filaments

Abstract

Filaments comprising a semi-crystalline polymer are disclosed herein. Also disclosed are processes for the production of filaments, cartridges comprising filaments, bioreactors comprising filaments, food products comprising filaments, cordage comprising filaments, woven materials comprising filaments, non-woven materials comprising filaments, and sutures comprising filaments.

Description

[0001] FILAMENTS

[0002] This application claims priority from GB 2412785.4 filed 30 August 2024, and GB 2413950.3 filed 23 September 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] 1.1 Field

[0004] In certain aspects and embodiments, the instant disclosure relates in part to filaments, as well as processes for their production and uses.

[0005] 1.2 BACKGROUND

[0006] US2198538 discloses “a process of manufacturing artificial fiber from protein contained in soybean”.

[0007] W02006138039A2 discloses “[A] process for the production of high quality fibers from wheat proteins and products made from wheat protein fibers”.

[0008] W02006083605A2 discloses “high quality and long natural cellulose fibers from rice straw and method of producing rice straw fibers”

[0009] WO2021150744A1 discloses “continuous production of keratin fibers”, wherein, “a process as described herein can comprise extruding a keratin solution into a first solution to form a first fiber; drawing the first fiber and oxidizing the first fiber to form a treated fiber; drawing the treated fiber and oxidizing the treated fiber one or more times; and setting the treated fiber to form the keratin fiber.”

[0010] GB202317377D0 discloses “fibres” and is incorporated herein by reference in its entirety.

[0011] 1.3 SUMMARY

[0012] The instant disclosure is based, at least in part, on the realisation that extruded or spun filaments comprising a semi-crystalline polymer comprising crosslinked polypeptides with a beta-sheet secondary structure, could enable the production of novel materials suitable for a wide range of applications. Such application may include without limitation, uses in cordage, textiles, composite materials, filtration media, sorbent materials, smart textiles, electronic textiles, reinforcement structures, biodegradable materials, thermal or acoustic insulation, protective gear or armour, decorative applications, biomedical materials, and / or as substrates for cell cultivation. Accordingly, in various aspects of the disclosure, provided are filaments comprising a semi-crystalline polymer comprising crosslinked polypeptides with a beta-sheet secondary structure. One challenge for producing regenerated protein fibres is that many current regenerated protein fibres swell significantly in an aqueous solution and become significantly weaker when wet. Such materials do not have the necessary, mechanical and / or chemical properties for use as textiles or composites. The filaments of many embodiments of this disclosure overcome these shortcomings by additionally providing a process comprising a novel combination of process steps.

[0013] One challenge associated with regenerated protein fibres comprising ester, thioester and / or amide crosslinked polypeptides, in general, is that ester, thioester and / or amide crosslinks can be broken by hydrolysis under certain conditions. Beta-sheets and beta-coil secondary structures in polypeptides may prevent the hydrolysis of ester, thioester and / or amide bonds. However, induced beta-sheets and betacoils can revert to amorphous structures in water or upon hydration under certain conditions.

[0014] As used herein, the term “beta-sheets” collectively refers to both beta-sheets and beta-coil secondary structures. As used herein, a polypeptide which comprises a beta-sheet secondary structure is a polypeptide which comprises a secondary structure which comprises a beta-sheet region and / or a betacoil region.

[0015] The disclosure is also based, at least in part, on the realisation that polymers comprising ester, thioester and / or amide crosslinked polypeptides with induced beta-sheet secondary structures may recrystalize upon being annealed, thereby relaxing the internal stresses within their crystal structures, such that the beta-sheet conformation becomes the stable micro-structure; a process by which is herein referred to as “protein annealing”. Consequently, the beta-sheet secondary structure of the crosslinked polypeptide polymer may remain intact upon rehydration and resist hydrolysis of the constituent ester, thioester and / or amide bond crosslinks.

[0016] As used herein, the term “polymer” refers to a macromolecule that comprises repeating subunits called monomers.

[0017] In one aspect, the disclosure provides a polymer material which comprises a semi-crystalline polymer. In some embodiments, the semi-crystalline polymer comprises a crosslinked polypeptide having a betasheet secondary structure.

[0018] A polymer material which comprises a semi-crystalline polymer, wherein the semi-crystalline polymer comprises a crosslinked polypeptide having a beta-sheet secondary structure, may be referred to as “Prokitein”. In some embodiments, the term “Prokitein” refers to a polymer material which comprises a semicrystalline polymer, wherein the semi-crystalline polymer comprises a polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptide having a beta-sheet secondary structure.

[0019] As used herein, a “semi-crystalline polymer” refers to a polymer that comprises molecular chains arranged in both crystalline and amorphous structures. In the crystalline regions, protein chains are ordered in repeating patterns. In the amorphous regions, protein chains are randomly orientated.

[0020] As used herein, a “regular semi-crystalline polymer” refers to a semi-crystalline polymer that comprises uniformly distributed crystal grain sizes and shapes, and / or grains that are orientated consistently relative to each other, within the crystalline phases of its molecular structure.

[0021] As used herein, a “polypeptide” refers to a polymer of amino acids.

[0022] As used herein, a “polysaccharide” refers to a polymer of monosaccharides.

[0023] As used herein, “crosslinking” refers to the process of chemically joining two or more molecules, or two or more regions of a single molecule. A “crosslink” is a chemical bond, or a short sequence of bonds, that connects one portion of a molecule to a different portion of the same molecule or to a separate molecule.

[0024] A crosslinking reagent is a chemical substance that can create chemical bonds between different polymer chains or between different regions of the same polymer chain, forming a three-dimensional network.

[0025] In some embodiments, the polypeptide is a crosslinked polypeptide. As used herein, a “crosslinked polypeptide” is a polypeptide which comprises a crosslink. Such a crosslink may join a portion (e.g., an amino acid / functional group / region) of one polypeptide to either (i) another portion (e.g., an amino acid / functional group / region) of the same polypeptide, or (ii) to another molecule (e.g., another polypeptide).

[0026] In some embodiments, the crosslink is an intra-polypeptide crosslink. As used herein, an “intrapolypeptide crosslink” is a crosslink that joins a portion (e.g., an amino acid / functional group / region) of a polypeptide to another portion (e.g., an amino acid / functional group / region) of the same polypeptide.

[0027] In some embodiments, the crosslink is an inter-polypeptide crosslink. As used herein, an “intra- polypeptide crosslink” is a crosslink that joins a portion of a polypeptide to another molecule (e.g., another polypeptide). In some embodiments, the polypeptide comprises an intra-polypeptide crosslink and / or an interpolypeptide crosslink.

[0028] In some embodiments, the crosslink is an intra-molecular crosslink. In some embodiments, the crosslink is an intermolecular crosslink. In some embodiments, the polypeptide comprises an intramolecular crosslink and / or an intermolecular crosslink.

[0029] In some embodiments, the crosslink is a covalent crosslink. In some embodiments, the crosslink is an ionic crosslink. In some embodiments, the polypeptide comprises a covalent crosslink and / or an ionic crosslink.

[0030] In some embodiments, the crosslinked polypeptide is a covalently-crosslinked polypeptide. As used herein, a “covalently-crosslinked polypeptide” is a polypeptide which comprises a covalent crosslink that joins a portion (e.g., an amino acid / functional group / region) of the polypeptide to either (i) another portion (e.g., an amino acid / functional group / region) of the polypeptide, or (ii) to another molecule (e.g., another polypeptide).

[0031] In some embodiments, the covalently-crosslinked polypeptide comprises an ester, thioester and / or amide bond crosslink. In some embodiments, the covalently-crosslinked polypeptide comprises an ester bond crosslink. In some embodiments, the covalently-crosslinked polypeptide comprises a thioester bond crosslink. In some embodiments, the covalently-crosslinked polypeptide comprises an amide bond crosslink.

[0032] As used herein, an “ester crosslink” or an “ester bond crosslink” is a crosslink comprising an ester bond. Ester bond crosslinks are known to the skilled person. For example, ester bonds between threonine and glutamine sidechains can form covalent crosslinks between polypeptide chains.

[0033] As used herein, a “thioester crosslink” or a “thioester bond crosslink” is a crosslink comprising a thioester bond. Thioester bond crosslinks are known to the skilled person. For example, thioester bonds between cysteine and glutamine sidechains can form covalent crosslinks between polypeptide chains.

[0034] As used herein, an “amide bond crosslink” or an “amide bond crosslink” is a crosslink comprising an amide bond. Amide bond crosslinks are known to the skilled person. An amide bond can be synthesized when the carboxyl group of one amino acid reacts with the amino group of another amino acid.

[0035] As used herein, the term “covalent ester, thioester and / or amide crosslinked polypeptide” means a polypeptide having at least one or more ester bond crosslink, one or more thioester bond crosslink and / or one or more amide bond crosslink. It is generally understood that any such ester, thioester and / or amide bond crosslink would be derived from a polycarboxylic acid (or a form thereof). As such, the term “polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides” encompasses any covalent ester, thioester and / or amide crosslinked polypeptide without the need for demonstrating that it was actually derived from a polycarboxylic acid (or a form thereof). As such, the terms “covalent ester, thioester and / or amide bond crosslink” and “polycarboxylic acid derived covalent ester, thioester and / or amide bond crosslink” are used interchangeably herein. Additionally, the terms “ester, thioester and / or amide bond crosslink” and “polycarboxylic acid derived ester, thioester and / or amide bond crosslink” are used interchangeably herein.

[0036] Accordingly, the term “covalent crosslink” may herein refer to a crosslink that comprises ester, thioester, and / or amide bonds.

[0037] As used herein, the term “polycarboxylic acid crosslinking reagent” may refer to a polycarboxylic acid (or a form thereof) that may be capable of forming a covalent crosslink. In some embodiments, the polycarboxylic acid crosslinking reagent is capable of forming a covalent crosslink. In certain embodiments, the polycarboxylic acid crosslinking reagent is capable of forming one or more ester, thioester and / or amide bond crosslinks. The polycarboxylic acid crosslinking reagent may be in the form of a free acid, partially or fully dissociated species, salt (e.g., alkali or alkaline earth metal salts), ester, amide, anhydride (cyclic or linear), lactone, polymeric or crosslinked structure, chelated or complexed form, or a buffered mixture thereof.

[0038] The disclosure is also based, at least in part, on the realisation that filaments comprising a semicrystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure may resist hydrolysis and could facilitate their use as materials for a plethora of applications. Such applications may include, without limitation, uses in cordage, textiles, composite materials, filtration media, sorbent materials, smart textiles, electronic textiles, reinforcement structures, biodegradable materials, thermal or acoustic insulation, protective gear or armour, decorative applications, biomedical materials, and / or as substrates for cell cultivation.

[0039] The disclosure is also based, at least in part, on the realisation that filaments comprising a regular semicrystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure may resist hydrolysis and could facilitate their use as materials for a plethora of applications. Such applications may include, without limitation, uses in cordage, textiles, composite materials, filtration media, sorbent materials, smart textiles, electronic textiles, reinforcement structures, biodegradable materials, thermal or acoustic insulation, protective gear or armour, decorative applications, biomedical materials, and / or as substrates for cell cultivation. The disclosure is also based, at least in part, on the realisation that the resistance to hydrolysis of Prokitein could facilitate its use as materials for a plethora of applications. Such applications may include, without limitation, uses in cordage, textiles, composite materials, filtration media, sorbent materials, smart textiles, electronic textiles, reinforcement structures, biodegradable materials, thermal or acoustic insulation, protective gear or armour, decorative applications, biomedical materials, and / or as substrates for cell cultivation.

[0040] In some embodiments, a filament of this disclosure comprises Prokitein.

[0041] One additional challenge, in general, is the production of porous filaments. Process steps, such as freeze-drying, are sometimes required to adjust the porosity of filamentous materials. The filaments of many embodiments of this disclosure may overcome this shortcoming by additionally providing a process by which filaments with porous structures may be formed without the use of freeze-drying.

[0042] The disclosure is also based, at least in part, on the realisation that filaments, as disclosed herein, can constitute a predominantly protein substrate or scaffold on which cells may be grown. Consequently, filaments of this disclosure can be used in the production of a structured cultivated meat food product with a fibrous structure, which can mimic the structure of natural muscle fibres.

[0043] A filament of this disclosure may comprise more than 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% protein (w / w).

[0044] In some embodiments, the filament comprises more than 50% protein (w / w). In some embodiments, the filament comprises more than 60% protein (w / w). In some embodiments, the filament comprises more than 70% protein (w / w). In some embodiments, the filament comprises more than 80% protein (w / w). In some embodiments, the filament comprises more than 90% protein (w / w). In some embodiments, the filament comprises more than 91 % protein (w / w). In some embodiments, the filament comprises more than 92% protein (w / w). In some embodiments, the filament comprises more than 93% protein (w / w). In some embodiments, the filament comprises more than 94% protein (w / w). In some embodiments, the filament comprises more than 95% protein (w / w). In some embodiments, the filament comprises more than 96% protein (w / w). In some embodiments, the filament comprises more than 97% protein (w / w). In some embodiments, the filament comprises more than 98% protein (w / w). In some embodiments, the filament comprises more than 99% protein (w / w).

[0045] The filaments of this disclosure are, in some aspects and / or embodiments, edible, non-cytotoxic and / or biocompatible, and in some embodiments, they can be ingested and digested as food by humans and / or animals. The disclosure is also based, at least in part, on the realisation that filaments as disclosed herein can, in some embodiments, constitute a predominantly protein substrate or scaffold on which cells may be grown. Consequently, filaments of this disclosure can be used in the production of a structured cultivated meat food product with a fibrous structure, which can mimic the structure of natural muscle fibres.

[0046] As used herein, a “lumen” is the widest cavity or channel that runs continuously along the long-axis of a tube, such as the bore of a hypodermic needle or a drinking straw.

[0047] In some embodiments, the lumen diameter at any point along its length is greater than 10 pm. In some embodiments, the lumen diameter at any point along its length is greater than 15 pm. In some embodiments, the lumen diameter at any point along its length is greater than 20 pm.

[0048] In one aspect, the disclosure provides a filament.

[0049] As used herein, a “filament” refers to a cylindrical structure that does not have a lumen. The term “filament” may alternatively be described as a “fibre”.

[0050] As used herein a “cylindrical structure” is defined as a cylinder; a three-dimensional solid that holds two bases joined by a curved surface.

[0051] As used herein, a "hollow fibre" refers to a cylindrical structure that comprises a lumen.

[0052] In some aspects and embodiments, the disclosure is also based, at least in part, on a hollow fibre having one or more of the properties of any filament described herein, with the distinction that the hollow fibre additionally comprises a lumen. A filament of this disclosure does not comprise a lumen as defined herein.

[0053] As defined herein, a filament is not a hollow fibre and conversely, a hollow fibre is not a filament.

[0054] It will be understood that, unless expressly stated otherwise, any use, feature and / or aspect described herein with respect to a filament is equally applicable to a hollow fibre of this disclosure.

[0055] The disclosure is further based, at least in part, on the realisation that the filaments of this disclosure can, in some embodiments, be suitable for use in any application where fibrous materials are required.

[0056] In one aspect, the disclosure provides a filament that is suitable for use in textiles.

[0057] In one aspect, the disclosure provides a filament that is suitable for use in yarns.

[0058] In one aspect, the disclosure provides a filament that is suitable for use in composites.

[0059] In one aspect, the disclosure provides a filament that is suitable for use as a substrate for cell growth. In one aspect, the disclosure provides a filament that is suitable for use in rope.

[0060] In one aspect, the disclosure provides a filament that is suitable for use in non-woven meshes.

[0061] In one aspect, the disclosure provides a filament that is suitable for use in woven meshes.

[0062] In one aspect, the disclosure provides a filament that is suitable for use in fabrics.

[0063] In one aspect, the disclosure provides a filament that is suitable for use in forming a composite thread.

[0064] In one aspect, the disclosure provides a filament that is suitable for use in filtration media.

[0065] In one aspect, the disclosure provides a filament that is suitable for use in sorbent materials.

[0066] In one aspect, the disclosure provides a filament that is suitable for use in smart textiles or electronic textiles.

[0067] In one aspect, the disclosure provides a filament that is suitable for use in reinforcement structures, (e.g., concrete reinforcement or polymer reinforcement).

[0068] In one aspect, the disclosure provides a filament that is suitable for use in biodegradable materials.

[0069] In one aspect, the disclosure provides a filament that is suitable for use in thermal or acoustic insulation.

[0070] In one aspect, the disclosure provides a filament that is suitable for use in protective gear or armour.

[0071] In one aspect, the disclosure provides a filament that is suitable for use in decorative applications.

[0072] The disclosure is further based, at least in part, on the realisation that filaments of this disclosure can, in some aspects or embodiments, be used as suture and / or materials for biomedical applications.

[0073] In one aspect, the disclosure provides a filament that is suitable for use as suture.

[0074] In one aspect, the disclosure provides a filament that is suitable for use in sutures for biomedical applications.

[0075] In one aspect, the disclosure provides a filament that is suitable for use in biomedical applications, including non-woven meshes and / or woven meshes.

[0076] In one aspect, the disclosure provides a filament that is suitable for use in medical devices.

[0077] (e.g., stents, scaffolds, or implants beyond sutures).

[0078] In one aspect, the disclosure provides a filament as a carrier.

[0079] In one aspect, the disclosure provides a filament as a carrier for drug delivery.

[0080] In one aspect, the disclosure provides a filament as a carrier in a capsule for drug delivery.

[0081] The disclosure is further based, at least in part, on the realisation that filaments described herein can, in some aspects or embodiments, be edible or be intended to be edible.

[0082] In one aspect, the disclosure provides an edible filament.

[0083] In another aspect, the disclosure provides a filament intended to be edible.

[0084] In a further aspect, the disclosure provides a filament that is not edible.

[0085] In one aspect, the disclosure provides a filament that is suitable for use in food products.

[0086] In one aspect, the disclosure provides a filament as a food product.

[0087] In one aspect, the disclosure provides a filament that is suitable as a carrier for food flavourings.

[0088] In one aspect, the disclosure provides a filament that is suitable as a carrier for pharmaceutical drugs. It will be understood that, unless expressly stated otherwise, any use or application described herein with respect to a filament is equally applicable to a hollow fibre of this disclosure.

[0089] A hollow fibre of this disclosure may be edible, intended to be edible, or non-edible.

[0090] In some embodiments, the hollow fibre is edible.

[0091] In some embodiments, the hollow fibre is a food product.

[0092] In some embodiments, the hollow fibre is intended to be edible.

[0093] In other embodiments, the hollow is non-edible.

[0094] The disclosure is also based, at least in part, on the realisation that hollow fibres, as disclosed herein, can constitute a predominantly protein substrate or scaffold on which cells may be grown. Consequently, hollow fibres of this disclosure can be used in the production of a structured cultivated meat food product with a fibrous structure, which can mimic the structure of natural muscle fibres.

[0095] The disclosure is also based, at least in part, on the realisation that filaments as disclosed herein can, in some embodiments, be used in conjunction with edible hollow fibres to act as an auxiliary scaffold for cell growth and / or as a means to alter the texture of cultivated meat food products.

[0096] The disclosure is further based, at least in part, on the realisation that filaments of this disclosure can, in some embodiments, be used in the production of bioreactor cartridges, both with and without edible hollow fibres, that are compatible with existing and novel bioreactor platforms.

[0097] The disclosure is further based, at least in part, on the realisation that the filaments of this disclosure, both with and without hollow fibres of this disclosure, could facilitate the realisation of a structured cultivated meat food product.

[0098] In one aspect, the disclosure provides a filament comprising one or more crosslinked polypeptides.

[0099] In one aspect, the disclosure provides a filament, comprising a semi-crystalline polymer comprising crosslinked polypeptides with a beta-sheet secondary structure.

[0100] In one aspect, the disclosure provides a filament, comprising a regular semi-crystalline polymer comprising crosslinked polypeptides with a beta-sheet secondary structure.

[0101] In one aspect, the disclosure provides a filament comprising one or more polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides.

[0102] In one aspect, the disclosure provides a filament, comprising a semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a betasheet secondary structure. In one aspect, the disclosure provides a filament, comprising a regular semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

[0103] In one aspect, the disclosure provides a filament comprising one or more ionically-crosslinked polypeptides.

[0104] In one aspect, the disclosure provides a filament, comprising a semi-crystalline polymer comprising ionically-crosslinked polypeptides with a beta-sheet secondary structure.

[0105] In one aspect, the disclosure provides a filament, comprising a regular semi-crystalline polymer comprising ionically-crosslinked polypeptides with a beta-sheet secondary structure.

[0106] In one aspect, the disclosure provides a filament comprising: (i) one or more polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides; and (ii) one or more ionically-crosslinked polypeptides.

[0107] In one aspect, the disclosure provides a filament, comprising a semi-crystalline polymer comprising: (i) polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a betasheet secondary structure; and / or (ii) ionically-crosslinked polypeptides with a beta-sheet secondary structure.

[0108] In one aspect, the disclosure provides a filament, comprising a regular semi-crystalline polymer comprising; (i) polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure; and / or (ii) ionically-crosslinked polypeptides with a beta-sheet secondary structure.

[0109] In some aspects and embodiments, a filament of this disclosure has an external diameter within the range of 50 - 6,600 pm.

[0110] In some aspects and embodiments, a filament of this disclosure is edible.

[0111] In some aspects and embodiments, a filament of this disclosure is intended to be edible.

[0112] In some aspects and embodiments, a filament of this disclosure is porous.

[0113] In another aspect, the disclosure provides a process for the production of a filament. In some embodiments, the process for the production of a filament is a process for the production of a plurality of filaments.

[0114] A process for the production of a filament described herein may be adapted as appropriate to become a process for the production of a plurality of filaments. Similarly, a process for the production of a plurality of filaments described herein may be adapted as appropriate to become a process for the production of a filament. The process for the production of a filament may be suitable for the production of a filament according to the present disclosure.

[0115] In some embodiments, the process is a process for the production of a filament comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

[0116] In some embodiments, the process comprises the step of: combining: i. a first composition comprising polypeptides, and ii. a second composition comprising a solvent, and one or more denaturing agents and / or reducing agents, to produce a third composition.

[0117] In some embodiments, the process comprises the step of: incubating the third composition under conditions sufficient to denature and / or reduce a polypeptide, to produce a fourth composition.

[0118] In some embodiments, the process comprises the step of: incubating a composition under conditions sufficient to denature and / or reduce a polypeptide.

[0119] In some embodiments, the process comprises the step of: extruding or spinning the fourth composition through an orifice, to produce a filament.

[0120] In some embodiments, the process comprises the step of: extruding or spinning a composition, to produce a filament.

[0121] In some embodiments, the process comprises the step of: extruding or spinning a composition through an orifice, to produce a filament.

[0122] In some embodiments, the process comprises the step of: treating a filament with a crosslinking reagent to form an inter-polypeptide and / or intrapolypeptide ester bond, thioester bond or amide crosslink in the filament.

[0123] In some embodiments, the process comprises the step of: treating a filament with a polycarboxylic acid crosslinking reagent to form an inter-polypeptide and / or intra-polypeptide ester bond, thioester bond or amide crosslink in the filament. In some embodiments, the process comprises the step of: treating a filament with an ionic crosslinking reagent to form an inter-polypeptide and / or intrapolypeptide ionic crosslink in the filament.

[0124] In some embodiments, the process comprises the step of: treating a filament with at least one post-production modification process.

[0125] In some embodiments, the process comprises the step of: treating a covalently-crosslinked filament with at least one post-production modification process.

[0126] In some embodiments, the post-production modification process comprises treating a filament with an organic solvent.

[0127] In some embodiments, the post-production modification process comprises treating a filament with an organic solvent to increase the relative abundance of beta-sheets in the filament.

[0128] In some embodiments, the post-production modification process comprises treating a filament with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptide.

[0129] In some embodiments, the post-production modification process comprises annealing a filament.

[0130] In some embodiments, the post-production modification process comprises treating a filament with a solvent.

[0131] In some embodiments, the post-production modification process comprises treating a filament with a solvent to increase porosity.

[0132] In some embodiments, the post-production modification process comprises treating a filament with a solvent to remove void inclusion elements from the filament.

[0133] In some embodiments, the post-production modification process comprises washing a filament with one or more wash solutions.

[0134] In some embodiments, the post-production modification process comprises coating a filament.

[0135] In some embodiments, the post-production modification process comprises modifying the surface topography of a filament. In some embodiments, the post-production modification process comprises drawing a filament.

[0136] In some embodiments, the post-production modification process comprises drawing a filament by stretching.

[0137] In some embodiments, the process comprises drying a filament.

[0138] In some embodiments, one or more void inclusion elements are introduced into the first, second, third and / or fourth composition. In some embodiments, a void inclusion element is introduced into the first composition. In some embodiments, a void inclusion element is introduced into the second composition.

[0139] In some embodiments, a void inclusion element is introduced into the third composition. In some embodiments, a void inclusion element is introduced into the fourth composition.

[0140] In one aspect, the disclosure provides a process for the production of a plurality of filaments comprising crosslinked polypeptides, the process comprising the steps: a. combining: i. a first composition comprising polypeptides, with ii. a second composition comprising a solvent and one or more denaturing agents and / or reducing agents, to produce a third composition; b. incubating the third composition under conditions sufficient to denature and / or reduce at least a fraction of the polypeptides, to produce a fourth composition; c. extruding or spinning the fourth composition through an orifice, to produce a plurality of filaments; d. treating the plurality of filaments with a crosslinking reagent in order to form inter-polypeptide and / or intra-polypeptide crosslinks within at least a fraction of the polypeptides in the filaments, to produce a plurality of crosslinked filaments; e. treating the crosslinked filaments with at least one post-production modification process selected from the group consisting of: i. treating the crosslinked filaments with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides; ii. annealing the crosslinked filaments; ill. treating the crosslinked filaments with a solvent to increase the porosity of the filaments; iv. washing the crosslinked filaments with one or more wash solutions; v. coating the crosslinked filaments; vi. modifying the surface topography of the crosslinked filaments; vii. drawing the crosslinked filaments; to produce a plurality of treated crosslinked filaments; and f. drying the treated crosslinked filaments, to produce dried, treated crosslinked filaments.

[0141] In another aspect, the disclosure provides a process for the production of a plurality of filaments, comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides, the process comprising the steps: a. combining: i. a first composition comprising polypeptides, with ii. a second composition comprising a solvent and one or more denaturing agents and / or reducing agents, to produce a third composition; b. incubating the third composition under conditions sufficient to denature and / or reduce at least a fraction of the polypeptides, to produce a fourth composition; c. extruding or spinning the fourth composition through an orifice, to produce a plurality of filaments; d. treating the plurality of filaments with a polycarboxylic acid crosslinking reagent in order to form inter-polypeptide and / or intra-polypeptide polycarboxylic acid derived covalent ester, thioester or amide bond crosslinks within at least a fraction of the polypeptides in the filaments, to produce a plurality of covalently-crosslinked filaments; e. treating the covalently-crosslinked filaments with at least one post-production modification process selected from the group consisting of: i. treating the covalently-crosslinked filaments with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides; ii. annealing the covalently-crosslinked filaments; ill. treating the covalently-crosslinked filaments with a solvent to increase the porosity of the filaments; iv. washing the covalently-crosslinked filaments with one or more wash solutions; v. coating the covalently-crosslinked filaments; vi. modifying the surface topography of the covalently-crosslinked filaments; vii. drawing the covalently-crosslinked filaments; to produce a plurality of treated covalently-crosslinked filaments; and f. drying the treated covalently-crosslinked filaments, to produce dried, treated covalently- crosslinked filaments. In yet another aspect, the disclosure provides a process for the production of a plurality of filaments comprising a semi-crystalline polymer comprising covalent ester, thioester and / or amide bond crosslinked polypeptides with a beta-sheet secondary structure, the process comprising the steps: a. combining: i. a first composition comprising polypeptides, with ii. a second composition comprising a solvent and one or more denaturing agents and / or reducing agents, to produce a third composition; b. incubating the third composition under conditions sufficient to denature and / or reduce at least a fraction of the polypeptides, to produce a fourth composition; c. extruding or spinning the fourth composition through an orifice, to produce a plurality of filaments; d. treating the plurality of filaments with one or more polycarboxylic acid crosslinking reagents in order to form inter-polypeptide and / or intra-polypeptide polycarboxylic acid derived covalent ester, thioester or amide bond crosslinks, within at least a fraction of the polypeptides in the filaments, to produce a plurality of crosslinked filaments; e. treating the crosslinked filaments with at least one post-production modification process selected from the group consisting of: i. treating the crosslinked filaments with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides; ii. annealing the crosslinked filaments; ill. treating the crosslinked filaments with a solvent to increase the porosity of the filaments; iv. washing the crosslinked filaments with one or more acid(s), alkali(s) and / or buffer solution(s); v. coating the crosslinked filaments; vi. modifying the surface topography of the crosslinked filaments; vii. drawing the crosslinked filaments; to produce a plurality of treated crosslinked filaments; and f. drying the treated crosslinked filaments, to produce dried, treated crosslinked filaments.

[0142] In yet a further aspect, the disclosure provides a process for the production of a plurality of filaments comprising a semi-crystalline polymer comprising ionically-crosslinked polypeptides with a beta-sheet secondary structure, the process comprising the steps: a. combining: i. a first composition comprising polypeptides, with ii. a second composition comprising a solvent and one or more denaturing agents and / or reducing agents, to produce a third composition; b. incubating the third composition under conditions sufficient to denature and / or reduce at least a fraction of the polypeptides, to produce a fourth composition; c. extruding or spinning the fourth composition through an orifice, to produce a plurality of filaments; d. treating the plurality of filaments with one or more ionic crosslinking reagents in order to form inter-polypeptide and / or intra-polypeptide ionic crosslinks within at least a fraction of the polypeptides in the filaments, to produce a plurality of ionically-crosslinked filaments; e. treating the ionically-crosslinked filaments with at least one post-production modification process selected from the group consisting of: i. treating the ionically-crosslinked filaments with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides; ii. annealing the ionically- crosslinked filaments; ill. treating the ionically-crosslinked filaments with a solvent to increase the porosity of the filaments; iv. washing the ionically-crosslinked filaments with one or more acid(s), alkali(s) and / or buffer solution(s); v. coating the ionically-crosslinked filaments; vi. modifying the surface topography of the ionically-crosslinked filaments; vii. drawing the ionically-crosslinked filaments; to produce a plurality of treated ionically-crosslinked filaments; and f. drying the treated ionically-crosslinked filaments, to produce dried, treated ionically- crosslinked filaments.

[0143] In yet an even further aspect, the disclosure provides a process for the production of a plurality of filaments comprising a semi-crystalline polymer comprising crosslinked polypeptides with a beta-sheet secondary structure, wherein the polypeptides are crosslinked with covalent ester, thioester and / or amide bond crosslinks and / or ionic crosslinks, the process comprising the steps: a. combining: i. a first composition comprising polypeptides, with ii. a second composition comprising a solvent and one or more denaturing agents and / or reducing agents, to produce a third composition; b. incubating the third composition under conditions sufficient to denature and / or reduce at least a fraction of the polypeptides, to produce a fourth composition; c. extruding or spinning the fourth composition through an orifice, to produce a plurality of filaments; d. treating the plurality of filaments with one or more crosslinking reagents in order to form interpolypeptide and / or intra-polypeptide crosslinks within at least a fraction of the polypeptides in the filaments, to produce a plurality of crosslinked filaments; e. treating the crosslinked filaments with at least one post-production modification process selected from the group consisting of: i. treating the crosslinked filaments with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides; ii. annealing the crosslinked filaments; ill. treating the crosslinked filaments with a solvent to increase the porosity of the filaments; iv. washing the crosslinked filaments with one or more acid(s), alkali(s) and / or buffer solution(s); v. coating the crosslinked filaments; vi. modifying the surface topography of the crosslinked filaments; vii. drawing the crosslinked filaments; to produce a plurality of treated crosslinked filaments; and f. drying the treated crosslinked filaments, to produce dried, treated crosslinked filaments comprising a semi-crystalline polymer comprising crosslinked polypeptides with a beta-sheet secondary structure, wherein the polypeptides are crosslinked with covalent ester, thioester and / or amide bond crosslinks and / or ionic crosslinks.

[0144] In yet another aspect, the disclosure provides a process for the production of a semi-crystalline polymer comprising ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure, the process comprising the steps: a. combining: i. a first composition comprising polypeptides, with ii. a second composition comprising a solvent and one or more denaturing agents and / or reducing agents, to produce a third composition; b. incubating the third composition to produce a fourth composition; c. shaping the fourth composition into the desired shape using one or more processes selected from extruding, spinning, casting, moulding or spraying; d. treating the fourth composition with a polycarboxylic acid crosslinking reagent in order to form inter-polypeptide and / or intra-polypeptide polycarboxylic acid derived covalent ester, thioester or amide bond crosslinks within at least a fraction of the polypeptides, to produce a polymer comprising covalently-crosslinked polypeptides; e. treating the polymer with at least one post-production modification process selected from the group consisting of: i. treating with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides; ii. annealing; ill. treating with a solvent to remove void inclusion elements; iv. washing with one or more acid(s), alkali(s) and / or buffer solution(s); v. coating; vi. modifying the surface topography; and vii. drawing; and f. drying.

[0145] A process for the production of a filament described herein may be adapted as appropriate to become a process for the production of a hollow fibre, wherein in Step (c), the fourth composition is extruded together with a bore solution through a co-axial orifice. In some embodiments, the process for the production of a filament is adapted as appropriate to become a process for the production of a hollow fibre. A process for the production of a filament described herein may be adapted as appropriate to become a process for the production of a plurality of hollow fibres.

[0146] In yet another aspect, the disclosure provides a process for the production of a hollow fibre that comprises a lumen and a semi-crystalline polymer comprising covalent ester, thioester and / or amide bond crosslinked polypeptide with a beta-sheet secondary structure, the process comprising the steps: a. combining: i. a first composition comprising polypeptides, with ii. a second composition comprising a solvent and one or more denaturing agents and / or reducing agents, to produce a third composition; b. incubating the third composition under conditions sufficient to denature and / or reduce at least a fraction of the polypeptides, to produce a fourth composition; c. extruding or spinning the fourth composition through a plurality of co-axial orifices, together with a bore solution, to produce a hollow fibre; d. treating the hollow fibre with one or more crosslinking reagents in order to form interpolypeptide and / or intra-polypeptide polycarboxylic acid derived ester, thioester or amide covalent bond crosslinks, within at least a fraction of the polypeptides in the hollow fibre, to produce a crosslinked hollow fibre; e. treating the crosslinked hollow fibre with at least one post-production modification process selected from the group consisting of: i. treating the crosslinked hollow fibre with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides; ii. annealing the crosslinked hollow fibre; iii. treating the crosslinked hollow fibre with a solvent to increase the porosity of the crosslinked hollow fibre; iv. washing the crosslinked hollow fibre with one or more acid(s), alkali(s) and / or buffer solution(s); v. coating the crosslinked hollow fibre; vi. modifying the surface topography of the crosslinked hollow fibre; vii. drawing the crosslinked hollow fibre; to produce a treated crosslinked hollow fibre; and f. drying the treated crosslinked hollow fibre, to produce a dried, treated crosslinked hollow fibre.

