Crosslinkable functionalized gelatin with low exothermic activity

Abstract

The present invention relates to a substituted crosslinkable carboxylic acid moiety R-(CH2) n The present invention relates to a gelatin functionalized with -COOH, where n is an integer between 1 and 10, and further having a lipopolysaccharide content of less than 100 EU / g, as well as a method for preparing said gelatin using N-hydroxysuccinimide esters of said substituted crosslinkable carboxylic acids.

Description

[Technical field]

[0001] This application relates generally to chemically functionalized gelatin, particularly gelatin functionalized with cross-linkable, substituted carboxylic acid. More particularly, the present invention relates to cross-linkable, substituted carboxylic acid functionalized gelatin having low pyrogenic activity, particularly low lipopolysaccharide content, hydrogels comprising cross-linkable, substituted carboxylic acid functionalized gelatin, methods for preparing cross-linkable, substituted carboxylic acid functionalized gelatin, and uses thereof. [Background technology]

[0002] Reconstruction of functional biological tissues, biological implants, and cell-based multi-organ models for clinical, diagnostic, or pharmaceutical research has attracted increasing attention. Hydrogels have emerged as prime candidates for various tissue engineering applications due to their similarity to the natural extracellular matrix.

[0003] Gelatin hydrogels are particularly attractive due to their biocompatibility and biodegradability. Gelatin is produced by partial hydrolysis of collagen, the most abundant protein in the body and the most common molecule of the extracellular matrix. The abundance of the cell recognition sequence arginine-glycine-aspartic acid (RGD) facilitates cell attachment and promotes their spreading and proliferation. This cell-matrix interaction is important for the construction of complex tissues. Gelatin has a long history as a reliable excipient in the pharmaceutical industry, meeting the highest standards of safety and regulatory compliance. Furthermore, the presence of free amino, hydroxyl, and carboxylate groups allows it to be chemically modified to obtain desired properties for specific applications.

[0004] Gelatin hydrogels are made by crosslinking gelatin polymers without prior modification of their side groups or after functionalization of their side groups. Addition of functional groups to the gelatin backbone is a crosslinking strategy with a high degree of control over the design and properties of the hydrogel. The most widely used and studied modification for crosslinking gelatin is methacryloylation. MA-modified gelatin is commonly referred to as gel-MA. A well-established alternative to methacryloyl gelatin is acryloyl gelatin (Billiet et al. 2013 "Quantitative Contrasts in the Photopolymerization of Acrylamide and Methacrylamide-Functionalized Gelatin Hydrogel Building Blocks" Macromolecular Bioscience 13:1531-45).

[0005] Other functionalized gelatins include, for example, tyramine-functionalized gelatin (Wang et al., 2010, Biomaterials. 31(6):1148-57). Wang et al. disclose the functionalization of gelatin by modification with a combination of 3-(4-hydroxyphenyl)-propionic acid (HPA), excess N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). WO 2006 / 010066 discloses the modification of gelatin using carbodiimide-mediated coupling of tyramine to gelatin. Other relevant publications include WO 2020 / 050779, which discloses hydrogels with tunable properties based on tyramine or hydroxyphenylpropionic acid-based crosslinkers. The compound 3-(4-hydroxyphenyl)propanoic acid (HOC6H4(CH2)2CO2H) is sometimes abbreviated as HPA and is also known as phloretic acid or desaminotyrosine (DAT). These designations may be used interchangeably.

[0006] Elvin et al. 2010, Biomaterials. 31(6):8323-8331 describes the creation of photopolymerizable gelatin-based materials based on increasing the content of the binding moiety, para-hydroxylphenyl (tyrosine-like), of gelatin by coupling free amines with 3-(4-hydroxyphenyl)propanoic acid. The DAT content was increased by reacting gelatin with Bolton-Hunter reagent (N-succinimidyl-3-[4-hydroxyphenyl]propionate). This is an example of carboxylic acid functionalized gelatin.

[0007] Gelatin-based hydrogels can be tailored in terms of their physical properties (crosslink density, swelling, and stiffness) depending on the degree of functionalization and polymer concentration, making this material a versatile platform for a variety of tissue engineering applications. Crosslinking can be initiated in a variety of ways. One of these ways is through radicals generated by UV or visible light depending on the photoinitiator used. Alternatively, it can be through thiol-ene photoclick chemistry, thiol-Michael addition, inverse electron demand Diels-Alder reaction, Diels-Alder click reaction, disulfide crosslinking, Schiff / base, π-π cycloaddition, photooxidation, and / or enzymatic crosslinking.

[0008] One of the main drawbacks of gelatin-based hydrogels for (bio)medical applications is the presence of endotoxins (also referred to herein as lipopolysaccharides) in conventionally produced gelatins. Endotoxins are large molecules that are highly immunogenic and are the main component of the outer membrane of gram-negative bacteria. Endotoxins are highly heat-resistant and therefore difficult to inactivate. When exposed to the immune system, endotoxins initiate an immune response that can lead to tissue inflammation, increased sensitivity to other allergens, and the risk of fatal shock. Most research is currently being carried out on cross-linked functionalized gelatins based on gelatins with high endotoxin levels.

[0009] Conventionally produced gelatin may also be contaminated by microbial components other than endotoxins, some of which, like endotoxins, may trigger adverse immune responses in humans. Nonendotoxin pyrogens include, for example, lipoteichoic acid (LTA) derived from gram-positive bacteria, as well as other compounds derived from fungi, yeasts, viruses, bacteria and parasites (Hasiwa et al. (2013) "Evidence for the detection of nonendotoxin pyrogens by the whole blood monocyte activation test. ALTEX 30:169-208). These nonendotoxin pyrogens, and preferably also common pyrogens or pathogen-associated molecular patterns (PAMPs), should be minimized in (bio)medical applications of gelatin-based hydrogels to prevent unwanted side effects upon activation of innate immune receptors.

[0010] Also, several reagents are used during the functionalization and crosslinking of gelatin to gelatin hydrogels. Residuals of reagents and reaction products may be present in the functionalized gelatin. For example, when gelatin is reacted with N-succinimidyl-3-[4-hydroxyphenyl]propionate as described by Elvin et al. (see above), hydrolysis products such as 3-[4-hydroxy]propionic acid (HPA, phloretic acid) and unreacted reagents such as N-succinimidyl-3-[4-hydroxyphenyl]propionate may be present in the functionalized gelatin composition. These residues are considered undesirable. Another problem to be solved with chemically modified gelatin is that the chemical process may result in degradation or hydrolysis of gelatin (reduction of Mw) or undesirable crosslinking (increase of molecular weight). Therefore, it is desirable for chemical modification to result in gelatin with approximately the same Mw before and after functionalization.

[0011] Conventional procedures rely on purifying the reaction mixture by dialysis against distilled water. Typically, dialysis takes place over a week. This time-consuming process is therefore not efficient for large-scale production of functionalized gelatin. Furthermore, during this dialysis period, the risk of microbial contamination and gelatin degradation increases. (Bio)medical applications of gelatin-based hydrogels require biocompatible (e.g., non-toxic and non-immunogenic to living tissues) functionalized gelatin that can tailor hydrogels with desired mechanical properties (e.g., strength and elasticity). There is also a demand to produce such gelatin by an efficient process that allows for industrial-scale production. Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention solves one or more of the above problems of the prior art. In particular, carboxylic acid-modified gelatin (modified gelatin comprising a primary amine functionalized with a substituted carboxylic acid) is provided that has low pyrogen content, especially low endotoxin content, and low contamination of reagents, by-products, and hydrolysis products. The carboxylic acid-functionalized gelatin has low pyrogen content, especially low endotoxin content, and therefore improved biocompatibility. The carboxylic acid-functionalized gelatin of the present invention is particularly useful for crosslinking, since it is less contaminated by reactants known to interfere with the crosslinking process. Also advantageously, the carboxylic acid-functionalized gelatin can be prepared by a simple manufacturing process that does not require lengthy dialysis steps. [Means for solving the problem]

