 1 ppb surfactants, based on the weight of the purified gelatin.

This purified gelatin may be used for a variety of applications. The purified 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 gelatin is yet a further aspect of the present invention. The hydrogel may for instance be obtained by cross-linking the gelatin.

Preferably, the purified gelatin has a weight-average molecular weight of 1500 Da or more, preferably of 100 kDa or more, such as 110 kDa or more, or 150 kDa or more. Most preferably within the range of 100 kDa to 300 kDa. A weight-average molecular weight of more than 100 kDa can advantageously be achieved by the method according to the present invention. Conventional purification methods relying on chromatography can decrease the weight average molecular weight of gelatin due to for example hydrolysis. The present invention, however, does not suffer from this drawback.

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.

The gelatin may also be used in films or bio-adhesive products, as described for instance in WO 2023/002017.

EXAMPLES

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

Example 1

A gelatin gel (about 25 g in about 490 g of MES buffer) having an endotoxin level of 138 EU/g was crushed into granules in the form of soft “jelly” particles having a size of 5 mm or less using a blender.

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 1. Table 1

After cycle nr. 6, the extraction solvent was decanted, and no further water was added. The gelatin granules were dried in an oven. LAL-testing showed an endotoxin level of < 50 EU/g.

Example 2

A gelatin gel having an endotoxin level of 317 EU/g was crushed into granules in the form of soft “jelly” particles having a size of 5 mm or less using a blender.

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.

Table 2

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

LAL-testing showed an endotoxin level of 105 EU/g.

Example 3 - influence of purification method on weight average molecular weight

A gelatin (weight average molecular weight 153000) was purified in a method according to Example 1.

In a comparative experiment, the same gelatin (weight average molecular weight 153000) was purified using ultrafiltration, using two Hydrosart ultrafiltration membranes (MWCO 10 kDa). The reaction mixture was purified for two days.

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

The results show that the weight average molecular weight of the gelatin remains nearly unaffected when using the method of the present invention, whereas it is reduced when using ultrafiltration. To this end, the gelatin was purified using an ultrafiltration system in which endotoxins were separated from the gelatin through a membrane with molecular weight cut off (MWCO) of 10 kDa. Table 3.

Claims

1. A method for purifying a gelatin, comprising the steps of: a) providing a gelatin gel comprising the gelatin and endotoxins; b) contacting the gelatin gel with an extraction solvent having a temperature of at most 10 °C, allowing at least a part of the endotoxins to dissolve in the extraction solvent; c) separating at least part of the extraction solvent with dissolved endotoxins from the gelatin gel to obtain a purified gelatin gel comprising a purified 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 according to the previous claim, wherein the extraction solvent comprises an organic solvent.

3. 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.

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

5. The method according to any of the previous claims, wherein the extraction solvent is 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.

6. The method according to any of the previous claims, wherein the extraction solvent is selected from the group consisting of ethanol, methanol, DMSO, DMF, ethyl acetate, chloroform, acetone, diethyl ether and combinations thereof.

7. The method according to any of the previous claims, wherein the extraction solvent comprises a combination of water and ethanol.

8. 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.

9. 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.

10. 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.

11. 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.

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

13. The method according to any of the previous claims, wherein the gelatin has a weight- average molecular weight within the range of 1500 Da to 300 kDa, preferably between 2000 Da and 300 kDa, most preferably between 100 kDa and 300 kDa.

14. The method according to any of the previous claims, wherein the purified 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.

15. The method according to any of the previous claims, wherein step a) of providing the gelatin gel comprising the gelatin, comprises the following steps: al) forming a solution, preferably an aqueous solution, of the 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.

16. The method according to the previous claim, wherein step al) comprises forming a solution of the gelatin and a chelating agent, wherein the chelating agent is preferably selected from the group consisting of ethylenediaminotetraacetic acid (EDTA), sodium citrate, and other chelating agents for divalent cations, and combinations thereof.

17. A purified gelatin product obtainable from the method according to any of the previous claims, wherein said purified gelatin product has an endotoxin level of less than 10 endotoxin units (EU)/g, as determined by the LAL-test, and is essentially free of surfactants.

18. The purified gelatin product according to claim 17, having a weight- average molecular weight of 100 kDa or more, more preferably 110 kDa or 150 kDa or more, most preferably within the range of 100 kDa to 300 kDa.

19. Product, such as a hydrogel, film or bio-adhesive, which is obtainable from a purified gelatin as prepared by the method according to any of claims 1-16.