[0147] 1.4 DETAILED DESCRIPTION OF THE DISCLOSURE

[0148] With a growing global population and the emergence of fast fashion trends, demands for the textiles is at an all-time high. In 2022, 1 13.8 million tonnes of chemical and textile fibres were produced globally, with global production more than doubling between 2000-2024 (Statista, 2024). As the global population is set to increase by 40% by 2050, the environmental impact of fast fashion and textiles is likely to increase accordingly. With the realisation of omnipresent microplastics and dwindling petrochemical resources, hitherto dependencies on non-renewable feedstocks and environmentally harmful process have been called into question, both publicly and legislatively (The Potential for Regenerated Protein Fibres within a Circular Economy: Lessons from the Past Can Inform Sustainable Innovation in the Textiles Industry, 2021 ). Animal-derived textiles have issues too, as wool fibre production accounted for 36% of the total carbon footprint in fibre production for clothing in the UK in 2009, while the farming of cashmere goats has been accredited for desertification of Mongolia (Referencing Historical Practices and Emergent Technologies in the Future Development of Sustainable Textiles: A Case Study Exploring “Ardil”, a UK- Based Regenerated Protein Fibre, 2022). Regenerated fibres offer a solution as a means for the production of textile fibres from renewable sources with a reduced environmental impact.

[0149] Regenerated cellulose fibres are an attractive alternative to petrochemical fibre materials as they may be produced from bio-based feedstocks. However, environmental concerns remain with the production of regenerated cellulose fibres. Viscose (rayon) production relies on a cellulose dissolution process, which requires strong carbon disulfide acid, followed by regeneration in a sulfuric acid bath, creating a heavy environmental burden. While lyocell production requires milder conditions, N-methylmorpholine-N-oxide, which is used to solubilise cellulose, must be recovered from water solidification baths, which creates a high electrical burden (Recent progress in regenerated fibers for “green” textile products, 2022).

[0150] Regenerated protein fibres present another alternative to petrochemical fibre materials that additionally enables circular manufacturing through the revalorisation of proteins from sources such as food and agricultural wastes. Historically, a number of regenerated protein fibres have been developed, including Vicara from soy protein and Lanital from milk protein. However, one challenge in the production of regenerated protein fibres is that most of these materials swell significantly in aqueous solutions and exhibit a reduction in strength of between 40-80 % when wet. Furthermore, harsh crosslinking reagents, such as formaldehyde, are typically used in their production. Given the acute toxicity, potential carcinogenicity and mutagenicity of formaldehyde, its use as a crosslinking reagent is non-desirable (The Potential for Regenerated Protein Fibres within a Circular Economy: Lessons from the Past Can Inform Sustainable Innovation in the Textiles Industry, 2021).

[0151] Additionally, regenerated protein fibres may find alternative applications outside of textiles. Recently, there has been significant interest in the development of cultivated meat, wherein animal cells are cultured in vitro and used for the production of meat. There are three broad advantages of cultured meat: sustainability, public health and animal welfare.

[0152] In the production of cultivated meat, cells are grown in in bioreactors. Cells may be grown in suspension in stirred tank reactors, and subsequently seeded onto an edible scaffold, which is required to give structure to the cultured meat food product. Alternatively, cells may be grown on scaffolds directly, such as in an edible hollow fibre bioreactor. Regenerated protein fibres may be used as a scaffold upon which cells grown in suspension may be seeded to create a structured cultivated meat food product. Alternatively, regenerated protein fibres may be integrated into an edible hollow fibre bioreactor, as an auxiliary scaffold for cells to grow on, and / or as a material used to alter the texture of the cultivated meat food product.

[0153] Furthermore, there is a growing need for substrates and suture for biomedical applications which enable cell attachment and subsequently act as a scaffold to facilitate cellular integration, as these can improve patient outcomes and prevent the development of post-care pathologies. As a predominantly protein substrate, regenerated protein fibres may be used in the production of next-generation biomedical substrates and suture.

[0154] Certain embodiments of the compositions and methods provided herein attempt to address and overcome some or all of these challenges.

[0155] In certain aspects and embodiments, the disclosure provides processes for the production of a plurality of filaments comprising a semi-crystalline polymer comprising crosslinked polypeptides with a beta-sheet secondary structure. In certain aspects and embodiments, the disclosure provides processes for the production of a plurality of filaments comprising a regular semi-crystalline polymer comprising crosslinked polypeptides with a betasheet secondary structure.

[0156] In certain aspects and embodiments, the disclosure provides processes for the production of a plurality of filaments comprising a semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

[0157] In certain aspects and embodiments, the disclosure provides processes for the production of a plurality of filaments comprising a regular semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

[0158] In certain aspects and embodiments, the disclosure provides processes for the production of a plurality of filaments comprising a semi-crystalline polymer comprising ionically-crosslinked polypeptides with a betasheet secondary structure.

[0159] In certain aspects and embodiments, the disclosure provides processes for the production of a plurality of filaments comprising a regular semi-crystalline polymer comprising ionically-crosslinked polypeptides with a beta-sheet secondary structure.

[0160] In certain aspects and embodiments, the disclosure provides processes for the production of a plurality of filaments comprising a semi-crystalline polymer comprising: (i) polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure; and / or (ii) ionically-crosslinked polypeptides with a beta-sheet secondary structure.

[0161] In certain aspects and embodiments, the disclosure provides processes for the production of a plurality of filaments comprising a regular semi-crystalline polymer comprising: (i) polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure; and / or (ii) ionically-crosslinked polypeptides with a beta-sheet secondary structure.

[0162] Step (a) comprises combining:

[0163] (i) a first composition comprising polypeptides, with

[0164] (ii) a second composition comprising a solvent and one or more denaturing agents and / or reducing agents, to produce a third composition.

[0165] The first composition comprises one or more polypeptides. The terms “polypeptide” and “protein” are used interchangeably herein. In some embodiments, the polypeptides of the filaments and / or methods of the disclosure are obtained or derived from plants, animals, bacteria, algae, archaea, and / or fungi.

[0166] In some preferred embodiments, the polypeptides of the filaments and / or methods of the disclosure are derived from one or more extracts and / or isolates obtained from plants, animals, bacteria, algae, archaea, and / or fungi.

[0167] In some embodiments, the polypeptides are plant polypeptides, wherein the term “plant” comprises legumes, nuts, seeds, cereals and / or tubers. Examples of such plant sources comprise:

[0168] • legumes: adzuki beans, anasazi beans, black beans, black-eyed peas, cannellini beans, chickpeas, cocoa beans, coffee beans, cranberry beans, edamame, fayot (flageolet) beans, fava beans (faba beams, broad beans), french green bean, gigante beans, great northern beans, green peas, kidney beans, lentils, lima beans, long beans, marrowfat peas, mung beans, navy beans, pinto beans, purple string beans, snow peas, soybeans, split peas, and / or sugar snap peas;

[0169] • nuts: almonds, walnuts, pistachios, cashews, brazil nuts, hazelnuts, macadamia nuts, pecans, pine nuts, peanuts, chestnuts and / or coconuts;

[0170] • seeds: sunflower, pumpkin, chia, flax, sesame, hemp, poppy, quinoa, cumin, fennel, coriander, mustard, caraway, cardamom, fenugreek, ajwain, anise, and / or nigella;

[0171] • cereals: wheat, rice, corn / maize, oats, barley, rye, millet, sorghum, quinoa, buckwheat, spelt, triticale, amaranth, teff, farro, kamut, freekeh, emmer, and / or fonio; and

[0172] • tubers: potatoes, sweet potatoes, yams, cassava, taro, jicama, Jerusalem artichoke, water chestnut, Chinese yam, malanga, arrowroot, ginger, turmeric, turnips and / or beets.

[0173] In some embodiments, the polypeptides are animal polypeptides, wherein the term “animal” comprises, mammals, marsupials, birds, fish, cephalopods, crustaceans and insects. Examples of such animal sources comprise:

[0174] • mammals: cattle, sheep, pig, horse, goat, deer, reindeer, bison, moose, elk, camel, wild boar, wildebeest, and / or guinea pig;

[0175] • marsupials: kangaroo, koala, wombat;

[0176] • birds: chicken, turkey, duck, goose, quail, pheasant, guineafowl, ostrich, emu, pigeon, partridge, grouse, snipe, and / or woodcock;

[0177] • fish: salmon, tuna, cod, trout, sardines, haddock, tilapia, catfish, mackerel, swordfish, halibut, dolphinfish, grouper, snapper, sea bass, anchovy, carp, perch, pike, flounder, sole, eel, herring, whitefish, and / or crawfish;

[0178] • cephalopods: squid, octopus, cuttlefish, and / or nautilus; • crustaceans: shrimp, crab, lobster, crawfish, prawn, krill, crayfish, mussel, oyster, clam, scallop, and / or cockle;

[0179] • reptiles: snakes, turtles, alligators, crocodiles, iguanas, and / or lizards; and

[0180] • insects: grasshopper, cricket, mealworm, beetle, ant, termite, cicada, caterpillar, silkworm, locust, waxworm, hornworm, bamboo worm, scorpion, and / or centipede.

[0181] Examples of bacteria include, but are not limited to, Escherichia coli., Bacillus subtilis, and Pseudomonas fluorescens.

[0182] In some embodiments, the polypeptides are algae polypeptides, wherein the term “algae” comprises Euglenophyta, Chrysophyta, Pyrrophyta, Chlorophyta, Rhodophyta, Paeophyta, and Xanthophyta.

[0183] Examples of algal sources of polypeptides include, but are not limited to: Euglena gracilis, Diatoms, Dinoflagellates pyrocystis, Netrium desmid, Chlamydomonas, Spirogyra, Volvox, Ulva, Chlorella, Chara, Corallina, Gelidium, Gracilaria, Laminaria, Fucus, Sargassum, and Vaucheria.

[0184] In some embodiments, the polypeptides are archaea polypeptides, wherein the term “archaea” comprises Euryarchaeota, Crenarchaeota and Korarchaeota.

[0185] Examples of archaea sources of polypeptides include, but are not limited to: Thermoproteus neutrophillus, Thermoproteus uzoniensis, Vulcanisaeta distributa, Vulcanisaeta moutnovskia, Metallosphaera cuprina, Metallosphaera sedula, Staphylothermus hellenicus, Staphylothermus marinus, Thermosphaera aggregans, Sulfolobus acidocaldarius, Sulfolobus islandicus, Desulfurococcus kamchatkensis, Hyperthermus butylicus, Thermus aqaticus, Archaeoglobus fulgidus, and Archaeoglobus veneficus

[0186] In some embodiments, the polypeptides are fungi polypeptides, wherein the term “fungi” comprises Ascomycota, Basidiomycota, Zygomycota, Chytridiomycota, Glomeromycota, and Deuteromycota.

[0187] Examples of fungi sources of polypeptides include, but are not limited to, mushrooms, truffle, yeast, penicillium, aspergillus, ergot, chanterelle, morel, bracket fungi, coral fungus, stinkhorn, puffball, bird’s nest fungi, and jelly fungi. Other examples include Saccharomyces cerevisiae and Pichia pastoris.

[0188] An extract or isolate may, for example, be an aqueous extract from a plant, animal, bacteria, algae, archaea, or fungi. This may be obtained, for example, by dissolving all or part of the organic source in an aqueous buffer (e.g., phosphate-buffered saline (PBS)), acid or alkali solution, optionally together with a surfactant, optionally with suitable mixing and / or homogenisation; and then isolating the aqueous extract or isolate from the undissolved material.

[0189] Solid protein may be recovered from the isolated sample through precipitation by adjusting the pH of the aqueous protein solution to the isoelectric point of the extracted protein through the addition of acid or alkali. The solid protein precipitate may then be recovered through centrifugation, and subsequently washed with water and heated to sterilise the protein extract. Finally, the extracted protein may be spray dried (Kaplan, 2010).

[0190] PROTEINS

[0191] In some embodiments, the extract or isolate from a plant, animal, bacteria, algae, archaea, or fungi is a protein extract or isolate.

[0192] Protein extracts can be obtained, inter alia, during the production of defatted beans, seeds or nut flakes. Suitable plant materials first undergo a process of cleaning, drying, conditioning, cracking, dehulling, solvent oil extraction and flash solvent removal, and the resulting proteins are then purified via alcohol washing, acid leaching and / or water leaching (Kaplan, 2010).

[0193] Additionally, bacteria, algae, archaea, or fungi may be genetically-modified to produce or excrete recombinant proteins. The recombinant protein may be purified through precipitation of the protein at its isoelectric point. Examples of proteins produced in this way include casein, lactoglobulin, and lactalbumin, which are extracted from recombinant proteins derived from bacteria such as Escherichia coli; yeasts such as Saccharomyces cerevisiae and P. pastoris; and fungi such as Rhizopus (Can recombinant milk proteins replace those produced by animals?, 2022).

[0194] Examples of proteins that may be used in the compositions and / or methods provided herein include, but are not limited to, soy proteins, such as glycinin and beta-conglycinin; wheat proteins, such as gliadin and glutenin; mung bean proteins, such as vignins, phaseolins, and globulins; maize proteins, such as zein; milk proteins, such as whey; egg proteins, such as albumin; epidermal proteins, such as keratin, gelatin and collagen; and insect proteins, such as resilin.

[0195] Examples of polypeptides derived from bacteria include, but are not limited to, casein, lactoglobulin and lactalbumin, which are extracted from the recombinant proteins derived from Escherichia coli. For example, chicken or cattle genes encoding casein, lactoglobulin or lactalbumin polypeptides may be expressed in E. coli (Can recombinant milk proteins replace those produced by animals?, 2022).

[0196] Examples of proteins derived from yeasts include, but are not limited to, casein, lactoglobulin and lactalbumin, which are extracted from recombinant proteins derived from Saccharomyces cerevisiae and P. pastoris. For example, chicken or cow genes encoding casein, lactoglobulin or lactalbumin polypeptides may be expressed in Saccharomyces cerevisiae or P. pastoris (Can recombinant milk proteins replace those produced by animals?, 2022).

[0197] Examples of proteins derived from other fungi include, but are not limited to, casein, lactoglobulin and lactalbumin, which are extracted from the recombinant proteins derived from the filamentous fungus Rhizopus. For example, chicken or cow genes encoding casein, lactoglobulin or lactalbumin polypeptides may be expressed in Rhizopus (Can recombinant milk proteins replace those produced by animals?, 2022).

[0198] In some embodiments, the polypeptides are used in a purified form or in combination with one or more other polypeptides and / or other materials.

[0199] POLYSACCHARIDES

[0200] The first composition may comprise one or more polysaccharides.

[0201] In some embodiments, the first composition comprises one or more polysaccharides.

[0202] In some embodiments, the first composition does not comprise polysaccharides.

[0203] One or more polysaccharides may be added to the compositions of Step (a) or Step (b). The polysaccharide may be present in the first composition, the second composition, or may be introduced after mixing. The present disclosure is not limited by the stage at which the polysaccharide is added.

[0204] The extract or isolate from a plant, animal, bacteria, algae, archaea, and / or fungi may comprise one or more polysaccharides.

[0205] In some embodiments, the extract or isolate from a plant, animal, bacteria, algae, archaea, and / or fungi comprises one or more polysaccharides.

[0206] There are many methods for extracting or isolating polysaccharides from biological materials that may be used in the compositions and / or methods provided herein. In one such example, polysaccharide extracts may be obtained during the production of defatted beans, seeds or nut flakes. Suitable plant materials first undergo a process of cleaning, drying, conditioning, cracking, dehulling, solvent oil extraction and flash solvent removal. Polysaccharides are subsequently obtained by removing the proteins via alcohol washing, acid leaching and / or water leaching (Kaplan, 2010). Examples of polysaccharides that could be used in addition to the polypeptides in the first composition include, but are not limited to, chitin, chitosan, starches derived from wheat, rice, potato or corn, alginate, agar, hyaluronic acid, dextran, chondroitin sulphate, carrageenan, carrageenan-kappa, carrageenan-iota, pullulan, xanthan gum, gellan gum, pectin, cellulose, microcrystalline cellulose, carboxymethyl cellulose, cellulose acetate, lignin, kraft lignin, alkali lignin, and organosolv lignin.

[0207] The first composition may comprise one or more salt derivatives of polysaccharides. Examples of salt derivatives of polysaccharides that could be used in addition to the polypeptides in the first composition include, but are not limited to, sodium alginate, potassium alginate, sodium carrageenan, and potassium carrageenan.

[0208] In some embodiments, the first composition comprises one or more salt derivatives of polysaccharides. In some embodiments, the first composition comprises sodium alginate, potassium alginate, sodium carrageenan, and / or potassium carrageenan.

[0209] In some embodiments, the first composition comprises sodium alginate.

[0210] LIPIDS

[0211] The first composition may comprise one or more lipids.

[0212] In some embodiments, the first composition comprises one or more lipids.

[0213] In some embodiments, the first composition does not comprise lipids.

[0214] The extract or isolate from a plant, animal, bacteria, algae, archaea, and / or fungi may comprise one or more lipids.

[0215] In some embodiments, the extract or isolate from a plant, animal, bacteria, algae, archaea, and / or fungi comprises one or more lipids.

[0216] Lipids are class of molecule that are highly diverse in their structure and function. Lipids are typically composed of fatty acids and glycerol, as well as other hydrocarbon chains.

[0217] Examples of lipids that could be used in addition to the polypeptides include, but are not limited to:

[0218] • plant oils: almond, avocado, canola, coconut, corn, flaxseed, grape seed, hemp seed, jojoba, mustard, olive, palm, peanut, pumpkin seed, rice bran, safflower, sesame, soybean, sunflower, walnut

[0219] • animal fats (including milks, butters, lards, and tallows): cattle, sheep, pig, horse, goat, deer, reindeer, bison, moose, elk, camel, wild boar, guinea pig, kangaroo, chicken, turkey, duck, goose, quail, pheasant, guineafowl, ostrich, emu, pigeon, partridge, grouse, snipe, woodcock, salmon, tuna, cod, trout, sardines, haddock, tilapia, catfish, mackerel, swordfish, halibut, dolphinfish, grouper, snapper, sea bass, anchovy, carp, perch, pike, flounder, sole, eel, herring, whitefish, and / or crawfish;

[0220] • faty acids: butyric acid (C4:0), caproic acid (C6:0), caprylic acid (C8:0), capric acid (C10:0), lauric acid (C12:0), myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), alpha-linolenic acid (C18:3), arachidonic acid (C20:4), eicosapentaenoic acid (C20:5) and / or docosahexaenoic acid (C22:6).

[0221] • salts of fatty acids;

[0222] • phospholipids;

[0223] • omega-3 oils;

[0224] • algal oils.

[0225] In some embodiments, the first composition comprises one or more plant oils.

[0226] POLYOLS

[0227] The first composition may comprise one or more polyols and / or polymers of polyols.

[0228] In some embodiments, the first composition comprises one or more polyols.

[0229] In some embodiments, the first composition comprises one or more polymers of polyols.

[0230] In some embodiments, the first composition comprises one or more polyols and / or polymers of polyols.

[0231] In some embodiments, the first composition does not comprise polyols or polymers of polyols.

[0232] Polyols are organic compounds characterised in having multiple hydroxyl (-OH) groups, commonly bonded to carbon atoms, in their molecular structure.

[0233] Examples of polyols may include, but are not limited to: Ethylene glycol, Glycerol, Erythritol, Threitol, Arabitol, Xylitol, Ribitol Mannitol, Sorbitol, Galactitol, Fucitol, Iditol, Inositol, Volemitol, Isomalt, Maltitol, Lactitol, Maltotriitol, Maltotetraitol, Polyglycitol, monoacylglycerols, diacylglycerols and / or triacylglycerols.

[0234] Polymers of polyols, also known as polymeric polyols, are polymers formed by polymerising monomeric polyols with reactive species, such as isocyanates, carboxylic acids, or epoxides. Examples of types of polymers of polyols include, but are not limited to, polyurethanes, polyesters, polyethers, polycarbonates, acrylic polyols, polybutadiene polyols, and polysulfide polyols.

[0235] The first compositions may comprise polyethers, including but not limited to polyethylene glycol, polypropylene glycol, and poly(tetramethylene ether) glycol.

[0236] In some embodiments, the first composition comprises polyethers including but not limited to polyethylene glycol, polypropylene glycol, and / or poly(tetramethylene ether) glycol.

[0237] In certain embodiments, the first composition comprises polyethylene glycol. In some preferred embodiments, commercially-available forms of protein extracts or isolates, polypeptides, polysaccharides, lipids, and / or polyols are used for the production of the filaments, for example as to avoid additional costs. However, extracts or isolates may also be derived from previous processing steps.

[0238] In another preferred embodiment, soy protein isolate (SPI), as obtained from soybeans, are used for the production of the filaments. SPI is the most highly-refined soy protein product commercially available, with some commercial products comprising more than 90 % protein (by mass). Soy protein extract is produced from defatted soybean flakes wherein the majority of fat, sugars and fibre have been removed to leave protein as the prominent remaining component. SPI may in some embodiments be produced through the extraction of protein in a mild alkali solution. The extract is then isolated through centrifugation and subsequently acidified to produce protein curd. The curd is washed to remove soluble sugars, neutralised and finally spray-dried (Kaplan, 2010). Other plant protein isolates may be produced and used in a similar manner.

[0239] Hence in one particularly-preferred embodiment, the polypeptides of the compositions and / or methods provided herein comprise soybean polypeptides. In certain embodiments the polypeptides consist essentially of soybean polypeptides.

[0240] In some embodiments, the first composition comprises, or consists essentially of, polypeptides, wherein at least 60 %, preferably at least 75 %, of the polypeptides are soybean polypeptides (e.g., extracted from soybeans).

[0241] 1.5 STEP (A)DISSOLVING IN A FIRST SOLVENT

[0242] Step (a), comprises combining a first composition, comprising polypeptides, with a second composition, comprising one or more solvents and one or more denaturing agents and / or reducing agents, to produce a third composition.

[0243] FIRST COMPOSITION

[0244] The first composition comprises one or more polypeptides.

[0245] The first composition may comprise one or more polysaccharides, lipids, polyols, polymers of polyols, and / or any combination thereof.

[0246] In some embodiments, the first composition comprises one or more polysaccharides.

[0247] In some embodiments, the first composition comprises one or more lipids.

[0248] In some embodiments, the first composition comprises one or more polyols and / or polymers of polyols.

[0249] In some embodiments, the first composition comprises one or more polysaccharides and lipids. In some embodiments, the first composition comprises one or more lipids, and polyols and / or polymers of polyols.

[0250] In some embodiments, the first composition is added to the second composition to a final concentration of 5 % to 60 % (w / v second composition); preferably, 7 % to 50 %; more preferably, 10 % to 40 %; even more preferably, 12 % to 30 %; and most preferably 15 % to 28 % (w / v second composition).

[0251] In some embodiments, the first composition is added to the second composition to a final concentration of 15 % to 28 % (w / v second composition).

[0252] In some embodiments, the percentage of polypeptide in the third composition ranges from 5 % to 10 %, 10 % to 20 %, 20 % to 35 %, 35 % to 45 %, 45 % to 55 %, or 55 % to 60 % (w / v second composition).

[0253] In some embodiments, polysaccharides are added to the first composition in a ratio that is within the range of 0.1 % to 10,000 % (w / w of polypeptide).

[0254] In some embodiments, polysaccharides are added to the first composition in a ratio that ranges from of 0.1 % to 1 %; 1 % to 5 %; 5 % to 10 %; 10 % to 25 %; 25 % to 50 %; 50 % to 100 %; 100 % to 250 %; 250 % to 500 %; 500 % to 1 ,000 %; 1 ,000 % to 2,500 %; 2,500 % to 5,000 %; or 5,000 % to 10,000% (w / w of polypeptide).

[0255] In some embodiments, polysaccharides are added to the first composition in a ratio that is within the range of 0.1 % to 10 % (w / w of polypeptide).

[0256] In some embodiments, polysaccharides are added to the first composition in a ratio that is within the range of 10 % to 20 % (w / w of polypeptide).

[0257] In some embodiments, lipids are added to the first composition in a ratio that is within the range of 0.1 % to 10,000 % (w / w of polypeptide).

[0258] In some embodiments, lipids are added to the first composition in a ratio that ranges from of 0.1 % to 1 %; 1 % to 5 %; 5 % to 10 %; 10 % to 25 %; 25 % to 50 %; 50 % to 100 %; 100 % to 250 %; 250 % to 500 %; 500 % to 1 ,000 %; 1 ,000 % to 2,500 %; 2,500 % to 5,000 %; or 5,000 % to 10,000% (w / w of polypeptide).

[0259] In some embodiments, polyols and / or polymers of polyols are added to the first composition in a ratio that is within the range of 0.001 % to 10,000 % (w / w of polypeptide). In some embodiments, polyols and / or polymers of polyols are added to the first composition in a ratio that ranges from of 0.001 % to 0.005 %; 0.005 % to 0.01 %; 0.01 % to 0.05 %; 0.05 % to 0.1 %; 0.1 % to 1 %; 1 % to 5 %; 5 % to 10 %; 10 % to 25 %; 25 % to 50 %; 50 % to 100 %; 100 % to 250 %; 250 % to 500 %; 500 % to 1 ,000 %; 1 ,000 % to 2,500 %; 2,500 % to 5,000 %; or 5,000 % to 10,000% (w / w of polypeptide).

[0260] In some embodiments, polyols and / or polymers of polyols are added to the first composition in a ratio that is within the range of 0.001 % to 1 % (w / w of polypeptide)).

[0261] In some embodiments, polyols and / or polymers of polyols are added to the first composition in a ratio that is within the range of 0.01 % to 10 % (w / w of polypeptide).

[0262] In some embodiments, polyols and / or polymers of polyols are added to the first composition in a ratio that is within the range of 10 % to 20 % (w / w of polypeptide).

[0263] SECOND COMPOSITION

[0264] The second composition comprises one or more solvents.

[0265] In some embodiments, the second composition also comprises one or more denaturing agents and / or reducing agents.

[0266] The second composition may dissolve at least some of the polypeptides and / or other components (e.g., polysaccharides, lipids, polyols, polymers of polyols, and / or salts) of the first composition.

[0267] In some embodiments, the second composition dissolves a portion of the polypeptides in the first composition. For example, the second composition may dissolve a small fraction, a portion, some, most, or all or substantially all of the polypeptides in the first composition.

[0268] In some embodiments, the second composition dissolves at least 0.1 %, 1 % , 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 99.99% or 100 % of the polypeptides in the first composition.

[0269] In some embodiments, the second composition dissolves, preferably, 0.1 % to 1 %; more preferably, 1 % to 5 %; even more preferably, 10 % to 20 %; and most preferably, 20 % to 30 % of the polypeptides in the first composition.

[0270] In some embodiments, the second composition dissolves, preferably, 30 % to 40 %; more preferably, 40 % to 50 %; even more preferably, 50 % to 60 %; and most preferably, 60 % to 70 % of the polypeptides in the first composition. In some embodiments, the second composition dissolves, preferably, 70 % to 80 %; more preferably, 80 % to 90 %; even more preferably, 90 % to 99.9 %; and most preferably, 99.99 % to 100 % of the polypeptides in the first composition.

[0271] In some embodiments, the second composition completely dissolves all of the polypeptides (and other components, e.g., polysaccharides, lipids, polyols, polymers of polyols, and / or salts) in the first composition.

[0272] In some embodiments, the second composition dissolves a portion of the polysaccharides in the first composition. For example, the second composition may dissolve a small fraction, a portion, some most, or all or substantially all of the polysaccharides in the first composition.

[0273] In some embodiments, the second composition dissolves at least 0.1 %, 1 % , 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 99.99% or 100 % of the polysaccharides in the first composition.

[0274] In some embodiments, the second composition dissolves, preferably, 0.1 % to 1 %; more preferably, 1 % to 5 %; even more preferably, 10 % to 20 %; and most preferably, 20 % to 30 % of the polysaccharides in the first composition.

[0275] In some embodiments, the second composition dissolves, preferably, 30 % to 40 %; more preferably, 40 % to 50 %; even more preferably, 50 % to 60 %; and most preferably, 60 % to 70 % of the polysaccharides in the first composition.

[0276] In some embodiments, the second composition dissolves, preferably, 70 % to 80 %; more preferably, 80 % to 90 %; even more preferably, 90 % to 99.9 %; and most preferably, 99.99 % to 100 % of the polysaccharides in the first composition.

[0277] As used herein, the term “dissolution” refers to a process wherein a substance, sometimes referred to as a “solute”, is dissolved into another substance, sometimes referred to as a “solvent”. This process typically includes “solvation”, wherein solvent molecules surround and interact with solute molecules or ions at the molecular level.

[0278] In some embodiments, dissolution can be experimentally quantified via visible light spectroscopy absorbance measurements. Absorbance measurements can be taken for multiple samples; complete dissolution can be verified if all of measurements are uniform. Conversely, partial dissolution can be determined by non-uniform measurements.

[0279] As used herein, the terms “dissolution”, “dissolving” and “solubilising” are used interchangeably. A person of skill in the art will readily be able to test the solubility of the polypeptides (and other components, e.g., polysaccharides, lipids, salts, when present) in the first composition in various second compositions to determine the suitability of the use of that second composition.

[0280] In some embodiments, the second composition is one which is capable of breaking disulphide bonds and / or hydrogen bonds in at least one polypeptide in the first composition.

[0281] Examples of solvents which may be used in the second composition include aqueous solutions and / or organic solutions.

[0282] Examples of aqueous solutions include, aqueous acids, aqueous alkalis and / or aqueous salt solutions.

[0283] Examples of organic solutions include, but are not limited to, alcohols, polyols and / or polymers of polyols.

[0284] In some embodiments, solvents used in the second composition comprise water, ethanol, acetic acid, glycerol, polyethylene glycol, iso-propanol, propanol, and / or formaldehyde, and / or mixtures thereof.

[0285] In other embodiments, a solvent used in the second composition is used either alone or in combination with other solvents.

[0286] In some preferred embodiments, the solvent used in the second composition is water.

[0287] In some embodiments, one or more salts are added to the second composition in order to aid dissolution of the first composition.

[0288] In some embodiments, salts added to the second composition comprise sodium chloride, potassium chloride, zinc chloride, sodium carbonate, potassium carbonate, sodium bicarbonate, zinc sulphate, lithium sulphate, sodium malonate, sodium maleate, potassium sodium tartrate, triammonium citrate, trilithium citrate, trisodium citrate, tripotassium citrate, disodium hydrogen phosphite, sodium hypophosphite, monosodium phosphate, dipotassium phosphate, sodium sulphate, sodium bisulphate, potassium sulphate, ammonium sulphate, and / or sodium sulphite.

[0289] In some embodiments, salts added to the second composition comprise sodium carbonate, potassium carbonate, and / or sodium bicarbonate.

[0290] In some embodiments, one or more salts are added to the second composition are hydrous and / or anhydrous. In some embodiments, the concentration of each salt which is added to the second composition is within the range of 0.01 % to 70 %; preferably, 0.1 % to 50 %; more preferably, 0.5 % to 10 %; even more preferably, 1 % to 7 %; and most preferably 1 % to 2 % (w / w first composition).

[0291] In some embodiments, concentration of each salt which is added to the second composition is within the range of 0.1 to 1 %, 1 % to 10 %, 10 % to 25 %, 25 % to 50 % or 50 % to 70 % (w / w first composition).

[0292] DENATURING AND REDUCING AGENTS

[0293] The second composition also comprises one or more denaturing agents and / or one or more reducing agents to solubilise and denature the polypeptides.

[0294] As used herein, the term “denaturing agent” refers to a substance that disrupts the native structure of a biomolecule, such as a polypeptide, by altering non-covalent interactions (e.g., hydrogen bonding, hydrophobic interactions, ionic interactions) and / or covalent structures, thereby causing loss of the molecule’s biological activity, conformation, or higher-order structure.

[0295] As used herein, the term “reducing agent” refers to a substance that donates electrons to another substance, such as a polypeptide, thereby reducing the oxidation state of the substance. A reducing agent may cleave disulfide bonds or otherwise modify covalent or non-covalent interactions, resulting in changes to the structure or activity of the polypeptide.

[0296] In some embodiments, the second composition comprises one or more denaturing agents.

[0297] In some embodiments, the second composition comprises one or more reducing agents.

[0298] In some embodiments, the second composition comprises one or more denaturing agents and one or more reducing agents.

[0299] In some embodiments, the second composition does not comprise reducing agents. In some embodiments, the second composition does not comprise denaturing agents.

[0300] The structure of a protein may be referred to in terms of its primary, secondary, and tertiary structures. The primary structure of a protein is the linear sequence of amino acids linked together via peptide bonds to form a polypeptide backbone. The secondary structure of a protein refers to the local folding of the polypeptide backbone into specific shapes and patterns that are stabilized by hydrogen bonding between amino acids which are close to each other in a three-dimensional space. The tertiary structure of a protein refers to three-dimensional shape of a protein which develops due to the interaction of distant amino acids in a three-dimensional space (Buxbaum, 2019). Secondary structures of proteins include alpha-helices, beta-sheets, beta-helices, and amorphous structures. Alpha-helices are spiral structures held together by hydrogen bonds between the amino acids in the polypeptide backbone. Beta-sheets are flat structures in which hydrogen bonds between adjacent protein chains result in the formation of pleated or accordion-like structures. Beta-helices are helical structures held together by hydrogen bonding between adjacent proteins (Buxbaum, 2019).

[0301] Amorphous, disordered, or denatured, used interchangeably herein, are protein structures which lack a well-defined or ordered secondary structure.

[0302] The primary, secondary and tertiary structures of polypeptides may be disrupted by applying appropriate agents to denature and / or reduce those polypeptides. However, in the production of the filaments of this disclosure, it is, in some embodiments, desirable to leave the primary structure intact, in order to maximise polypeptide chain entanglement. Alkali and acid solvents have traditionally been used to solubilise and denature polypeptides, but these solvents may also disrupt the primary structure, in addition to the secondary and tertiary structures. By contrast, aqueous urea may solubilise polypeptides, and when used together with reducing agents may denature the secondary and tertiary structures, without disrupting the primary structure (Preparation and characterisation of bioplastics made from cottonseed protein, 2012).