[0013] The invention is particularly expressed by any one or any combination of one or more of the following numbered aspects and embodiments (i) to (xv): (i) A carboxylic acid moiety R-(CH2) via an amide bond n1. A gelatin functionalized with -COOH, where n is an integer from 1 to 10, and further having a lipopolysaccharide content of less than 100 EU / g, preferably less than 50 EU / g, more preferably less than 20 EU / g, even more preferably less than 10 EU / g, even more preferably less than 5 EU / g, even more preferably less than 2 EU / g, and most preferably less than 1 EU / g. (ii) Gelatin according to (i) containing reactants and reagents, preferably less than 100 ppm of free substituted carboxylic acid. (iii) Gelatin according to (i) or (ii), which has a low content of pyrogens derived from gram-positive bacteria, pyrogens derived from flagellated bacteria, single-stranded viral RNA, and bacterial DNA rich in unmethylated CpG motifs, preferably has a low content of pyrogens derived from gram-positive bacteria and flagellated bacteria, and more preferably has a low content of pyrogens derived from gram-positive bacteria. (iv) The gelatin according to any one of (i) to (iii), wherein the gelatin is type A gelatin. (v) Gelatin according to any one of (i) to (iv), having a carboxylic acid substitution degree of 5% to 100%, preferably 25% to 100%, more preferably 50% to 100%, and even more preferably 80% to 100%. (vi) The gelatin according to any one of (i) to (v), which is further modified with a (meth)acryloyl group or moiety, an acetyl group or moiety, a phenol group or moiety, a thiol group or moiety, a norbornene group or moiety, a tetrazine group or moiety, an azide group or moiety, a furan group or moiety, an allyl group or moiety, a maleimide group or moiety, or any combination thereof. (vii) A hydrogel comprising gelatin modified with a carboxylic acid according to any one of (i) to (vi) above, and a crosslinking agent. (viii) A film comprising the hydrogel according to (vii) or the gelatin according to any one of (i) to (vi). (ix) A method for providing a functionalized gelatin according to any one of (i) to (vi), comprising the steps of: a. In the reaction medium, the carboxylic acid moiety R-(CH2) n N-hydroxysuccinimide esters of —COOH (wherein n is an integer from 1 to 10) (preferably R—(CH n modifying gelatin by reacting the gelatin with a reagent containing b. lowering the pH of the reaction medium to a value between 2.0 and 4.0, preferably between 2.5 and 3.5, more preferably between 3.0 and 3.5; c. adding 0.01-1.5 w / w% of a micelle-forming surfactant to the acidic reaction medium; d. contacting the micelle-containing medium with an adsorbent, preferably a solid adsorbent, preferably a solid adsorbent, preferably activated carbon; e. separating the adsorbent from the medium; f. recovering the medium containing the functionalized gelatin; A method comprising: (x) The method according to (ix), wherein the micelle-forming detergent comprises a non-ionic detergent, preferably the detergent is Triton X-100 or Triton X-102, or a mixture thereof. (xi) The method according to (ix) or (x), wherein the solid adsorbent is a hydrophobic adsorbent, preferably activated carbon. (xii) The method according to any one of (ix) to (xi), further comprising the step of drying the medium containing the carboxylic acid modified gelatin. (xiii) A gelatin according to any one of (i) to (vi), or a hydrogel according to (vii), for use in medicine. (xiv) The in vitro or ex vivo use of a gelatin according to any one of (i) to (vi) or a hydrogel according to (vii) for producing a biological construct such as a tissue or organ, or a part thereof, a coating, a scaffold, or a controlled release dosage form. (xv) Use of a gelatin according to any one of (i) to (vi), or a hydrogel according to (vii), as a bio-ink or bio-resin. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a graphical representation of the purification of 3-(4-hydroxyphenyl)-propionic acid modified gelatin using dialysis, which is reduced by 98% at T=5 after dialysis. [Diagram 2] FIG. 2 is a graphical representation of the purification of 3-(4-hydroxyphenyl)-propionic acid modified gelatin using micelle formation, absorption, and filtration. [Diagram 3] Figure 3 shows the HPLC signal detected for the AC filtered sample. No PA was detected. [Figure 4A] FIG. 4 shows the MW distribution of the starting gelatin (1), and the MW distribution resulting from modification via a route using an in situ mixture of NHS, HPA and EDC followed by purification using one-step dialysis (2) and two-step dialysis (3). [Figure 4B] Shown is the MW distribution of the same starting gelatin (1) and the MW distribution resulting from modification via a route using NHS-HPA in the essential absence of EDC followed by a two-step purification using micelle formation-based purification. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, have the meanings commonly understood by those skilled in the art to which the present invention belongs. By way of further guidance, definitions of terms are included to better understand the teachings of the present invention. As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0016] As used herein, the terms "comprising, comprises" and "comprised of" are synonymous with "including, includes" and "containing, contains" and are inclusive or open-ended and do not exclude additional unrecited components, elements, or method steps. When referring to an embodiment including an element or step, the reference also encompasses an embodiment consisting essentially of the recited element or step. Numerical ranges recited by endpoints include all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. The term "about" as used herein when referring to a measurable value, such as a parameter, amount, duration, or the like, is meant to encompass a variation of the particular value of no more than + / -10%, preferably no more than + / -5%, more preferably no more than + / -1%, and even more preferably no more than + / -0.1%, provided such variation is appropriate for the practice of the disclosed invention. It is to be understood that the value referred to by the modifier "about" is itself specific and preferred and disclosed. All documents cited herein are incorporated by reference in their entirety.

[0017] This application relates generally to functionalized gelatin, for example for crosslinking gelatin for the purpose of preparing gelatin-based hydrogels and films. More particularly, this application relates to carboxylic acid functionalized gelatin.

[0018] Gelatin is a mixture of water-soluble proteins derived from collagen. Gelatin is obtained, for example, by partial or enzymatic hydrolysis of collagen obtained by aqueous extraction under acid or alkaline conditions from, for example, bovine, porcine, poultry or fish skin, tendons, ligaments, bones, etc., as known in the art. Gelatin obtained by acid treatment is called "type A gelatin" and "type B gelatin" is derived from an alkali-based process. The isoelectric points (IEP) of type A and type B gelatin are at pH 7.0-9.0 and pH 4.9-5.1, respectively, due to the higher deamination of asparagine and glutamine in type B gelatin, which allows them to be positively and negatively charged at neutral physiological pH. In a preferred embodiment, the present invention relates to type A gelatin.

[0019] Gelatin does not constitute a uniform protein molecule, but contains a variable amount of protein molecules of variable length. Preferably, the gelatin used herein has an average molecular weight in the range of 1500 Da to 300 kDa, preferably 2000 Da to 300 kDa, 4000 Da to 300 kDa, 5000 Da to 300 kDa, 10 kDa to 300 kDa, or 20 kDa to 300 kDa, more preferably 50 kDa to 300 kDa, most preferably 100 kDa to 300 kDa, for example 100 kDa to 275 kDa or 100 kDa to 250 kDa. The molecular weight distribution of gelatin is usually measured by size-exclusion high performance liquid chromatography (HPLC) technique, the eluted fraction is detected by UV adsorption, and the measurement data is evaluated by appropriate software. All techniques are known in the art, see Olijve et. al. (2000. Journal of Colloid and Interface Science 243:476-482).

[0020] As used herein, the term "gelatin" also encompasses "gelatin derivatives", including chemically modified gelatin. As used herein, the expression "gelatin modified with chemical groups or moieties (e.g., carboxylic acid groups or moieties, tyramine groups or moieties, methacryloyl groups or moieties, acryloyl groups or moieties, acetyl groups or moieties, phenolic groups or moieties, etc.)" refers to gelatin that contains said chemical groups or moieties (e.g., carboxylic acid groups or moieties, tyramine groups or moieties, methacryloyl groups or moieties, acryloyl groups or moieties, acetyl groups or moieties, phenolic groups or moieties, etc.) bonded to at least one amine group, at least one hydroxyl group, at least one carboxyl group, and / or at least one phenolic group of gelatin.

