Hollow fiber

Abstract

This disclosure provides extruded or spun semipermeable porous hollow fibers, comprising covalent esters, thioesters, and / or amide-crosslinked polypeptides, as well as processes for producing the same. The hollow fibers may be produced from proteins, protein extracts, and / or protein isolates derived from plants, animals, bacteria, algae, archaea, and / or fungi, and in certain embodiments are intended to be suitable for human and / or animal ingestion. In some embodiments, the hollow fibers may be designed for use in the production of cartridges compatible with existing and / or novel bioreactor platforms for housing cell cultures in the production of cultured meat.

Description

[Technical Field]

[0001] 1.1 Fields In certain aspects and embodiments, this disclosure relates in part to hollow fibers and processes for their production and use.

[0002] 1.2 Overview In some aspects and embodiments of the present disclosure, extruded or spun semipermeable porous hollow fibers comprising covalent esters, thioesters, and / or amide-crosslinked polypeptides are provided along with their production processes. The hollow fibers of the present disclosure may be produced from proteins, protein extracts, and / or protein isolates derived from plants, animals, bacteria, algae, archaea, and / or fungi, and in certain embodiments are intended to be suitable for human and / or animal ingestion. In some embodiments, the hollow fibers of the present disclosure may be designed for use in the production of cartridges compatible with existing and / or novel bioreactor platforms for housing cell cultures in the production of cultured meat. [Background technology]

[0003] 1.3 Background U.S. Patent Application Publication US2013 / 0192459 discloses "a hollow fiber membrane having a support layer and an inner (i.e., the side facing the lumen of the support layer) or outer separation layer, and a process for manufacturing such a hollow fiber membrane."

[0004] PCT application WO2019158494A1 discloses "edible fibers containing biopolymers and plasticizers, wherein the weight ratio of biopolymers to plasticizers is about 1:0.25 to about 1:3, and the fibers have a diameter of about 0.5 μm to about 1 mm."

[0005] European Patent Application No. EP0077098A2 discloses "a general ultrafiltration and dialysis process, more specifically, a chitosan hollow fiber for use in renal dialysis, and a process for preparing the same."

[0006] WO2016 / 007879A1 discloses that "a bioprotein tube having an outer diameter of about 1 mm and an inner diameter of about 0.80 mm can be prepared by extruding a bioprotein precursor solution containing 50 mg / ml of alginate and gelatin in a 3:1 ratio through an orifice into a solution of about 5 mg / ml of calcium chloride, and exposing the tube to a washing solution within about 10 seconds."

[0007] WO2018 / 011805A2 discloses "a system for culturing cells that can be used to produce edible meat in some embodiments of the present invention." It also states that "culturing is performed on an edible hollow fiber cartridge." Furthermore, it states that "the plant-derived matrix is ​​derived from grasses, legumes (Fabaceae) or pseudocereals." It also states that "legume plants are soybeans or peas."

[0008] WO2022 / 038241A1 discloses "closed continuous, semi-continuous, or batch culture systems for cell proliferation and differentiation and subsequent tissue proliferation, for example, for the production of clean meat."

[0009] WO2009 / 035414A1 discloses that "the present invention relates to chitosan constructs and methods for preparing them. Chitosan constructs may have higher mechanical strength. In particular, the present invention relates to chitosan fibers or chitosan hollow fibers." It also states that "in particular, the constructs may have a sponge-like porous structure. The porosity of the constructs may be up to 80%. For example, the porosity may be about 20-80%."

[0010] WO2022 / 038240A2 discloses "a method for producing structured clean meat products using hollow fibers, cartridges, and a bioreactor."

[0011] WO2023 / 021213A1 discloses "a method for producing crosslinked edible porous hollow fibers and sheet membranes suitable for the manufacture of clean meat products, hollow fibers and sheet membranes produced thereby, and methods for using them." Herein, "the novelty of the present invention lies in the use of physical crosslinking, which is made via an energy source such as heat, gamma, electron beam, beta, X-ray, or UV."

[0012] Modrzejewska and Eckstein, Biopolymers, 73:61-68 (2004), concluded that "it is possible to form chitosan hollow fibers by wet weaving using one type of water coagulant (sodium hydroxide)."

[0013] JP2022072917A discloses "a cultured meat complex comprising a hollow fiber membrane or its degradation products or lysates, and cultured meat containing a population of animal cells present along the hollow fiber membrane or its degradation products or lysates." Here, "the cultured meat complex obtained by using a hollow fiber membrane made of collagen may be subjected to heat treatment to gelatinize the collagen."