[0303] The disruption of the secondary and tertiary structures may enable the solvation and stabilisation of the polypeptide backbone structures. The linear nature of the unfolded molecular backbones may facilitate the lamination of the molecules in solution, which decreases the viscosity of the solution. On extrusion, the linear nature of the molecules in solutions may ensure chain entanglement and alignment to enable the realisation of appropriate mechanical properties. By maximising polypeptide chain length, the degree of polypeptide chain entanglement may be maximised (Preparation and characterisation of bioplastics made from cottonseed protein, 2012).

[0304] In some embodiments, solubilisation and denaturation of polypeptides with reducing agents is preferred.

[0305] The denaturation and reduction of polypeptides may be achieved by treatment with a number of chemical agents.

[0306] Urea is widely used to denature polypeptides for proteomic and metabolomic research, as the secondary and tertiary structures may be disrupted without the destruction of the molecular backbone. Urea may solubilise polypeptides through the interaction of hydrophobic motifs on the surface of the polypeptide tertiary structure to expose and solvate hydrophilic motifs in the protein structure (The molecular basis for the chemical denaturation of proteins by urea, 2003). In some embodiments, the second composition comprises urea.

[0307] The concentration of urea in the second composition may be within the range of 0 to 9.074 mol / L.

[0308] In some embodiments, the concentration of urea in the second composition is within the range of 0 mol / L to 9.074 mol / L.

[0309] In some embodiments, the concentration of urea in the second composition is within the range of 0 mol / L to 1 mol / L; preferably, 0.01 mol / L to 0.9 mol / L; more preferably, 0.1 mol / L to 0.5 mol / L; even more preferably, 0.15 mol / L to 0.3 mol / L; and most preferably, 0.2 mol / L to 0.25 mol / L.

[0310] In some embodiments, the concentration of urea in the second composition is within the range of 1 mol / L to 9.074 mol / L; preferably, 4 mol / L to 9 mol / L; more preferably, 6 mol / L to 8.2 mol / L; even more preferably, 7 mol / L to 8.1 mol / L; and most preferably, 7.5 mol / L to 8 mol / L.

[0311] In some embodiments, the concentration of urea in the second composition is within the range of 0 mol / L to 0.01 mol / L, 0.01 mol / L to 0.1 mol / L, 0.1 mol / L to 0.2 mol / L, 0.2 mol / L to 0.3 mol / L, 0.3 mol / L to 0.4 mol / L, 0.4 mol / L to 0.5 mol / L, 0.5 mol / L to 0.6 mol / L, 0.6 mol / L to 0.7 mol / L, 0.7 mol / L to 0.8 mol / L, 0.8 mol / L to 0.9 mol / L or 0.9 mol / L to 1 mol / L.

[0312] In some embodiments, the concentration of urea in the second composition is within the range of 1 mol / L to 2 mol / L, 2 mol / L to 3 mol / L, 3 mol / L to 4 mol / L, 4 mol / L to 5 mol / L, 5 mol / L to 6 mol / L, 6 mol / L to 7 mol / L, 7 mol / L to 8 mol / L, 8 mol / L to 9 mol / L, or 9 mol / L to 9.074 mol / L.

[0313] Treatment with acid or alkali may also be used to denature and solubilise polypeptides, such as those in the solvent of the second composition in Step (a). However, the use of acid and alkali chemical treatments may disrupt the primary structure of the treated polypeptides (Spinnability and rheological properties of globular soy protein solution, 2019).

[0314] Examples of acids which may be used as denaturing agents include, but are not limited to, oxalic acid, malic acid, succinic acid, adipic acid, tartaric acid, citric acid, malonic acid, acetic acid, formic acid, sulphuric acid, phosphoric acid, nitric acid, and / or hydrochloric acid.

[0315] Examples of alkalis which may be used as denaturing agents include, but are not limited to, sodium hydroxide, potassium hydroxide, ammonia, calcium hydroxide, magnesium hydroxide, sodium bicarbonate and / or sodium carbonate. Examples of other denaturing agents which may be used include, but are not limited to, guanidine hydrochloride, sodium dodecyl sulphate (SDS), Triton X-100, ethanol, acetone, dimethyl sulfoxide (DMSO), polysorbates, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sophorolipids, and chaotropic salts (e.g., ammonium sulphate).

[0316] It is also contemplated that the polypeptides of this disclosure may be denatured by treatment with enzymes and / or by the application of heat.

[0317] Reducing agents may disrupt disulphide bonds in the polypeptides of the first composition and prevent their reformation by inhibiting their oxidation, thereby aiding the disruption of the secondary and tertiary structures and the formation of a linear molecular backbone.

[0318] Examples of suitable reducing agents which may be used include, but are not limited to, N-acetyl- cysteine, L-cysteine, glutathione, ascorbic acid, citric acid, tartaric acid, malic acid, sodium borohydride, sodium sulphite, sodium bisulphite, sodium metabisulphite, sodium hypophosphite, sodium hydrosulphite, mercaptoethanol and dithiothreitol.

[0319] In some embodiments, one or more denaturing agents, reducing agents, and / or combinations thereof, are included with the solvent in the second composition in order to aid the solvation of the polypeptides of the first composition to form the third composition.

[0320] In some embodiments, the concentration of each denaturing and / or reducing agent are within the range of 0.01 to 50 % (by mass) of the polypeptides in the first composition and are included with the solvent of the second composition.

[0321] In some embodiments, the concentration of the denaturing and / or reducing agents in the second composition is within the range of 0.01 to 0.1 %, 0.1 to 1 %, 1 % to 10 %, 10 % to 25 %, or 25 % to 50 % (by mass) of the polypeptides in the first composition.

[0322] In some embodiments, the concentration of each denaturing and / or reducing agent are within the range of 0.01 to 1500 % (by mass) of the polypeptides in the first composition and are included with the solvent of the second composition.

[0323] In some embodiments, the concentration of the denaturing and / or reducing agents in the second composition is within the range of 50 to 75 %, 75 to 100 %, 100 % to 200 %, 200 % to 300 %, 300 % to 400 %, 400 % to 500 %, 500 % to 600 %, 600 % to 700 %, 700 % to 800 %, 800 % to 900 %, 900 % to 1000 %, or 1000 % to 1500 %, (by mass) of the polypeptides in the first composition. In some embodiments, the pH of the second composition is within the range of 0 to 14; preferably, within the range of 5.01 to 9.99; and most preferably, within the range of 8.5 to 9.99.

[0324] In some embodiments, the pH of the second composition is within the range of 0 to 1 ; 1 to 2; 2 to 3; 3 to 4; 4 to 5; 5 to 6; 6 to 7; 7 to 8; 8 to 9; 9 to 10; 10 to 1 1 ; 11 to 12; 12 to 13; or 13 to 14.

[0325] In some embodiments, the pH of the second composition is within the range of 9.0 to 9.99, 9.1 to 9.8, 9.15 to 9.7, or 9.2 to 9.4.

[0326] In some embodiments, the pH of the second composition is within the range of 8.0 to 8.5; 8.5 to 9.0; 9.0 to 9.5; 9.5 to 9.99; 9.99 to 10.5; 10.5 to 1 1 ; 11 to 11 .5; 11 .5 to 12; or 12 to 14.

[0327] In some embodiments, in Step (a), the first composition comprises, consists essentially of, or consists of, soybean polypeptides.

[0328] In some embodiments, in Step (a), the solvent in the second composition is water.

[0329] In some embodiments, in Step (a), the denaturing agents and / or reducing agents in the second composition comprise urea, N-acetyl-cysteine and / or sodium sulphite.

[0330] In some embodiments, Step (a) comprises combining:

[0331] (i) a first composition comprising, consisting essentially of, or consisting of, soybean polypeptides with,

[0332] (ii) a second composition comprising water, and one or more salts, denaturing and / or reducing agents selected from urea, sodium bicarbonate, N-acetyl-cysteine and sodium sulphite, at a pH that is within the range of 8.0 to 9.5; to produce a third composition.

[0333] In some embodiments, Step (a) comprises combining:

[0334] (i) a first composition comprising, consisting essentially of, or consisting of, mung bean polypeptides with,

[0335] (ii) a second composition comprising water, and one or more salts, denaturing and / or reducing agents selected from urea, sodium bicarbonate, N-acetyl-cysteine and sodium sulphite, at a pH that is within the range of 8.0 to 9.5; to produce a third composition.

[0336] In some embodiments, Step (a) comprises combining: (i) a first composition comprising, consisting essentially of, or consisting of, chickpea polypeptides with,

[0337] (ii) a second composition comprising water, and one or more salts, denaturing and / or reducing agents selected from urea, sodium bicarbonate, N-acetyl-cysteine and sodium sulphite, at a pH that is within the range of 8.0 to 9.5; to produce a third composition.

[0338] In some embodiments, Step (a) comprises combining:

[0339] (i) a first composition comprising, consisting essentially of, or consisting of, sunflower seed polypeptides with,

[0340] (ii) a second composition comprising water, and one or more salts, denaturing and / or reducing agents selected from urea, sodium bicarbonate, N-acetyl-cysteine and sodium sulphite, at a pH that is within the range of 8.0 to 9.5;

[0341] In some embodiments, Step (a) comprises combining:

[0342] (i) a first composition comprising, consisting essentially of, or consisting of, faba bean polypeptides with,

[0343] (ii) a second composition comprising water, and one or more salts, denaturing and / or reducing agents selected from urea, sodium bicarbonate, N-acetyl-cysteine and sodium sulphite, at a pH that is within the range of 8.0 to 9.5;

[0344] Any of the first, second, third and / or fourth compositions disclosed herein may be used in the production of any filament, hollow fibre, polymer, or Prokitein of this disclosure.

[0345] In some embodiments, any of the first, second, third and / or fourth compositions may be used in the production of a filament.

[0346] In some embodiments, any of the first, second, third and / or fourth compositions may be used in the production of a hollow fibre.

[0347] In some embodiments, any of the first, second, third and / or fourth compositions may be used in the production of a semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

[0348] In some embodiments, any of the first, second, third and / or fourth compositions may be used in the production of a Prokitein.

[0349] 1.6 STEP (B)INCUBATING / AGEING THE THIRD COMPOSITION

[0350] Step (b) may comprise incubating the third composition under conditions that may be sufficient to solubilise, denature and / or reduce at least a fraction of the polypeptides of the first composition, to form the fourth composition. As used herein, the term “incubating” refers to a controlled period of time during which a solution or composition (e.g. the third composition) is maintained under defined conditions (e.g., temperature, agitation).

[0351] In Step (b), the viscosity of the third composition may be decreased, and the secondary and tertiary structures of the polypeptides of the first composition may be disrupted.

[0352] In some embodiments, in Step (b), the viscosity of the third composition is decreased, and the secondary and tertiary structures of the polypeptides of the first composition are disrupted.

[0353] In some embodiments, in Step (b), the third composition is incubated under conditions that are sufficient to solubilise, denature and / or reduce at least a fraction of the polypeptides of the first composition, to form the fourth composition.

[0354] In some embodiments, the third composition is incubated for a duration that is within the range of 0 minutes to 7 days; preferably, 10 minutes to 4 days; more preferably, 15 minutes to 1 days; most preferably, 20 minutes to 6 hours.

[0355] In some embodiments, the third composition is incubated for a period of time that is within the range of 0 minutes to 5 minutes, 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 min to 1 hour, 1 hour to 3 hours, 3 hours to 6 hours, 6 hours to 12 hours, 12 hours to 1 day, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, or 6 days to 7 days.

[0356] In some embodiments, the third composition is incubated at a temperature that is within the range of -25 °C to 150 °C; or within the range of 15 °C to 90 °C; or within the range of 20 °C to 90 “C.

[0357] In some embodiments, the third composition is incubated at a temperature that is within the range of -25 °C to 0 °C, 0 °C to 25 °C, 25 °C to 50 °C, 50 °C to 75 °C, 75 °C to 100 °C, 100 °C to 125 °C, or 125 °C to 150 °C.

[0358] In some embodiments, the third composition is incubated at room temperature.

[0359] Room temperature may refer a temperature within the range of 15 °C to 30 °C. In some embodiments, room temperature is a temperature within the range of 15 °C to 30 °C, preferably 18 °C to 25 °C, more preferably 19 °C to 22 °C, and most preferably 20 °C. In some embodiments, in Step (b), the third composition is incubated at a temperature between 18 °C and 25 °C.

[0360] In some embodiments, in Step (b), the third composition is incubated for 2 days to 4 days at a temperature between 18 °C and 25 °C.

[0361] In some embodiments, the pH of the third composition is within the range of 0 to 14; preferably, within the range of 5.01 to 9.99; and most preferably, within the range of 8.5 to 9.99.

[0362] In some embodiments, the pH of the third composition is within the range of 0 to 1 ; 1 to 2; 2 to 3; 3 to 4; 4 to 5; 5 to 6; 6 to 7; 7 to 8; 8 to 9; 9 to 10; 10 to 1 1 ; 1 1 to 12; 12 to 13; or 13 to 14.

[0363] In some embodiments, the pH of the third composition is within the range of 9.0 to 9.99, 9.1 to 9.8, 9.2 to 9.7, or 9.3 to 9.4.

[0364] In some embodiments, the pH of the third composition is within the range of 9.99 to 10.5; 10.5 to 11 ; 1 1 to 11.5; 1 1.5 to 12; or 12 to 14.

[0365] As used interchangeably herein, the terms “mixing” and “blending”, may refer to the physical combination of two or more substances or components, which may involve dissolution, and may produce homogenous or heterogeneous systems including, but not limited to, suspensions, emulsions, or multiphasic mixtures of solids and liquids. Mixing may be carried out mechanically.

[0366] In Step (b), the third composition may be mixed. Mixing may be carried out using equipment such as, but not limited to, mechanical stirrers, helical ribbon impellers, blenders, homogenisers, ribbon blenders, planetary mixers, sigma blade mixers, vacuum mixers, colloid mills, twin-screw extruders and / or high- shear mixers, depending on the viscosity and nature of the third composition.

[0367] In some embodiments, the third composition is mixed in Step (b). In certain embodiments, mixing is performed using a helical ribbon impeller or a homogeniser. In other certain embodiments, mixing is performed using a vacuum mixer.

[0368] In some embodiments, the third composition is not mixed in Step (b).

[0369] In some embodiments, the third composition is mixed for a period of time that is within the range of 0 minutes to 5 minutes, 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 min to 1 hour, 1 hour to 3 hours, 3 hours to 6 hours, 6 hours to 12 hours, 12 hours to 1 day, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, or 6 days to 7 days. In some other embodiments, the third composition is mixed throughout a portion of, the majority of, or the entirety of the incubation step.

[0370] In some embodiments, the third composition is mixed at a speed that is within the range of 0 rpm to 20,000 rpm; preferably, 50 rpm to 10,000 rpm; more preferably, 100 rpm to 5,000 rpm; even more preferably, 200 rpm to 2,500 rpm; and most preferably, 400 rpm to 1 ,000 rpm.

[0371] In some other embodiments, the third composition is mixed at a speed that is within the range of 0 rpm to 5 rpm, 5 rpm to 50 rpm, 50 rpm to 100 rpm, 100 rpm to 150 rpm, 150 rpm to 200 rpm, 200 rpm to 300 rpm, 300 rpm to 400 rpm, 400 rpm to 500 rpm, 500 rpm to 600 rpm, 600 rpm to 700 rpm, 700 rpm to 800 rpm, 800 rpm to 900 rpm, 900 rpm to 1 ,000 rpm, 1 ,000 rpm to 1 ,100 rpm, 1 ,100 rpm to 1 ,200 rpm, 1 ,200 rpm to 1 ,300 rpm, 1 ,300 rpm to 1 ,400 rpm, 1 ,400 rpm to 1 ,500 rpm, 1 ,500 rpm to 1 ,600 rpm, 1 ,600 rpm to 1 ,700 rpm, 1 ,700 rpm to 1 ,800 rpm, 1 ,800 rpm to 1 ,900 rpm, 1 ,900 rpm to 2,000 rpm, 2,000 rpm to 2,100 rpm, 2,100 rpm to 2,200 rpm, 2,200 rpm to 2,300 rpm, 2,300 rpm to 2,400 rpm, 2,400 rpm to 2,500 rpm,

[0372] 2.500 rpm to 2,600 rpm, 2,600 rpm to 2,700 rpm, 2,700 rpm to 2,800 rpm, 2,800 rpm to 2,900 rpm, 2,900 rpm to 3,000 rpm, 3,000 rpm to 3,100 rpm, 3,100 rpm to 3,200 rpm, 3,200 rpm to 3,300 rpm, 3,300 rpm to 3,400 rpm, 3,400 rpm to 3,500 rpm, 3,500 rpm to 3,600 rpm, 3,600 rpm to 3,700 rpm, 3,700 rpm to 3,800 rpm, 3,800 rpm to 3,900 rpm, 3,900 rpm to 4,000 rpm, 4,000 rpm to 4,500 rpm, 4,500 rpm to 5,000 rpm, 5,000 rpm to 5,500 rpm, 5,500 rpm to 6,000 rpm, 6,000 rpm to 6,500 rpm, 6,500 rpm to 7,000 rpm, 7,000 rpm to 7,500 rpm, 7,500 rpm to 8,000 rpm, 8,000 rpm to 8,500 rpm, 8,500 rpm to 9,000 rpm, 9,000 rpm to

[0373] 9.500 rpm, 9,500 rpm to 10,000 rpm, 10,000 rpm to 10,500 rpm, 10,500 rpm to 1 1 ,000 rpm, 11 ,000 rpm to 11 ,500 rpm, 11 ,500 rpm to 12,000 rpm, 12,000 rpm to 12,500 rpm, 12,500 rpm to 13,000 rpm, 13,000 rpm to 13,500 rpm, 13,500 rpm to 14,000 rpm, 14,000 rpm to 14,500 rpm, 14,500 rpm to 15,000 rpm, 15,000 rpm to 15,500 rpm, 15,500 rpm to 16,000 rpm, 16,000 rpm to 16,500 rpm, 16,500 rpm to 17,000 rpm, 17,000 rpm to 17,500 rpm, 17,500 rpm to 18,000 rpm, 18,000 rpm to 18,500 rpm, 18,500 rpm to

[0374] 19,000 rpm, 19,000 rpm to 19,500 rpm, or 19,500 rpm to 20,000 rpm.

[0375] In some preferred embodiments, the third composition is mixed at a speed that is within the range of 100 rpm to 400 rpm.

[0376] In some preferred embodiments, the third composition is mixed at a speed that is within the range of 400 rpm to 1000 rpm.

[0377] In some preferred embodiments, the third composition is mixed at a speed that is within the range of 1 ,000 rpm to 2,500 rpm.

[0378] In some embodiments, the third composition is mixed at room temperature. In some embodiments, the third composition is mixed at a temperature that is within the range of -25 °C to 150 °C; or within the range of 15 °C to 90 °C; or within the range of 20 °C to 90 “C.

[0379] In some embodiments, the third composition is mixed at a temperature that is within the range of -25 °C to 0 °C, 0 °C to 25 °C, 25 °C to 50 °C, 50 °C to 75 °C, 75 °C to 100 °C, 100 °C to 125 °C, or 125 °C to 150 °C.

[0380] In some embodiments, the third composition is mixed at a temperature within the range of 15 °C to 30 °C; preferably 18 °C to 25 °C, more preferably 19 °C to 22 °C and most preferably 20 °C.

[0381] The third composition may be mixed at the local atmospheric pressure, or at pressures below that of the local atmospheric pressures, such that the environment constitutes a vacuum.

[0382] In some embodiments, a vacuum is considered to comprise any pressure below 101 ,325 Pa.

[0383] In some embodiments, the third composition is mixed at the local atmospheric pressure.

[0384] In some embodiments, the third composition is mixed under vacuum.

[0385] In some embodiments, the third composition is mixed at pressures that are within the range of 10-8Pa to 10-5Pa (ultra-high vacuum); 10-5Pa to 0.1 Pa (high vacuum); 0.1 Pa to 100 Pa (fine vacuum); and / or 100 Pa to 100,000 Pa (rough vacuum).

[0386] In some embodiments, the third composition is mixed at pressures that are within the range of 10-8Pa to 10’7Pa; 10’7Pa to 10’6Pa; 10’6Pa to 10’5Pa; 10’5Pa to 10’4Pa; 10’4Pa to 10’3Pa; 10’3Pa to 0.01 Pa; 0.01 Pa to 0.1 Pa; 0.1 Pa to 1 Pa; 1 Pa to 10 Pa; 10 Pa to 100 Pa; 100 Pa to 1 ,000 Pa; 1 ,000 Pa to 10,000 Pa; 10,000 Pa to 25,000 Pa; 25,000 Pa to 50,000 Pa; 50,000 Pa to 100,000 Pa; and / or 100,000 Pa to 125,000 Pa.

[0387] In some other preferred embodiments, during incubation at room temperature, the third composition is mixed a speed that is 400 rpm for 20 minutes with a helical ribbon impeller.

[0388] In some other preferred embodiments, during incubation at room temperature, the third composition is mixed a speed that is 1 ,000 rpm for 10 minutes with a helical ribbon impeller.

[0389] In some other preferred embodiments, during incubation at room temperature, the third composition is mixed a speed that is 2,000 rpm for 10 minutes with a helical ribbon impeller. In some embodiments, the viscosity of the fourth composition, at 25 °C and a shear rate of 2 s-1, is within the range of 100 cP to 200,000 cP, 100 cP to 1000 cP, 1000 cP to 5,000 cP, 5,000 cP to 10,000 cP, 10,000 cP to 50,000 cP, 50,000 cP to 100,000 cP or 100,000 cP to 200,000 cP, 200,000 cP to 300,000 cP, 300,000 cP to 400,000 cP, 400,000 cP to 500,000 cP, 500,000 cP to 600,000 cP, 600,000 cP to 700,000 cP, 700,000 cP to 800,000 cP, 800,000 cP to 900,000 cP, 900,000 cP to 1 ,000,000 cP, 1 ,000,000 cP to 1 ,100,000 cP, 1 ,100,000 cP to 1 ,200,000 cP, 1 ,200,000 cP to 1 ,300,000 cP, 1 ,300,000 cP to 1 ,400,000 cP, 1 ,400,000 cP to 1 ,500,000 cP, 1 ,500,000 cP to 1 ,600,000 cP, 1 ,600,000 cP to 1 ,700,000 cP, 1 ,700,000 cP to 1 ,800,000 cP, 1 ,800,000 cP to 1 ,900,000 cP, 1 ,900,000 cP to 2,000,000 cP, 2,000,000 cP to 3,000,000, 3,000,000 cP to 4,000,000, 4,000,000 cP to 5,000,000, 5,000,000 cP to 10,000,000, 10,000,000 cP to 15,000,000, 15,000,000 cP to 20,000,000, 20,000,000 cP to 25,000,000, 25,000,000 cP to 30,000,000, or 30,000,000 cP to 35,000,000

[0390] Brookfield rotational viscometers are the most common device used in the evaluation of sample viscosities. Brookfield rotational viscometers measure the torque required to rotate a spindle in a fluid. For a Newtonian fluid, as the rate of spindle rotation or the surface area of the spindle used increases, the torque required will increase linearly. By evaluating the viscosity of a fluid at a given shear rate and temperature, a characteristic apparent viscosity may be measured. How the relationship between viscosity and shear rate develops determines the rheological characteristic of a fluid. Fluids may be Newtonian, in which shear rate and viscosity are linearly proportional; shear thinning, where the apparent viscosity decreases with an increase in shear rate; shear thickening, where the apparent viscosity increases with an increase in shear rate; a Bingham plastic fluid, which requires a finite yield stress before they begin to flow but then exhibit a linear shear rate and apparent viscosity profile; or a Bingham pseudoplastic fluid, which require a finite yield stress before they flow and exhibit a non-linear shear rate and apparent viscosity profile (More Solutions to Sticky Problems: A guide to getting more from your Brookfield Viscometer, 2005).

[0391] Rotational viscometers produced by other manufacturers may be used to evaluate viscosity.

[0392] In some embodiments, the fraction of the polypeptides in the third composition that are denatured and / or reduced is higher than 0 %; preferably, higher than 1 %; more preferably, higher than 5 %; and most preferably, higher than 10 %.

[0393] In some embodiments, the fraction of the polypeptides in the third composition that are denatured and / or reduced is higher than 10 %; preferably, higher than 20 %; more preferably, higher than 30 %; and most preferably, higher than 40 %. In some embodiments, the fraction of the polypeptides in the third composition that are denatured and / or reduced is higher than 40 %; preferably, higher than 50 %; more preferably, higher than 60 %; and most preferably, higher than 70 %.

[0394] In some embodiments, the fraction of the polypeptides in the third composition that are denatured and / or reduced is higher than 70 %; preferably, higher than 80 %; more preferably, higher than 90 %; and most preferably, higher than 99.99 %.

[0395] In some embodiments, the fraction of the polypeptides in the third composition that are denatured and / or reduced is within the range of 0 % to 5 %, 5 % to 10 %, 10 % to 20 %, 20 % to 30 %, %, 30 % to 40 %, or 40 % to 50 %.

[0396] In some embodiments, the fraction of the polypeptides in the third composition that are denatured and / or reduced is within the range of 50 % to 60 %, 60 % to 70 %, 70 % to 80 %, 80 % to 90 %, or 90 % to 99.99 %.

[0397] The extent of denaturation and reduction of the polypeptides in the third composition may be quantified through the use of Fourier transform infrared (FTIR) spectroscopy, circular dichroism, wide-angle X-ray scattering (WAXS) or Raman spectroscopy. By recording representative spectra of the starting organic species, changes in band intensity associated with disulphide bonds may be used to evaluate the degree of reduction. Similarly, changes in band intensity associated with alpha-helices and beta-sheets may be used to determine changes in protein secondary structure.

[0398] OTHER ADDITIVES

[0399] VOID INCLUSION ELEMENTS

[0400] One or more void inclusion elements may be introduced into the first, second, third or fourth compositions to aid in the controlled formation of pores in the filaments of this disclosure. Void inclusion elements may impart porosity by acting as pore templates. This may occur as the dope solution undergoes phase inversion and solidifies around particles of the void inclusion element. Upon the removal of the void inclusion elements, the filaments may be left with a microstructure that consists of a pore morphology that is a negative cast of the original particles of the void inclusion elements. The void inclusion elements may be in the form of a solid, liquid gas, or a mixture thereof.

[0401] As used herein, the term “pore volume” refers to the total volume of open space or pores within the material. The term “porosity” refers to the fraction of material’s total volume that is occupied by open space or pores. Both pore volume and / or porosity may be characterised using methods such as mercury porosimetry.

[0402] The pore volume and / or porosity of a material may also be determined by image analysis. Images which may be used to determine the porosity of materials include those produced through scanning electron microscopy. By considering the ratio of black and white pixels in a binarized image, the porosity may be determined by one skilled in the art.

[0403] In some embodiments, one or more void inclusion elements are introduced into the first, second, third and / or fourth compositions to aid in the controlled formation of pores in the filaments of this disclosure.

[0404] In some embodiments, void inclusion elements include, but are not limited, to powdered or ground calcium carbonate.

[0405] In some embodiments, void inclusion elements include, but are not limited to, crushed or ground ice.

[0406] In some embodiments, void inclusion elements include, gases such as, but are not limited, air, nitrogen, oxygen, carbon dioxide, helium, neon, argon, xenon, argon, and / or mixtures thereof.

[0407] In some embodiments, void inclusion elements include salts such as, but are not limited to, sodium chloride, and potassium chloride.

[0408] Polysaccharides may be used as void inclusion elements. Examples of polysaccharides that may be used as void inclusion elements include, but are not limited to, chitin; chitosan; starches derived from wheat; rice; potato or corn; alginate; agar; hyaluronic acid; dextran; chondroitin sulphate; carrageenan; carrageenan-kappa; carrageenan-iota; pullulan; xanthan gum; gellan gum; pectin; cellulose; microcrystalline cellulose; carboxymethyl cellulose; cellulose acetate; lignin; kraft lignin; alkali lignin; and organosolv lignin.

[0409] In some embodiments, one or more polysaccharides are used as void inclusion elements.

[0410] In some particular embodiments, sodium alginate is used as a void inclusion element.

[0411] In some embodiments, void inclusion elements include lipids such as, but are not limited to, plant oils (e.g., almond, avocado, canola, coconut, corn, flaxseed, grape seed, hemp seed, jojoba, mustard, olive, palm, peanut, pumpkin seed, rice bran, safflower, sesame, soybean, sunflower, walnut).

[0412] In some embodiments, wherein lipids are used as void inclusion elements, the lipids have secondary process functions that include, but are not limited to, acting as flavour enhancers and / or as plasticisers. In some embodiments, void inclusion elements include one or more polyols and / or polymers of polyols.

[0413] In some embodiments, void inclusion elements include water soluble polymers such as, but are not limited to polyvinyl alcohol.

[0414] In some embodiments, void inclusion elements are added to the first composition in Step (a).

[0415] In other embodiments, void inclusion elements are added to the second composition in Step (a).

[0416] In other embodiments, void inclusion elements are added to the third composition before or during the incubation step in Step (b).

[0417] In other embodiments, void inclusion elements are added to the third composition after the incubation step in Step (b), and then mixed.

[0418] In other embodiments, void inclusion elements are added to the fourth composition in Step (b).

[0419] In a preferred embodiment, void inclusion elements are added to the fourth composition in Step (b).

[0420] In one embodiment, void inclusion elements are added to the compositions in Step (a) or Step (b), such that the void inclusion elements comprise 1 % to 80 % of the fourth composition; preferably 20 % to 75 %; more preferably 30 % to 70 %; and most preferably 35 % to 65 % of the fourth composition.

[0421] In some embodiments, void inclusion elements are added to the compositions in Step (a) or Step (b), such that the void inclusion elements comprise 1 % to 5 %, 5 % to 10 %, 10 % to 15 %, 15 % to 20 %, 20 % to 25 %, 25 % to 30 %, 30 % to 35 %, 35 % to 40 %, 40 % to 45 %, 45 % to 50 %, 50 % to 55 %, 55 % to 60 %, 60 % to 65 %, 65 % to 70 %, 70 % to 75 %, and / or 75 % to 80 % of the fourth composition.

[0422] In some embodiments, the third composition and / or fourth compositions is mixed, following the addition of one or more void inclusion elements, under conditions (mixer type, mixing duration, temperature, pressure, and mixing speed) sufficient to influence or control the pore characteristics (e.g., pore size, pore volume, and porosity) of a filament of this disclosure.

[0423] PLASTICISERS

[0424] One or more components may be added as plasticisers to any of the compositions of Step (a) or Step (b).

[0425] As used herein, the term "plasticiser" refers to a substance added to a material to increase its flexibility, workability and / or extensibility by reducing intermolecular forces between polymer chains. A plasticiser may function by increasing the free volume within the polymer matrix, thereby enhancing chain mobility and lowering the glass transition temperature (Tg) of the material. In some embodiments, one or more plasticisers are added to one or more of the compositions of Step (a) and / or (b).

[0426] Plasticisers may include, but are not limited to, lipids (e.g., plant oils, epoxidised plant oils), polyols (e.g., glycerol, sorbitol), polymers of polyols (e.g., polyethylene glycol), esters (e.g., phthalates, citrates), and other compounds capable of plasticising natural or synthetic polymers.

[0427] Polysaccharides, such as dextran, pullulan or low-molecular-weight chitosan may also be used as plasticisers.

[0428] In some embodiments, one or more polysaccharides, lipids, polyols, polymers of polyols or esters are added to the compositions of Step (a) and / or Step (b) as plasticisers.

[0429] In some embodiments, one or more polysaccharides are added to one or more of the compositions of Step (a) and / or (b) as plasticisers.

[0430] In some embodiments, one or more lipids are added to one or more of the compositions of Step (a) and / or (b) as plasticisers.

[0431] In some embodiments, one or more polyols and / or polymers of polyols are added to one or more of the compositions of Step (a) and / or (b) as plasticisers.

[0432] In some embodiments, one or more esters are added to one or more of the compositions of Step (a) and / or (b) as plasticisers.

[0433] In some embodiments, one or more plasticisers are added to one or more of the compositions of Step (a) and / or Step (b) at any stage.

[0434] In some embodiments, one or more components added to a composition of Step (a) or Step (b) acts as a plasticiser, even if plasticisation is not their primary intended function.

[0435] In a preferred embodiment, one or more plasticisers is added to the fourth composition in Step (b).

[0436] In some embodiments, plasticisers are not added to the compositions of Step (a) and / or Step (b).

[0437] The plasticisers may also have secondary process functions that include, but are not limited to, acting as flavour enhancers and / or void inclusion elements. In some embodiments, a plasticiser added to a composition of Step (a) and / or Step (b) is also able to act as a flavour enhancer.

[0438] In some embodiments, a plasticiser added to a composition of Step (a) and / or Step (b) is also able to act as a void inclusion element.

[0439] In some embodiments, one or more plasticisers are added to one or more of the compositions in Step (a) and / or Step (b), such that they each comprise 0.01 % to 80 %; preferably 0.05% to 1 %; more preferably 0.1 % to 0.75 %; and most preferably 0.2 % to 0.5 % (w / v) of the fourth composition.

[0440] In some embodiments, one or more plasticisers are added to the compositions in Step (a) or Step (b), such that they each comprise 0.01 % to 0.05 %, 0.01 % to 0.05 %, 0.05 % to 0.1 %, 0.1 % to 0.2 %, 0.2 % to 0.3 %, 0.3 % to 0.4 %, 0.4 % to 0.5 %, 0.5 % to 0.6 %, 0.6 % to 0.7 %, 0.7 % to 0.8 %, 0.8 % to 0.9 %, 0.9 % to 1 %, 1 % to 5 %, 5 % to 10 %, 10 % to 15 %, 15 % to 20 %, 20 % to 25 %, 25 % to 30 %, 30 % to 35 %, 35 % to 40 %, 40 % to 45 %, 45 % to 50 %, 50 % to 55 %, 55 % to 60 %, 60 % to 65 %, 65 % to 70 %, 70 % to 75 %, and / or 75 % to 80 % (w / v) of the fourth composition.

[0441] FOOD ADDITIVES

[0442] One or more food additives may be added to any of the compositions of Step (a) and / or Step (b).

[0443] In some embodiments, wherein a filament of this disclosure is edible or intended to be edible, the inclusion of additives in one or more compositions of Step (a) and / or Step (b) may enhance one or more food-related or processing-related properties, such as, but not limited to, flavour, texture, colour, aroma, shelf life, nutritional content, mouthfeel, workability, processability, or compatibility with other food ingredients.