[0021] As used herein, "gelatin modified with carboxylic acid groups" or "gelatin modified with crosslinkable substituted carboxylic acid groups", also referred to herein as "carboxylic acid modified gelatin", "carboxylic acid substituted gelatin" or "carboxylic acid-gelatin" or "gelatin carboxylic acid", is defined as gelatin having at least one free amine substituted with a carboxylic acid group, preferably via an amide bond. Gelatin contains amino acids, some of which have side chains containing terminal amines (e.g., lysine, arginine, asparagine, glutamine) or have side chains containing hydroxyls (e.g., serine, threonine, aspartic acid, glutamic acid, tyrosine, hydroxyproline). In addition, gelatin also contains N-terminal amines. All of these terminal amines can be substituted with carboxylic acid groups to produce functionalized gelatin containing carboxylic acid groups. It is preferred to substitute the N-terminal amine with a carboxylic acid to form an amide bond.

[0022] The carboxylic acids used for the functionalization of the gelatin of the present invention have the formula R-(CH2) n It is a carboxylic acid having -COOH (wherein n is an integer of 1 to 10).

[0023] In an embodiment, in the carboxylic acid used in the functionalization of gelatin, R is selected from the group consisting of 4-phenol, norbornenyl, or SH. In an embodiment, n is 1 to 5, preferably 2 or 3. In a preferred embodiment, in the carboxylic acid used in the functionalization of gelatin, the carboxylic acid moiety is 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.

[0024] The "degree of functionalization (DoF)" of gelatin generally refers to the ratio of functionalized primary amine groups to the total primary amine groups. As used herein, the "degree of carboxylic acid substitution" refers to the ratio of free amine groups in gelatin that are substituted with carboxylic acid groups. The degree of functionalization, such as the degree of carboxylic acid substitution, of gelatin can be quantified by methods known per se.

[0025] For example, the Fe(III)-acetohydroxamic acid method can be used to quantify the substitution at hydroxyl groups. The Habeeb method (trinitrobenzenesulfonic acid (TNBS)-based spectrophotometric determination of (meth)acrylamides, Habeeb 1996. Anal. Biochem. 14:328-336), 1H-NMR and fluoroaldehyde assay (also known as o-Phthaldialdehyde (OPA)-based fluorometric determination of free amines) can be used to quantify the carboxylic acid substitution at amine groups. A combination of the aforementioned methods can also be used, such as a combination of the fluoroaldehyde assay to quantify amine group conversion and the Fe(III)-acetohydroxamic acid method to quantify hydroxyl groups. In an embodiment, the fluoroaldehyde assay is used to quantify the degree of carboxylic acid substitution by quantifying free amines.

[0026] In the carboxylic acid functionalized gelatins disclosed herein, the carboxylic acid functionalization occurs preferably at free amine groups. In an embodiment, the carboxylic acid functionalized gelatin has a degree of carboxylic acid substitution of 20% to 100%, preferably 50% to 100%, more preferably 80% to 100%, such as 85% to 100%, 90% to 100 or 95% to 100%. In an embodiment, the carboxylic acid functionalized gelatin has a degree of carboxylic acid substitution of less than 20%, preferably less than 10%, 9%, 8%, 7% or 6%, more preferably less than 5%, 4%, 3%, 2% or 1%.

[0027] The carboxylic acid functionalized gelatin disclosed herein may be further modified and / or functionalized. In embodiments, the carboxylic acid-gelatin is further modified with (meth)acryloyl groups or moieties, acetyl groups or moieties, phenol groups or moieties, thiol groups or moieties, norbornene groups or moieties, tetrazine groups or moieties, azide groups or moieties, furan groups or moieties, allyl groups or moieties, maleimide groups or moieties, or any combination thereof, preferably acetyl groups. Dual chemically functionalized gelatin may be produced as described in Hoch et al. (2012. Chemical tailoring of gelatin to adjust its chemical and physical properties for functional bioprinting. J. Mater. Chem. B 1:5675).

[0028] The carboxylic acid-gelatins disclosed herein have low pyrogenic activity. Pyrogenic activity can be measured using the Monocyte Activation Test (MAT) assay known in the art. The MAT assay allows for the quantification of both endotoxin and non-endotoxin pyrogen levels, but does not distinguish between types of PAMP contamination. To detect and quantify various PAMPs, as well as various non-endotoxins, a cell-based pyrogen detection assay (PAMP assay) was developed by the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB. This cell-based test system uses human Toll-like receptors (TLRs) to detect PAMPs (Burger-Kentischer et al. (2010) A new cell-based innate immune receptor assay for the examination of receptor activity, ligand specificity, signaling pathways and the detection of pyrogens. J Immunol Methods, 358:93-103). More specifically, cells, such as NIH3T3 cells, express different TLR combinations that allow the detection of different PAMPs. For example, cell lines with receptor combinations TLR1 / 2, TLR2 / 6, TLR4 / CD14, TLR5, TLR7, and TLR9 allow the detection of pyrogens from gram-positive bacteria (TLR1 / 2, TLR2 / 6), pyrogens from flagellated bacteria (TLR5), single-stranded viral RNA (TLR7), cell lines TLR4 / CD14 allow the detection of endotoxins from gram-negative bacteria, and TLR9 cell lines detect bacterial DNA rich in unmethylated CpG motifs. In embodiments, the HPA-gelatins disclosed herein have a low content of pyrogens from gram-positive bacteria, pyrogens from flagellated bacteria, single-stranded viral RNA, endotoxins from gram-negative bacteria, and bacterial DNA rich in unmethylated CpG motifs.

[0029] In a particular embodiment, the carboxylate-gelatin has a low content of pyrogens from gram-positive bacteria and flagellated bacteria, in particular a low content of pyrogens from gram-positive bacteria. In a particular embodiment, the carboxylate-gelatin disclosed herein is characterized by a low content of endotoxins or lipopolysaccharides (LPS), in particular an LPS content of less than 100 EU / g, more preferably less than 50 EU / g, even more preferably less than 20 EU / g, even more preferably less than 10 EU / g, even more preferably less than 5 EU / g, even more preferably less than 2 EU / g5, and most preferably less than 1 EU / g. In a further particular embodiment, the carboxylate-gelatin disclosed herein is (derived from) type A gelatin and is characterized by an LPS content of less than 100 EU / g, more preferably less than 50 EU / g, even more preferably less than 20 EU / g, even more preferably less than 10 EU / g, even more preferably less than 5 EU / g, even more preferably less than 2 EU / g, and most preferably less than 1 EU / g. In further particular embodiments, the carboxylate-gelatin disclosed herein is (derived from) type B gelatin and is characterized by an LPS content of less than 20 EU / g, preferably less than 10 EU / g, more preferably less than 5 EU / g, even more preferably less than 2 EU / g, and most preferably less than 1 EU / g. The term EU is known in the art and stands for "endotoxin unit". 1 EU is approximately equal to 100 pg of E. coli lipopolysaccharide, the amount present in approximately 104-105 bacteria. As used herein, the term EU / g stands for the number of EU per dry weight of carboxylate-functionalized gelatin. The Limulus assay (LAL) is a bioassay known in the art for measuring LPS down to sub-picogram amounts. Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amebocytes) from the American horseshoe crab, Limulus polyphemus. LAL reacts with bacterial endotoxins or lipopolysaccharides. This reaction is the basis of the LAL test and is used to detect and quantify bacterial endotoxins.For example, a suitable LAL method to quantify LPS levels is the chromogenic Endosafe method from Charles River, USA. Another accepted and recommended method is the Endozyme Recombinant Factor C method from Hyglos GmbH (Germany). Both methods give similar or identical measurements, so both methods can be used interchangeably.