[0014] WO2023 / 152492A1 discloses "a substrate assembly for culturing cells, wherein the substrate assembly comprises a plurality of edible fibers, each fiber having an internal channel extending along its length." Here, "the plurality of fibers may comprise alginates." In this case, "calcium chloride acts as a crosslinking agent for the alginates."

[0015] WO2023 / 152493A1 discloses "a plurality of edible fibers, each fiber having internal channels extending along its length." Here, "calcium chloride acts as a crosslinking agent for alginates," and "a freeze-drying step may also be used to adjust the porosity of the final fiber material." Furthermore, "the freeze-drying step has been found to be particularly useful when the fibers contain alginates."

[0016] WO2024 / 038281A1 discloses "a method for culturing muscle cells for food products, comprising seeding muscle cells onto one or more porous hollow fibers." [Overview of the project]

[0017] 1.4 Overview This disclosure is at least partially based on the recognition that extruded or spun semi-permeable porous hollow fibers containing polycarboxylic acid-derived covalent esters, thioesters, and / or amide-crosslinked polypeptides can enable the production of edible high-protein cultured meat products.

[0018] This disclosure also relates, at least in part, to the recognition that hollow fibers, as disclosed herein, can, in some embodiments, primarily constitute a protein substrate, thereby eliminating the need to later separate cells grown on the substrate or scaffold. As a result, the need for an additional seeding step onto a tertiary scaffold, which is typically required in conventional culture methods, can be eliminated.

[0019] Separating cells from the extracapillary space of existing semipermeable hollow fiber bioreactors is a difficult step and often requires the use of multiple enzymatic washes. This separation step may not be necessary if the hollow fibers themselves are edible and can be ingested.

[0020] This disclosure is at least partially based on the recognition that extruded or spun semi-permeable porous hollow fibers, including covalent esters, thioesters, and / or amide-crosslinked polypeptides, can be used to produce cartridges and modules compatible with existing and novel bioreactor platforms. The hollow fibers of this disclosure may enable high-density cell culture and cost reductions associated with reduced use of growth factors in hollow fiber bioreactors. Taken together, these advantages could significantly reduce costs in the production of cultured meat.

[0021] The extruded or spun semi-permeable porous hollow fibers of the present disclosure, comprising covalent esters, thioesters, and / or amide-crosslinked polypeptides, are, in some aspects and / or embodiments, edible, non-cytotoxic and / or biocompatible, and digestible when ingested as food by humans and / or animals. Furthermore, the hollow fibers of some aspects and embodiments of the present disclosure can be formed and processed to enhance the texture and / or flavor of food products.

[0022] A bioreactor platform comprising cartridges and / or modules containing hollow fibers of the present disclosure may, in many embodiments, be used to produce an edible food containing a mixture of cultured cells and the hollow fibers of the present disclosure. The edible food thus produced may, in certain embodiments, be ingested and digested by humans and / or animals as food without the risk of containing non-edible substrate contaminants.

[0023] However, one challenge in producing extruded or spun semipermeable porous hollow fibers containing covalent esters, thioesters, and / or amide-crosslinked polypeptides is that most edible materials are water-soluble, swell significantly in aqueous solutions, and / or have low protein content. Such materials lack the separation, mechanical, and / or chemical properties necessary for the operation of semipermeable hollow fiber bioreactors. Many embodiments of the hollow fibers of this disclosure overcome these drawbacks by further providing processes that include novel combinations of process steps.

[0024] One of the problems associated with polymers derived from ester, thioester, and / or amide cross-linked polypeptides is generally that ester, thioester, and / or amide linkages can be broken by hydrolysis under certain conditions. The secondary structures of beta-sheets and beta-coils in polypeptides can prevent hydrolysis of ester bonds, thioester bonds, and / or amide bonds. However, the induced beta-sheets and beta-coils can return to an amorphous structure upon hydration in water or under certain conditions.

[0025] As used herein, the term "beta-sheet" may collectively refer to both the secondary structures of beta-sheets and beta-coils.

[0026] The present disclosure is also based, at least in part, on the recognition that polymers comprising esters, thioesters, and / or amide cross-linked polypeptides in which a beta-sheet secondary structure is induced may recrystallize upon annealing, thereby relaxing internal stresses within their crystal structures and resulting in a beta-sheet conformation that is stable in a microstructure. This process is referred to herein as "protein annealing." As a result, the beta-sheet secondary structure of the cross-linked polypeptide polymer remains intact upon rehydration and can resist hydrolysis of the constituent ester, thioester, and / or amide linkages. Accordingly, one object of the present disclosure is to provide a polymeric material comprising a regular semi-crystalline polymer comprising an ester, thioester, and / or amide cross-linked polypeptide having a beta-sheet secondary structure, which is referred to herein as Prokitein.