[0444] In some embodiments, additives which may be added to a composition of Step (a) or Step (b) include those listed in EU Regulation (EC) No 1333 / 2008 and Annexes thereof. Such additives include: sweeteners; colouring agents; preservatives; antioxidants; carriers; acids; acidity regulators; anti-caking agents; anti-foaming agents; bulking agents; emulsifiers; emulsifying salts; firming agents; flavour enhancers (flavouring agents); foaming agents; gelling agents; glazing agents; humectants; modified starches; packaging gases; propellants; raising agents; sequestrants; stabilisers; thickeners; and flour treatment agents. These additives may be present any combination.

[0445] In some embodiments, additives which may be added to a composition of Step (a) or Step (b) include those on the GRAS Substances (SCOGS) Database or in Title 21 of the Code of Federal Regulations (CFR), Parts 182 through 184, or any other additive considered GRAS by the FDA as of the 1st January 2024. These additives include: sweeteners; colouring agents; preservatives; antioxidants; carriers; acids; acidity regulators; anti-caking agents; anti-foaming agents; bulking agents; emulsifiers; emulsifying salts; firming agents; flavour enhancers (flavouring agents); foaming agents; gelling agents; glazing agents; modified starches; raising agents; sequestrants; stabilisers; and thickeners. These additives may be present any combination.

[0446] In some embodiments, food additives are not added to the compositions of Step (a) or Step (b).

[0447] One or more components may be added to any of the compositions of Step (a) and / or Step (b) to act as flavouring agents, which may improve the flavour of the filaments of this disclosure.

[0448] In some embodiments, one or more components are added to one or more of the compositions in Step (a) and / or Step (b) to act as flavouring agents.

[0449] One or more components may be added to any of the compositions in Step (a) and / or Step (b) to act as flavouring agents, such that the filaments of this disclosure may act as flavour carrying agents.

[0450] In some embodiments, one or more components are added to one or more of the compositions in Step (a) and / or Step (b) to act as flavouring agents, such that the filaments of this disclosure act as flavour carrying agents.

[0451] In some embodiments, the flavouring agents added to one or more of the compositions in Step (a) and / or Step (b) comprise polysaccharides, lipids, polyols, and / or polymers of polyols.

[0452] In some embodiments, the flavouring agents added to the compositions in Step (a) and / or Step (b) comprise those listed by the European Commission in (EC) No 1334 / 2008 established in 2012 with regulation EU 872 / 2012 and non-food sourced flavourings added in amendments in accordance with Regulation EU 2018 / 1259.

[0453] In some embodiments, flavouring agents are not added to the compositions in Step (a) and / or Step (b).

[0454] In some embodiments, the compositions of Step (a) and / or Step (b) are mixed following the addition of at least one additive, including but not limited to, void inclusion elements, plasticisers, and / or food additives.

[0455] DEGASSING

[0456] In some embodiments, the compositions of Step (a) and / or Step (b) are degassed.

[0457] As used herein, the term “degassing”, also referred to as “degasification”, refers to the partial or complete removal of gas bubbles and / or dissolved gases from any of the compositions disclosed herein. In some embodiments, the third composition is degassed.

[0458] In some embodiments, the fourth composition is degassed.

[0459] In some embodiments, the compositions of Step (a) and / or Step (b) are not degassed.

[0460] Degassing may be achieved via one or more methods which may include, but are not limited to, pressure reduction, thermal regulation, membrane degasification, ultrasonic degassing, freeze-pump-thaw cycling, and / or centrifugation.

[0461] In some embodiments, degassing is achieved via pressure reduction.

[0462] In some embodiments, degassing is achieved via pressure reduction under vacuum.

[0463] In some embodiments, degassing is achieved via thermal regulation.

[0464] In some embodiments, degassing is achieved via membrane degasification.

[0465] In some embodiments, degassing is achieved via ultrasonic degassing.

[0466] In some embodiments, degassing is achieved via freeze-pump-thaw cycling.

[0467] In some embodiments, degassing is achieved via centrifugation.

[0468] In some embodiments, Step (b) comprises adding one or more void inclusion elements into the fourth composition.

[0469] In some embodiments, undissolved components of lower densities in the third composition may be removed prior to Step (c) by employing methods such as, but not limited to, centrifugal density-based separation (density gradient centrifugation) as performed by centrifugation.

[0470] In another embodiment, by employing centrifugal density-based separation, gases and undissolved components of lower densities in the third composition arise to the surface of the third composition, which may then be mechanically removed.

[0471] In some preferred embodiments, the fourth composition is mixed.

[0472] In some preferred embodiments, the fourth composition is mixed for 20 minutes.

[0473] In some preferred embodiments, the fourth composition is degassed.

[0474] In some preferred embodiments, one or more polyol is added to the third composition. In the subsequent ageing process, the third composition is mixed at 1000 rpm and at room temperature with an overhead mixer equipped with a helical blade impeller for 10 minutes to form the fourth composition. The fourth composition is then degassed via centrifugation. In some preferred embodiments, one or more polysaccharide is added to the third composition. In the subsequent ageing process, the third composition is mixed at 1000 rpm and at room temperature with an overhead mixer equipped with a helical blade impeller for 10 minutes to form the fourth composition. The fourth composition is then degassed via centrifugation.

[0475] In some preferred embodiments, one or more polysaccharide and one or more polyol is added to the third composition. In the subsequent ageing process, the third composition is mixed at 1000 rpm and at room temperature with an overhead mixer equipped with a helical blade impeller for 10 minutes to form the fourth composition. The fourth composition is then degassed via centrifugation.

[0476] In some preferred embodiments, one or more lipid is added to the third composition. In the subsequent ageing process, the third composition is mixed at 400 rpm and at room temperature with an overhead mixer equipped with a helical blade impeller for 20 minutes to form the fourth composition. The fourth composition is then degassed via centrifugation.

[0477] In some preferred embodiments, one or more lipid is added to the third composition. In the subsequent ageing process, the third composition is mixed at 1000 rpm and at room temperature with an overhead mixer equipped with a helical blade impeller for 20 minutes to form the fourth composition. The fourth composition is then degassed via centrifugation.

[0478] In some preferred embodiments, one or more lipid is added to the third composition. In the subsequent ageing process, the third composition is mixed at 2000 rpm and at room temperature with an overhead mixer equipped with a helical blade impeller for 20 minutes to form the fourth composition. The fourth composition is then degassed via centrifugation.

[0479] In some embodiments, the fourth composition of any embodiments of this disclosure can be used in the production of Prokitein and / or filaments of this disclosure.

[0480] 1.7 STEP (C)EXTRUDINGZSPINNING

[0481] Step (c) may comprise extruding or spinning the fourth composition through an orifice to produce a plurality of filaments.

[0482] In some embodiments, Step (c) comprises extruding or spinning the fourth composition through an orifice to produce a plurality of filaments. As used herein, the term “dope solution” refers to a fourth composition that is being, has been, or is intended to be extruded or spun. Accordingly, a fourth composition in any embodiment of this disclosure may be referred to as a dope solution.

[0483] A die, sometimes referred to as a spinneret and used interchangeably herein, consists of one or more orifices fed by a corresponding number of inlet streams.

[0484] A die may be in the form of a spinneret or a plurality of orifices.

[0485] In some embodiments, a die comprises a single orifice.

[0486] In some embodiments, a die comprises a co-axial orifice.

[0487] In some embodiments, a die comprises a plurality of orifices.

[0488] In some embodiments, a die comprises a plurality of co-axial orifices.

[0489] A die may be in the form of a spinneret or a plurality of co-axial orifices.

[0490] In some embodiments, a die is in the form of a spinneret or a plurality of co-axial orifices.

[0491] Extrusion may be used to create an extruded product which can be treated to produce filaments of a fixed cross-sectional profile by extruding the fourth composition through one or more orifices (e.g., a die) of the desired cross-section.

[0492] In some embodiments, Step (c) comprises extruding and / or spinning the fourth composition through a die comprising one or more orifices to produce a plurality of filaments.

[0493] The filaments may be cylindrical in shape.

[0494] In some embodiments, the filaments are cylindrical in shape.

[0495] In some embodiments, the dimensions of the die are selected such as to produce filaments having the dimensions specified herein.

[0496] In some embodiments, the fourth composition is extruded through an orifice at a rate that is within the range of 0.1 mL / hour to 100 mL / hour; preferably, within the range of 5 mL / hour to 75 mL / hour; more preferably, within the range of 10 mL / hour to 50 mL / hour; and most preferably at 12 mL / hour.

[0497] In some embodiments, the fourth composition is extruded through an orifice at a rate that is within the range of 0.1 mL / hour to 0.5 mL / hour, 0.5 mL / hour to 1 mL / hour, 1 mL / hour to 5 mL / hour, 5 mL / hour to 10 mL / hour, 10 mL / hour to 15 mL / hour, 15 mL / hour to 20 mL / hour, 20 mL / hour to 25 mL / hour, 25 mL / hour to 30 mL / hour, 30 mL / hour to 35 mL / hour, 35 mL / hour to 40 mL / hour, 40 mL / hour to 45 mL / hour, 45 mL / hour to 50 mL / hour, 50 mL / hour to 60 mL / hour, 60 mL / hour to 70 mL / hour, 70 mL / hour to 80 mL / hour, 80 mL / hour to 90 mL / hour, or 90 mL / hour to 100 mL / hour.

[0498] Each orifice of the die may have profiles that are circular, square, triangular, pentagonal, hexagonal and / or other polygons in shape.

[0499] In some embodiments, each orifice of the die has a profile that is circular, square, triangular, pentagonal, hexagonal and / or other polygons in shape.

[0500] Each orifice of the die may have an internal diameter within the range of 15 pm to 6600 pm.

[0501] In some embodiments, each orifice of the die has an internal diameter within the range of 15 pm to 6600 pm; preferably, 20 pm to 2000 pm; and most preferably, 50 pm to 500 pm.

[0502] In some embodiments, each orifice of the die has an internal diameter within the range of 50 pm to 100 pm.

[0503] In some embodiments, each orifice of the die has an internal diameter within the range of 20 pm to 100 pm.

[0504] In some embodiments, each orifice of the die has an internal diameter within the range of 15 pm to 20 pm, 20 pm to 30 pm, 30 pm to 40 pm, 40 pm to 50 pm, 50 pm to 100 pm, 100 pm to 200 pm, 200 pm to 300 pm, 300 pm to 400 pm, or 400 pm to 500 pm.

[0505] In some embodiments, each orifice of the die has an internal diameter within the range of 500 pm to 1 ,000 pm, 1 ,000 pm to 1 ,500 pm, 1 ,000 pm to 1 ,500 pm, 1 ,500 pm to 2,000 pm, 2,000 pm to 2,500 pm, 2,500 pm to 3,000 pm, 3,000 pm to 3,500 pm, 3,500 pm to 4,000 pm, 4,000 pm to 4,500 pm, 4,500 pm to 5,000 pm, 5,000 pm to 5,500 pm, or 5,500 pm to 6,000 pm, or 6,000 pm to 6,600 pm,

[0506] Dry or wet spinning systems may also be used depending on the composition of the spinning solutions that form the fourth composition.

[0507] As used interchangeably herein, the terms “spinning”, “wet spinning”, and “extruding”, are used interchangeably herein and refer to the production of a filament through a process in which a polymer solution may be passed through a die into a coagulation bath in which the polymer solution may solidify into a filament. The process may be referred to as “wet spinning” as the polymer solution is extruded into a liquid bath. The terms “spinning”, “wet spinning”, and / or “extruding” may therefore refer to a process which comprises: (i) the preparation of a polymer solution; (ii) die / spinneret assembly; (iii) extrusion of the polymer solution; (iv) coagulation of the extruded polymer solution; (v) drawing in which the extruded solidified filaments are stretched; (vi) washing, in which residual solvents are removed; and (vii) drying in which the filaments produced are dried and wound onto a spool for storage and future application.

[0508] In some embodiments, the fourth composition is extruded or spun into air and / or a gaseous atmosphere to produce filaments. The extruded or spun filaments are then directed either into a coagulation bath or into an atmosphere that coagulates, precipitates, and / or dries the filaments.

[0509] In some embodiments, the fourth composition is extruded or spun into an atmosphere and then into a coagulation bath.

[0510] In one preferred embodiment, the fourth composition is extruded through a die directly into a coagulation bath.

[0511] In some embodiments, Step (c) comprises extruding or spinning the fourth composition through a plurality of orifices to form one or more filaments with cylindrical geometry directly into a coagulation bath.

[0512] In some embodiments, Step (c) comprises extruding the fourth composition through an orifice of circular profile. The fourth composition is extruded directly into a coagulation bath filled with coagulation bath solution. The coagulation bath solution comprises sodium citrate and sodium hypophosphite in an aqueous solution at a pH between 8 and 8.5.

[0513] In some embodiments, Step (c) comprises extruding the fourth composition through an orifice of circular profile. The fourth composition is extruded directly into a coagulation bath filled with coagulation bath solution. The coagulation bath solution comprises sodium citrate in an aqueous solution at a pH between 8 and 8.5.

[0514] In some embodiments, Step (c) comprises extruding the fourth composition through an orifice of circular profile. The fourth composition is extruded directly into a coagulation bath filled with coagulation bath solution. The coagulation bath solution comprises sodium malate and sodium hypophosphite in an aqueous solution at a pH between 8 and 8.75.

[0515] In some embodiments, Step (c) comprises extruding the fourth composition through an orifice of circular profile. The fourth composition is extruded directly into a coagulation bath filled with coagulation bath solution. The coagulation bath solution comprises sodium malate in an aqueous solution at a pH between 8 and 8.75. In some embodiments, Step (c) does not comprise extruding or spinning the fourth composition through an orifice to produce a plurality of filaments; and instead comprises shaping the fourth composition using one or more processes comprising extruding, casting, moulding and / or spraying.

[0516] BORE SOLUTION

[0517] In some embodiments, Step (c) comprises extruding or spinning the fourth composition through a plurality of co-axial orifices, together with a bore solution, to form a hollow fibre instead of a filament.

[0518] As used herein, the term “bore solution” refers to a solution which may fill the lumen of a hollow fibre at the moment of extrusion.

[0519] In some embodiments, the bore solution is a solution that fills the lumen of a hollow fibre.

[0520] A bore solution may comprise an aqueous solution.

[0521] In some embodiments, a bore solution comprises an aqueous solution.

[0522] In some embodiments, a bore solution may be formulated to facilitate the covalent crosslinking of polypeptides.

[0523] A bore solution may comprise one or more polycarboxylic acid crosslinking reagents.

[0524] In some embodiments, a coagulation bath solution comprises one or more polycarboxylic acid crosslinking reagents.

[0525] In some embodiments, a coagulation bath solution comprises one or more polycarboxylic acid crosslinking reagents as defined herein.

[0526] In some embodiments, a bore solution comprises sodium citrate.

[0527] In some embodiments, a bore solution comprises sodium malate.

[0528] A bore solution may have the same composition and / or one or more properties (e.g., pH) of a coagulation bath solution as described herein.

[0529] In some embodiments, the bore solution and the coagulation bath solution have the same composition and / or one or more shared properties (e.g., pH). In certain embodiments, the bore solution and the coagulation bath solution are the same solution. In other embodiments, the bore solution differs from the coagulation bath solution in composition, properties (e.g., pH), or both. In some other embodiments, the bore solution comprises thickening agents and / or viscosity modifiers to help maintain the lumen of the hollow fibres.

[0530] In some embodiments, a bore solution comprises one or more polysaccharides as thickening agents and / or viscosity modifiers, wherein the polysaccharides may comprise chitin, chitosan, starches (e.g., those derived from wheat, rice, potato, or corn), alginate, agar, hyaluronic acid, dextran, chondroitin sulphate, carrageenan (e.g., kappa- or iota-carrageenan), pullulan, xanthan gum, gellan gum, and / or pectin.

[0531] In some embodiments, the bore solution is removed after Step (d).

[0532] A bore solution may be extruded through an orifice at a rate that is within the range of 0.001 mL / h to 30 mL / h.

[0533] In some embodiments, the bore solution is extruded through an orifice at a rate that is within the range of 0.001 mL / hour to 10 mL / hour; more preferably, within the range of 0.05 mL / hour to 5 mL / hour; and most preferably at 4 mL / hour.

[0534] In some embodiments, a bore solution is extruded through a die at a rate that is within the range 0.001 mL / h to 0.01 mL / h, 0.01 mL / h to 0.1 mL / h, 0.1 mL / h to 0.5 mL / h, 0.5 mL / h to 1 mL / h, 1 mL / h to 5 mL / h, or 5 mL / h to 10 mL / h.

[0535] In some embodiments, the bore solution is extruded through the die at a rate that is within the range of 10 mL / hour to 15 mL / hour, 15 mL / hour to 20 mL / hour, 20 mL / hour to 25 mL / hour, or 25 mL / hour to 30 mL / hour.

[0536] In some embodiments, the plurality of co-axial orifices comprises 2 to 4 orifices; preferably, 2 to 3 orifices; and most preferably, 2 orifices.

[0537] In some embodiments, the plurality of co-axial orifices comprises 2 orifices, 3 orifices, or 4 orifices.

[0538] SYNTHETIC TEXTILE POLYMERS

[0539] In some embodiments, Step (c) comprises extruding or spinning the fourth composition together with one or more synthetic textile polymers.

[0540] As used herein, the term “composite filament” refers to a filament that comprises one or more semicrystalline polymers comprising crosslinked polypeptides with a beta-sheet secondary structure, and one or more synthetic textile polymers. In some embodiments, a filament of this disclosure is a composite filament.

[0541] In some embodiments, a filament of this disclosure is not a composite filament.

[0542] In some embodiments, a composite filament comprises one or more semi-crystalline polymers comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a betasheet secondary structure; and one or more synthetic textile polymers.

[0543] In some embodiments, a composite filament comprises one or more synthetic textile polymers selected from the group comprising: Polyethylene Terephthalate (PET), Polyester, Polytrimethylene Terephthalate (PTT), Polybutylene Terephthalate (PBT), Nylon 6, Nylon 6,6, Polyurethane, Spandex (Lycra, Elastane), Polyvinyl Chloride (PVC), Acrylic, Modacrylic, Polypropylene, Polytetrafluoroethylene (PTFE or Teflon), Aramids (Kevlar, Nomex), Polybenzimidazole (PBI), Polyvinyl Alcohol (PVA), Polylactic Acid (PLA), Elastomers (rubber fibers), Chlorofibre, Elastolefin, Elastoester, Olefin (Polyolefin), Polyacrylonitrile, Polyhydroxyalkanoates (PHAs), Regenerated Cellulose (Viscose / Rayon, Lyocell / Tencel, Modal, Cupro, Bamboo Viscose, SeaCell), and / or Triacetate.

[0544] In some embodiments, Step (c) comprises extruding or spinning the fourth composition together with a synthetic textile polymer, in a core-and-shell configuration, to produce a core-and-shell configuration filament.

[0545] As used herein, the term “core-and-shell configuration filament” refers to a composite filament that comprises two distinct layers; a central core and an outer shell or sheath. In this configuration each layer comprises different materials.

[0546] In some embodiments, a composite filament is a core-and-shell configuration filament.

[0547] In some embodiments, a composite filament is not a core-and-shell configuration filament.

[0548] In some embodiments, a core-and-shell configuration filament comprises a synthetic textile polymer core and an outer shell comprising a semi-crystalline polymer comprising crosslinked polypeptides with a betasheet secondary structure.

[0549] In some embodiments, a core-and-shell configuration filament comprises a synthetic textile polymer core and an outer shell comprising a polymer, comprising a semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

[0550] In some embodiments, a core-and-shell configuration filament comprises a synthetic textile polymer core and an outer shell comprising a polymer, comprising a regular semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a betasheet secondary structure.

[0551] In some embodiments, a core-and-shell configuration filament comprises a core comprising a semicrystalline polymer comprising crosslinked polypeptides with a beta-sheet secondary structure, and a synthetic textile polymer outer shell.

[0552] In some embodiments, the bore solution comprises a synthetic textile polymer.

[0553] In some embodiments, the bore solution does not comprise a synthetic textile polymer.

[0554] In some embodiments, the bore solution comprises Polyethylene Terephthalate (PET), Polyester, Polytrimethylene Terephthalate (PTT), Polybutylene Terephthalate (PBT), Nylon 6, Nylon 6,6, Polyurethane, Spandex (Lycra, Elastane), Polyvinyl Chloride (PVC), Acrylic, Modacrylic, Polypropylene, Polytetrafluoroethylene (PTFE or Teflon), Aramids (Kevlar, Nomex), Polybenzimidazole (PBI), Polyvinyl Alcohol (PVA), Polylactic Acid (PLA), Elastomers (rubber fibers), Chlorofibre, Elastolefin, Elastoester, Olefin (Polyolefin), Polyacrylonitrile, Polyhydroxyalkanoates (PHAs), Regenerated Cellulose (Viscose / Rayon, Lyocell / Tencel, Modal, Cupro, Bamboo Viscose, SeaCell), and / or Triacetate.

[0555] The mass percent of any one component in a composite filament may be within the range of 0.01 % to 99.99 % (w / w composite filament).

[0556] In some embodiments, the mass percent of any one component in a composite filament is within the range of 1 % to 5 % (w / w composite filament).

[0557] In some embodiments, the mass percent of any one component in a composite filament is within the range of 5 % to 10 % (w / w composite filament).

[0558] In some embodiments, the mass percent of any one component in a composite filament is within the range of 10 % to 90 % (w / w composite filament).

[0559] In some embodiments, the mass percent of any one component in a composite filament is within the range of 20 % to 80 % (w / w composite filament).

[0560] In some embodiments, the mass percent of any one component in a composite filament is within the range of 30 % to 70 % (w / w composite filament).

[0561] In some embodiments, the mass percent of any one component in a composite filament is within the range of 40 % to 60 % (w / w composite filament).

[0562] In some embodiments, the mass percent of any one component in a composite filament is within the range of 0.01 % to 2.5 %, 2.5 % to 5 %, 5 % to 10 %, 10 % to 20 %, 20 % to 30 %, 30 % to 40 %, 40 % to 50 %, 50 % to 60 %, 60 % to 70 %, 70 % to 80 %, 80 % to 90 %, or 90 % to 99.99 %, (w / w composite filament). 1.8 STEP (D)COAGULATION AND CROSSLINKING

[0563] Step (d) comprises treatment with a crosslinking reagent to form inter-polypeptide and / or intrapolypeptide crosslinks.

[0564] Step (d) may comprise treating the plurality of filaments produced in Step (c) with one or more crosslinking reagents which may form inter-polypeptide and / or intra-polypeptide crosslinks within at least a fraction of the polypeptides comprising the filaments, to produce a plurality of crosslinked filaments.

[0565] Step (d) may comprise treating a plurality of filaments produced in Step (c) with one or more polycarboxylic acid crosslinking reagents. In some embodiments, Step (d) comprises treating the plurality of filaments produced in Step (c) with one or more polycarboxylic acid crosslinking reagents.

[0566] Step (d) may comprise treating the plurality of filaments produced in Step (c) with one or more ionic crosslinking reagents. In some embodiments, Step (d) comprises treating the plurality of filaments produced in Step (c) with one or more ionic crosslinking reagents.

[0567] Step (d) may comprise treating the plurality of filaments produced in Step (c) with one or more polycarboxylic acid crosslinking reagents and / or ionic crosslinking reagents. In some embodiments, Step (d) comprises treating the plurality of filaments produced in Step (c) with one of more polycarboxylic acid crosslinking reagents and / or ionic crosslinking reagents.

[0568] In some embodiments, Step (d) comprises treating the plurality of filaments, produced in Step (c), with one of more polycarboxylic acid crosslinking reagents and / or ionic crosslinking reagents.

[0569] Step (d) may comprise treating the plurality of filaments, produced in Step (c), with one of more polycarboxylic acid crosslinking reagents which may form inter-polypeptide and / or intra-polypeptide polycarboxylic acid derived ester, thioester or amide covalent bond crosslinks within at least a fraction of the polypeptides comprising the filaments, to produce a plurality of covalently-crosslinked filaments.

[0570] In some embodiments, Step (d) comprises treating the plurality of filaments with one of more polycarboxylic acid crosslinking reagents to form inter-polypeptide and / or intra-polypeptide polycarboxylic acid derived ester, thioester or amide covalent bond crosslinks within at least a fraction of the polypeptides comprising the filaments, to produce a plurality of covalently-crosslinked filaments.

[0571] Step (d) may comprise treating the plurality of filaments with one or more ionic crosslinking reagents which may form inter-polypeptide and / or intra-polypeptide ionic crosslinks within at least a fraction of the polypeptides comprising the filaments, to produce a plurality of ionically-crosslinked filaments. In some embodiments, Step (d) comprises treating the plurality of filaments with one or more ionic crosslinking reagents to form inter-polypeptide and / or intra-polypeptide ionic crosslinks within at least a fraction of the polypeptides comprising the filaments, to produce a plurality of ionically-crosslinked filaments.

[0572] The terms “crosslink”, "crosslinks" or “crosslinked’ may herein refer to any covalent, ionic, and / or both ionic and covalent crosslink, crosslinks or crosslinked materials of the present disclosure.

[0573] In some embodiments, the terms “crosslink”, "crosslinks" or “crosslinked’ herein refer to any covalent, ionic, and / or both ionic and covalent crosslink, crosslinks or crosslinked materials of the present disclosure.

[0574] In some embodiments, the terms “crosslink”, "crosslinks" or “crosslinked’ herein refer to any covalent crosslink, crosslinks or crosslinked materials of the present disclosure.

[0575] In some embodiments, the terms “crosslink”, "crosslinks" or “crosslinked’ herein refer to any ionic crosslink, crosslinks or crosslinked materials of the present disclosure.

[0576] In some embodiments, the terms "crosslinks" or “crosslinked’ herein refer to both ionic and covalent crosslinks or crosslinked materials of the present disclosure.

[0577] In some embodiments, the term “crosslinks” herein refers to covalent crosslinks.

[0578] In some embodiments, the term “crosslinks” herein refers to ionic crosslinks.

[0579] In some embodiments, the term “crosslinks” herein refers to ionic and covalent crosslinks.

[0580] In some embodiments, “covalent” herein refers to a type of bond formed between two atoms wherein electrons are shared between the atoms via the interaction of atomic orbitals.

[0581] In some embodiments, “ionic” herein refers to a type of bond formed between two atoms wherein electrons are transferred completely from one atom to another atom (or group of atoms), forming ions of opposite charge. These ions then attract each other electrostatically to form a bond.

[0582] Step (d) may be performed in a coagulation bath comprising a coagulation bath solution, as defined herein. In some embodiments, Step (d) is performed in a coagulation bath comprising a coagulation bath solution.

[0583] Step (d) may be carried out using multiple and / or consecutive coagulation baths, comprising coagulation bath solutions of the same composition and / or of different compositions. In some embodiments, Step (d) is carried out using multiple and / or consecutive coagulation baths, comprising coagulation bath solutions of the same composition and / or of different compositions.

[0584] During precipitation and coagulation, a thermodynamically-stable solution of the polypeptides may transform from a liquid state into a solid state in a controlled manner.

[0585] Immersing a polypeptide-rich solution (non-solvent), such as the extruded or spun fourth composition from Step (c), in a polypeptide-lean solution (solvent), such as the coagulation bath solution, may form a mixture that will begin to separate into different phases. The phase rich in polypeptides may start to solidify through processes such as gelation, crosslinking, vitrification and / or crystallisation. Separation may be induced in a number of ways, including: (i) immersion precipitation, whereby liquid-liquid interactions between the solvent and non-solvent may lead to the precipitation of the polypeptide; (ii) controlled evaporation of the solvent to potentially isolate the polypeptides; and (iii) gelation, in which bond formation between the polypeptide molecules may be induced during liquid-liquid transfer between the solvent and non-solvent by applying either crosslinking reagents and sometimes a catalyst, or altering the isoelectric point with salts (Understanding and guiding the phase inversion process for synthesis of solvent resistant nanofiltration membranes, 2015) or through salting out with Hoffmeister series salts (Hofmeister Series: Insights of Ion Specificity from Amphiphilic Assembly and Interface Property, 2020).

[0586] Crosslinking polypeptides with a crosslinking reagent may lead to the formation of larger polypeptide aggregates. The formation of larger polypeptide aggregates in the extruded or spun filaments may decrease both the water solubility and elasticity of the filaments and increase both their material stiffness and tensile strength (Kaplan, 2010).

[0587] The precipitation of the solid phase in the coagulation bath may form porous structures in the solid phase. The principal parameters which may define the porosity of the precipitate are the concentration of the polymer in the polymer-rich phase (i.e. , the concentration of the polypeptides in the fourth composition) and the rate at which the precipitate forms. In a process which may be referred to as non-solvent precipitation, the rapid de-mixing of a binary solvent and solute system, upon the addition of a miscible tertiary non-solvent liquid and under favourable thermodynamic conditions may lead to spinodal precipitation potentially resulting in pore formation. Typically, rapid de-mixing and precipitation of the solute into the solid phase yields a more porous precipitate. Such an occurrence may be characterised by a rapid colour change or onset of turbidity in the solute rich phase (Understanding and guiding the phase inversion process for synthesis of solvent resistant nanofiltration membranes, 2015) (Mulder, 1996).

[0588] Additionally, in a process that may be referred to as “pore templating”, void inclusion elements may be added to the polymer solution prior to extrusion, and subsequently removed from the precipitate to potentially increase the void fraction of the precipitate (Rapid Production of a Porous Cellulose Acetate Membrane for Water Filtration using Readily Available Chemicals, 2017). If the void inclusion element is a lipid, such as sunflower oil, and is used to form an oil-in-water emulsion with the polymer solution to create controlled porous structures, the process may be referred to as “emulsion templating”.

[0589] Covalent crosslinking may be carried out through the reaction of polycarboxylic acids with the reactive groups on the polypeptide backbone, including amine (-NH2), hydroxide (-OH), carboxyl (-COOH) and thiol (-SH) groups, to potentially form ester, thioester and / or amide covalent bond crosslinks.

[0590] In some embodiments, covalent crosslinking is carried out through the reaction of polycarboxylic acids with the reactive groups on the polypeptide backbone, including amine (-NH2), hydroxide (-OH), carboxyl (-COOH) and thiol (-SH) groups, to form ester, thioester and / or amide covalent bond crosslinks.

[0591] Ionic crosslinking may be carried out through the ionic interactions of charged regions of polymers (e.g. polypeptides and / or polysaccharides) with multi-valent ions, anions, cations, carbocations and / or carbanions, to potentially form ionic crosslinks.

[0592] In some embodiments, ionic crosslinking is carried out through the ionic interactions of charged regions of polymers (e.g. polypeptides and / or polysaccharides) with multi-valent ions, anions, cations, carbocations, and / or carbanions, to form ionic crosslinks.

[0593] As used herein, the term “covalent ester, thioester and / or amide bond crosslinked polypeptide” means a polypeptide having at least one or more ester, thioester and / or amide crosslinking bond. It is generally understood that any such ester, thioester and / or amide crosslinking bond would be derived from a polycarboxylic acid. As such, the term “polycarboxylic acid derived covalent ester, thioester and / or amide bond crosslinked polypeptides” encompasses any covalent ester, thioester and / or amide bond crosslinked polypeptide without the need for demonstrating that it was actually derived from a polycarboxylic acid. As such, the terms “covalent ester, thioester and / or amide bond crosslinked polypeptide” and “polycarboxylic acid derived covalent ester, thioester and / or amide bond crosslinked polypeptides” are used interchangeably herein.

[0594] In some preferred embodiments, the covalent crosslinking of the polypeptides in the filaments produced in Step (c) is performed via non-specific esterification, thio-esterification or amidation reactions involving the use of one or more polycarboxylic acid crosslinking reagents as non-harsh chemical crosslinking reagents.

[0595] In some preferred embodiments, the covalent crosslinking of the polypeptides in the filaments produced in Step (c) is performed via non-specific esterification, thio-esterification or amidation reactions involving the use of one or more polycarboxylic acid crosslinking reagents as chemical crosslinking reagents.

[0596] In some embodiments, the ionic crosslinking of the polypeptides in the filaments produced in Step (c) is performed via non-specific ionic crosslinking involving the use of multi-valent ions, anions, cations, carbocations and / or carbanions, to form ionic crosslinks as crosslinking reagents.

[0597] Covalent crosslinking may be carried out via the reaction of polycarboxylic acids with the reactive amine (- NH2), hydroxide (-OH), carboxyl (-COOH) and thiol (-SH) groups of the polypeptides, polysaccharides, and / or polyols.

[0598] Ionic crosslinking may be carried through the ionic interactions of charged regions of polypeptides, polysaccharides, and / or polyols.

[0599] Step (d) may comprise covalent crosslinking, ionic crosslinking, or a combination thereof. In some embodiments, Step (d) comprises covalent crosslinking. In some embodiments, Step (d) comprises ionic crosslinking. In some embodiments, Step (d) comprises both covalent crosslinking and ionic crosslinking.

[0600] Crosslinking may be carried out between polypeptides, polysaccharides, and / or polyols, to potentially form polypeptide-polypeptide, polypeptide-polysaccharide, polypeptide-polyol, polysaccharidepolysaccharide, polysaccharide-polyol crosslinks.

[0601] In some embodiments, the covalent crosslinking of polypeptides and polysaccharides and / or polyols in the filaments produced in Step (c) is performed via non-specific esterification, thio-esterification or amidation reactions involving the use of polycarboxylic acid salts as chemical crosslinking reagents.

[0602] In some embodiments, the covalent crosslinking of polypeptides and polyols in the filaments produced in Step (c) is performed via non-specific esterification, thio-esterification or amidation reactions involving the use of polycarboxylic acid salts as chemical crosslinking reagents.

[0603] In some other embodiments, the covalent crosslinking of polypeptides and polysaccharides in the filaments produced in Step (c) is performed via non-specific esterification, thio-esterification or amidation reactions involving the use of polycarboxylic acid salts as chemical crosslinking reagents. The polypeptides, polysaccharides, polyols and / or polymers of polyols of the filaments of this disclosure may be covalently crosslinked and / or ionically crosslinked. In some embodiments, the polypeptides, polysaccharides, polyols and / or polymers of polyols of the filaments of this disclosure are covalently and / or ionically crosslinked.

[0604] In some embodiments, the term “non-harsh chemical” herein refers to a chemical which is designated as GRAS.

[0605] In some other embodiments, the covalent crosslinking of polypeptides and polysaccharides and / or polyols in the filaments produced in Step (c) is performed via non-specific esterification, thio-esterification or amidation reactions involving the use of polycarboxylic acid salts as non-harsh chemical crosslinking reagents.

[0606] In some embodiments, the crosslinking reagents used in Step (d) are considered to be “non-harsh chemicals”.

[0607] In some embodiments, the crosslinking reagents used in Step (d) are GRAS.

[0608] In some embodiments, the polycarboxylic acid crosslinking reagents used in Step (d) are considered to be “non-harsh chemicals”.