[0030] Also advantageously, the carboxylic acid-gelatin disclosed herein is substantially free of reagents and reaction products. In an embodiment, the carboxylic acid-gelatin disclosed herein contains low levels of free carboxylic acid, i.e., carboxylic acid not bound to gelatin, and contains less than 150 ppm carboxylic acid, or less than 100 ppm carboxylic acid, or less than 50 ppm carboxylic acid, preferably less than 30 ppm carboxylic acid, more preferably less than 25 ppm carboxylic acid, and even more preferably less than 20 ppm carboxylic acid. In an embodiment, the carboxylic acid-gelatin disclosed herein contains less than 150 ppm carboxylic acid, such as less than 140 ppm carboxylic acid, less than 130 ppm carboxylic acid, less than 120 ppm carboxylic acid, or less than 110 ppm carboxylic acid, preferably less than 100 ppm carboxylic acid, such as less than 90 ppm carboxylic acid, less than 80 ppm carboxylic acid, less than 70 ppm or less than 60 ppm carboxylic acid, where preferably the carboxylic acid content is determined in a sample of the carboxylic acid-gelatin dissolved in 50 mM phosphate buffer (pH 9.5). As detailed in the Examples, carboxylic acids can be measured by dissolving samples of carboxylic acid-gelatin in water or 50 mM phosphate buffer (pH 9.5), ultrafiltration of the dissolved sample (e.g., using a 10 kDa Amicon Ultra Centrifugal Filter), and HPLC analysis of the filtrate. Residual carboxylic acids in carboxylic acid-functionalized gelatin can prevent crosslinking of carboxylic acid-functionalized gelatin molecules. Thus, the carboxylic acid-functionalized gelatin disclosed herein is particularly suitable for crosslinking to form hydrogels or films due to its low carboxylic acid content. Therefore, a further aspect relates to crosslinked carboxylic acid-gelatin or hydrogels comprising the crosslinked carboxylic acid-gelatin disclosed herein. Further disclosed herein are products derived from said hydrogels, such as films, bioadhesives, etc. The term "hydrogel" as used herein refers to a network of hydrophilic polymer chains, such as crosslinked carboxylic acid-gelatin, that form a gel. The term "gel" refers to a substantially dilute crosslinked system that does not exhibit flow when in a steady state.

[0031] The carboxylic acid-gelatin crosslinking process is well known in the art. Usually, upon exposure to light in the presence of a photoinitiator, the carboxylic acid group of one gelatin molecule can react with the carboxylic acid group of another gelatin molecule to crosslink the carboxylic acid-gelatin.

[0032] The term "photoinitiator" as used herein refers to any chemical compound, or mixture of compounds, that decomposes into free radicals when exposed to light, e.g., ultraviolet (UV) or visible light (VIS). Non-limiting examples of ultraviolet photoinitiators include 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one (also known by the trade name Irgacure® 2959), and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Visible light photoinitiators generate free radicals when exposed to visible light. Exemplary ranges of visible light useful for exciting visible light photoinitiators include green, blue, indigo, and violet. Preferably, the visible light has a wavelength within the range of 450-550 nm. Non-limiting examples of visible light photoinitiators include eosin Y, riboflavin / triethanolamine, vinylcaprolactam, dl-2,3-diketo-1,7,7-trimethylnorcamphane (CQ), 1-phenyl-1,2-propanedione (PPD), 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819), 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 4,4'-bis(dimethyl ... ) benzophenone, 2-chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl 1 (2,4,6 trimethylbenzoyl) phosphine oxide / 2-hydroxy-2-methylpropiophenone (50 / 50 blend), dibenzosuberenone, (benzene)tricarbonylchromium, resazurin, resorufin, 5 benzoyltrimethylgermane (Ivocerin®), derivatives thereof, and any combination thereof.

[0033] The light irradiation time may be any appropriate time that allows crosslinking of the polymer. For example, the irradiation time may be in the range of 10 seconds to 20 minutes, preferably 1 minute to 20 minutes, and more preferably 2 to 15 minutes (e.g., 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes).

[0034] The mechanical properties of gelatin-based hydrogels can be tailored for different applications by varying, for example, the molecular weight of the gelatin used, the degree of HPA substitution, the carboxylic acid-gelatin concentration, the amount of photoinitiator, and the light exposure time.

[0035] As used herein, the concentration of carboxylate-substituted gelatin is defined as the weight of carboxylate-substituted gelatin divided by the volume of solvent (w / v) and expressed as a percentage. The solvent may be a pharma- ceutically acceptable carrier. In hydrogel embodiments, the carboxylate-substituted gelatin is present at a concentration of 5%-25% (w / v), 17%-25% (w / v), 17%-23% (w / v), or about 20% (w / v). In some embodiments, the carboxylate-substituted gelatin is present at a concentration of 5%-15% (w / v), 8%-12% (w / v), or about 10% (w / v). In some embodiments, the carboxylic acid substituted gelatin is present in a concentration of 10%-40% (w / v), 15%-35% (w / v), 20%-30% (w / v), or about 5%, 10%, 15%, 20%, or 25% (w / v).

[0036] The chemically modified gelatins, particularly carboxylic acid-gelatins, and hydrogels according to the present invention may be used in a variety of applications, including but not limited to the manufacture or repair of human or non-human animal tissues (e.g., cartilage, soft tissues) and as bioinks or bioresins for the 3D biofabrication or 3D bioprinting of biological constructs. The biological constructs may be any animal tissue or organ or part thereof that can be manufactured using biofabrication or bioprinting techniques, including, for example, cell-containing scaffolds, which may be porous or non-porous.

[0037] The term "bio-ink" refers to a cytocompatible hydrogel that can be 3D printed, 3D plotted, or fabricated into a particular shape or construct. The hydrogel may or may not incorporate living cells and / or growth factors, etc. The term "bio-resin" refers to a cytocompatible hydrogel that can be 3D printed or fabricated into a particular shape or construct using laser or light projection based photo stereolithography, or similar lithography techniques. The hydrogel may or may not incorporate living cells, drugs, and / or growth factors, etc.

[0038] A related aspect of Carboxylic Acid A relates to the in vitro or ex vivo use of the carboxylate-gelatin disclosed herein or a hydrogel comprising the carboxylate-gelatin disclosed herein to manufacture a tissue or organ or a portion thereof. In an embodiment, the tissue or organ is selected from bone tissue, cartilage, and vascular tissue. Also disclosed herein are tissue engineered tissues or organs or portions thereof comprising the carboxylate-gelatin disclosed herein or a hydrogel comprising the carboxylate-gelatin disclosed herein.

[0039] Another aspect relates to the in vitro or ex vivo use of the carboxylate-gelatin disclosed herein or of a hydrogel comprising the carboxylate-gelatin disclosed herein for the manufacture of sustained release dosage forms or biological constructs, which may for example be a coating for a (solid) support suitable for cell attachment and proliferation, or a scaffold suitable for containing cells or drugs and / or mediators.

[0040] Yet a further aspect relates to the use of the carboxylate-gelatin disclosed herein, or a hydrogel comprising the carboxylate-gelatin disclosed herein, as a bio-ink or bio-resin.

[0041] In further embodiments, the bio-ink or bio-resin is used for 3D biofabrication or 3D bioprinting of biological constructs. The biological construct may be an animal tissue or organ, or a part thereof. The biological construct may also be a scaffold suitable for containing cells, or a scaffold suitable for containing, for example, drugs and / or mediators (e.g., drug delivery / gene therapy applications). The biological construct may also be a bioabsorbable screw, or other biomaterial (e.g., bioadhesive). In an embodiment, the biological construct is a scaffold suitable for containing cells. In an embodiment, the biological construct is a coating. Also disclosed herein are biological constructs comprising carboxylate-gelatin as disclosed herein, or hydrogels comprising carboxylate-gelatin as disclosed herein. Yet a further aspect relates to a method for preparing carboxylate-gelatin as disclosed herein. The inventors have surprisingly found that purification of the aqueous reaction medium of carboxylic acid-gelatin according to a modified version of the method described in WO 2016 / 085345 not only removes lipopolysaccharides from the medium, but also removes other pyrogens, as well as free carboxylic acids and other reactants formed during or remaining after the carboxylic acid functionalization of the gelatin reaction. Thus, dialysis of the reaction medium is not necessary, resulting in a faster process to provide carboxylic acid-gelatin with low LPS content, low pyrogen content and low carboxylic acid content, as specified elsewhere herein.