[0609] In some embodiments, the polycarboxylic acid crosslinking reagents used in Step (d) are GRAS.

[0610] A polycarboxylic acid crosslinking reagent used in Step (d) may be in the form of a free acid, partially or fully dissociated species, salt (e.g., alkali or alkaline earth metal salts), ester, amide, anhydride (cyclic or linear), lactone, polymeric or crosslinked structure, chelated or complexed form, or a buffered mixture thereof.

[0611] In some embodiments, a polycarboxylic acid crosslinking reagent used in Step (d) is one or more form, wherein the forms are selected from the group comprising, a free acid, partially or fully dissociated species, salt (e.g., alkali or alkaline earth metal salts), ester, amide, anhydride (cyclic or linear), lactone, polymeric or crosslinked structure, chelated or complexed form, or a buffered mixture thereof.

[0612] In certain embodiments, a polycarboxylic acid crosslinking reagent used in Step (d) is in the form of a free acid (i.e. , a polycarboxylic acid).

[0613] In some embodiments, a polycarboxylic acid crosslinking reagent used in Step (d) is in the form of a partially or fully dissociated species.

[0614] In some embodiments, a polycarboxylic acid crosslinking reagent used in Step (d) is is in the form of a salt (i.e., a polycarboxylic acid salt). In some embodiments, the polycarboxylic acid salts used in Step (d) comprise sodium oxalate, potassium oxalate, sodium malate, potassium malate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, sodium tartrate, potassium tartrate, potassium citrate, sodium citrate, sodium malonate, potassium malonate, and / or combinations thereof.

[0615] In some preferred embodiments, the polycarboxylic acid salt used in Step (d) is sodium citrate.

[0616] In some other preferred embodiments, the polycarboxylic acid salt used in Step (d) is sodium malate.

[0617] In some preferred embodiments, the polycarboxylic acid salt used in Step (d) is potassium citrate.

[0618] In some other preferred embodiments, the polycarboxylic acid salt used in Step (d) is potassium malate.

[0619] In some other embodiments, covalent crosslinking with polycarboxylic salts with more than one carboxyl groups is carried out under alkaline conditions leading to the formation of non-specific ester, thioester or amide -bond covalent crosslinks (Alkali-Catalyzed Low Temperature Wet Crosslinking of Plant Proteins Using Carboxylic Acids, 2009).

[0620] The use of alkaline conditions may allow for the covalent crosslinking of polypeptides at low temperatures, which may increase the energy efficiency of the production process and negate the potential yellowing of materials seen when operating at higher temperatures (Alkali-Catalyzed Low Temperature Wet Crosslinking of Plant Proteins Using Carboxylic Acids, 2009).

[0621] In some further embodiments, polycarboxylic acid salts with more than one carboxyl group form crosslinks in a solution comprising other salts, including, but not limited to, sodium hypophosphite, in which sodium hypophosphite acts as a catalyst or is integrated into the crosslinks (Formation of Cyclic Anhydride Intermediates and Esterification of Cotton Cellulose by Multifunctional Carboxylic Acids: An Infrared Spectroscopy Study, 1996).

[0622] Step (d) comprises a polycarboxylic acid crosslinking reagent, an ionic crosslinking reagent, or a combination of both. In some embodiments, Step (d) comprises polycarboxylic acid crosslinking reagents. In some embodiments, Step (d) comprises ionic crosslinking reagents. In some embodiments, Step (d) comprises polycarboxylic acid crosslinking reagents and ionic crosslinking reagents.

[0623] In some embodiments, the concentration of each polycarboxylic acid crosslinking reagent in a coagulation bath solution of Step (d) is within the range of 0.01 % to 50 %; preferably, within the range of 0.05 % (w / v) to 35 %; more preferably, within the range of 0.1 % to 30 %; even more preferably, within the range of 1 % to 25 %; and most preferably, within the range of 5 % to 15 % (w / v solution). In some preferred embodiments, the concentration of each polycarboxylic acid crosslinking reagent in a coagulation bath solution of Step (d) is within the range of 20 % to 35 % (w / v solution).

[0624] In some other embodiments, the concentration of each polycarboxylic acid crosslinking reagent in a coagulation bath solution of Step (d) is within the range of 0.01 % to 1 %, 1 % to 5 % , 5 % to 10 % , 10 % to 15 % , 15 % to 20 % , 20 % to 25 % , 25 % to 30 % , 30 % to 35 % , 35 % to 40 % , 40 % to 45 % , and / or 45 % to 50 % (w / v solution).

[0625] In some embodiments, the concentration of each ionic crosslinking reagent in a coagulation bath solution of Step (d) is within the range of 0.01 % to 50 %; preferably, within the range of 0.05 % (w / v) to 35 %; more preferably, within the range of 0.1 % to 25 %; even more preferably, within the range of 0.5 % to 15 %; and most preferably, within the range of 1 % to 5 % (w / v solution).

[0626] In some preferred embodiments, the concentration of each ionic crosslinking reagent in a coagulation bath solution of Step (d) is within the range of 1 % to 5 % (w / v solution).

[0627] In some other embodiments, the concentration of each ionic crosslinking reagent in a coagulation bath solution of Step (d) is within the range of 0.01 % to 0.05 %, 0.05 % to 1 %, 1 % to 1 .1 %, 1.1 % to 1 .2 %, 1 .2 % to 1 .3 %, 1 .3 % to 1 .4 %, 1 .4 % to 1 .5 %, 1 .5 % to 1 .6 %, 1 .6 % to 1 .7 %, 1 .7 % to 1 .8 %, 1 .8 % to 2.0 %, 2.0 % to 2.5 %, 2.5 % to 5 %, 5 % to 10 %, 10 % to 15 %, 15 % to 20 %, 20 % to 25 %, 25 % to 30 %, 30 % to 35 %, 35 % to 40 %, 40 % to 45 %, and / or 45 % to 50 % (w / v solution).

[0628] Step (d) may be carried out temperatures between 5 °C and 95 °C. In some embodiments, Step (d) is carried out at room temperature, or at temperatures that are either higher or lower than room temperature.

[0629] In some embodiments, Step (d) is carried out at a temperature within the range of 5 °C to 95 °C; preferably, 10 °C to 75 °C; even more preferably, 15 °C to 65 °C; yet even more preferably, 20 °C to 60 °C; and most preferably, in the range of 30 °C to 55 °C.

[0630] In some embodiments, Step (d) is carried out at a temperature within the range of 10 °C to 30 °C.

[0631] In some embodiments, Step (d) is carried out at a temperature within the range of 5 °C to 10 °C, 10 °C to 20 °C, 20 °C to 30 °C, 30 °C to 50 °C, 50 °C to 75 °C, and / or 75 °C to 95 °C.

[0632] Step (d) may be carried out at atmospheric pressure or at pressures that are either higher or lower than atmospheric pressure. In some embodiments, Step (d) is carried out at atmospheric pressure or at pressures that are either higher or lower than atmospheric pressure. In some embodiments, Step (d) is carried out at a pressure within the range of 90,000 Pa to 109,000 Pa; preferably, 96,400 Pa to 108,400 Pa; and most preferably, in the range of 100,825 Pa to 101 ,825 Pa.

[0633] In some embodiments, Step (d) is carried out at a pressure within the range of 90,000 Pa to 109,000 Pa, 95,000 Pa to 105,000 Pa, and / or 100,000 Pa to 102,000 Pa.

[0634] In some embodiments, Step (d) is carried out at atmospheric pressure.

[0635] Step (d) may be carried out with pH in the range of 0 to 14.

[0636] In some embodiments, Step (d) is carried out with pH in the range of O to 14; preferably, within the range of 5.01 to 9.99; and most preferably, within the range of 8.5 to 9.99.

[0637] In some embodiments, Step (d) is carried out with pH in the range of 0 to 1 ; 1 to 2; 2 to 3; 3 to 4; 4 to 5; 5 to 6; 6 to 7; 7 to 8; 8 to 9; 9 to 10; 10 to 1 1 ; 11 to 12; 12 to 13; or 13 to 14.

[0638] In some embodiments, Step (d) is carried out with pH in the range of 7.01 to 9.99, 8.0 to 9.5, 8.1 to 9.0, or

[0639] 8.15 to 8.5.

[0640] In some embodiments, Step (d) is carried out with pH in the range of 7.01 to 9.99; preferably, 8.0 to 9.0; and most preferably, 8.0 and 8.5.

[0641] In some embodiments, Step (d) is carried out with pH in the range of 7.01 to 7.5, 7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, and / or 9.5 to 9.99.

[0642] In some embodiments, Step (d) is carried out with pH in the range of 3.0 to 6.0, 3.5 to 5.5, 4 to 5, or 4.2 to 4.8.

[0643] In some embodiments, Step (d) is carried out with pH in the range of 3.0 to 6.0; preferably, 4.0 to 5.0; and most preferably, 4.2 and 4.8.

[0644] In some embodiments, Step (d) is carried out with pH in the range of 3.0 to 6.0, 3.5 to 5.5, 4.2 to 4.8, 4.8 to 8.0, 8.0 to 8.5, 9.0 to 9.5, 9.5 to 9.99, 9.99 to 10.5, 10.5 to 1 1 , 11 to 11 .5, 11 .5 to 12, or 12 to 14.

[0645] The duration of Step (d) may be within the range of 30 seconds and 7 days. In some embodiments, the duration of Step (d) is within the range of 30 seconds and 6 hours; preferably, within the range of 10 minutes and 6 hours; more preferably, within the range of 20 minutes and 3 hours; even more preferably, within the range of 30 minutes and 2 hours; and most preferably, within the range of 45 minutes and 1 hour.

[0646] In some other embodiments, the duration of Step (d) is within the range of 30 seconds to 60 seconds, 1 minute to 30 minutes, 30 minutes to 60 minutes, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, and / or 5 hours to 6 hours.

[0647] In some embodiments, the duration of Step (d) is within the range of 5 minutes to 90 minutes; preferably, within the range of 20 minutes to 80 minutes; and most preferably, within the range of 45 minutes to 65 minutes.

[0648] In some embodiments, the duration of Step (d) is within the range of 5 minutes to 15 minutes, 15 minutes to 30 minutes, 30 minutes to 45 minutes, 45 minutes to 60 minutes, or 60 minutes to 100 minutes.

[0649] In some other embodiments, the duration of Step (d) is within the range of 30 seconds to 7 days; 24 hours to 96 hours; 30 seconds to 6 hours; 6 hours to 12 hours; 12 hours to 24 hours; 24 hours to 48 hours; 48 hours to 72 hours; 72 hours to 96 hours; and / or 96 hours to 7 days.

[0650] Step (d) may be carried out at multiple conditions (e.g., coagulation bath solution composition, temperature, pressure, pH, duration), either in the same coagulation bath, or across multiple and / or consecutive coagulation baths.

[0651] COAGULATION BATH

[0652] As used interchangeably herein, the terms “coagulation bath” or “coagulation bath solution” refer to a liquid medium used to solidify or precipitate a dissolved or dispersed material. A coagulation bath may be configured to facilitate crosslinking.

[0653] In some embodiments, a coagulation bath facilitates crosslinking.

[0654] In certain embodiments, a coagulation bath facilitates the formation of polycarboxylic acid derived ester, thioester or amide covalent bond crosslinks.

[0655] In certain embodiments, a coagulation bath facilitates the formation of ionic crosslinks.

[0656] In certain embodiments, a coagulation bath facilitates the formation of both polycarboxylic acid derived ester, thioester or amide covalent bond crosslinks; and ionic crosslinks. A coagulation bath solution of Step (d) may comprise one or more polycarboxylic acid crosslinking reagent and / or ionic crosslinking agents dissolved in an aqueous solvent or in a mixture of multiple solvents.

[0657] In some embodiments, a coagulation bath solution of Step (d) comprises at least one polycarboxylic acid crosslinking reagent dissolved in an aqueous solvent or in a mixture of multiple solvents.

[0658] In some embodiments, a coagulation bath solution of Step (d) contains aqueous solvents, such as water.

[0659] In some embodiments, a coagulation bath solution of Step (d) comprises at least one alkali, acid, alcohol, catalyst, organic solvent, salt, polyol, polymer of polyol and / or any combination thereof.

[0660] In some embodiments, a coagulation bath solution of Step (d) comprises one or more polycarboxylic acid salts, such as sodium oxalate, potassium oxalate, sodium malate, potassium malate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, sodium tartrate, potassium tartrate, potassium citrate, sodium citrate, sodium malonate, potassium malonate, and / or any combination thereof.

[0661] In some embodiments, a coagulation bath solution of Step (d) comprises trisodium citrate. Herein, sodium citrate, and trisodium citrate are used interchangeably.

[0662] A polycarboxylic acid crosslinking reagent may be added to a coagulation bath solution of Step (d) in the form of a free acid, partially or fully dissociated species, salt (e.g., alkali or alkaline earth metal salts), ester, amide, anhydride (cyclic or linear), lactone, polymeric or crosslinked structure, chelated or complexed form, or a buffered mixture thereof.

[0663] Upon addition to a coagulation bath solution, a polycarboxylic acid crosslinking reagent may partially or fully dissolve, and may undergo a change in form, such as dissociation from a free acid or salt into one or more species, depending on factors including its chemical structure, solubility, and the composition and conditions (e.g., pH, temperature) of the bath solution.

[0664] In some embodiments, a coagulation bath solution of Step (d) comprises a polycarboxylic acid crosslinking reagent in one or more forms, wherein the forms are selected from the group comprising, a free acid, partially or fully dissociated species, salt (e.g., alkali or alkaline earth metal salts), ester, amide, anhydride (cyclic or linear), lactone, polymeric or crosslinked structure, chelated or complexed form, or a buffered mixture thereof.

[0665] In some embodiments, a coagulation bath solution of Step (d) comprises a polycarboxylic acid crosslinking reagent in the form of a free acid (i.e . , a polycarboxylic acid). In some embodiments, a coagulation bath solution of Step (d) comprises a polycarboxylic acid crosslinking reagent in the form of a partially or fully dissociated species.

[0666] In some embodiments, a coagulation bath solution of Step (d) comprises a polycarboxylic acid crosslinking reagent in the form of a salt (i.e., a polycarboxylic acid salt).

[0667] The concentration of each polycarboxylic acid crosslinking reagent in a coagulation bath solution of Step (d) may be within the range of 0.1 % to 40 % (w / v coagulation bath solution).

[0668] In some embodiments, the concentration of each polycarboxylic acid crosslinking reagent in a coagulation bath solution of Step (d) is within the range of 0.1 % to 40 %; preferably, within the range of 10 % to 30 %; more preferably, within the range of 20 % to 28 %; and most preferably at 25 % (w / v coagulation bath solution).

[0669] In some other embodiments, the concentration of each polycarboxylic acid crosslinking reagent within a coagulation bath solution of Step (d) is within the range of 0.1 % to 1 %; 1 % to 5 %; 5 % to 10 %; 10 % to 20 %; 20 % to 40 %; 40 % to 60 %; or 60 % to 75 %; (w / v coagulation bath solution).

[0670] In some embodiments, a coagulation bath solution of Step (d) comprises one or more ionic crosslinking reagents, such as calcium chloride (CaCI2), magnesium chloride (MgCI2), sodium tripolyphosphate, aluminium sulphate (AI2(SO4)3), zinc chloride (ZnCI2), ferric chloride (FeCI3), barium chloride (BaCI2), and / or any combination thereof.

[0671] In some embodiments, a coagulation bath solution of Step (d) comprises one or more ions, including, Ca2+, Mg2+, Fe3+, Al3+, Zn2+, and / or any combination thereof.

[0672] In some embodiments, the concentration of each ionic crosslinking reagent in a coagulation bath solution of Step (d) is within the range of 0.01 % to 50 %; preferably, within the range of 0.05 % (w / v) to 35 %; more preferably, within the range of 0.1 % to 25 %; even more preferably, within the range of 0.5 % to 15 %; and most preferably, within the range of 1 % to 5 % (w / v solution).

[0673] In some preferred embodiments, the concentration of each ionic crosslinking reagent in a coagulation bath solution of Step (d) is within the range of 1 % to 5 % (w / v coagulation bath solution).

[0674] In some other embodiments, the concentration of each ionic crosslinking reagent in a coagulation bath solution of Step (d) is within the range of 0.01 % to 0.05 %, 0.05 % to 1 %, 1 % to 1 .1 %, 1.1 % to 1 .2 %, 1 .2 % to 1 .3 %, 1 .3 % to 1 .4 %, 1 .4 % to 1 .5 %, 1 .5 % to 1 .6 %, 1 .6 % to 1 .7 %, 1 .7 % to 1 .8 %, 1 .8 % to 2.0 %, 2.0 % to 2.5 %, 2.5 % to 5 %, 5 % to 10 %, 10 % to 15 %, 15 % to 20 %, 20 % to 25 %, 25 % to 30 %, 30 % to 35 %, 35 % to 40 %, 40 % to 45 %, and / or 45 % to 50 % (w / v coagulation bath solution). A coagulation bath solution of Step (d) may comprise one or more catalysts, such as zinc sulphate, sodium sulphite, sodium bisulphite, sodium meta-bisulphite, sodium hypophosphite and / or ammonium sulphate.

[0675] In some embodiments, a coagulation bath solution of Step (d) comprises one or more catalysts, such as zinc sulphate, sodium sulphite, sodium bisulphite, sodium meta-bisulphite, sodium hypophosphite, and ammonium sulphate.

[0676] In some embodiments, the concentration of each catalyst within a coagulation bath solution of Step (d) is within the range of 0.1 % to 40 %; preferably, within the range of 5 % to 15 %; more preferably, within the range of 10 % to 14 %; and most preferably at 12.5 % (w / v coagulation bath solution).

[0677] In some other embodiments, the concentration of each catalyst within a coagulation bath solution of Step (d) is within the range of 0.1 % to 1 %; 1 % to 5 %; 5 % to 10 %; 10 % to 20 %; and / or 20 % to 40 % (w / v coagulation bath solution).

[0678] In some embodiments, a coagulation bath solution of Step (d) comprises one or more alkalis, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium carbonate, ammonium hydroxide, potassium carbonate, and sodium bicarbonate.

[0679] In some embodiments, a coagulation bath solution of Step (d) comprises one or more acids, such as ascorbic acid, acetic acid, adipic acid, citric acid, formic acid, hydrochloric acid, lactic acid, malic acid, malonic acid, oxalic acid, succinic acid, sulphuric acid, and tartaric acid.

[0680] The pH of a coagulation bath solution of Step (d) may be within the range of 3 to 14.

[0681] In some embodiments, the pH of a coagulation bath solution is within the range of 3.0 to 5.5, 3.5 to 5.0, 4.0 to 4.9, or 4.2 to 4.8.

[0682] In some embodiments, the pH of a coagulation bath solution of Step (d) is within the range of 3 to 14; preferably, within the range of 5.01 to 9.99; and most preferably, within the range of 8.5 to 9.99.

[0683] In some embodiments, the pH of a coagulation bath solution of Step (d) is within the range of 3 to 4; 4 to

[0684] 5; 5 to 6; 6 to 7; 7 to 8; 8 to 9; 9 to 10; 10 to 11 ; 11 to 12; 12 to 13; or 13 to 14.

[0685] In some embodiments, the pH of a coagulation bath solution of Step (d) is within the range of 7.01 to

[0686] 9.99, 8.0 to 9.5, 8.1 to 9.0, or 8.15 to 8.5.

[0687] In some embodiments, the pH of a coagulation bath solution of Step (d) is within the range of 7.01 to 9.99; preferably, 8.0 to 9.0; and most preferably, 8.0 and 8.5.

[0688] In some embodiments, the pH of a coagulation bath solution of Step (d) is within the range of 7.01 to 7.5, 7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, and / or 9.5 to 9.99. In some embodiments, the pH of a coagulation bath solution of Step (d) is within the range of 3.0 to 6.0, 3.5 to 5.5, 4 to 5, or 4.2 to 4.8.

[0689] In some embodiments, the pH of a coagulation bath solution of Step (d) is within the range of 3.0 to 6.0; preferably, 4.0 to 5.0; and most preferably, 4.2 and 4.8.

[0690] In some embodiments, a coagulation bath solution of Step (d) comprises alcohols, such as methanol, ethanol, propanol, iso-propanol, butanol and / or iso-butanol.

[0691] In some other embodiments, the concentration of each alcohol within a coagulation bath solution of Step (d) is within the range 0.5 % to 5 %, 5 % to 10 %, 10 % to 30 %, 20 % to 30 %, 30 % to 40 %, 40 % to 50 %, 50 % to 60 %, 60 % to 70 %, 70 % to 80 %, 80 % to 90 %, or 90 % to 99.99 % (v / v coagulation bath solution).

[0692] In some embodiments, a coagulation bath solution of Step (d) comprises one or more salts including, but not limited to, ammonium sulphate, disodium hydrogen phosphite, dipotassium phosphate, lithium sulphate, monosodium phosphate, potassium carbonate, potassium chloride, potassium sulphate, sodium bicarbonate, sodium bisulphate, sodium carbonate, sodium chloride, sodium hypophosphite, sodium malonate, sodium maleate, sodium meta-bisulphite, sodium sulphate, sodium sulphite, trilithium citrate, triammonium citrate, trisodium citrate, tripotassium citrate, zinc chloride, and / or zinc sulphate.

[0693] In some embodiments, the concentration of each salt within a coagulation bath solution of Step (d) is within the range of 0.01 % to 40 %; preferably, within the range of 0.1 % to 10 %; more preferably, within the range of 0.5 % to 5 %; and most preferably, at 1 % (w / v coagulation bath solution).

[0694] In some other embodiments, the concentration of each salt within a coagulation bath solution of Step (d) is within the range of 0.01 % to 0.1 %, 0.1 % to 1 %, 1 % to 5 %, 5 % to 10 %, 10 % to 20 %, and / or 20 % to 40 % (w / v coagulation bath solution).

[0695] In some embodiments, a coagulation bath solution of Step (d) comprises polyols.

[0696] In some embodiments, a coagulation bath solution of Step (d) comprises polyols and / or polymers of polyols.

[0697] In some embodiments, the temperature of a coagulation bath solution of Step (d) is room temperature.

[0698] In some embodiments, the temperature of a coagulation bath solution of Step (d) is at temperatures that are either higher or lower than room temperature. In some embodiments, the temperature of a coagulation bath solution of Step (d) is in the range of 5 °C to 95 °C; preferably, 10 °C to 75 °C; more preferably, 15 °C to 65 °C; even more preferably, 20 °C to 60 °C; and most preferably, 20 °C to 35 °C.

[0699] In some embodiments, the temperature of a coagulation bath solution of Step (d) is within the range of 5 °C to 10 °C, 10 °C to 20 °C, 20 °C to 30 °C, 30 °C to 50 °C, 50 °C to 75 °C, or 75 °C to 95 °C.

[0700] COAGULATION BATH SOLUTION

[0701] In some preferred embodiments, a coagulation bath solution of Step (d) comprises water, which also contains 33.1 % sodium citrate (w / v of the coagulation bath) and is at a pH within the range of 7.01 to 9.5 at room temperature.

[0702] In some preferred embodiments, a coagulation bath solution of Step (d) comprises water, sodium citrate and glycerol, and is at a pH within the range of 7.01 to 14, at room temperature.

[0703] In other preferred embodiments, a coagulation bath solution of Step (d) comprises water and sodium malate, and is at a pH within the range of 7.01 to 10.0, at room temperature.

[0704] In other preferred embodiments, a coagulation bath solution of Step (d) comprises water, 22 % sodium malate (w / v) and glycerol, and is at a pH within the range of 7.01 to 14, at room temperature.

[0705] In some preferred embodiments, a coagulation bath solution of Step (d) does not have more than 5 % (w / v) polypeptides prior to the immersion of the extruded or spun filaments.

[0706] In some preferred embodiments, a coagulation bath solution of Step (d) does not comprise more than 1 % (w / v) polypeptides prior to the immersion of the extruded or spun filaments.

[0707] For wet spinning, a suitable coagulation bath solution may comprise a solvent, or multiple solvents, together with one or more crosslinking reagents in a mixture, that may also comprise a mixture of catalysts and / or salts. The coagulation bath solution may be formulated to facilitate the crosslink- mediated gelation and / or precipitation of the crosslinked filaments. Consequently, wet spinning may facilitate the formation of the extruded or spun filaments, comprising crosslinked polypeptides.

[0708] For wet spinning, a suitable coagulation bath solution may comprise a solvent, or multiple solvents, together with one or more polycarboxylic acid crosslinking reagents in a mixture, that may also comprise a mixture of catalysts and / or salts. The coagulation bath solution may be formulated to facilitate the polycarboxylic acid derived ester, thioester or amide covalent bond crosslink mediated gelation, precipitation of the covalently-crosslinked filaments. Consequently, this may facilitate the formation of the extruded or spun filaments, comprising covalent ester, thioester and / or amide crosslinked polypeptides.

[0709] In some embodiments, the fraction of the polypeptides in the extruded or spun filaments which are covalently-crosslinked in Step (d) is at least 10 %, preferably, at least 20 % more preferably, at least 40 %, even more preferably, at least 60 % and most preferably, at least 80 %.

[0710] In some embodiments, the fraction of the polypeptides in the extruded or spun filaments which are covalently-crosslinked in Step (d) ranges from 10 % to 30 %, 20 % to 30 %, 30 % to 40 %, 40 % to 50 %, 50 to 60 %, 60 to 70 %, 70 to 80 %, 80 to 90 %, or 90 % to 99.99 %.

[0711] In some embodiments, the fraction of the polypeptides in the extruded or spun filaments which are ionically crosslinked in Step (d) is at least 10 %, preferably, at least 20 % more preferably, at least 40 %, even more preferably, at least 60 % and most preferably, at least 80 %.

[0712] In some embodiments, the fraction of the polypeptides in the extruded or spun filaments which are ionically crosslinked in Step (d) ranges from 10 % to 30 %, 20 % to 30 %, 30 % to 40 %, 40 % to 50 %, 50 to 60 %, 60 to 70 %, 70 to 80 %, 80 to 90 %, or 90 % to 99.99 %.

[0713] In some embodiments, the fraction of the polypeptides in the extruded or spun filaments which are both covalently and ionically crosslinked in Step (d) is at least 10 %, preferably, at least 20 % more preferably, at least 40 %, even more preferably, at least 60 % and most preferably, at least 80 %.

[0714] In some embodiments, the fraction of the polypeptides in the extruded or spun filaments which are both covalently and ionically crosslinked in Step (d) ranges from 10 % to 30 %, 20 % to 30 %, 30 % to 40 %, 40 % to 50 %, 50 to 60 %, 60 to 70 %, 70 to 80 %, 80 to 90 %, or 90 % to 99.99 %.

[0715] The extent by which crosslinking has occurred in the polypeptides of the filaments of this disclosure may be readily determined by a person of skill in the art by methods that include Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry, Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy.

[0716] To evaluate the degree of covalent-crosslinking with SDS-PAGE, a covalently-crosslinked sample is run through an SDS-PAGE gel alongside a non-crosslinked sample and a channel of proteins with known masses. The theoretical mass of emergent bands in the crosslinked sample and non-crosslinked sample channels may be calculated by comparing the distance of the bands along the channels relative to that of the bands in the channel with proteins of known mass. By evaluating the relative masses of the bands in the crosslinked and non-crosslinked sample channels, the degree of crosslinking associated with each band may be calculated.

[0717] Structural changes associated with crosslinking may be evaluated by the change in band intensities associated with various functional groups as determined by FTIR or Raman spectroscopy.

[0718] In some preferred embodiments, Step (d) comprises treating the filaments produced in Step (c) in a coagulation bath filled with an aqueous coagulation bath solution, comprising sodium citrate and sodium hypophosphite, at a pH between 8.0 and 8.5, room temperature, and atmospheric pressure, for a duration of 1 hour.

[0719] Any embodiment of Step (d) of this disclosure may be used in any embodiment of a process for producing a hollow fibre according to this disclosure. In particular, the crosslinking reagents, process conditions (e.g., temperature, pressure, pH, and duration), and / or coagulation baths described in any embodiment of Step (d) may be applied in the formation of such a hollow fibre.

[0720] In certain embodiments, a hollow fibre of any embodiment of this disclosure is treated with one or more polycarboxylic acid crosslinking agents to form inter-polypeptide and / or intra-polypeptide polycarboxylic acid derived ester, thioester or amide covalent bond crosslinks within at least a fraction of the polypeptides within the hollow fibres, thereby producing a covalently-crosslinked hollow fibre.

[0721] In some embodiments, a coagulation bath described in any embodiment of Step (d) is used in the formation of a hollow fibre. In some embodiments, the process conditions of any embodiment of Step (d) are used in the formation of a hollow fibre. In some embodiments, the polycarboxylic acid crosslinking agents of any embodiment of Step (d) are used in the formation of a hollow fibre.

[0722] Any embodiment of Step (d) of this disclosure may be used in any embodiment of a process for producing a Prokitein according to this disclosure. In particular, the crosslinking reagents, process conditions (e.g., temperature, pressure, pH, and duration), and / or coagulation baths described in any embodiment of Step (d) may be applied in the formation of such a Prokitein.

[0723] 1.9 STEP (E) POST PRODUCTION MODIFICATIONS

[0724] Step (e) comprises at least one post-production modification process selected from the group consisting of: i. treating with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides; ii. annealing; iii. treating with a solvent to remove void inclusion elements; iv. washing with one or more wash solutions; v. coating; vi. modifying surface topography; and vii. drawing.

[0725] Step (e) may comprise treating the crosslinked filaments of Step (d) with at least one post-production modification process selected from the group consisting of: i. treating the crosslinked filaments with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides; ii. annealing the crosslinked filaments; ill. treating the crosslinked filaments with a solvent to increase the porosity of the filaments; iv. washing the crosslinked filaments with one or more wash solutions; v. coating the crosslinked filaments; vi. modifying the surface topography of the crosslinked filaments; and vii. drawing the crosslinked filaments; to produce a plurality of treated crosslinked filaments.

[0726] Step (e) may comprise treating the crosslinked filaments produced in Step (d) with at least one postproduction modification process. In some embodiments, Step (e) comprises treating the filaments with at least one post-production modification process. In other embodiments, Step (e) comprises treating the filaments with at least two post-production modification processes.

[0727] The crosslinked filaments may be further treated as part of their production, or as a post-production modification, in order to potentially enhance one or more of the mechanical properties, porosity, and water stability of the filaments.

[0728] In some embodiments, the crosslinked filaments are further treated as part of their production, or as a post-production modification, to enhance one or more of the mechanical properties, porosity, and water stability of the filaments.

[0729] Step (e) may comprise treating the hollow fibres of any embodiment of this disclosure with at least one post-production modification process. In some embodiments, Step (e) comprises treating the hollow fibres of any such embodiment with at least one post-production modification process. A post-production modification process described in any embodiment of Step (e) may be applied to hollow fibres according to any other embodiment. Step (e) may comprise treating a Prokitein of any embodiment of this disclosure with at least one postproduction modification process. In some embodiments, Step (e) comprises treating the Prokitein of any such embodiment with at least one post-production modification process. A post-production modification processes described in any embodiment of Step (e) may be applied to a Prokitein according to any other embodiment.

[0730] 1.10 STEP (E.i) MACROMOLECULE SECONDARY STRUCTURE MODIFICATION

[0731] Step (e.i) comprises treating with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides.

[0732] Step (e.i) may comprise treating the crosslinked filaments with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides.

[0733] Step (e.i) may comprise treating the covalently-crosslinked filaments with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides.

[0734] In some embodiments, Step (e.i) comprises treating crosslinked filaments with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides.

[0735] In some embodiments, Step (e.i) comprises treating the covalently-crosslinked filaments with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides.

[0736] In particular, Step (e.i) may comprise treating the covalently-crosslinked filaments with organic solvents to dehydrate them and alter the secondary macromolecular structures of the polypeptides of the filaments.

[0737] In some embodiments, Step (e.i) comprises treating the crosslinked filaments with organic solvents to dehydrate them and alter the secondary macromolecular structures of the polypeptides of the filaments.

[0738] The aim of Step (e.i) is to potentially alter the secondary macromolecular structure of the polypeptides in the crosslinked filaments, for the purpose of increasing the abundance of beta-sheets and beta-coils, relative to amorphous random coils and alpha-helices. This change in the secondary structure may result in an increase in water stability of the filaments, such that their mechanical properties may remain relatively unaltered following continuous submersion in an aqueous solution for 3 or more days.

[0739] In some embodiments, in Step (e.i), the alteration of the secondary macromolecular structure of the polypeptides in the covalently-crosslinked filaments is achieved with organic solvents, either through solution washes and / or vapour exposure. In some embodiments, in Step (e.i), the alteration of the secondary macromolecular structure of the polypeptides in the crosslinked filaments is achieved with organic solvents, either through solution washes and / or vapour exposure.

[0740] In some embodiments, in Step (e.i), the alteration of the secondary macromolecular structure of the polypeptides in the covalently-crosslinked filaments is achieved with organic solvents in an aqueous solution.

[0741] In some embodiments, in Step (e.i), the alteration of the secondary macromolecular structure of the polypeptides in the crosslinked filaments is achieved with organic solvents in an aqueous solution.

[0742] In some embodiments, in Step (e.i), the alteration of the secondary macromolecular structure of the polypeptides in the crosslinked filaments is achieved with supercritical CO2.

[0743] In some embodiments, solvents used in Step (e.i) comprise acetone, ethanol, ethanol, propanol, isopropanol, butanol, iso-butanol, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, and / or mixtures thereof.

[0744] In some embodiments, solvents used in Step (e.i) comprise polyols

[0745] In some embodiments, solvents used in Step (e.i) comprise polyols and / or polymers of polyols.

[0746] In some embodiments, solvents used in Step (e.i) comprise polyols and / or polymers of polyols in an aqueous solution.

[0747] In some embodiments, solvents used in Step (e.i) comprise water.

[0748] In some embodiments, solvents used in Step (e.i) comprise water and ethanol.

[0749] In some embodiments, solvents used in Step (e.i) comprise water, organic solvents, polyols and / or polymers of polyols.

[0750] In some embodiments, in Step (e.i), the alteration of the secondary macromolecular structure of the polypeptides in the covalently-crosslinked filaments is achieved with supercritical CO2.

[0751] In some embodiments, the covalently-crosslinked filaments are treated with one or more fluids comprising supercritical CO2. Herein, supercritical CO2 is considered to function analogously to an organic solvent when above its critical point, and is referred herein to as such. Accordingly, the term “organic solvent” as used herein is intended to include supercritical CO2.

[0752] In some embodiments, solvents used in Step (e.i) comprise polycarboxylic acid salts.