[0042] Therefore, in a further aspect, the present invention relates to a method for preparing carboxylic acid-gelatin as disclosed herein, said method comprising the steps of: a. In the reaction medium, the carboxylic acid moiety R-(CH2) n N-hydroxysuccinimide ester of —COOH (wherein n is an integer from 1 to 10) (R—(CH n modifying gelatin by reacting the gelatin with a reagent containing b. lowering the pH of the reaction medium to a value between 2.0 and 4.0, preferably between 2.5 and 3.5, more preferably between 3.0 and 3.5; c. adding 0.01 to 1.5 w / w% of a micelle-forming surfactant to the acidic reaction medium; d. contacting the micelle-containing medium with an adsorbent, preferably activated carbon; e. separating the adsorbent from the medium; f. recovering the medium containing the functionalized gelatin; g. Optionally, drying the medium containing the functionalized gelatin of step f). The present invention relates to a method comprising the steps of:

[0043] An alternative method of preparing carboxylic acid functionalized gelatin-gelatin disclosed herein comprises the following steps: a) In a reaction medium, a carboxylic acid moiety R-(CH2) n N-hydroxysuccinimide ester of —COOH (wherein n is an integer from 1 to 10) (R—(CH n modifying gelatin by reacting the gelatin with a reagent containing b1) lowering the pH of the reaction medium to a value between 4.0 and 9.0, preferably between 4.0 and 6.0, more preferably between 4.0 and 6.0, and even more preferably between 4.5 and 5.5; c) adding 0.01 to 1.5 w / w% of a micelle-forming surfactant to the reaction medium; b2) lowering the pH of the reaction medium to a value between 2.0 and 4.0, preferably between 2.5 and 3.5, more preferably between 3.0 and 3.5; d) contacting the medium of step b2) with a solid adsorbent; e) separating the solid sorbent of step d) from said medium; f) recovering said medium containing the functionalized gelatin; g) optionally drying the medium containing the carboxylic acid functionalized gelatin-gelatin of step f); Includes.

[0044] Any type of gelatin can be used in the methods described herein, for example type A or type B gelatin, such as gelatin of bovine, porcine, poultry or fish origin. In an embodiment, type A gelatin is used. In another embodiment, type B gelatin is used.

[0045] Carboxylic acid functionalization of gelatin with N-hydroxy-succinimidyl-activated carboxylic acids can be carried out by reacting the respective N-hydroxy-succinimidyl-activated carboxylic acids with gelatin in a suitable buffer, e.g., carbonate buffer or phosphate-buffered saline (PBS) at pH 9.0, at a temperature of 50° C. for 60-180 min, e.g., 60 min or 120-180 min. The degree of carboxylic acid functionalization of gelatin can be adjusted by varying the ratio of N-hydroxy-succinimidyl-activated carboxylic acid reagent to gelatin, as described in Shirahama et al. (2016). During the reaction, free carboxylic acids are formed along with the carboxylic acid-gelatin.

[0046] In an embodiment, the N-hydroxy-succinimidyl activated carboxylic acid used for the functionalization of the gelatin of the present invention is of the formula R-(CH2) preferably essentially in the absence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). n N-hydroxy-succinimidyl-(NHS) activated carboxylic acid having the formula -COO-NHS, where n is an integer from 1 to 10.

[0047] In an embodiment, in the N-hydroxy-succinimidyl-(NHS)-activated carboxylic acid used in the functionalization of gelatin, R is selected from the group consisting of 4-phenol, 5-norbornenyl, or SH. In an embodiment, n is 1-10, preferably 1-5, preferably 2 or 3. In a preferred embodiment, in the N-hydroxy-succinimidyl-(NHS)-activated carboxylic acid used in the functionalization of gelatin, the carboxylic acid moiety is 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. In a preferred embodiment, the N-hydroxy-succinimidyl-(NHS) activated carboxylic acid used for the functionalization of gelatin is selected from the group consisting of N-hydroxy-succinimidyl-3-(4-hydroxyphenyl)-propionate, N-hydroxy-succinimidyl-3-(SH)-propionate and N-hydroxy-succinimidyl-2-(5-norbornenyl)-acetyl, preferably N-hydroxy-succinimidyl-3-(4-hydroxyphenyl)-propionate.

[0048] The pH of the reaction medium may be lowered to a value of 2.0 to 5.0, preferably 2.0 to 4.0, more preferably 3.0 to 4.0, for example to a value of about 3.5 or 2.0 to 3.5, for example to a value of about 3.0, even more preferably to 3.0 to 3.5, before the addition of the micelle-forming surfactant (step b) in the method described herein). Alternatively, the pH of the reaction medium may be lowered to a value of 4.0 to 9.0, preferably 4.0 to 6.0, more preferably 4.0 to 6.0, even more preferably 4.5 to 5.5, before the addition of the micelle-forming surfactant (step b1) in the method described herein), and after the addition of the micelle-forming surfactant, before the medium is contacted with the solid adsorbent, may be further lowered to a value of 2.0 to 4.0, more preferably 3.0 to 4.0, for example to a value of about 3.5 or 2.0 to 3.5, for example to a value of about 3.0, even more preferably to 3.0 to 3.5 (step b2) in the method described herein). Advantageously, performing the subsequent process steps at a lower pH results in more efficient removal of LPS, pyrogens and carboxylic acids.

[0049] In the method described herein, a micelle-forming surfactant is added to the reaction medium. The micelle-forming surfactant can form micelles (soluble aggregates) in solution. Micelle-forming surfactants are known in the art. The micelle-forming surfactant can be an ionic surfactant, such as a cationic or anionic surfactant. Preferably, the surfactant is a non-ionic surfactant, which tends to form micelles at lower concentrations compared to ionic surfactants. Furthermore, ionic surfactants can interact with gelatin through ionic bonds, making them more difficult to remove.

[0050] Preferably, the micelle-forming non-ionic surfactant is an ethoxylated surfactant, preferably an alkylphenol ethoxylate, preferably having the formula C x H 2x-1Alkylphenol ethoxylates of the formula -C6H4-O-(C2H4O)nH, where x is 4-12, n is 7.5-14, X is preferably 8, n is preferably 8-13, more preferably 8.5-12.5, most preferably 9-12, in particular Triton X-100, Triton X-102, or mixtures thereof. The Triton X series of nonionic surfactants are prepared by the reaction of octylphenol with ethylene oxide. The products are generally of the type described as alkylaryl polyether alcohols, and are represented by the formula C8H4-O-(C2H4O)nH. 17 C6H5(CH2CH2O) 9.7 (Triton X-100), and C9H 19 It has the structural formula C6H5(CH2CH2O)12.3 (Triton X-102). Other suitable non-ionic surfactants include nonylphenoxypolyethoxyethanol C 15 H 24 O(C2H4O) n where n is 3 to 40, such as nonoxynol-4, nonoxynol-15, and nonoxynol-30; or 12 -C 18 Polyethylene glycol sorbitan monoesters of fatty acids, such as TWEEN. CHAPSO (3-([3-cholamidopropyl]dimethylammonio)-2-hydroxyl-1-propanesulfonate) is another suitable nonionic surfactant.

[0051] It is believed that as a result of the addition of the micelle-forming surfactant, LPS is monomerized, and said monomer interacts with the surfactant to form a micellar complex comprising the surfactant and LPS. To allow efficient removal of LPS and preferably also other pyrogens, in particular pyrogens from gram-positive bacteria and flagellated bacteria, especially pyrogens from gram-positive bacteria, the weight ratio of gelatin, in particular carboxylic acid-gelatin, to the surfactant added, in particular non-ionic surfactant, is preferably 2000:1 or less, more preferably 500:1 or less, even more preferably 250:1 or less, most preferably 50:1 or less. Indeed, at higher weight ratios, i.e. when a relatively large amount of gelatin is present, not all LPS is bound by the surfactant. Preferably, the surfactant is added to the reaction medium at a concentration of 0.01 to 1.5 w / w%, preferably 0.015 to 1.0 w / w%, more preferably 0.020 to 0.50 w / w%.