[0753] In some embodiments, solvents used in Step (e.i) comprise multivalent ions In some embodiments, solvents used in Step (e.i) comprise calcium chloride (CaCI2), magnesium chloride (MgCI2), sodium tripolyphosphate, aluminium sulphate (AI2(SO4)3), zinc chloride (ZnCI2), ferric chloride (FeCI3), barium chloride (BaCI2), and / or any combination thereof.

[0754] In some embodiments, solvents used in Step (e.i) comprise one or more polycarboxylic acid salts, including, sodium oxalate, potassium oxalate, sodium malate, potassium malate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, sodium tartrate, potassium tartrate, potassium citrate, sodium citrate, sodium malonate, potassium malonate, and / or any combination thereof.

[0755] In some embodiments, a solvent of Step (e.i) comprises a polycarboxylic acid crosslinking reagent in one or more forms, wherein the forms are selected from the group comprising, a free acid, partially or fully dissociated species, salt (e.g., alkali or alkaline earth metal salts), ester, amide, anhydride (cyclic or linear), lactone, polymeric or crosslinked structure, chelated or complexed form, or a buffered mixture thereof.

[0756] In some embodiments, solvents used in Step (e.i) comprise one or more ions, including, Ca2+, Mg2+, Fe3+, Al3+, Zn2+, and / or any combination thereof.

[0757] The concentration of any singular component in the solvent mixture used in Step (e.i) may be in the range of O % to 100 %.

[0758] In some embodiments, the concentration of any singular component in the solvent mixture used in Step (e.i) is within the range of 0 % to 100 %; preferably, within the range of 10 % to 75 %; and most preferably, within the range of 20 % to 60 % (w / v), relative to the total solvent mixture volume.

[0759] In some other embodiments, the concentration of any singular component in the solvent mixture used in Step (e.i) is within the range of 0 % to 5 %, 5 % to 10 %, 10 % to 15 %, 15 % to 20 %, 20 % to 25 %, 25 % to 30 %, 30 % to 35 %, 35 % to 40 %, 40 % to 45 %, 45 % to 50 %, 50 % to 55 %, 55 % to 60 %, 60 % to 65 %, 65 % to 70 %, 70 % to 75 %, 75 % to 80 %, 80 % to 85 %, 85 % to 90 %, 90 % to 95 %, or 95 % to 100 % (w / v), relative to the total solvent mixture volume.

[0760] Step (e.i) may be carried out at temperatures that are within the range of 0 °C to 300 °C.

[0761] In some embodiments, Step (e.i) is carried out at a temperature that is within the range of 0 °C to 100 °C; preferably, within the range of 10 °C to 80 °C; more preferably, within the range of 20 °C to 70 °C; even more preferably, within the range of 30 °C to 60 °C; and most preferably, at room temperature. In some other embodiments, Step (e.i) is carried out at a temperature that is within the range of 10 °C to 20 °C, 20 °C to 30 °C, 30 °C to 40 °C, 40 °C to 50 °C, 50 °C to 60 °C, 60 °C to 70 °C, 70 °C to 80 °C, or 80 °C to 90 °C, 90 °C to 100 °C, 100 °C to 110 °C, 110 °C to 120 °C, 120 °C to 130 °C, 130 °C to 140 °C, 140 °C to 150 °C, 150 °C to 160 °C, 160 °C to 170 °C, and / or 170 °C to 180 °C.

[0762] In some other embodiments, Step (e.i) is carried out at a temperature that is within the range of 15 to 300 °C, preferably, within the range of 100 °C to 250 °C; more preferably, within the range of 120 °C to 200 °C; even more preferably, within the range of 130 °C to 150 °C; and most preferably, at 135 °C to 145 °C.

[0763] In some other embodiments, Step (e.i) is carried out across a range of temperatures.

[0764] Step (e.i) may be carried out at atmospheric pressure, at pressures that are either higher or lower than atmospheric pressure, or at any pressure between 0.09 MPa and 25 MPa.

[0765] In some embodiments, Step (e.i) is carried out at atmospheric pressure, or at pressures that are either higher or lower than atmospheric pressure; preferably, in the range of 90,000 Pa to 109,000 Pa; more preferably, in the range of 96,400 Pa to 108,400 Pa; and most preferably, in the range of 100,825 Pa to 101 ,825 Pa.

[0766] In some other embodiments, the pressure used in Step (e.i) is in the range of 90,000 Pa to 109,000 Pa, 95,000 Pa to 105,000 Pa, or 100,000 Pa to 102,000 Pa.

[0767] In some embodiments, Step (e.i) is carried out at a pressure that is within the range of 0.1 MPa to 100 MPa; preferably, in the range of 7 MPa to 50 MPa; and more preferably, in the range of 10 MPa to 25 MPa.

[0768] Step (e.i) may be carried out in a pressure vessel.

[0769] In some embodiments, Step (e.i) is carried out in a pressure vessel.

[0770] Step (e.i) may be carried out over a duration that is within the range of 1 second to 10 days.

[0771] In some embodiments, Step (e.i) is carried out over a duration that is within the range of 1 second to 10 days; preferably, within the range of 10 minutes and 3 hours; more preferably, within the range ranging from 40 and 90 minutes; and most preferably for 1 hour. In some embodiments, Step (e.i) is carried out over a duration that is within the range of 1 second to 1 hour; preferably, within the range of 30 seconds to 30 minutes; more preferably, within the range of 45 seconds to 15 minutes; and most preferably within the range of 1 minute to 5 minutes.

[0772] Step (e.i) is carried out over a duration that is within the range of 1 second to 10 seconds, 10 seconds to 20 seconds, 20 seconds to 30 seconds, 30 seconds to 1 minute, 1 minute to 2 minutes, 2 minutes to 5 minutes, 5 minutes to 10 minutes, 10 minutes to 15 minutes, 15 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, and 50 minutes to 1 hour.

[0773] Changes in the secondary macromolecular structure of the polypeptides in the crosslinked filaments may be determined by evaluating the changes in intensity of peaks associated with amorphous random coils, alpha-helices, beta-helices, beta-sheets and disordered structures. This type of analysis may be performed by a person of skill in the art using methods that include Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), wide-angle X-ray scattering (WAXS), and Raman spectroscopy (RS).

[0774] In some preferred embodiments, Step (e.i) comprises treating the crosslinked filaments produced in Step (d) in a solvent bath comprising at least 35 % aqueous ethanol at room temperature and atmospheric pressure for one hour.

[0775] In some preferred embodiments, Step (e.i) comprises treating the crosslinked filaments produced in Step (d) in a solvent bath comprising at least 35 % aqueous ethanol and at least 0.1 mol / L trisodium citrate, at room temperature and atmospheric pressure for one hour.

[0776] In some preferred embodiments, Step (e.i) comprises treating the crosslinked filaments produced in Step (d) in a solvent bath comprising at least at room temperature and atmospheric pressure for a duration lasting up to 5 minutes.

[0777] In some particularly preferred embodiments, Step (e.i) comprises treating the crosslinked filaments produced in Step (d) in a solvent bath comprising at least 40 % aqueous ethanol at room temperature and atmospheric pressure for one hour.

[0778] In some embodiments, a coagulation bath solution of Step (d) comprises an organic solvent, such that Step (d) is effectively combined with Step (e.i), and beta-sheet formation in the secondary structure of the polypeptides occurs concurrent to the coagulation, solidification, and crosslinking of the polypeptides.

[0779] The polypeptides in the filaments treated in Step (e.i) may comprise a secondary structure that comprise beta-sheets. In some embodiments, some of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0780] In some embodiments, part of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0781] In some embodiments, most of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0782] In some embodiments, almost all of the polypeptides in the treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0783] In some embodiments, more than 0 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0784] In some embodiments, more than 5 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0785] In some embodiments, more than 10 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0786] In some embodiments, more than 20 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0787] In some embodiments, more than 30 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0788] In some embodiments, more than 40 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0789] In some embodiments, more than 50 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0790] In some embodiments, more than 60 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0791] In some embodiments, more than 70 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0792] In some embodiments, more than 80 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0793] In some embodiments, more than 90 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets.

[0794] In some embodiments, more than 95 % of the polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets. In some embodiments, the proportion of polypeptides in the filaments treated in Step (e.i) comprise a secondary structure that comprise beta-sheets is within the range of 0.01 % to 10 %, 10 % to 20 %, 20 % to 30 %, 30 % to 40 %, 40 % to 50 %, 50 % to 60 %, 60 % to 70 %, 70 % to 80 %, 80 % to 90 %, 90 % to 95 %, or 95 % to 99.99 %.

[0795] Step (e.i) may comprise treating the hollow fibres of any embodiment of this disclosure with an organic solvent. In some embodiments, Step (e.i) comprises treating the hollow fibres of any such embodiment with an organic solvent. The solvents and conditions described in any embodiment of Step (e.i) may be applied in the treatment of hollow fibres of any other embodiment. In certain embodiments, treating the hollow fibres of any embodiment of this disclosure with an organic solvent may result in an increase the relative abundance of beta-sheets in the secondary structure of the polypeptides in the hollow fibres.

[0796] Step (e.i) may comprise treating a Prokitein of any embodiment of this disclosure with an organic solvent. In some embodiments, Step (e.i) comprises treating a Prokitein of any such embodiment with an organic solvent. The solvents and conditions described in any embodiment of Step (e.i) may be applied in the treatment of a Prokitein of any other embodiment. In certain embodiments, treating a Prokitein of any embodiment of this disclosure with an organic solvent may result in an increase the relative abundance of beta-sheets in the secondary structure of the polypeptides in the Prokitein.

[0797] 1.11 STEP (E.ii) ANNEALING

[0798] Step (e.ii) comprises annealing.

[0799] Step (e.ii) may comprise annealing the crosslinked filaments.

[0800] In some embodiments, Step (e.ii) comprises annealing the crosslinked filaments.

[0801] Step (e.ii) may comprise annealing the covalently-crosslinked filaments.

[0802] In some embodiments, Step (e.ii) comprises annealing the covalently-crosslinked filaments.

[0803] The annealing process typically allows crystalline structures to rearrange into more stable forms which may be used to decrease internal stresses in the crosslinked filaments. More specifically, the annealing process may allow the beta-sheets formed in Step (e.i) to become the stable micro-structure state of the filaments. Consequently, the beta-sheet micro-structure may remain intact upon rehydration of the filaments and prevent the hydrolysis of the crosslinks formed in Step (d). Collectively, the consecutive crosslinking, beta-sheet formation and annealing steps may grant the filaments long-term water stability.

[0804] In some embodiments, the annealing process allows crystalline structures to rearrange into more stable forms to decrease internal stresses in the crosslinked filaments. More specifically, the annealing process results in the beta-sheets formed in Step (e.i) to become the stable micro-structure state of the filaments. Consequently, the beta-sheet micro-structure remains intact upon rehydration of the filaments and prevents the hydrolysis of the ester crosslinks formed in Step (d). Collectively, the consecutive crosslinking, beta-sheet formation and annealing steps grant the filaments long-term water stability.

[0805] The treated crosslinked filaments may be partially or completely dried prior to annealing, in a process herein referred to as “pre-drying”.

[0806] In some embodiments, the treated crosslinked filaments are partially dried prior to annealing, such that some water still remains within the microstructure of the filaments.

[0807] In some embodiments, the treated crosslinked filaments are completely dried prior to annealing, such that, ostensibly, all of the water is removed from the microstructure of the filaments.

[0808] In some embodiments, the treated crosslinked filaments are not dried prior to annealing.

[0809] In some embodiments, the pre-drying process is carried out at temperatures that range from 5 to 90 °C; preferably, within the range of 15 °C to 80 °C; more preferably, within the range of 40 °C to 70 °C; and most preferably, within the range of 50 °C to 70 °C.

[0810] In some embodiments, the pre-drying process is carried out at temperatures that are within the range of 5 to 90 °C; preferably, 15 °C to 75 °C; more preferably, 20 °C to 60 °C; even more preferably, 30 to 50 °C; and most preferably, at 40 °C.

[0811] In some other embodiments, the pre-drying process is carried out at temperatures that range from 5 °C to 10 °C, 10 °C to 15 °C, 15 °C to 20 °C, 20 °C to 25 °C, 25 °C to 30 °C, 30 °C to 35 °C, 35 °C to 40 °C, 40 °C to 45 °C, 45 °C to 50 °C, 50°C to 55 °C, 55 °C to 60 °C, 60 °C to 65 °C, 65 °C to 70 °C, 70 °C to 75 °C, 75 °C to 80 °C, 80 °C to 85 °C, and / or 85 °C to 90 °C.

[0812] In some embodiments, the filaments of this disclosure are dried prior to annealing with freeze-drying.

[0813] In some embodiments, the filaments are not freeze-dried.

[0814] In some embodiments, the filaments are not dried prior to annealing with freeze-drying.

[0815] In some embodiments, the pre-drying process is carried out at temperatures that range from 5 to -90 °C; preferably, within the range of -15 °C to -90 °C; more preferably, within the range of -40 °C to -90 °C; even more preferably, within the range of -60 °C to -90 °C; and most preferably, within the range of -80 °C to -86 °C. In some other embodiments, the pre-drying process is carried out at temperatures that range from 5 °C to 0 °C, 0 °C to -10 °C, -10 °C to -20 °C, -20 °C to -30 °C, -30 °C to - 0 °C, -40 °C to -50 °C, -50 °C to -60 °C, -60 °C to -70 °C, -70 °C to -80 °C, -80 °C to -86 °C, or -80 °C to -90 °C.

[0816] In some embodiments, the filaments are dried under conditions below the Triple point of water.

[0817] In some embodiments, the pre-drying process is carried out over a duration within the range of 0 minutes to 7 days; preferably, 20 minutes to 5 days; more preferably, 40 minutes to 3 days; even more preferably, 1 hour to 2 days; and most preferably, within the range of 2 hours to 1 day.

[0818] In some embodiments, the pre-drying process is carried out over a duration that ranges from 0 minutes to 6 hours; preferably, 20 minutes to 5 hours; more preferably, 40 minutes to 4 hours; even more preferably, 1 hour to 3 hours; and most preferably, for 2 hours.

[0819] In some other embodiments, the pre-drying process is carried out over a duration within the range 0 minutes to 5 minutes, 5 minutes to 10 minutes, 10 minutes to 15 minutes, 15 minutes to 20 minutes, 20 minutes to 25 minutes, 25 minutes to 30 minutes, 30 minutes to 35 minutes, 35 minutes to 40 minutes, 40 minutes to 45 minutes, 45 minutes to 50 minutes, 50 minutes to 55 minutes, 55 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 12 hours, 12 hours to 18 hours, 18 hours to 24 hours, 24 hours to 30 hours, 30 hours to 36 hours, 36 hours to 42 hours, 42 hours to 48 hours, 48 hours to 54 hours, 54 hours to 60 hours, 60 hours to 66 hours, 66 hours to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, or 6 days to 7 days.

[0820] The pre-drying process may be performed with convection, an infra-red heat source, and / or on a heated surface.

[0821] In some embodiments, the pre-drying process is performed with convection, radiation, microwave, an infra-red heat source, and / or on a heated surface.

[0822] The annealing step may be performed in different atmospheres, liquids and / or mixtures thereof.

[0823] In some embodiments, the annealing step is performed in different atmospheres, liquids, fluids and / or mixtures thereof.

[0824] The filaments may be annealed in a convection oven, in an autoclave or pressure cooker, and / or in a hot oil bath.

[0825] In some embodiments, the filaments are annealed in a convection oven, in an autoclave or pressure cooker, and / or in a hot oil bath. Atmospheres used in the annealing step may comprise air, carbon dioxide, steam, inert gases (such as nitrogen, helium, neon, argon, and xenon), vapours (such as ethanol, plant oils, water) and / or a mixture thereof.

[0826] In some embodiments, atmospheres used in the annealing step comprise air, carbon dioxide, steam, inert gases (such as nitrogen, helium, neon, argon, and xenon), vapours (such as ethanol, plant oils, water) and / or a mixture thereof.

[0827] Fluids used in the annealing step may comprise ethanol, plant oils, and / or a mixture thereof.

[0828] In some embodiments, fluids used in the annealing step comprise ethanol, plant oils, and / or a mixture thereof.

[0829] Fluids used in the annealing step may comprise alcohols such as, methanol, ethanol, propanol, isopropanol, butanol, and / or a mixture thereof.

[0830] In some embodiments, fluids used in the annealing step comprise alcohols such as, methanol, ethanol, propanol, iso-propanol, butanol, and / or a mixture thereof.

[0831] Fluids used in the annealing step may comprise polyols and / or polymers of polyols.

[0832] In some embodiments, fluids used in the annealing step comprise polyols and / or polymers of polyols.

[0833] In some embodiments, fluids used in the annealing step do not comprise polyols and / or polymers of polyols.

[0834] In some embodiments, fluids used in the annealing step do not comprise polyols.

[0835] In some embodiments, fluids used in the annealing step do not comprise polymers of polyols.

[0836] Fluids used in the annealing step may comprise supercritical fluids, such as supercritical CO2. In some embodiments, fluids used in the annealing step comprise supercritical fluids, such as supercritical CO2.

[0837] In some preferred embodiments, the annealing step is conducted in an atmosphere of air.

[0838] In some embodiments, the annealing step is carried out at a temperature that is within the range of 50 °C to 180 °C; preferably, 75 °C to 160 °C; more preferably, 110 °C to 140 °C; and most preferably, 120 °C to 130 °C.

[0839] In some other embodiments, the annealing step is carried out at temperatures that range from 50 °C to 80 °C, 80 °C to 100 °C, 100 °C to 120 °C, 120 °C to 140 °C, 140 °C to 160 °C, or 160 °C to 180 °C. In some embodiments, the annealing step is carried out at a temperature that is within the range of

[0840] 180 °C to 300 °C; preferably, 200 °C to 280 °C; more preferably, 220 °C to 260 °C; and most preferably, 240 °C to 250 °C.

[0841] In some other embodiments, the annealing step is carried out at temperatures that range from 180 °C to 200 °C, 200 °C to 220 °C, 220 °C to 240 °C, 240 °C to 260 °C, 260 °C to 280 °C, or 280 °C to 300 °C.

[0842] In some embodiments, the duration of the annealing step is within the range of 30 minutes to 6 hours; preferably, 10 minutes and 4 hours; more preferably, 30 minutes and 3 hours, even more preferably, 45 hours and 2 hours; and most preferably, 1 hours and 1 .5 hours.

[0843] In some embodiments, the duration of the annealing step is within the range of 45 minutes and 1 .25 hours.

[0844] In some other embodiments the duration of the annealing step is within the range of 5 minutes to 15 minutes, 15 minutes to 30 minutes, 30 minutes to 45 minutes, 45 minutes to 1 hour, 1 hour to 1 .25 hours, 1 .25 hours to 1 .5 hours, 1 .5 hours to 1 .75 hours, 1 .75 hours to 2 hours, 2 hours to 2.25 hours, 2.25 hours to 2.5 hours, 2.5 hours to 2.75 hours, 2.75 hours to 3 hours, 3 hours to 3.25 hours, 3.25 hours to 3.5 hours, 3.5 hours to 3.75 hours, 3.75 hours to 4 hours, 4 hours to 4.25 hours, 4.25 hours to 4.5 hours, 4.5 hours to 4.75 hours, 4.75 hours to 5 hours, 5 hours to 5.25 hours, 5.25 hours to 5.5 hours, 5.5 hours to 5.75 hours, and / or 5.75 hours to 6 hours.

[0845] The annealed filaments may be hydrated in water and / or an aqueous solution, in a process herein referred to as rehydration.

[0846] In some embodiments, the annealed filaments are rehydrated in water and / or an aqueous solution.

[0847] In some embodiments, the annealed filaments are not rehydrated in water and / or an aqueous solution.

[0848] In some embodiments, the aqueous rehydration solutions of Step (e.ii) comprise water, alcohols, polyols, acids, alkalis, salts and / or combinations thereof.

[0849] In some preferred embodiments, the aqueous rehydration solutions of Step (e.ii) comprise polyols.

[0850] In some preferred embodiments, Step (e.ii) comprises annealing the filaments at 130 °C in air for two hours. In some particularly preferred embodiments, Step (e.ii) comprises drying the filaments at 40 °C with convection for 2 hours, followed by annealing at 130 °C in air for 1 hour, and subsequent rehydration in an aqueous solution containing 50 % glycerol (v / v).

[0851] Step (e.ii) may comprise the process of protein annealing.

[0852] In some embodiments, Step (e.ii) comprises the process of protein annealing.

[0853] Step (e.ii) may comprise annealing the hollow fibres of any embodiment of this disclosure. In some embodiments, Step (e.ii) comprises annealing the hollow fibres of any such embodiment. The pre-drying and annealing conditions described in any embodiment of Step (e.ii) may be applied in the treatment of hollow fibres of any other embodiment.

[0854] In certain embodiments, a hollow fibre of any embodiment of this disclosure is annealed to alter and / or tailor its materials properties, such as, but not limited to, its Young’s modulus, ultimate tensile stress, and / or ultimate tensile strain.

[0855] Step (e.ii) may comprise annealing a Prokitein of any embodiment of this disclosure. In some embodiments, Step (e.ii) comprises annealing a Prokitein of any such embodiment. The pre-drying and annealing conditions described in any embodiment of Step (e.ii) may be applied in the treatment of a Prokitein of any other embodiment.

[0856] In certain embodiments, a Prokitein of any embodiment of this disclosure is annealed to alter and / or tailor its materials properties, such as, but not limited to, its Young’s modulus, ultimate tensile stress, and / or ultimate tensile strain.

[0857] 1.12 STEP (E.iii) REMOVAL OF VOID INCLUSION ELEMENTS

[0858] Step (e.iii) comprises treating with one or more solvents to increase porosity.

[0859] Step (e.iii) may comprise treating with one or more solvents to remove void inclusion elements.

[0860] In some embodiments, Step (e.iii) comprises treating with one or more solvents to remove void inclusion elements, thereby imparting porosity.

[0861] Step (e.iii) may comprise treating the covalently-crosslinked filaments with one or more solvents to remove void inclusion elements in the filaments, which may impart porosity.

[0862] In some embodiments, Step (e.iii) comprises treating the covalently-crosslinked filaments with one or more solvents to remove void inclusion elements in the filaments, thereby, imparting porosity. Solvents used in Step (e.iii) may comprise one or more aqueous solutions, organic solutions and / or supercritical fluids.

[0863] In some embodiments, Step (e.iii) comprises treating filaments with an aqueous solution.

[0864] In some embodiments, Step (e.iii) comprises treating filaments with an organic solution.

[0865] In some embodiments, Step (e.iii) comprises treating filaments with aqueous solutions and / or organic solutions.

[0866] In some embodiments, Step (e.iii) comprises treating filaments with supercritical fluids.

[0867] In some embodiments, Step (e.iii) comprises treating filaments with supercritical CO2.

[0868] In some embodiments, one or more aqueous solutions of Step (e.iii) comprise water, acids, alkalis and / or salt solutions.

[0869] In some other embodiments, Step (e.iii) comprises treating filaments with water.

[0870] An aqueous solution of Step (e.iii) may comprise acids, such as oxalic acid, malic acid, succinic acid, adipic acid, tartaric acid, citric acid, malonic acid, acetic acid and / or formic acid.

[0871] In some embodiments, one or more aqueous solutions of Step (e.iii) comprise acids, such as oxalic acid, malic acid, succinic acid, adipic acid, tartaric acid, citric acid, malonic acid, acetic acid and / or formic acid; preferably citric acid and / or malic acid.

[0872] In some embodiments, wherein the void inclusion element is powdered calcium carbonate, the filaments are treated with an aqueous acid solution, such as citric acid. Hydrogen ions react with the calcium carbonate embedded in the filaments, to form calcium salts and carbon dioxide. As the carbon dioxide is released and the salts dissolve, voids are created in the walls of the filaments, in the spaces previously occupied by calcium carbonate; thereby, increasing the void fraction in the filaments.

[0873] An organic solution of Step (e.iii) may comprise ethanol, propanol, iso-propanol, butanol, iso-butanol, acetone, dimethyl sulfoxide, dimethylformamide, and / or dimethylacetamide.

[0874] In some embodiments, one or more organic solutions of Step (e.iii) comprise ethanol, propanol, isopropanol, acetone butanol, iso-butanol, dimethyl sulfoxide, dimethylformamide, and / or dimethylacetamide; preferably ethanol.

[0875] A solvent of Step (e.iii) may comprise a polyols and / or polymers of polyols.

[0876] In some embodiments, a solvent of Step (e.iii) comprises a polyols and / or polymers of polyols.

[0877] A solvent of Step (e.iii) may comprise aqueous solutions and organic solutions in combination to form a solvent mixture. In some embodiments, aqueous solutions and organic solutions may be used in combination to form a solvent mixture.

[0878] In some embodiments, wherein the void inclusion element is a lipid, the plurality of filaments is exposed to an organic solvent, such as iso-propanol. As the oil droplets mix with the organic phase and is subsequently removed, voids are created in the walls of the filaments in the spaces previously occupied by the oil droplets; thereby, imparting porosity onto the filaments.

[0879] In some embodiments, Step (e.iii) comprises treating the filaments with ethanol.

[0880] In some embodiments, Step (e.iii) comprises treating the filaments with iso-propanol.

[0881] The concentration of any singular component in a solvent mixture of Step (e.iii) may be within the range of 0 % to 100 % (v / v total solvent mixture volume).

[0882] In some embodiments, the concentration of any singular component in a solvent mixture of Step (e.iii) is within the range of 0 % to 100 %; preferably, 10 % to 75 %; and most preferably, 20 % to 60 % (v / v total solvent mixture volume).

[0883] In some embodiments, the concentration of any singular component in a solvent mixture of Step (e.iii) is within the range of 0 % to 5 %, 5 % to 10 %, 10 % to 15 %, 15 % to 20 %, 20 % to 25 %, 25 % to 30 %, 30 % to 35 %, 35 % to 40 %, 40 % to 45 %, 45 % to 50 %, 50 % to 55 %, 55 % to 60 %, 60 % to 65 %, 65 % to 70 %, 70 % to 75 %, 75 % to 80 %, 80 % to 85 %, 85 % to 90 %, 90 % to 95 %, or 95 % to 100 % (v / v total solvent mixture volume).

[0884] In some embodiments, Step (e.iii) does not comprise treating with any solvents.

[0885] In some embodiments, Step (e.iii) does not comprise treating the filaments with any solvents.

[0886] A solvent or solvent mixture of Step (e.iii) may have a pH within the range of 0 to 14.

[0887] In some embodiments, the pH of a solvent or solvent mixture of Step (e.iii) is within the range of 0 to 14. In some embodiments, the pH of a solvent or solvent mixture of Step (e.iii) is within the range of 0 to 1 ; 1 to 2; 2 to 3; 3 to 4; 4 to 5; 5 to 6; 6 to 7; 7 to 8; 8 to 9; 9 to 10; 10 to 1 1 ; 11 to 12; 12 to 13; or 13 to 14.

[0888] Step (e.iii) may be performed at a temperature that is within the range of 1 °C to 95 °C.

[0889] In some embodiments, Step (e.iii) is performed at a temperature that is within the range of 1 °C to 95 °C; preferably, 10 °C to 80 °C; more preferably, 15 °C to 50 °C; even more preferably, 15 °C to 30 °C; and most preferably, at room temperature.

[0890] In some other embodiments, Step (e.iii) is performed at a temperature that is within the range of 10 °C to 20 °C, 20 °C to 30 °C, 30 °C to 40 °C, 40 °C to 50 °C, 50 °C to 60 °C, 60 °C to 70 °C, or 70 °C to 80 °C. The duration of Step (e.iii) may be within the range of 1 second to 10 days.

[0891] In some embodiments, the duration of Step (e.iii) is ranges from of 1 second to 10 days; preferably, from 5 minutes to 1 day; more preferably, from 10 minutes and 6 hours; and most preferably for up to 1 hour.

[0892] In some other embodiments, Step (e.iii) is carried out over a duration that is within the range of 1 second to 5 minutes, 5 minutes to 15 minutes, 15 minutes to 30 minutes, 30 minutes to 1 hour, 1 hour to 2 hours,

[0893] 2 hours to 4 hours, 4 hours to 6 hours, 6 hours to 12 hours, 12 hours to 1 day, 1 day to 2 days, 2 days to

[0894] 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 7 days, or 7 days to 10 days.

[0895] Wherein a solvent is not used, Step (e.iii) may comprise pyrolysis-induced pore formation.

[0896] Herein, the term “pyrolysis-induced pore formation” herein refers to a process of thermal decomposition of one or more void inclusion elements to remove said void inclusion elements, thereby, imparting porosity.

[0897] In some embodiments, wherein a solvent is not used, Step (e.iii) comprises subjecting the filaments to pyrolysis-induced pore formation to impart porosity in the filament.

[0898] In some embodiments, Step (e.iii) is performed at a temperature that is higher than 95 °C;

[0899] In some embodiments, Step (e.iii) is not performed.

[0900] In some preferred embodiments, Step (e.iii) comprises treating the covalently-crosslinked filaments, comprising lipids, with ethanol at room temperature for 30 minutes.

[0901] In some preferred embodiments, Step (e.iii) comprises treating the filaments, comprising lipids, with isopropanol at room temperature for at least 20 minutes.

[0902] It is also contemplated that one or more void inclusion elements may be removed in processing steps that are distinct from Step (e.iii), including, but are not limited to, Step (d), Step (e.i), and / or Step (e.iv). For example, water-soluble void inclusion elements could dissolve in a coagulation bath solution of Step (d).

[0903] In another example, alcohol-soluble void inclusion elements could dissolve in a coagulation bath solution of Step (d) comprising aqueous solvents and one or more alcohols.

[0904] In some embodiments, one or more void inclusion elements are removed from in processing steps that are distinct from Step (e.iii), including, but are not limited to, Step (d), Step (e.i), and / or Step (e.iv).

[0905] In some embodiments, the solvent used to remove void inclusion elements in any of the embodiments of this disclosure can be used in the production of Prokitein and / or filaments of this disclosure. Step (e.iii) may comprise treating the hollow fibres of any embodiment of this disclosure with one or more solvents to remove void inclusion elements, which may impart porosity. In some embodiments, Step (e.iii) comprises treating the hollow fibres with one or more solvents to remove void inclusion elements, thereby imparting porosity. The solvents and conditions described in any embodiment of Step (e.iii) may be applied in the treatment of hollow fibres of any other embodiment.

[0906] Step (e.iii) may comprise treating a Prokitein of any embodiment of this disclosure with one or more solvents to remove void inclusion elements, which may impart porosity. In some embodiments, Step (e.iii) comprises treating a Prokitein with one or more solvents to remove void inclusion elements, thereby imparting porosity. The solvents and conditions described in any embodiment of Step (e.iii) may be applied in the treatment of a Prokitein of any other embodiment.

[0907] 1.13 STEP (E.iv) WASH

[0908] Step (e.iv) comprises washing with one or more wash solutions.

[0909] Step (e.iv) may comprise washing the crosslinked filaments with one or more wash solutions.

[0910] In some embodiments, Step (e.iv) comprises washing the crosslinked filaments with one or more wash solutions.

[0911] Step (e.iv) may comprise washing the crosslinked filaments with one or wash solutions to potentially decrease at least one of, the Young’s modulus, ultimate tensile strength and / or ultimate tensile strain of the filaments.

[0912] In some embodiments, Step (e.iv) comprises washing the crosslinked filaments with one or more wash solutions to decrease at least one of, the Young’s modulus, ultimate tensile strength and / or ultimate tensile strain of the filaments.

[0913] In some embodiments, Step (e.iv) comprises washing the covalently-crosslinked filaments with wash solutions to decrease at least one of, the Young’s modulus, ultimate tensile strength and / or ultimate tensile strain of the filaments.

[0914] The wash solutions of Step (e.iv) may comprise one or more acids, alkalis and / or buffer solutions.

[0915] The wash solutions of Step (e.iv) may comprise water.

[0916] In some embodiments, the filaments are treated in Step (e.iv) with water.

[0917] In some embodiments, a wash solutions of Step (e.iv) comprises one or more acids, alkalis and / or buffer solutions. In some embodiments, the filaments are treated in Step (e.iv) with one or more acids.

[0918] Acids used in Step (e.iv) may comprise ascorbic acid, acetic acid, adipic acid, citric acid, formic acid, hydrochloric acid, lactic acid, malic acid, malonic acid, oxalic acid, succinic acid, sulphuric acid and / or tartaric acid.

[0919] In some embodiments, one or more acids used in Step (e.iv) comprise ascorbic acid, acetic acid, adipic acid, citric acid, formic acid, hydrochloric acid, lactic acid, malic acid, malonic acid, oxalic acid, succinic acid, sulphuric acid and / or tartaric acid.

[0920] In some embodiments, the filaments are treated in Step (e.iv) with one or more alkalis.

[0921] Alkalis used in Step (e.iv) may comprise sodium hydroxide, potassium hydroxide, ammonia, calcium hydroxide, magnesium hydroxide sodium bicarbonate and / or sodium carbonate.

[0922] In some embodiments, one or more alkalis used in Step (e.iv) comprise sodium hydroxide, potassium hydroxide, ammonia, calcium hydroxide, magnesium hydroxide and / or sodium carbonate.

[0923] In some embodiments, the filaments are washed in Step (e.iv) with a buffer solution.

[0924] Buffer solutions used Step (e.iv) may comprise MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Tris or Trizma®, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, Phosphate, Citric Acid - Na2HPO4, Citric Acid - Sodium Citrate, Sodium Acetate - Acetic Acid, Na2HPO4 - NaH2PO4, Imidazole (glyoxaline) - HCI and / or Sodium Carbonate - Sodium Bicarbonate.

[0925] In some embodiments, buffer solutions used Step (e.iv) comprise MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Tris or Trizma®, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, Phosphate, Citric Acid - Na2HPO4, Citric Acid - Sodium Citrate, Sodium Acetate - Acetic Acid, Na2HPO4 - NaH2PO4, Imidazole (glyoxaline) - HCI and / or Sodium Carbonate - Sodium Bicarbonate.

[0926] In some embodiments, the filaments are washed in Step (e.iv) with a sodium carbonate buffer solution.

[0927] The pH of the wash solution may be within the range of 0 to 14. In some embodiments, the pH of the wash solution is within the range of 0 to 14; preferably, within the range of 0.01 to 5; and most preferably, within the range of 8.5 to 14.

[0928] In some embodiments, the pH of the wash solution is within the range of 0 to 1 ; 1 to 2; 2 to 3; 3 to 4; 4 to 5; 5 to 6; 6 to 7; 7 to 8; 8 to 9; 9 to 10; 10 to 11 ; 11 to 12; 12 to 13; or 13 to 14.

[0929] The duration of Step (e.iv) may be within the range of 10 minutes to 3 hours.

[0930] In some embodiments, the duration of Step (e.iv) is within the range of 10 minutes to 3 hours; preferably, 15 minutes to 2 hours; and most preferably, 30 minutes to 1 hour.

[0931] In some embodiments, the duration of Step (e.iv) is within the range of 10 minutes to 15 minutes, 15 minutes to 30 minutes, 30 minutes to 45 minutes, 45 minutes to 1 hour, 1 hour to 2 hours, or 2 hours to 3 hours.

[0932] Step (e.iv) may be performed at a temperature that is within the range of 10 °C to 80 °C.

[0933] In some embodiments, Step (e.iv) is performed at a temperature that is within the range of 10 °C to 80 °C; preferably, 20 °C to 60 °C; more preferably, 30 °C to 50 °C; and most preferably, at room temperature.

[0934] In some other embodiments, Step (e.iv) is performed at a temperature that is within the range of 10 °C to 20 °C, 20 °C to 30 °C, 30 °C to 40 °C, 40 °C to 50 °C, 50 °C to 60 °C, 60 °C to 70 °C, or 70 °C to 80 °C.

[0935] In some embodiments, Step (e.iv) is not performed.

[0936] In some particularly preferred embodiments, Step (e.iv) comprises washing the covalently-crosslinked filaments with an aqueous solution of sodium carbonate buffer with a pH of 1 1 .0 at 37 °C for 60 minutes.

[0937] Step (e.iv) may comprise washing the hollow fibres of any embodiment of this disclosure with one or more wash solutions. In some embodiments, Step (e.iii) comprises washing the hollow fibres with one or more wash solutions. The wash solutions and conditions described in any embodiment of Step (e.iv) may be applied in the treatment of hollow fibres of any other embodiment. In certain embodiments, treating the hollow fibres of any embodiment of this disclosure with a wash solution may result in a decrease in at least one of, the Young’s modulus, ultimate tensile strength and / or ultimate tensile strain of the hollow fibres.

[0938] Step (e.iv) may comprise washing a Prokitein of any embodiment of this disclosure with one or more wash solutions. In some embodiments, Step (e.iii) comprises washing a Prokitein with one or more wash solutions. The wash solutions and conditions described in any embodiment of Step (e.iv) may be applied in the treatment of a Prokitein of any other embodiment.

[0939] 1.14 STEP (E.v) COATINGS

[0940] Step (e.v) comprises coating.

[0941] Step (e.v) may comprise coating the crosslinked filaments.

[0942] Step (e.v) may comprise coating the covalently-crosslinked filaments.

[0943] In some embodiments, Step (e.v) comprises coating the crosslinked filaments.

[0944] In some embodiments, Step (e.v) comprises coating the covalently-crosslinked filaments.

[0945] In some embodiments, Step (e.v) comprises coating the covalently-crosslinked filaments with one or more coatings, wherein the composition and / or method of application of the coating is selected based on the intended end-use application (e.g., textiles). The coating may be employed to modify surface characteristics, enhance performance, or impart additional functionalities, including, without limitation, hydrophobicity, oleophilicity, antimicrobial activity, electrical or thermal conductivity, flame resistance, UV- resistance, enhanced adhesion, decorative finishes, or controlled degradation. Suitable application techniques may comprise dip-coating, spray-coating, in-situ polymerisation, plasma treatment, and layer- by-layer deposition.

[0946] Coatings are widely used in conjunction with traditional cell culture techniques to aid cell attachment and / or proliferation. Coatings which may be used in Step (e.v) comprise protein complexes, polypeptides, cell attachment peptide sequences (CAPs), and / or growth factors (Discovering Cell-Adhesion Peptides in Tissue Engineering: Beyond RGD, 2018; Recent advances in post-modification strategies of polymeric electrospun membranes, 2018).

[0947] Step (e.v) may comprise protein complexes to potentially enhance cell attachment to the filaments of this disclosure. Protein complexes which may used in Step (e.v) comprise collagen, gelatine, fibrinogen, fibronectin and / or laminin.

[0948] In some embodiments, protein complexes are used in Step (e.v) to enhance cell attachment to the filaments of this disclosure.

[0949] In some embodiments, protein complexes used in Step (e.v) comprise collagen, gelatine, fibrinogen, fibronectin and / or laminin. In some embodiments, protein complexes are not used in Step (e.v).

[0950] CAPs are short sequences of amino acids that embody the minimum motif required for cell attachment (Discovering Cell-Adhesion Peptides in Tissue Engineering: Beyond RGD, 2018). In some embodiments, CAPs are used in Step (e.v) to enhance cell attachment to the filaments of this disclosure.

[0951] Step (e.v) may comprise CAPS to potentially enhance cell attachment to the filaments of this disclosure. CAPS used in Step (e.v) may comprise AELDVP, CGGNGEPRGDTYRAY, CFALRGDNP, CKKQRFRHRNRKG, CNYYSNS, CSVTCG, DGEA, ELVTDFPTDLPAT, FHRRIKA, FQGVLQNVRFVF, GACRGDCLGA (cyclic), GFOGER, GFRGDGQ, GRGDS, GRGDAC, GTFALRGDNGQ, IDAPS, IKLLI, IKVAV, IWKHKGRDVILKKDVRFYC, KAFDITYVRLKF, KLDAPT (FN5), KQAGDV, KRSR, LIGRKK, LGTIPG, LRE, LRGDN, MNYYSNS, NPWHSIYITRFG, PDGSR, PHRSN, PKRGDL, PRARI, REDV, RGD, SIGFRGDGQTC, SIKVAV, SINNNR, SPPRRARV, SVVYGLR, TWYKIAFQRNRK, VALDEP, VGVAPG, VPGIG, WQPPRARI, and / or YIGSR.

[0952] In some embodiments, CAPs are used in Step (e.v) to enhance cell attachment to the filaments of this disclosure.

[0953] In some embodiments, CAPS used in Step (e.v) comprise AELDVP, CGGNGEPRGDTYRAY, CFALRGDNP, CKKQRFRHRNRKG, CNYYSNS, CSVTCG, DGEA, ELVTDFPTDLPAT, FHRRIKA, FQGVLQNVRFVF, GACRGDCLGA (cyclic), GFOGER, GFRGDGQ, GRGDS, GRGDAC, GTFALRGDNGQ, IDAPS, IKLLI, IKVAV, IWKHKGRDVILKKDVRFYC, KAFDITYVRLKF, KLDAPT (FN5), KQAGDV, KRSR, LIGRKK, LGTIPG, LRE, LRGDN, MNYYSNS, NPWHSIYITRFG, PDGSR, PHRSN, PKRGDL, PRARI, REDV, RGD, SIGFRGDGQTC, SIKVAV, SINNNR, SPPRRARV, SVVYGLR, TWYKIAFQRNRK, VALDEP, VGVAPG, VPGIG, WQPPRARI, and / or YIGSR.

[0954] In some embodiments, CAPs are not used in Step (e.v).

[0955] Step (e.v) may comprise growth factors to potentially enhance cell attachment to the filaments of this disclosure. Growth factors which may be used in Step (e.v) comprise basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and / or vascular endothelial growth factor (VEGF).

[0956] In some embodiments, growth factors are used in Step (e.v) to enhance cell attachment to the filaments of this disclosure. In some embodiments, growth factors used in Step (e.v) comprise basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and / or vascular endothelial growth factor (VEGF).

[0957] In some embodiments, heparin is used in Step (e.v) to enhance cell attachment to the filaments of this disclosure.

[0958] In some embodiments, growth factors are not used in Step (e.v).

[0959] In some embodiments, protein complexes, CAPs and / or growth factors are used in Step (e.v).

[0960] Chemical methods, such as grafting, may be used to coat the surface of the filaments with protein complexes, CAPS and / or growth factors to enhance cell attachment to the filaments.

[0961] In some embodiments, chemical methods are used to coat the surface of the filaments with protein complexes, CAPS and / or growth factors to enhance cell attachment to the filaments.

[0962] Peptide conjunction methods may be used to coat the surface filaments with protein complexes, CAPS and / or growth factors to enhance cell attachment to the filaments. Exemplary protein conjunction methods as contemplated by the present disclosure comprise those described in WO2023152492A1 , which is incorporated herein by reference in its entirety.

[0963] In some embodiments, Peptide conjunction methods are used to coat the surface of the filaments with protein complexes, CAPS and / or growth factors to enhance cell attachment to the filaments.

[0964] In some embodiments, the surface of the filaments are not coated with protein complexes CAPS and / or growth factors to enhance cell attachment to the filaments.

[0965] In some embodiments, Step (e.v) is not performed.

[0966] In some preferred embodiments, Step (e.v) comprises leaving the filaments uncoated.

[0967] Step (e.v) may comprise coating the hollow fibres of any embodiment of this disclosure. In some embodiments, Step (e.v) comprises coating the hollow fibres. The coatings and conditions described in any embodiment of Step (e.v) may be applied in the treatment of hollow fibres of any other embodiment. Step (e.v) may comprise coating a Prokitein of any embodiment of this disclosure. In some embodiments, Step (e.v) comprises coating a Prokitein. The coatings and conditions described in any embodiment of Step (e.v) may be applied in the treatment of a Prokitein of any other embodiment.

[0968] 1.15 STEP (E.vi) SURFACE TOPOGRAPHIC MODIFICATION

[0969] Step (e.vi) comprises modifying surface topography.

[0970] Step (e.vi) may comprise modifying the surface topography of the crosslinked filaments.

[0971] In some embodiments, Step (e.vi) comprises modifying the surface topography of the crosslinked filaments.

[0972] In some embodiments, Step (e.vi) comprises modifying the surface topography of the covalently- crosslinked filaments.

[0973] Step (e.vi) may comprise stretching the filaments to create tears in the microstructure, thereby potentially forming porous structures.

[0974] In some embodiments, Step (e.vi) comprises stretching the filaments to create tears in the microstructure, forming porous structures.

[0975] Step (e.vi) may comprise modifying the surface topography of the filaments.

[0976] In some embodiments, the surface topography of the filaments is modified.

[0977] In some embodiments, the surface topography of the filaments is not modified.

[0978] Methods which may be used to modify the surface topography of the filaments comprise physical methods such as plasma treatment, physical vapour deposition, ultra-sonification and mechanical etching.

[0979] In some embodiments, methods which are used to modify the surface topography of the filaments comprise physical methods such as plasma treatment, physical vapour deposition, ultra-sonification and mechanical etching.

[0980] Mechanical etching may be used to impart striations along the surface of the filaments.

[0981] In some embodiments, mechanical etching is used to impart striations along the surface of the filaments. The die or spinneret orifice geometry, used in Step (c), may be used to impart striations along the surface of the filaments.

[0982] In some embodiments, the die or spinneret orifice geometry, used in Step (c), is used to impart striations along the surface of the filaments.

[0983] In one particularly preferred embodiment, Step (e.vi) comprises leaving the surface topography of the filaments unmodified.

[0984] Step (e.vi) may comprise modifying surface topography of the hollow fibres of any embodiment of this disclosure. In some embodiments, Step (e.vi) comprises modifying surface topography the hollow fibres. The methods of modifying surface topography described in any embodiment of Step (e.v) may be applied in the treatment of hollow fibres of any other embodiment.

[0985] Step (e.vi) may comprise modifying surface topography of a Prokitein of any embodiment of this disclosure. In some embodiments, Step (e.vi) comprises modifying surface topography a Prokitein. The methods of modifying surface topography described in any embodiment of Step (e.v) may be applied in the treatment of a Prokitein of any other embodiment.

[0986] 1.16 STEP (E.vii) DRAWING

[0987] Step (e.vii) comprises drawing.

[0988] As used herein, the term “drawing” refers to a mechanical process in which a material, such as a filament, fibre or polymer, is stretched under tension.

[0989] In some embodiments, drawing may be used to orient or align the molecular chains of a filament of this disclosure, which may, improve its mechanical properties, and / or reduce its cross-sectional area.

[0990] Step (e.vii) may comprise drawing the crosslinked filaments.

[0991] In some embodiments, Step (e.vi) comprises drawing the crosslinked filaments.

[0992] In some embodiments, the crosslinked filaments are not drawn.

[0993] Drawing the crosslinked filaments may comprise stretching and thinning.

[0994] In some embodiments, the filaments are stretched under tension.

[0995] Drawing a crosslinked filament may align its molecular chains, increase crystallinity, enhance its mechanical properties, improve water stability, and / or reduce time-dependent deformation (creep). In some embodiments, drawing also increases the length of the filament and correspondingly reduces its cross-sectional area. In some embodiments, drawing aligns the molecular chains in the filaments.

[0996] In some embodiments, drawing increases the crystallinity in the filaments.

[0997] In some embodiments, drawing enhances mechanical properties of the filaments.

[0998] In some embodiments, drawing increases the water-stability of the filaments.

[0999] In some embodiments, drawing decreases creep (time-dependent deformation) of the filaments.

[1000] In some embodiments, drawing increases filament length.

[1001] In some embodiments, drawing reduces the cross-sectional area of the filaments.

[1002] Drawing may be achieved through the controlled winding and unwinding of spooled filaments such that the rate of winding and unwinding are different to impart a draw ratio.

[1003] In some embodiments, drawing is achieved through the controlled winding and unwinding of spooled filaments such that the rate of winding and unwinding are different to impart a draw ratio.

[1004] Stretching and / or drawing may be achieved through the lengthening of a filament fixed between a fixed anchor point and a secondary displaced fixed point.

[1005] In some embodiments, drawing is achieved through the lengthening of a filament fixed between a fixed anchor point and a secondary displaced fixed point.

[1006] Drawing may be achieved through the lengthening of a filament fixed between two anchor points which are progressively separated from each other.

[1007] In some embodiments, drawing may be achieved through the lengthening of a filament fixed between two anchor points which are progressively separated from each other.

[1008] As used herein, the term “draw ratio” is defined as the ratio of the length of a filament before and after the drawing process of Step (e.vii).For example, a draw ratio of 1 :4 means the filament has been stretched to 4 times its original length.

[1009] The draw ratio may be within the range of 1 :1 .1 to 1 :10.

[1010] In some embodiments, the draw ratio is 1 :1 .1 , 1 :1 .2, 1 :1 .3, 1 :1 .4, 1 :1 .5, 1 :1 .6, 1 :1 .7, 1 :1 .8, 1 :1 .9, 1 :2, 1 :3, 1 :3.5, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10.

[1011] In some embodiments, the draw ratio is in the range of 1 :1 .1 to 1 :1.5, 1 :1 .5 to 1 :2, 1 :2 to 1 :3, 1 :3 to 1 :4, 1 :4 to 1 :5, 1 :5 to 1 :6, 1 :6 to 1 :7, 1 :7 to 1 :8, 1 :8 to 1 :9, or 1 :9 to 1 :10.

[1012] Drawing a covalently-crosslinked filament may increase ultimate tensile strength, increase its Young’s modulus and / or decrease its ultimate tensile strain.

[1013] In some embodiments, drawing increases the ultimate tensile strength of the filaments.

[1014] In some embodiments, drawing increases the Young’s modulus of the filaments. In some embodiments, drawing decreases the ultimate tensile strain of the filaments.

[1015] Step (e.vii) may comprise drawing the hollow fibres of any embodiment of this disclosure. In some embodiments, Step (e.vii) comprises drawing the hollow fibres. The methods of drawing described in any embodiment of Step (e.vii) may be applied in the treatment of hollow fibres of any other embodiment.

[1016] Step (e.vii) may comprise drawing and / or stretching a Prokitein of any embodiment of this disclosure. In some embodiments, Step (e.vii) comprises drawing and / or stretching a Prokitein. The methods of drawing described in any embodiment of Step (e.vii) may be applied in the treatment of a Prokitein of any other embodiment.

[1017] The post-production modification processes of Step (e), described herein in Steps (e.i) to (e.vii), may be performed in any combination and in any sequence.

[1018] In some embodiments, Step (e.i) is performed.

[1019] In some embodiments, Step (e.i) is not performed.

[1020] In some embodiments, Step (e.ii) is performed.

[1021] In some embodiments, Step (e.ii) is not performed.

[1022] In some embodiments, Step (e.iii) is performed.

[1023] In some embodiments, Step (e.iii) is not performed

[1024] In some embodiments, Step (e.iv) is performed.

[1025] In some embodiments, Step (e.iv) is not performed.

[1026] In some embodiments, Step (e.v) is performed.

[1027] In some embodiments, Step (e.v) is not performed.

[1028] In some embodiments, Step (e.vi) is performed.

[1029] In some embodiments, Step (e.vi) is not performed.

[1030] In some embodiments, Step (e.vii) is performed.

[1031] In some embodiments, Step (e.vii) is not performed.

[1032] In some preferred embodiments, Step (e.i) and Step (e.ii) are performed sequentially in the order specified.

[1033] In some embodiments, Step (e.i), Step (e.ii) and Step (e.iii) are performed sequentially in the order specified.

[1034] In some embodiments, Step (e.i), Step (e.iii) and Step (e.ii) are performed sequentially in the order specified.

[1035] In some embodiments, one or more of any of Step (e.i), to Step (e.vi) are performed sequentially in the order specified.

[1036] In some embodiments, Step (e.i), Step (e.ii), Step (e.iii) and Step (e.iv) are performed sequentially in the order specified. In some embodiments, Step (e.i), Step (e.ii), Step (e.iii), Step (e.iv) and Step (e.v) are performed sequentially in the order specified.

[1037] In some embodiments, Step (e.i), Step (e.ii), Step (e.iii), Step (e.iv), Step (e.v) and Step (e.vi) are performed sequentially in the order specified.

[1038] In some embodiments, Step (e.i), Step (e.ii), Step (e.iii), Step (e.iv), Step (e.v) , Step (e.vi) and Step (e.vii) are performed sequentially in the order specified.

[1039] In some embodiments, Step (e.i), Step (e.ii), Step (e.iv), Step (e.v) and Step (e.vi) are performed sequentially in the order specified.

[1040] In some embodiments, Step (e.i), Step (e.ii), Step (e.iii), Step (e.v) and Step (e.vi) are performed sequentially in the order specified.

[1041] In some embodiments, Step (e.i), Step (e.ii), Step (e.iii), Step (e.iv), and Step (e.vi) are performed sequentially in the order specified.

[1042] In some embodiments, Step (e.i), Step (e.ii), Step (e.iii), Step (e.iv), and Step (e.v) are performed sequentially in the order specified.

[1043] In some embodiments, Step (e.i), Step (e.ii) and Step (e.iii) are performed sequentially in the order specified.

[1044] In some embodiments, Step (e.i), Step (e.ii), and Step (e.iv), are performed sequentially in the order specified.

[1045] In some embodiments, Step (e.i), Step (e.ii), and Step (e.v) are performed sequentially in the order specified.

[1046] In some embodiments, Step (e.i), Step (e.ii), and Step (e.vi) are performed sequentially in the order specified.

[1047] In some embodiments, Step (e.i), Step (e.ii), and Step (e.vii) are performed sequentially in the order specified.

[1048] In some embodiments, Step (e.i), Step (e.ii) and Step (e.iii) are performed within a single process step.

[1049] 1.17 STEP (F) DRYING AND STORAGE

[1050] Step (f) comprises drying and / or storing.

[1051] Step (f) may comprise drying the treated crosslinked filaments, to produce dried, treated crosslinked filaments.

[1052] Step (f) may comprise drying the treated covalently-crosslinked filaments, to produce dried, treated covalently-crosslinked filaments.

[1053] In some embodiments, Step (f) comprises drying the treated crosslinked filaments, to produce dried, treated covalently-crosslinked filaments. In some embodiments, Step (f) comprises drying the treated covalently-crosslinked filaments, to produce dried, treated crosslinked filaments.

[1054] The treated crosslinked filaments may be partially dried or completely dried.

[1055] In some embodiments, the treated crosslinked filaments are partially dried; and preferably completely dried.

[1056] In some embodiments, the treated crosslinked filaments are not dried.

[1057] Step (f) may comprise drying the filaments with convection, radiation, freeze-drying, a microwave source, an infra-red heat source, and / or on a heated surface.

[1058] In some embodiments, Step (f) comprises drying the filaments with convection, radiation, freeze-drying, a microwave source, an infra-red heat source, and / or on a heated surface.

[1059] In some embodiments, freeze-drying is used modify the porosity of the crosslinked filaments.

[1060] In some embodiments, freeze-drying is additionally used modify the porosity of the crosslinked filaments.

[1061] In some embodiments, freeze-drying is not used modify the porosity of the crosslinked filaments.

[1062] In some embodiments, Step (f) does not comprise freeze-drying.

[1063] Step (f) may comprise drying at temperatures that are within the range of 5 °C to 90 °C.

[1064] In some embodiments, Step (f) comprises drying at temperatures that range from 5 °C to 90 °C; preferably, 15 °C to 80 °C; more preferably, 40 °C to 70 °C; and most preferably, 50 °C to 70 °C.

[1065] In some embodiments, Step (f) comprises drying at temperatures that range from 5 to 90 °C; preferably, 15 °C to 75 °C; more preferably, 20 °C to 60 °C; even more preferably, 30 to 50 °C; and most preferably, at 40 °C.

[1066] In some other embodiments, Step (f) comprises drying at room temperature.

[1067] In some other embodiments, Step (f) comprises drying at temperatures that range from 5 °C to 10 °C, 10 °C to 15 °C, 15 °C to 20 °C, 20 °C to 25 °C, 25 °C to 30 °C, 30 °C to 35 °C, 35 °C to 40 °C, 40 °C to 45 °C, 45 °C to 50 °C, 50 °C to 55 °C, 55 °C to 60 °C, 60 °C to 65 °C, 65 °C to 70 °C, 70 °C to 75 °C, 75 °C to 80 °C, 80 °C to 85 °C, and / or 85 °C to 90 °C.

[1068] Step (f) may comprise freeze-drying, which can aid in the formation of pores and / or modification of the microstructure of the crosslinked filaments. In some embodiments, Step (f) comprises freeze-drying.

[1069] In some embodiments, freeze-drying is used modify the porosity of the filaments.

[1070] In some embodiments, freeze-drying is additionally used modify the porosity of the filaments.

[1071] In other embodiments, Step (f) does not comprise freeze-drying.

[1072] In some other embodiments, Step (f) comprises drying at temperatures that range from -80 °C to 0 °C,

[1073] Step (f) may comprise chemical drying with solvents. As used herein, the term “chemical drying” refers to the process of removing water from a material by using a chemical agent that reacts with, absorbs, or adsorbs water, rather than relying primarily on heat or mechanical evaporation. Solvents which may be used for chemical drying comprise acetone, ethanol, ethanol, propanol, iso-propanol, butanol, iso-butanol, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, and / or mixtures thereof.

[1074] In some embodiments, the filaments are chemically-dried with solvents.

[1075] In certain embodiments, the filaments are air-dried following chemical drying.

[1076] In other embodiments, Step (f) does not comprise chemical drying.

[1077] In some embodiments, Step (f) comprises drying under conditions defined herein for the pre-drying process of Step (e.ii).

[1078] The crosslinked filaments may be stored.

[1079] In some embodiments, the crosslinked filaments are stored.

[1080] In some embodiments, crosslinked filaments are stored after they have been dried.

[1081] The treated crosslinked filaments may be stored in air, inert gases (such as nitrogen, helium, neon, argon, and xenon), CO2, under vacuum and / or in a PBS buffer solution, water, an aqueous solution, organic solution, and / or a mixture thereof.

[1082] In some embodiments, the filaments are stored in air.

[1083] In some embodiments, the filaments are stored in an inert gas, such as nitrogen, helium, neon, argon, xenon and / or mixtures thereof.

[1084] In some embodiments, the filaments are stored in CO2.

[1085] In some embodiments, the filaments are stored under vacuum.

[1086] In some embodiments, the filaments are stored in PBS buffer solution.

[1087] In some embodiments, the treated crosslinked filaments are stored in water and / or an aqueous solution. Suitable components which are present within the aqueous solution could include, but are not limited to, water, alcohols, polyols, acids, alkalis, salts and / or combinations thereof. In some embodiments, the treated crosslinked filaments are stored in water and / or an aqueous solution. Suitable components which are present within the aqueous solution could include, but are not limited to, water, alcohols, polyols, acids, alkalis, salts and / or combinations thereof.

[1088] In some embodiments, the treated crosslinked filaments are stored in an organic solvent.

[1089] In other embodiments, the filaments are stored in a mixture of organic and aqueous solvents, such as, but not limited to, water and ethanol.

[1090] The treated crosslinked filaments may be stored at temperatures that are within the range of -90 °C to 90 °C.

[1091] In some embodiments, Step (f) comprises storing the filaments at temperatures that range from -90 °C to 90 °C; preferably, -20 °C to 50 °C; more preferably, 0 °C to 37 °C; and most preferably, at room temperature.

[1092] In some embodiments, the filaments are stored at room temperature.

[1093] In some embodiments, the filaments are stored in refrigerated conditions, at temperatures between 0 “C and 8 °C.

[1094] In some embodiments, the filaments are stored in frozen conditions, at temperatures between -90 “C and 0 °C.

[1095] In some preferred embodiments, Step (f) comprises partially drying the treated crosslinked filaments through exposure to heat, and subsequently storing them in air.

[1096] In some preferred embodiments, Step (f) comprises partially drying the treated crosslinked filaments through exposure to heat, and subsequently rehydrating them in an aqueous 45 % glycerol solution, and then storing them in air.

[1097] Step (f) may comprise drying and / or storing the hollow fibres of any embodiment of this disclosure. In some embodiments, Step (f) comprises drying and / or storing the hollow fibres. The methods of drying and storing described in any embodiment of Step (f) may be applied to the hollow fibres of any other embodiment.

[1098] Step (f) may comprise drying and / or storing the hollow fibres of any embodiment of this disclosure. In some embodiments, Step (f) comprises drying and / or storing the hollow fibres. The methods of drying and storing described in any embodiment of Step (f) may be applied to the hollow fibres of any other embodiment. Step (f) may comprise drying and / or storing a Prokitein of any embodiment of this disclosure. In some embodiments, Step (f) comprises drying and / or storing a Prokitein. The methods of drying and storing described in any embodiment of Step (f) may be applied to a Prokitein of any other embodiment.

[1099] In some preferred embodiments, the process steps are carried out sequentially in the (a)-(f) order specified.

[1100] In some preferred embodiments, the process for the production of a plurality of filaments, comprising covalent ester, thioester and / or amide crosslinked polypeptides comprises sequentially carrying out Step (a), Step (b), Step (c), Step (d), Step (e.i), Step (e.ii) and Step (e.iii) in the order specified.

[1101] In some preferred embodiments, the process for the production of a plurality of filaments, comprising covalent ester, thioester and / or amide crosslinked polypeptides comprises sequentially carrying out Step (a), Step (b), Step (c), Step (d), Step (e.i), Step (e.iii) and Step (e.ii) in the order specified.

[1102] In some preferred embodiments, the process for the production of a plurality of filaments, comprising covalent ester, thioester and / or amide crosslinked polypeptides comprises sequentially carrying out Step (a), Step (b), Step (c), Step (d), Step (e.i), Step (e.ii), and Step (e.vii) in the order specified.

[1103] The Applicant also provides a plurality of filaments comprising covalent ester, thioester and / or amide crosslinked polypeptides, that are obtained or are obtainable by a process of the application.

[1104] The Applicant also provides a polymer comprising covalent ester, thioester and / or amide crosslinked polypeptides, that is obtained or are obtainable by a process ofthe application.

[1105] The Applicant also provides a plurality of filaments, wherein said filaments comprise a semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

[1106] The Applicant also provides a plurality of filaments, wherein said filaments comprise a regular semicrystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

[1107] In some embodiments, the drying conditions used in any of the embodiments of this disclosure can be used in the production of Prokitein and / or filaments of this disclosure. 1.18 PHYSICAL STRUCTURES OF THE COVALENTLY-CROSSLINKED FILAMENTS

[1108] In certain aspects and embodiments, the filaments of this disclosure comprise one or more covalent ester, thioester and / or amide crosslinked polypeptides, wherein:

[1109] (a) the external diameter of the filament is 50 - 6,600 pm;

[1110] In certain aspects and embodiments, the filaments of this disclosure comprise one or more covalent ester, thioester and / or amide crosslinked polypeptides, wherein:

[1111] (a) the external diameter of the filament is 50 - 6,600 pm; and

[1112] (b) the porosity of the filament is 30-75 %.

[1113] In certain aspects and embodiments, the filaments of this disclosure comprise one or more covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

[1114] In certain aspects and embodiments, the filaments of this disclosure comprise a regular semi-crystalline polymer comprising ionically crosslinked polypeptides with a beta-sheet secondary structure.

[1115] In certain aspects and embodiments, the filaments of this disclosure comprise a semi-crystalline polymer comprising ionically crosslinked polypeptides with a beta-sheet secondary structure.

[1116] In certain aspects and embodiments, the filaments of this disclosure comprise a regular semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides, and ionically crosslinked polypeptides, with a beta-sheet secondary structure.

[1117] A filament of this disclosure may have a cross-section that is a circle, near-circle, oval, square, rectangle, triangle, pentagon, hexagon, or other polygon.

[1118] In some embodiments, a filament of this disclosure has a cross-section that is a circle, near-circle, oval, square, rectangle, triangle, pentagon, hexagon, or other polygon.

[1119] In some embodiments, a filament of this disclosure has a circular cross-section.

[1120] A filament of this disclosure may be in the form of a cylinder, preferably a circular cylinder.

[1121] In some embodiments, a filament of this disclosure is in the form of a cylinder, preferably a circular cylinder.

[1122] A filament of this disclosure may be of any desired length.

[1123] In some embodiments, a filament of this disclosure is of any desired length.

[1124] The external diameter of a filament of this disclosure may be within the range of 15 pm and 6600 pm. In some embodiments, the external diameter of a filament of this disclosure is within the range of 15 pm and 6600 pm; preferably, 25 pm to 700 pm, more preferably, 50 pm to 500 pm; and most preferably, 100 pm to 250 pm.

[1125] In some other embodiments, the external diameter of a filament of this disclosure is within the range of range 15 pm to 20 pm, 20 pm to 30 pm, 30 pm to 40 pm, 40 pm to 50 pm, 50 pm to 100 pm, 100 pm to 200 pm, 200 pm to 300 pm, 300 pm to 400 pm, 400 pm to 500 pm, 500 pm to 600 pm, 600 pm to 700 pm, 700 pm to 800 pm, 800 pm to 900 pm, 900 pm to 1000 pm, 1000 pm to 1100 pm, 1 100 pm to 1200 pm, 1200 pm to 1300 pm, 1300 pm to 1400 pm, 1400 pm to 1500 pm, 1500 pm to 1600 pm, 1600 pm to 1700 pm, 1700 pm to 1800 pm, 1800 pm to 1900 pm, 1900 pm to 2000 pm, 2000 pm to 2100 pm, 2100 pm to 2200 pm, 2200 pm to 2300 pm, 2300 pm to 2400 pm, 2400 pm to 2500 pm, 2500 pm to 2600 pm, 2600 pm to 2700 pm, 2700 pm to 2800 pm, 2800 pm to 2900 pm, 2900 pm to 3000 pm, 3000 pm to 3100 pm, 3100 pm to 3200 pm, 3200 pm to 3300 pm, 3300 pm to 3400 pm, 3400 pm to 3500 pm, 3500 pm to 3600 pm, 3600 pm to 3700 pm, 3700 pm to 3800 pm, 3800 pm to 3900 pm, 3900 pm to 4000 pm, 4000 pm to 4100 pm, 4100 pm to 4200 pm, 4200 pm to 4300 pm, 4300 pm to 4400 pm, 4400 pm to 4500 pm, 4500 pm to 4600 pm, 4600 pm to 4700 pm, 4700 pm to 4800 pm, 4800 pm to 4900 pm, 4900 pm to 5000 pm, 5000 pm to 5100 pm, 5100 pm to 5200 pm, 5200 pm to 5300 pm, 5300 pm to 5400 pm, 5400 pm to 5500 pm, 5500 pm to 5600 pm, 5600 pm to 5700 pm, 5700 pm to 5800 pm, 5800 pm to 5900 pm, 5900 pm to 6000 pm, 6000 pm to 6100 pm, 6100 pm to 6200 pm, 6200 pm to 6300 pm, 6300 pm to 6400 pm, 6400 pm to 6500 pm, or 6500 pm to 6600 pm.

[1126] Scanning electron microscopy (SEM) may be used to produce images of a material with a wide range of magnifications from 10 to more than 500,000 times by scanning the surface of the material with a beam of electrons. SEM image analysis can be used by a person of skill in the art to quantify various structural characteristics of the filaments of this disclosure, including external diameter, surface pore diameter, and inner structure (Reimer, 1998; Ul-Hamid, 2018).

[1127] The filaments of this disclosure may semi-permeable.

[1128] In some embodiments, the filaments of this disclosure are semi-permeable, such that fluids and some solutes comprising nutrients, can pass through the filaments, but soluble growth factors, proteins and cells cannot.

[1129] In some embodiments, the filaments of this disclosure are not semi-permeable.

[1130] In some embodiments, the filaments of this disclosure are permeable, such that fluids, nutrients, soluble growth factors, proteins and cells can pass through the filaments. Soluble growth factors that may be of particular interest in cellular agriculture include, but are not limited to, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) (Bioprocessing technology of muscle stem cells: Implications for cultured meat, 2021).

[1131] Soluble growth factors may vary in size. For example, bFGF ranges between 18-34 kDa, depending on its form. HGF is cleaved to form two active polypeptide chains, which are 69 kDa and 34 kDa in size.

[1132] Whereas, IGF-1 is 7.6 kDa; EGF is 6 kDa; and VEGF, has 17 isoforms which range in size from 16-45 kDa.

[1133] The filaments of this disclosure may be semi-permeable such that solutes smaller than 7 kDa may pass through, but solutes larger than 7 kDa may not.

[1134] In some embodiments, the filaments of this disclosure are semi-permeable such that solutes smaller than 7 kDa may pass through, but some solutes larger than 7 kDa may not.

[1135] In some embodiments, the filaments of this disclosure are porous, semi-permeable such that solutes of sizes within the range 7 kDa to 12 kDa, 12 kDa to 17 kDa, 17 kDa to 22 kDa, 22 kDa to 27 kDa, 27 kDa to 32 kDa, 32 kDa to 37 kDa, 37 kDa to 42 kDa, 42 kDa to 47 kDa, 47 kDa to 52 kDa, 52 kDa to 57 kDa, 57 kDa to 62 kDa, 62 kDa to 67 kDa, 67 kDa to 72 kDa, 72 kDa to 77 kDa, or larger than 77 kDa can pass through, but solutes outside of these ranges cannot.