[0052] In step d) of the method described herein, the medium of step c) or the medium of step b2), which may contain soluble aggregates comprising detergents, LPS and its monomers and / or other pyrogens, in particular pyrogens from gram-positive bacteria and pyrogens from flagellated bacteria, especially pyrogens from gram-positive bacteria, is contacted with a (solid) adsorbent, which, in addition to binding detergents and preferably also LPS, also preferably binds other pyrogens, in particular pyrogens from gram-positive bacteria, pyrogens from flagellated bacteria, single-stranded viral RNA and bacterial DNA rich in unmethylated CpG motifs, and HPA.

[0053] The solid sorbent can be any suitable sorbent known to those skilled in the art, such as a hydrophobic sorbent, capable of binding surfactants, preferably also LPS, other pyrogens and carboxylic acids. The sorbent is preferably insoluble, suitable sorbents include clays, such as (activated) diatomaceous earth or clays, phyllosilicates, such as aluminum phyllosilicate, smectite minerals and hydrophobic sorbents, such as activated carbon, such as Norit SX Plus or Norit ROX 0.8 (Cabot, The Netherlands), or 3M Zeta Carbon Filter Cartridges, such as types R55S or R30L3S (3M, USA). Also mixtures of one or more sorbents can be applied. In a preferred embodiment, the solid sorbent is activated carbon.

[0054] Contacting the medium with a solid sorbent can be accomplished, for example, by adding a particulate sorbent to the medium, by passing the medium through a filter element containing the sorbent, by passing the medium through a column stacked with the sorbent, or by incubating the medium with a carrier having the sorbent present on its exterior surface.

[0055] The solid sorbent is preferably added to the medium in a weight ratio to the surfactant of at least 2.5:1, more preferably at least 3.0:1, and most preferably at least 3.5:1. The solid sorbent is preferably added to the medium in a concentration of 0.1-3 w / w%, preferably 0.5-1 w / w%. If a filter element or filter system is used, it may be preferable to use a similar amount of sorbent in the filter system.

[0056] By carrying out the contact step for a sufficient period of time, the surfactant is appropriately adsorbed, and as a result, the surfactant and LPS and / or other pyrogens bound to the surfactant, particularly pyrogens derived from gram-positive bacteria and flagellated bacteria, especially pyrogens derived from gram-positive bacteria, are removed, and preferably HPA, LPS and other pyrogens bound to the adsorbent, particularly pyrogens derived from gram-positive bacteria, flagellated bacteria, single-stranded viral RNA, and bacterial DNA rich in unmethylated CpG motifs, and methacrylic acid are adsorbed. Preferably, the adsorbent is contacted with the (aqueous) medium for 5 minutes to 1 hour, more preferably for 10 to 30 minutes.

[0057] In the next step (e)) of the method described herein, the solid sorbent is removed from the medium. The skilled person will be aware of suitable methods for contacting the aqueous medium with the solid sorbent and separating the sorbent from the medium. The separation can include, for example, centrifugation or filtration if the sorbent is added to the medium as particulates, with filtration being preferred from the standpoint of industrial applicability. For example, the solid sorbent can be added to the medium (of step c) or step b2), surfactants can be bound to the sorbent, and preferably LPS, other pyrogens and carboxylic acids can also be bound to the sorbent, and the sorbent can then be removed, for example, by filtration, sedimentation or centrifugation. In another example, the solid sorbent can be present in a filter, and the medium (of step c) or step b2) of the method described herein) can be passed through said filter, or a series of such filters, while optionally washing the filter in order to maximize the yield of filtrate. In this way, steps d), e) and f) of the method described herein can be combined in a single filtration step. Following separation of the solid adsorbent, the medium containing the carboxylate-gelatin is recovered.

[0058] Preferably, steps c) to f) of the method described herein are carried out at a temperature below the cloud point of the surfactant used. As used herein, the term "cloud point" refers to the temperature at which the surfactant forms insoluble aggregates in the medium. The temperature depends on the conditions of the medium, such as the salt concentration. If no specific conditions are given, the cloud point is defined herein as the temperature at which a 1 w / w% aqueous solution forms insoluble aggregates. Thus, when a temperature is stated to be below the cloud point of a surfactant, the temperature is 68-69°C (i.e., for a 1 w / w% Triton X-100 solution), whereas for a 16-25 w / w% NaCl solution, the cloud point is room temperature. The cloud point can be conveniently determined in a given situation by measuring the absorbance of the solution at 620 nm without adding surfactant and checking whether the absorbance increases when the expected amount of surfactant is added. The absorbance increases above the cloud point.

[0059] In an embodiment, steps c) to f) are performed at a temperature of 65° C. or less, more preferably 62° C. or less, and even more preferably 60° C. or less. In an embodiment, steps c) to f) are performed at a temperature of 30° C. to 65° C., preferably 30° C. to 60° C., more preferably 30° C. to 50° C. or 30° C. to 40° C., and even more preferably 30° C. to 35° C.

[0060] In some embodiments, the pH of the medium throughout steps c) to f) of the method is 2.0 to 5.0, preferably 2.0 to 4.0, more preferably 3.0 to 4.0, such as about 3.5, or 2.0 to 3.5, such as about 3.0, even more preferably 3.0 to 3.5. At such a pH, the temperature is preferably below 35°C, more preferably 30°C to 35°C, such as about 30°C.

[0061] In another embodiment, the pH of the medium is 2.0 to 5.0, preferably 2.0 to 4.0, more preferably 3.0 to 4.0, for example about 3.5, or 2.0 to 3.5, for example about 3.0, even more preferably 3.0 to 3.5 throughout steps d) to f) of the method. At such a pH, the temperature is preferably below 35°C, more preferably 30°C to 35°C, for example about 30°C.

[0062] Following recovery of the medium containing carboxylate-gelatin, the method may further comprise a step of increasing the pH of the medium to 3.5-9.0, preferably 4.0-8.0, more preferably 5.0-7.0. As mentioned above, the method described herein results in carboxylate-gelatin with a low LPS content, in particular less than 100 EU / g, more preferably less than 50 EU / g, even more preferably less than 20 EU / g, even more preferably less than 10 EU / g, even more preferably less than 5 EU / g, even more preferably less than 2 EU / g, most preferably less than 1 EU / g. In particular, the carboxylate-gelatin-gelatin contains at least 50 times less LPS, preferably at least 100 times less, more preferably at least 150 times less, even more preferably at least 200 times less, most preferably at least 250 times less LPS, compared to the LPS content of the gelatin used as starting material in step a). The LPS count can be quantified in the recovered medium, for example using the LAL assay described elsewhere herein.

[0063] The resulting carboxylic acid-gelatin is further characterized by a low content of non-endotoxin pyrogens, in particular low contents of pyrogens from gram-positive bacteria, pyrogens from flagellated bacteria, single-stranded viral RNA, endotoxins from gram-negative bacteria and bacterial DNA rich in unmethylated CpG motifs, in particular low contents of pyrogens from gram-positive bacteria and flagellated bacteria, and more particularly low contents of pyrogens from gram-positive bacteria. In particular, the carboxylic acid-gelatin contains at least 10 times, preferably at least 20 times, more preferably at least 50 times, even more preferably at least 100 times, at least 150 times, at least 200 times or at least 250 times less pyrogens from gram-positive bacteria than the content of pyrogens from gram-positive bacteria of the gelatin used as starting material in step a). In particular, the carboxylic acid-gelatin contains at least 10 times less, preferably at least 20 times less, more preferably at least 50 times less, even more preferably at least 100 times less, at least 150 times less, at least 200 times less, or at least 250 times less, pyrogens derived from flagellated bacteria, compared to the content of pyrogens derived from flagellated bacteria in the gelatin used as the starting material in step a).In particular, the carboxylic acid-gelatin contains at least 10 times less, preferably at least 20 times less, more preferably at least 50 times less, even more preferably at least 100 times less, at least 150 times less, at least 200 times less, or at least 250 times less, bacterial DNA rich in unmethylated CpG motifs, compared to the content of bacterial DNA rich in unmethylated CpG motifs in the gelatin used as the starting material in step a).Low single-stranded viral RNA.