[1136] In some embodiments, the filaments of this disclosure are semi-permeable such that solutes of sizes within the range 7 kDa to 12 kDa, 12 kDa to 17 kDa, 17 kDa to 22 kDa, 22 kDa to 27 kDa, 27 kDa to 32 kDa, 32 kDa to 37 kDa, 37 kDa to 42 kDa, 42 kDa to 47 kDa, 47 kDa to 52 kDa, 52 kDa to 57 kDa, 57 kDa to 62 kDa, 62 kDa to 67 kDa, 67 kDa to 72 kDa, 72 kDa to 77 kDa, or larger than 77 kDa can pass through, but solutes outside of these ranges cannot.

[1137] The filaments of this disclosure may be porous.

[1138] In some embodiments, the filaments of this disclosure are porous.

[1139] Materials may be classified as being macroporous (> 50 nm), mesoporous (2 - 50 nm) or microporous (< 2 nm) depending on the range of the pore sizes in the material. Microporous materials may also be referred to as nanoporous. A material is classified by the largest pore size present. For example, a porous material comprising pores which are smaller than 50 nm, as well as pores which are larger than 50 nm, would be classified as macroporous.

[1140] The filaments of this disclosure may be macroporous, mesoporous and / or microporous. In some embodiments, the filaments of this disclosure are macroporous, mesoporous and / or microporous.

[1141] The filaments of this disclosure may have pores with diameters that are within the range of 1 nm to 100 pm.

[1142] In some embodiments, the filaments of this disclosure have pores with diameters that are within the range of 1 nm to 100 pm; preferably, 5 nm to 15 pm; more preferably, 10 nm to 5 pm; even more preferably, 50 nm to 3 pm; and most preferably, 100 nm to 2 pm.

[1143] In some other embodiments, the filaments of this disclosure have pores with diameters that are within the range of 1 nm to 10 nm, 10 nm to 50 nm, 50 nm to 100 nm,100 nm to 500 nm, 500 nm to 1 pm, 1 pm to 5 pm, 5 pm to 10 pm, 10 pm to 50 pm, and / or 50 pm to 100 pm.

[1144] In some embodiments, there is provided a combination of filaments of this disclosure, which comprises:

[1145] (i) a plurality of filaments having pore sizes that are within the range of 1 pm to 15 pm; and

[1146] (ii) a plurality of filaments having pore sizes that are within the range of 1 nm to 10 nm.

[1147] A filament of this disclosure may have a porosity that is within the range of 1 % to 95 %.

[1148] In some embodiments, a filament of this disclosure has a porosity that is within the range of 1 % to 95 %; preferably, 50 % to 90 %; and most preferably, 60 % to 80 %.

[1149] In some embodiments, a filament of this disclosure has a porosity that is within the range of 1 % to 20 %, 20 % to 40 %, 40 % to 60 %, 60 % to 80 %, or 80 % to 90 %.

[1150] In some other embodiments, a filament of this disclosure has a porosity that is below 1 %.

[1151] Mercury porosimetry is the gold standard for the measurement of pore volume, porosity and pore size of semi-permeable porous filaments. Based on the capillary law for small pores, porosity may be determined by the total penetrating volume of a non-wetting fluid per unit volume of sample. Pore size may be calculated from the Washburn equation. Modern mercury porosimetry may measure pore diameters ranging between 900 pm to 0.005 pm (Webb, 2001). Additionally, surface pore diameter may be measured through SEM image analysis (Reimer, 1998; Ul-Hamid, 2018). Such analytical methods may be applied to the filaments of this disclosure, by someone of skill in the art.

[1152] As used herein, the term “Molecular Weight Cut-Off’ (MWCO) is defined as the lowest molecular weight at which more than 90 % of the solute with a known molecular weight is retained by filament. Typically, polyethylene glycols, poly(ethylene oxide) or bovine serum albumin are used as marker molecules to determine the MWCO (Bradley Ladewig, 2016). The change in abundance of these marker molecules in the feed and permeate streams, can be measured with refractive index signal intensity separated by size exclusion chromatography with a high-performance liquid chromatography analyser. This method may be used by someone skilled in the art to determine the molecular cut off weight of the filaments of this disclosure.

[1153] The MWCO of a filament of this disclosure may be within the range of 5 kDa to 100 kDa.

[1154] In some embodiments, the MWCO of a filament of this disclosure is within the range of 5 kDa to 100 kDa; preferably, 5 kDa to 7 kDa; more preferably, 7 kDa and 10 kDa; even more preferably, 10 kDa and 20 kDa; and most preferably, 20 kDa and 30 kDa.

[1155] In some other embodiments, the MWCO of a filament of this disclosure is within the range of 5 kDa to 10 kDa, 10 kDa to 15 kDa, 15 kDa to 20 kDa, 20 kDa to 25 kDa, 25 kDa to 30 kDa, 30 kDa to 35 kDa, 35 kDa to 40 kDa, 40 kDa to 45 kDa, 45 kDa to 50 kDa, 50 kDa to 55 kDa, 55 kDa to 60 kDa, 60 kDa to 65 kDa, 65 kDa to 70 kDa, 70 kDa to 75 kDa, 75 kDa to 80 kDa, 80 kDa to 85 kDa, 85 kDa to 90 kDa, 90 kDa to 95 kDa, or 95 kDa to 100 kDa.

[1156] HOLLOW FIBRES

[1157] In any embodiment of this disclosure, a hollow fibre may possess one or more of the same physical structures, characteristics, and / or properties as a filament described herein, with the distinction that the hollow fibre further comprises a lumen. In some embodiments, a hollow fibre possesses one or more of the same physical structures, characteristics, and / or properties as a filament described herein, and further comprises a lumen, which is characteristic of hollow fibres. The physical structures, characteristics, and / or properties shared between a filament and a hollow fibre may comprise one or more of the following: external diameter, porosity, cross-sectional shape, length, permeability, and MWCO.

[1158] In some embodiments, a hollow fibre of this disclosure is in the form of a tubular cylinder, preferably a circular tubular cylinder.

[1159] In some embodiments, the lumen of a hollow fibre of this disclosure is open at one or both ends.

[1160] In a preferred embodiment, the lumen of a hollow fibre of this disclosure is open at both ends.

[1161] The wall thickness of a hollow fibre of this disclosure may be within the range of 1 pm to 400 pm.

[1162] The wall thickness of a hollow fibre of this disclosure may be within the range of 20 pm to 1100 pm In some embodiments, the wall thickness of a hollow fibre of this disclosure is within the range of 20 pm to 50 pm, 50 pm to 100 pm, 100 pm to 200 pm, 200 pm to 300 pm, 300 pm to 400 pm, 400 pm to 500 pm, 500 pm to 600 pm, 600 pm to 700 pm, 700 pm to 800 pm, 800 pm to 900 pm, 900 pm to 1000 pm, or 1000 pm to 1 100 pm.

[1163] The lumen diameter of a hollow fibre of this disclosure may be within the range of 20 pm and 6,600 pm.

[1164] In some embodiments, the lumen diameter of a hollow fibre of this disclosure is within the range of 20 pm to 500 pm; preferably, within the range of 50 pm to 400 pm; more preferably, within the range of 100 pm to 300 pm; and most preferably, within the range of 150 pm to 275 pm.

[1165] In some embodiments, the lumen diameter of a hollow fibre of this disclosure is within the range of 20 pm to 50 pm, 50 pm to 100 pm, 100 pm to 150 pm, 150 pm to 200 pm, 200 pm to 250 pm, 250 pm to 300 pm, 300 pm to 400 pm, 400 pm to 500 pm, 500 pm to 1000 pm, 1000 pm to 2500 pm, 2500 pm to 5000 pm, or 5000 pm to 6600 pm.

[1166] In any embodiment of this disclosure, a Prokitein may possess one or more of the same physical structures, characteristics, and / or properties as a filament described herein, In some embodiments, a Prokitein possesses one or more of the same physical structures, characteristics, and / or properties as a filament described herein. The physical structures, characteristics, and / or properties shared between a filament and a Prokitein may comprise one or more of the following: external diameter, porosity, cross- sectional shape, length, permeability, and MWCO.

[1167] 1.19 COMPOSITION OF FILAMENTS

[1168] In certain aspects and embodiments, the filaments of this disclosure comprise one or more covalent ester, thioester and / or amide crosslinked polypeptides.

[1169] In certain aspects and embodiments, the filaments of this disclosure comprise a polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a betasheet secondary structure.

[1170] In some embodiments, the term “polymer” herein refers to a macromolecule that comprises repeating subunits called monomers.

[1171] In some embodiments, the term “polymer” herein refers to a macromolecule of non-repeating structures.

[1172] In some embodiments, the filaments of this disclosure comprise a semi-crystalline polymer. In some embodiments, the filaments of this disclosure comprise a semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a betasheet secondary structure.

[1173] In some embodiments, a filament of this disclosure comprises a regular semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

[1174] In some embodiments, the filaments of this disclosure comprise a polymer comprising ionically crosslinked polypeptides with a beta-sheet secondary structure.

[1175] In some embodiments, the filaments of this disclosure comprise a semi-crystalline polymer comprising ionically crosslinked polypeptides with a beta-sheet secondary structure.

[1176] In some embodiments, the filaments of this disclosure a regular semi-crystalline polymer comprising ionically crosslinked polypeptides with a beta-sheet secondary structure.

[1177] In some embodiments, the filaments comprise Prokitein.

[1178] In some embodiments, the covalent ester, thioester and / or amide crosslinked polypeptides may be multimers, such that they comprise multiple chains of those polypeptides.

[1179] In some embodiments, a minority of the polypeptides in a filament of this disclosure are crosslinked.

[1180] In some embodiments, some of the polypeptides in a filament of this disclosure are crosslinked.

[1181] In some embodiments, a majority of the polypeptides in a filament of this disclosure are crosslinked.

[1182] In some embodiments, most of the polypeptides in a filament of this disclosure are crosslinked.

[1183] In some embodiments, almost all of the polypeptides in a filament of this disclosure are crosslinked.

[1184] In some embodiments, all of the polypeptides in a filament of this disclosure are crosslinked.

[1185] The proportion of polypeptides in a filament of this disclosure that are crosslinked may be within the range of 1 % to 99.99 % (w / w polypeptides).

[1186] In some embodiments, at least 50 % (w / w) of the polypeptides in a filament of this disclosure are crosslinked. In some embodiments, the proportion of polypeptides in a filament of this disclosure that are crosslinked is at least 1 %, 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or 99.99 % (w / w polypeptides).

[1187] In some embodiments, the proportion of polypeptides in a filament of this disclosure that are crosslinked is within the range of 1 % to 5 %, 5 % to 10 %, 10 % to 20 %, 20 % to 30 %, 30 % to 40 %, 40 % to 50 %, 50 % to 60 %, 60 % to 70 %, 70 % to 80 %, 80 % to 90 %, or 90 % to 99.99 % (w / w polypeptides). filaments of this disclosure may comprise polypeptides that are covalently crosslinked.

[1188] In some embodiments, none of the polypeptides in a filament of this disclosure are covalently crosslinked.

[1189] In some embodiments, a minority of the polypeptides in a filament of this disclosure are covalently crosslinked.

[1190] In some embodiments, some of the polypeptides in a filament of this disclosure are covalently crosslinked.

[1191] In some embodiments, a majority of the polypeptides in a filament of this disclosure are covalently crosslinked.

[1192] In some embodiments, most of the polypeptides in a filament of this disclosure are covalently crosslinked.

[1193] In some embodiments, almost all of the polypeptides in a filament of this disclosure are covalently crosslinked.

[1194] In some embodiments, all of the polypeptides in a filament of this disclosure are covalently crosslinked. The proportion of polypeptides in a filament of this disclosure that are covalently crosslinked may be within the range of 1 % to 99.99 % (w / w polypeptides).

[1195] In some embodiments, at least 50 % (w / w) of the polypeptides in a filament of this disclosure are covalently crosslinked.

[1196] In some embodiments, the proportion of polypeptides in a filament of this disclosure that are covalently crosslinked is at least 1 %, 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or 99.99 % (w / w polypeptides).

[1197] In some embodiments, the proportion of polypeptides in a filament of this disclosure that are covalently crosslinked is within the range of 1 % to 5 %, 5 % to 10 %, 10 % to 20 %, 20 % to 30 %, 30 % to 40 %, 40 % to 50 %, 50 % to 60 %, 60 % to 70 %, 70 % to 80 %, 80 % to 90 %, or 90 % to 99.99 % (w / w polypeptides).

[1198] The filaments of this disclosure may comprise polypeptides that are ionically crosslinked.

[1199] In some embodiments, none of the polypeptides in a filament of this disclosure are ionically crosslinked.

[1200] In some embodiments, a minority of the polypeptides in a filament of this disclosure are ionically crosslinked.

[1201] In some embodiments, some of the polypeptides in a filament of this disclosure are ionically crosslinked. In some embodiments, a majority of the polypeptides in a filament of this disclosure are ionically crosslinked.

[1202] In some embodiments, most of the polypeptides in a filament of this disclosure are ionically crosslinked.

[1203] In some embodiments, almost all of the polypeptides in a filament of this disclosure are ionically crosslinked.

[1204] In some embodiments, all of the polypeptides in a filament of this disclosure are ionically crosslinked.

[1205] The proportion of polypeptides in a filament of this disclosure that are ionically crosslinked may be within the range of 1 % to 99.99% (w / w polypeptides).

[1206] In some embodiments, at least 50 % (w / w) of the polypeptides in a filament of this disclosure are ionically crosslinked.

[1207] In some embodiments, the proportion of polypeptides in a filament of this disclosure that are ionically crosslinked is at least 1 %, 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 % or 99.99 % (w / w polypeptides).

[1208] In some embodiments, the proportion of polypeptides in a filament of this disclosure that are ionically crosslinked is within the range of 1 % to 5 %, 5 % to 10 %, 10 % to 20 %, 20 % to 30 %, 30 % to 40 %, 40 % to 50 %, 50 % to 60 %, 60 % to 70 %, 70 % to 80 %, 80 % to 90 %, or 90 % to 99.99 % (w / w polypeptides).

[1209] The filaments of this disclosure may comprise polypeptides that are covalently and / or ionically crosslinked.

[1210] The filaments of this disclosure may comprise polysaccharides, lipids, polyols and / or polymers of polyols.

[1211] In some embodiments, the filaments of this disclosure comprise polysaccharides.

[1212] In some embodiments, the filaments of this disclosure comprise polysaccharides that are each present in a mass ratio that is within the range of 0.1 % to 10,000 % (w / w polypeptide).

[1213] In some embodiments, the filaments of this disclosure comprise lipids.

[1214] In some embodiments, the filaments of this disclosure comprise lipids that are each present in a mass ratio that is within the range of 0.1 % to 10,000 % (w / w polypeptide).

[1215] In some embodiments, the filaments of this disclosure comprise polyols and / or polymers of polyols.

[1216] In some embodiments, the filaments of this disclosure comprise polyols and / or polymers of polyols that are each present in a mass ratio that is within the range of 0.1 % to 10,000 % (w / w polypeptide).

[1217] In some embodiments, the filaments of this disclosure comprise at least one of polysaccharides, lipids, polyols and / or polyols, that are each present in a mass ratio that is within the range of 1 % to 10,000 % (w / w polypeptide). The filaments of this disclosure may comprise polypeptides and / or polysaccharides that are crosslinked with a combination of intramolecular (within individual molecules) or intermolecular (between multiple molecules) covalent ester, thioester or amide bonds, and / or ionic bonds to form polypeptide-polypeptide, polypeptides-polysaccharide, polysaccharide-polysaccharide ester, thioester and / or amide covalent crosslinks, and / or ionic crosslinks.

[1218] In some embodiments, the polypeptides in the filaments of this disclosure are crosslinked with a combination of intramolecular and intermolecular covalent ester, thioester or amide bonds.

[1219] In some embodiments, the polypeptides in the filaments of this disclosure are crosslinked with a combination of intramolecular and intermolecular ionic bonds.

[1220] In some embodiments, wherein the filaments of this disclosure comprise more than one type of polypeptide, the crosslinking is between all or substantially all of the types of polypeptides of the filaments.

[1221] In some embodiments, wherein the filaments of this disclosure comprise one or more types of polysaccharides, the crosslinking is between all, or substantially all, of the types of polypeptides and / or polysaccharides of the filaments.

[1222] In some embodiments, Prokitein comprises polypeptides obtained from a single source (e.g. soybeans or faba beans).

[1223] The disclosure is also based, at least in part, on the realisation that Prokitein comprising polypeptides derived from multiple sources (e.g., soybeans and faba beans) may have different properties to Prokitein comprising polypeptides derived from just a single source.

[1224] As used herein, a “Prokitein alloy” refers to a Prokitein comprising polypeptides derived from multiple different sources.

[1225] In some embodiments, the polypeptides of Prokitein, Prokitein alloy, and / or methods of the disclosure are obtained or derived from plants, animals, bacteria, algae, archaea, and / or fungi.

[1226] A Prokitein alloy may comprise polypeptides derived from two, three, four, five, six or more than six sources.

[1227] In some embodiments, a Prokitein alloy comprises polypeptides derived from two sources; three sources; four sources; five sources; six sources; or more than six sources. In some other embodiments, Prokitein comprises a Prokitein alloy.

[1228] Methods for producing polycarboxylic acid derived ester, thioester and / or amide crosslinked polypeptides are discussed herein.

[1229] The polypeptides in a filament of this disclosure may be covalently-crosslinked via esterification, thioesterification and / or amidation reactions involving the use of one or more polycarboxylic acid crosslinking reagents.

[1230] In some embodiments, the polypeptides in a filament of this disclosure are crosslinked via esterification, thioesterification and / or amidation reactions with one or more polycarboxylic acids.

[1231] In some embodiments, the polypeptides in a filament of this disclosure are crosslinked via esterification, thioesterification and / or amidation reactions with one or more polycarboxylic acid salts.

[1232] In some embodiments, the polypeptides in a filament of this disclosure are crosslinked via esterification, thioesterification and / or amidation reactions with one or more partially and / or fully dissociated polycarboxylic acid crosslinking reagents.

[1233] The polypeptides in a filament of this disclosure may be crosslinked via esterification, thioesterification and / or amidation reactions with one or more polycarboxylic acid salts that comprise sodium oxalate, potassium oxalate, sodium malate, potassium malate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, sodium tartrate, potassium tartrate, potassium citrate, sodium citrate, sodium malonate, potassium malonate, and / or combinations thereof; and preferably, sodium citrate and / or sodium malate.

[1234] In some embodiments, the polypeptides in a filament of this disclosure are crosslinked via esterification, thioesterification and / or amidation reactions with one or more polycarboxylic acid salts that comprise sodium oxalate, potassium oxalate, sodium malate, potassium malate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, sodium tartrate, potassium tartrate, potassium citrate, sodium citrate, sodium malonate, potassium malonate, and / or combinations thereof.

[1235] In some preferred embodiments, the polypeptides in a filament of this disclosure are crosslinked with sodium citrate.

[1236] In some preferred embodiments, the polypeptides in a filament of this disclosure are crosslinked with potassium citrate. In some preferred embodiments, the polypeptides in a filament of this disclosure are crosslinked with sodium malate.

[1237] In some preferred embodiments, the polypeptides in a filament of this disclosure are crosslinked with potassium malate.

[1238] Polycarboxylic acids with more than one carboxyl groups may be used to form inter-polypeptide (i.e. , in between two polypeptide chains) and / or intra-polypeptide (i.e., in between two locations along the one polypeptide chain) covalent ester, thioester or amide crosslinks (Alkali-Catalyzed Low Temperature Wet Crosslinking of Plant Proteins Using Carboxylic Acids, 2009).

[1239] In some embodiments, polycarboxylic acids with more than one carboxyl groups are used to form inter- polypeptide and / or intra-polypeptide covalent ester, thioester or amide crosslinks.

[1240] Polycarboxylic acids with more than one carboxyl groups may be used to form inter-polypeptide and / or intra-polypeptide covalent ester crosslinks in the presence of a secondary catalyst, such as sodium hypophosphite. In such cases, the two carboxyl groups may initially form a cyclic anhydride at high temperatures (Infrared Spectroscopic Studies of the Nonformaldehyde Durable Press Finishing of Cotton Fabrics by Use of Polycarboxylic Acids, 1991 ; Formation of Cyclic Anhydride Intermediates and Esterification of Cotton Cellulose by Multifunctional Carboxylic Acids: An Infrared Spectroscopy Study, 1996). The cyclic anhydride may then react with one of the amine (-NH2), hydroxide (-OH), or thiol (-SH) functional groups present on the polypeptide chain to potentially form a polypeptide ester, thioester or amide. A secondary addition reaction between the polycarboxyl derivative and sodium hypophosphite may then form an intermediate which may react with a second polypeptide ester, thioester or amide thereby potentially creating a crosslink. Other reaction pathways with bi-functional polycarboxylic acids may be possible, and catalysts may also be used (Cross-Linking Cotton Cellulose by the Combination of Maleic Acid and Sodium Hypophosphite. 1. Fabric Wrinkle Resistance, 2010; Effects of chemical structures of polycarboxylic acids on molecular and performance manipulation of hair keratin, 2016).

[1241] In some embodiments, polycarboxylic acids with more than one carboxyl groups are used to form inter- polypeptide and / or intra-polypeptide covalent ester crosslinks in the presence of a secondary catalyst, such as sodium hypophosphite.

[1242] Polycarboxylic acids with two carboxyl groups may be used to form inter-polypeptide and / or intra- polypeptide covalent ester, thioester or amide crosslinks. In some embodiments, polycarboxylic acids with two carboxyl groups are used to form inter-polypeptide and / or intra-polypeptide covalent ester, thioester or amide crosslinks.

[1243] Covalent crosslinking with polycarboxylic salts with two carboxyl groups may be carried out under alkaline conditions leading to the formation of non-specific ester, thioester or amide -bond covalent crosslinks.

[1244] In some embodiments, covalent crosslinking with polycarboxylic salts with two carboxyl groups is carried out under alkaline conditions leading to the formation of non-specific ester, thioester or amide -bond covalent crosslinks.

[1245] Polycarboxylic acids with two carboxyl groups may be used to form inter-polypeptide and / or intra- polypeptide covalent ester crosslinks in the absence of a secondary catalyst. In such cases, the two carboxyl groups may initially form a cyclic anhydride. The cyclic anhydride may then react with one of the amine (-NH2), hydroxide (-OH), or thiol (-SH) functional groups present on the polypeptide chain to potentially form a polypeptide ester, thioester or amide. Under alkali conditions the free carboxyl may convert to a carboxylate which may then react with a one of the amine (-NH2), hydroxide (-OH), or thiol (- SH) functional groups present on a second polypeptide chain to potentially form a polycarboxylic acid derived ester, thioester or amide crosslinked polypeptide (Formation of Cyclic Anhydride Intermediates and Esterification of Cotton Cellulose by Multifunctional Carboxylic Acids: An Infrared Spectroscopy Study, 1996).

[1246] Polycarboxylic acids with more than two carboxyl groups may be used to form inter-polypeptide and / or intra-polypeptide covalent ester crosslinks. In such cases, initially two of the carboxyl groups on the polycarboxylic acid may be dehydrated to form a cyclic anhydride. The cyclic anhydride may then react with one of the amine (-NH2), hydroxide (-OH) or thiol (-SH) functional groups present on the polypeptide chain to potentially form a polypeptide ester, thioester or amide with two or more carboxyl groups. Two of the carboxyl groups on the polypeptide ester, thioester or amide may then be dehydrated again to potentially form a cyclic anhydride. Finally, the polypeptide ester with a cyclic anhydride may then react with a functional group on a second polypeptide chain to potentially form a polycarboxylic acid derived ester, thioester or amide crosslinked polypeptide (Alkali-Catalyzed Low Temperature Wet Crosslinking of Plant Proteins Using Carboxylic Acids, 2009; Low-Temperature Wet-Cross-linking of Silk with Citric Acid, 2011 ).

[1247] In some embodiments, polycarboxylic acid salts with more than two carboxyl groups are used to form inter-polypeptide and / or intra-polypeptide covalent ester, thioester or amide crosslinks. In some embodiments, polycarboxylic acid salts with more than two carboxyl groups are used to form inter-polypeptide and / or intra-polypeptide covalent ester, thioester or amide crosslinks in the presence of a catalyst.

[1248] Polycarboxylic acids with more than two carboxyl groups may be used to form inter-polypeptide and / or intra-polypeptide covalent ester crosslinks in the presence of a secondary catalyst, such as sodium hypophosphite. In such cases, the two carboxyl groups may initially be dehydrated to potentially form a cyclic anhydride. Subsequent acylation between the polycarboxylic acid derived anhydride and sodium hypophosphite may result in the formation of an intermediate. Nucleophilic substitution of the anhydride and sodium hypophosphite intermediate with one of the amine (-NH2) hydroxide (-OH), and thiol (-SH) functional groups present on the polypeptide chain may then produce a polypeptide ester, thioester or amide with two or more carboxyl groups. Two of the carboxyl groups on the polypeptide polycarboxylic acid derived ester, thioester or amide may then be dehydrated again to potentially form a polypeptide ester, thioester or amide cyclic anhydride intermediate. Acylation of this intermediate with sodium hypophosphite potentially forms yet another intermediate, which may undergo nucleophilic substitution with another functional group present on a polypeptide chain to potentially form a polycarboxylic acids derived polypeptide ester, thioester or amide crosslink and the reformation of the sodium hypophosphite such that it acts as a catalyst and is not consumed (Green and Sustainable Technology for High- Efficiency and Low- Damage Manipulation of Densely Crosslinked Proteins, 2017).

[1249] In some embodiments, catalysts used to catalyse the formation of polycarboxylic acid derived inter- polypeptide and / or intra-polypeptide covalent ester, thioester or amide crosslinks include, but are not limited to, sodium sulphite, sodium bisulphite, sodium metabisulphite, and / or sodium hypophosphite.

[1250] The polypeptides in a filament of this disclosure may be crosslinked in the presence of a catalyst.

[1251] In some embodiments, the polypeptides in a filament of this disclosure are crosslinked in the presence of a catalyst.

[1252] In some embodiments, the polypeptides in a filament of this disclosure are crosslinked in the absence of a catalyst.

[1253] In some embodiments, ions with more than one valent electron are used to form inter-polypeptide and / or intra-polypeptide ionic crosslinks.

[1254] In some preferred embodiments, the polypeptides in a filament of this disclosure are crosslinked with sodium citrate in the presence of sodium hypophosphite. A person skilled in the art may be able to determine the chemical composition and structure of the polypeptides in the filaments of this disclosure, as well as the polypeptides from which they are derived. Suitable analytical methods include Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS), Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Fourier Transform Infrared (FTIR) Spectroscopy, Circular Dichroism (CD), Wide-Angle X-ray Scattering (WAXS), and Raman Spectroscopy (RS).

[1255] NMR may be used to determine whether a filament comprises polypeptides crosslinked with polycarboxylic acid-derived ester, thioester, or amide covalent bonds. In particular,1H proton NMR can identify carboxylic acid derivatives through the presence of characteristic carbonyl group resonances. Carbonyl protons typically resonate at 2.0-3.0 ppm, protons on carbons attached to the alkoxide oxygen in esters at 3.5-4.5 ppm, and protons on carbons attached to sulphur in thioesters at 2.0-3.0 ppm

[0044] . As these groups are not native in polypeptides, identification of these groups in a filament may therefore be used as evidence that given filaments are filaments of this disclosure.

[1256] Similarly, 13C NMR may be used to determine the presence of carboxylic acid derivatives based on differences in the resonant frequencies of the carbonyl carbon in carboxylic acids (160-180 ppm) and derived esters (50-90 ppm), aldehydes and ketones (180-220 ppm). In carboxylic acids, the carbonyl carbon is strongly deshielded by the adjacent highly electronegative double-bounded oxygen. As the carboxyl carbon in derivatives of carboxylic acids experience different levels of deshielding, they exhibit varying resonance frequencies. The carbons in other functional groups also exhibit different resonant frequencies, such as the carbon bond to nitrogen in amides (20-65 ppm) and those bonded to sulphur in thioesters (20-45 ppm)

[0044] . Similarly, as these groups are not native in polypeptides, identification of these groups in a filament may therefore be used as evidence that given filaments constitute a filament of this disclosure.

[1257] Using these NMR techniques, filament samples comprising polypeptides crosslinked with polycarboxylic acid-derived ester, thioester, or amide covalent bonds can be distinguished from polypeptides crosslinked by other means, such as thermal physical crosslinking. Samples within the scope of this disclosure exhibit characteristic and typically stronger signals in the1H or13C NMR spectral ranges described above, corresponding to these non-native functional groups.

[1258] MS is commonly used to determine primary and higher-order protein structures, including those in the filaments of this disclosure. MS analysis is generally performed on proteins samples that are either fully intact ("top-down”) or on proteins that have initially been digested into fragments (“bottom-up”). In both approaches, samples are initially converted into an ionised gas, through methods such as electrospray ionization or matrix-assisted laser desorption / ionization (MADLI). The mass(es) of the ionised sample or sample fragments may then be determined with a time of flight (TOF) detector. Peptide sequences may then be determined by comparison of recorded sample fragments and predicted peptide sequence masses. To avoid complex mixtures of proteins being evaluated at once, methods such as SDS-PAGE, high-performance liquid chromatography (HPLC), or gas chromatography (GC) may be used to separate protein mixtures prior to analysis (Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications, 2020). These methods can be applied to a filament to identify its constituent polypeptides and confirm structural modifications. Identification of such constituent polypeptides and structural modifications in a filament may therefore be used as evidence that given filaments constitute a filament of this disclosure.

[1259] MS may also be used to detect carboxylic acid derivatives by identify a peak associated with an acylium ion (R-CO+), where “R” represents the variable organic group bonded to the carbonyl group. However, due to the variable mass of the body (R-) bonded to the acylium, ion the theoretical mass of these ions must be calculated on a case-by-case basis

[0044] . This approach can therefore assist in confirming the presence of polycarboxylic acid-derived crosslinks in the disclosed filaments.

[1260] Due to the non-standard peak number associated with carboxylic acid derivatives measured with mass spectrometry, NMR analysis is generally preferred for definitive identification of polycarboxylic acid- derived cros...

Claims

CLAIMS1 . A filament comprising a semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

2. A process for the production of a filament comprising a semi-crystalline polymer which comprises a polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptide with a beta-sheet secondary structure, the process comprising the steps: a. combining: i. a first composition comprising polypeptides, with ii. a second composition comprising a solvent, and one or more denaturing agent and / or reducing agent, to produce a third composition; b. incubating the third composition under conditions sufficient to denature and / or reduce a polypeptide, to produce a fourth composition; c. extruding or spinning the fourth composition through an orifice, to produce a filament; d. treating the filament with a polycarboxylic acid crosslinking reagent to form an interpolypeptide and / or intra-polypeptide polycarboxylic acid derived covalent ester bond, thioester bond or amide bond crosslink in the filament, to produce a covalently-crosslinked filament; e. treating the covalently-crosslinked filament with at least one post-production modification process selected from the group consisting of: i. treating the covalently-crosslinked filament with an organic solvent to increase the relative abundance of beta-sheets; ii. annealing the covalently-crosslinked filament; ill. treating the covalently-crosslinked filament with a solvent to increase porosity; iv. washing the covalently-crosslinked filament with one or more wash solutions; v. coating the covalently-crosslinked filament; vi. modifying the surface topography of the covalently-crosslinked filament; and vii. drawing the covalently-crosslinked filament; to produce a treated covalently-crosslinked filament. f. drying the treated covalently-crosslinked filament.

3. The process of Claim 2, wherein the second composition comprises at least a solvent, a denaturing agent, and / or a reducing agent.

4. The process according to any one of Claims 2 to 3, wherein the second composition comprises urea.

5. The process according to any one of Claims 2 to 4, wherein the second composition comprises sodium sulphite.

6. The process according to any one of Claims 2 to 5, wherein the second composition comprises N-acetylcysteine.

7. The process according to any one of Claims 2 to 6, wherein the second composition comprises L- cysteine.

8. The process according to any one of Claims 2 to 7, wherein the pH of the second composition is within the range of 5.01 to 9.99.

9. The process according to any one of Claims 2 to 7, wherein the pH of the second composition is within the range of 9.99 to 14.

10. The process according to any one of Claims 2 to 9, wherein the coagulation bath solution comprises sodium malate, and / or potassium malate.11 . The process according to any one of Claims 2 to 10, wherein the coagulation bath solution comprises sodium citrate, and / or potassium citrate.

12. The process according to any one of Claims 2 to 1 1 , wherein the annealing process of Step (e.ii) is carried out at a temperature that is within the range of 110 °C to 180 °C.

13. The process according to any one of Claims 2 to 12, wherein the organic solvent used in step (e.i) comprises methanol, ethanol, propanol, iso-propanol, butanol, iso-butanol, dimethyl sulfoxide, dimethylformamide, and / or dimethylacetamide.

14. The process according to any one of Claims 2 to 13, wherein the organic solvent used in step (e.i) comprises water and ethanol.

15. The process according to any one of Claims 2 to 14, wherein a void inclusion element is introduced into the first, second, third or fourth composition.

16. A filament obtained or obtainable by the process of any one of Claims 2 to 15.

17. A filament according to any one of Claims 1 or 16, wherein the polymer is a regular semicrystalline polymer.

18. A filament according to any one of Claims 1 or 16 to 17, wherein the materials of the filament is Generally Recognized As Safe (GRAS).

19. A filament according to any one of Claims 1 or 16 to 18, wherein the filament is intended to be edible.

20. A filament according to any one of Claims 1 or 16 to 19, wherein the filament is edible.21 . A filament of any one of Claims 16 to 20, wherein said filament comprises a semi-crystalline polymer comprising polycarboxylic acid derived covalent ester, thioester and / or amide crosslinked polypeptides with a beta-sheet secondary structure.

22. A filament according to any one of Claims 1 or 16 to 21 , wherein the porosity is 45 % to 85 %.

23. A filament according to any one of Claims 1 or 16 to 22, wherein the filament is water-stable for 3 or more days.

24. A cartridge comprising a filament of any one of Claims 1 or 16 to 23.

25. A bioreactor comprising a filament of any one of Claims 1 or 16 to 23.

26. A food product comprising a filament of any one of Claims 1 or 16 to 23.

27. A food product of Claim 26, wherein the food product further comprises a plurality of cells.

28. A food product of Claim 26 or Claim 27, wherein the food product is a cultured meat product.

29. Cordage comprising a filament of any one of Claims 1 or 16 to 23.

30. Woven material comprising a filament of any one of Claims 1 or 16 to 23.31 . Non-woven material comprising a filament of any one of Claims 1 or 16 to 23.

32. A suture comprising a filament of any one of Claims 1 or 16 to 23.

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