[0064] Furthermore, the methods described herein result in carboxylic acid modified gelatins with low free (non-gelatin bound) carboxylic acid content, in particular less than 100 ppm, preferably less than 50 ppm, more preferably less than 30 ppm of carboxylic acid.To quantify the free (non-gelatin bound) 3-(4-hydroxyphenyl)-propionic acid content, the low carboxylic acid functionalized gelatins were dissolved in 50 mM phosphate buffer (pH 9.5).

[0065] Preferably, the carboxylic acid is less than 150 ppm, preferably less than 100 ppm, when determined in a sample of carboxylic acid-gelatin dissolved in water, when determined in a sample of carboxylic acid-gelatin dissolved in 50 mM phosphate buffer, pH 9.5.

[0066] Therefore, according to this method, purified carboxylate-gelatin can be provided by carrying out the above steps a) to f) (of six or seven steps) once.

[0067] The method described herein preferably does not include a dialysis step, since it provides carboxylated-gelatin with low carboxylated content without the need for a dialysis step. Since such dialysis steps are generally time consuming, the method of the present invention is more efficient, especially for large-scale production of purified carboxylated-gelatin.

[0068] In an embodiment, the method further comprises the step of drying the collection medium comprising the carboxylic acid-gelatin, optionally after increasing the pH of said medium, for example by freeze-drying to a white porous foam (Van Den Bulcke et al., 2000., Biomacromolecules, 1:31-38).

[0069] The invention will now be further illustrated by the following non-limiting examples. EXAMPLES

[0070] Example 1: Preparation method of 3-(4-hydroxyphenyl)-propionate functionalized gelatin and properties of 3-(4-hydroxyphenyl)-propionate functionalized gelatin Materials and Methods A solution of 15-20% gelatin (pig skin, type A, Bloom 200) in 1.25 M carbonate buffer at pH 9.0 was reacted with N-hydroxysuccinimide-3-(4-hydroxyphenyl)-propionate in a ratio of 1:1 with respect to the total available amino groups in the gelatin at 50°C for 120-180 minutes. During the reaction, 3-(4-hydroxyphenyl)-propionic acid (PA) was formed. The pH of the reaction medium was lowered to pH 3.5. HCl was used to remove the carbonate. 1.12% (with respect to the weight of the gelatin) of Triton X100 was added to the medium and the medium was maintained under stirring for 1 hour. The medium was then filtered through a column packed with activated charcoal. Other N-hydroxysuccinimide esters of carboxylic acids were reacted in the same manner.

[0071] Quantification of LPS content The LPS content was quantified using the Endozyme II Assay Kit (Hyglos) according to the manufacturer's instructions. Briefly, ultrapure aqueous solutions of functionalized gelatin were prepared at 2.50% (w / w) and dilutions of 10x, 20x, 50x, 100x, etc., depending on the expected LPS content. 100 μL of each sample was added to a well of a multi-well plate and mixed with 100 μL of assay reagent prepared according to the manufacturer's instructions. The fluorescence intensity of the resulting mixture was monitored with a microplate fluorescence reader (BopTek, excitation / emission = 380 nm / 455 nm). A dilution series of 50 EU / mL endotoxin standard (E. coli 055:B5) reconstituted in endotoxin-free water was used as calibration standard, and endotoxin-free water was used as blank for functionalized gelatin (GelDAT) samples in aqueous solution with 8% (w / w) 4-hydroxyphenyl-propionic acid content or 50 mM phosphate buffer solution (pH 9.5). Samples were allowed to swell for 15 min at room temperature and then placed at 50°C for 30 min until the samples were completely dissolved in appearance. 0.5 mL of sample solution was added to a 10 kDa Amicon Ultra centrifugal filter (Millipore), placed at 50°C (oven) for 10 min, and then centrifuged at 12000xg for 30 min at 40°C. The filtrate was then collected for HPLC analysis. A 1% (w / w) aqueous solution of 4-hydroxyphenyl-propionic acid or a 50 mM phosphate buffer solution (pH 9.5) was prepared, and a dilution series of 4-hydroxyphenyl-propionic acid in the range of 0.1 ppm to 100 ppm was prepared by diluting it two-fold with water or 50 mM phosphate buffer solution (pH 9.5) and used as a standard solution.

[0072] Quantification of DNA content Salmon testis dsDNA standard solutions were prepared in TE buffer (10 mM Tris-HCl aqueous solution containing 1 mM EDTA, pH=8) with a stock concentration of 0.1 mg / mL and further diluted to a stock concentration of 10 μg / mL. Quantitative standards were prepared in TE buffer ranging from 10 μg / mL to 0 μg / mL. 10% (w / w) gelatin samples were prepared in TE buffer, and the samples were allowed to swell for 30 min and dissolved at 40 °C using a water bath. 95 μl of master mix (100x) containing 94 μL TE buffer and 1 μL Cyber ​​Green and 5 μl of sample or standard were added to wells of a black well fluorescent 96-well plate and mixed by pipetting up and down. The 96-well plate was covered and incubated at 37 °C, 700 rpm for 30 min. Fluorescence was measured at Ex535nm / Em617nm at 25°C using a Synergy™ Mx fluorometer (BioTek) according to the manufacturer's instructions.

[0073] Example 2: Comparison of functionalized gelatin prepared by a method according to an embodiment of the present invention with functionalized gelatin prepared by a dialysis-based method. Materials and Methods Gelatin was modified according to the method described herein. The starting gelatin was contaminated with 2229 endotoxin units (EU) per gram of gelatin (Hyglos Endozyme). 3-(4-hydroxyphenyl)-propionic acid residues and LPS contamination were measured in ppm and EU per gram of gelatin, respectively, as described in Example 1. To quantify the 3-(4-hydroxyphenyl)-propionic acid content, the functionalized gelatin was dissolved in 50 mM phosphate buffer (pH 9.5). In a comparative example method, the 3-(4-hydroxyphenyl)-propionic acid by-product was removed from the functionalized gelatin solution by dialysis against ultrapure water (Milli-Q, Merck Millipore). Aliquots of 20 milliliters of the 3-(4-hydroxyphenyl)-propionic acid solution were transferred into five dialysis tubes (dialysis tubing cellulose membrane, MWCO 14 kDa, catalog number: D9527-100FT, Sigma-Aldrich) and each of the dialysis tubes was immersed in 2.5 liters of water. Dialysis proceeded at 40° C. and the water was changed every 24 hours. At T1=day 1, T2=day 2, T3=day 3, T4=day 4, and T5=day 7, one tube was removed and the contents of 3-(4-hydroxyphenyl)-propionic acid and LPS were measured as described in Example 1. To quantify the 3-(4-hydroxyphenyl)-propionic acid content, the functionalized gelatin was dissolved in 50 mM phosphate buffer (pH 9.5). In a method according to one embodiment of the present invention, five 40 ml aliquots of the functionalized gelatin solution were adjusted to pH 2.0, 2.5, 3.0, 3.5 and 4.0 by adding dilute HCl (all measured at 40° C.) and 0.1% Triton X100 (relative to gelatin content) was added to each aliquot. Each aliquot was then split into two, one of which was used as a negative control for the effect of lowering pH and adding Triton X100 on methacrylic acid content and LPS contamination. Five grams of Norit activated charcoal powder (S268, activated charcoal Norit SX Plus 8013-1) were added to each of the experimental tubes and the tubes were incubated for 1 hour at 40° C. on a rotary shaker.The tubes were then centrifuged at 2000 rpm for 30 min and the supernatants were filtered (0.45 μm) and analyzed for 3-(4-hydroxyphenyl)-propionic acid and LPS content as described in Example 1. To quantify the content of free (non-gelatin-bound) 3-(4-hydroxyphenyl)-propionic acid, the functionalized gelatin was dissolved in 50 mM phosphate buffer (pH 9.5).

[0074] result Table 1 shows the 3-(4-hydroxyphenyl)-propionic acid (PA) content (ppm) and LPS content (EU / g) of purified functionalized gelatin obtained by the methods disclosed elsewhere herein, and is shown graphically in Figure 1.

[0075] [Table 1]

[0076] Purification via micelle formation and filtration Table 2 shows the 3-(4-hydroxyphenyl)-propionic acid content (PA, ppm) and LPS content (EU / g) of purified functionalized gelatins obtained by dialysis-based method or by a method according to an embodiment of the present invention. A graphical representation is shown in Figure 2. HPLC is shown in Figure 3.

[0077] [Table 2]

[0078] Figure 4 shows in Figure 4A the MW distribution of the starting gelatin (1) and the MW distribution resulting from modification via a route using an in situ mixture of NHS, HPA and EDC followed by purification using one-step dialysis (2) and two-step dialysis (3). The modified gelatin shows a broader MW distribution and undesired cross-linking compared to the starting gelatin.

[0079] Figure 4B shows the MW distribution of the same starting gelatin in Figure 4A (1) and the MW distribution resulting from modification via the NHS-HPA route in the essential absence of EDC and subsequent purification using micelle formation-based purification. The modified gelatin has a nearly identical Mw distribution to the starting gelatin, indicating successful derivatization in the essential absence of undesired crosslinking.

[0080] Derivatization with NHS-HPA in the absence of EDC leads to a product with a different Mw profile that is closer to the original gelatin compared to derivatization with a mixture of NHS, HPA and EDC. Purification via micelle formation leads to a product with less LPS and less HPA, improving the quality.

Claims

1. Gelatin functionalized with a carboxylic acid moiety R-(CH ２ ) ｎ -COOH via an amide bond, R is selected from the group consisting of 4-phenol, norbornenyl, or SH, n is an integer from 1 to 10, the functionalized gelatin has a lipopolysaccharide content of less than 100 EU / g, the functionalized gelatin has an amount of free carboxylic acid R-(CH₂)n-COOH of less than 100 ppm, where the amount of the free carboxylic acid R-(CH₂)n-COOH is quantified by high performance liquid chromatography of the filtrate obtained by ultrafiltration of a sample in which the functionalized gelatin is dissolved in water or a 50 mM phosphate buffer with a pH of 9.5, gelatin.

2. The gelatin according to claim 1, wherein n is an integer from 1 to 5.

3. The gelatin according to claim 1, wherein the carboxylic acid moiety is one or more of 3-(4-hydroxyphenyl)-propionic acid, 3-(SH)-propionic acid, and 2-(5-norbornenyl)-acetic acid.

4. The gelatin according to claim 1, wherein the degree of modification of the functionalized gelatin by the carboxylic acid moiety is 5% to 100%.

5. The gelatin according to claim 1, wherein the content of free carboxylic acid R-(CH₂)n-COOH of the functionalized gelatin is less than 50 ppm.

6. The gelatin according to claim 5, wherein the content of free carboxylic acid R-(CH₂)n-COOH of the functionalized gelatin is less than 30 ppm.

7. The gelatin according to claim 1, wherein the functionalized gelatin is type A gelatin.

8. The gelatin according to claim 1, wherein the functionalized gelatin is further modified with a (meth)acrylate group or (meth)acrylate moiety, an acetyl group or acetyl moiety, a phenol group or phenol moiety, a thiol group or thiol moiety, a norbornene group or norbornene moiety, a tetrazine group or tetrazine moiety, an azide group or azide moiety, a furan group or furan moiety, an allyl group or allyl moiety, a maleimide group or maleimide moiety, or any combination thereof.

9. A method for providing the functionalized gelatin according to claim 1, comprising: a. In a reaction medium, a step of modifying gelatin by reacting gelatin with a reagent containing an N-hydroxysuccinimide ester of a carboxylic acid moiety R-(CH ２ )( ｎ -COOH (wherein n is an integer of 1 to 10)), R-(CH ２ )( ｎ -COO-NHS); and b. a step of reducing the pH of the reaction medium to a value of 2.0 to 3.5; c. a step of adding 0.01 to 1.5 w / w% of a micelle-forming surfactant to the acidic reaction medium; d. a step of contacting the medium containing micelles with an adsorbent; e. A step of separating the adsorbent from the medium; f. A step of recovering the medium containing the functionalized gelatin; comprising; wherein R is selected from the group consisting of 4-phenol, norbornenyl, and SH; Method.

10. A method for preparing the functionalized gelatin according to claim 1, comprising: a) In a reaction medium, a step of modifying gelatin by reacting gelatin with a reagent containing an N-hydroxysuccinimide ester of a carboxylic acid moiety R-(CH ２ )( ｎ )-COOH (wherein n is an integer of 1 to 10) (R-(CH ２ )( ｎ )-COO-NHS); and b1) A step of reducing the pH of the reaction medium to a value of 4.0 to 9.0; c) A step of adding 0.01 to 1.5 w / w% of a micelle-forming surfactant to the reaction medium; b2) A step of reducing the pH of the reaction medium to a value of 2.0 to 3.5; d) A step of contacting the medium of step b2) with a solid adsorbent; e) A step of separating the solid adsorbent of step d) from the medium; f) A step of recovering the medium containing the functionalized gelatin; comprising; wherein R is selected from the group consisting of 4-phenol, norbornenyl, and SH; Method.

11. The method according to claim 9 or claim 10, wherein n is an integer from 1 to 5.

12. The carboxylic acid moiety R-(CH ２ ), ｎ -COOH (wherein n is an integer from 1 to 10), the reagent containing the N-hydroxysuccinimide ester of which is R-(CH ２ ), ｎ -COO-NHS, the method according to claim 9 or claim 10.

13. The method according to claim 9 or claim 10, wherein the carboxylic acid moiety is one or more of 3-(4-hydroxyphenyl)-propionic acid, 3-(SH)-propionic acid, and 2-(5-norbornenyl)-acetic acid.

14. The method according to claim 9 or claim 10, wherein the primary amine group of the gelatin is modified with the carboxylic acid moiety.

15. The method according to claim 9 or claim 10, wherein the primary amine group of the functionalized gelatin is coupled to the carboxylic acid moiety via an amide bond.

16. The method according to claim 9 or claim 10, which does not include a dialysis step.

17. The method according to claim 9 or claim 10, wherein the micelle-forming surfactant contains a nonionic surfactant.

18. The method according to claim 9 or claim 10, wherein the lipopolysaccharide content of the functionalized gelatin is less than 100 EU / g.

19. The method according to claim 9 or claim 10, wherein the degree of modification of the functionalized gelatin with the carboxylic acid moiety is 5% to 100%.

20. The free carboxylic acid R-(CH ２ ) ｎ -COOH contained in the functionalized gelatin is less than 100 ppm, where the amount of the free carboxylic acid R-(CH2)n-COOH is determined by high performance liquid chromatography of the filtrate obtained by ultrafiltration of a sample in which the functionalized gelatin is dissolved in water or a 50 mM phosphate buffer having a pH of 9.

5. The method according to claim 9 or claim 10.

21. The method according to claim 9 or claim 10, wherein the functionalized gelatin is further modified with a (meth)acrylate group or (meth)acrylate moiety, an acetyl group or acetyl moiety, a phenol group or phenol moiety, a thiol group or thiol moiety, a norbornene group or norbornene moiety, a tetrazine group or tetrazine moiety, an azide group or azide moiety, a furan group or furan moiety, an allyl group or allyl moiety, a maleimide group or maleimide moiety, or any combination thereof.

22. The method according to claim 9 or claim 10, comprising the step of drying the medium containing the functionalized gelatin in step f).

23. A hydrogel comprising the functionalized gelatin according to any one of claims 1 to 8 and a crosslinking agent.

24. A film comprising a hydrogel comprising the functionalized gelatin according to any one of claims 1 to 8 and a crosslinking agent, or comprising the functionalized gelatin according to claims 1 to 8.

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