[0027] As used herein, the term "semi-crystalline polymer" refers to a polymer comprising molecular chains arranged in both a crystalline structure and an amorphous structure. In the crystalline regions, the protein chains can be aligned in a repeating pattern. In the amorphous regions, the protein chains can be randomly oriented.

[0028] As used herein, "regular semicrystalline polymer" refers to a semicrystalline polymer in which the size and shape of the crystal grains are uniformly distributed and / or uniformly oriented relative to one another within the crystalline phase(s) of its molecular structure.

[0029] This disclosure is also, at least in part, based on the recognition that the use of Prokitein can be promoted as a material that can be used for many applications, such as for the production of hollow fibers for long-term cell culture required for the production of cultured meat products, due to Prokitein's resistance to hydrolysis. In addition, the extruded or spun semipermeable porous hollow fibers of this disclosure, comprising covalent esters, thioesters, and / or amide-crosslinked polypeptides, include Prokitein in some aspects and / or embodiments.

[0030] One of the further challenges in producing porous hollow fibers is generally the formation of pores. Process steps such as freeze-drying are sometimes required to adjust the porosity of the hollow fiber material. Many embodiments of the present disclosure can overcome this drawback by further providing a process that can form hollow fibers having a porous structure without using freeze-drying.

[0031] Therefore, one object of this disclosure is to provide extruded or spun semi-permeable porous hollow fibers containing covalent esters, thioesters, and / or amide-crosslinked polypeptides, suitable for use as cell growth substrates in bioreactors and as edible foods.

[0032] Another object of this disclosure is to provide a process for producing extruded or spun semi-permeable porous hollow fibers suitable for use as food, comprising covalent esters, thioesters, and / or amide-crosslinked polypeptides.

[0033] A further object of this disclosure is to provide a cartridge containing a plurality of extruded or spun semipermeable porous hollow fibers comprising covalent esters, thioesters, and / or amide-crosslinked polypeptides.

[0034] A further object of this disclosure is to provide a bioreactor comprising one or more hollow fiber cartridges, each containing a plurality of extruded or spun semipermeable porous hollow fibers.

[0035] A further object of this disclosure is to provide a food product comprising a combination of cultured cells and extruded or spun semipermeable porous hollow fibers containing covalent esters, thioesters, and / or amide-crosslinked polypeptides.

[0036] Another object of this disclosure is to provide a production process for combinations of extruded or spun semipermeable porous hollow fibers and cultured cells, comprising covalently bonded esters, thioesters, and / or amide-crosslinked polypeptides, suitable for use as food.

[0037] Therefore, in one embodiment, the present disclosure provides an edible semi-permeable porous hollow fiber comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, wherein (a) The outer diameter of the hollow fiber is 50 to 6600 μM. (b) The wall thickness of the hollow fiber is 20 to 800 μM. (c) The inner diameter of the hollow fiber is 20 to 5000 μM. (d) The pore volume of the hollow fiber is 1-95%.

[0038] Edible semi-permeable porous hollow fiber comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, wherein (a) The outer diameter of the hollow fiber is 50 to 6600 μm. (b) The wall thickness of the hollow fiber is 20 to 800 μm. (c) The inner diameter of the hollow fiber is 20 to 5000 μm. (d) The porosity of the hollow fibers is 1-95%.

[0039] In another aspect, the disclosure also provides semipermeable porous hollow fibers comprising covalent esters, thioesters, and / or amide-linked polypeptides, wherein, (a) The outer diameter of the hollow fiber is 50 to 6600 μM. (b) The wall thickness of the hollow fiber is 20 to 800 μM. (c) The inner diameter of the hollow fiber is 20 to 5000 μM. (d) The pore volume of the hollow fiber is 1-95%.

[0040] In another aspect, the disclosure also provides semipermeable porous hollow fibers comprising covalent esters, thioesters, and / or amide-linked polypeptides, wherein, (a) The outer diameter of the hollow fiber is 50 to 6600 μm. (b) The wall thickness of the hollow fiber is 20 to 800 μm. (c) The inner diameter of the hollow fiber is 20 to 5000 μm. (d) The porosity of the hollow fibers is 1-95%.

[0041] In yet another aspect, the Disclosure provides a process for producing a plurality of semipermeable porous hollow fibers comprising covalent esters, thioesters, and / or amide-linked polypeptides, the process being a. i. A first composition comprising a polypeptide and ii. A second composition comprising a solvent and one or more denaturing agents and / or reducing agents. The steps include: combining these to produce a third composition, b. Incubate the third composition under conditions sufficient to denaturate and / or reduce at least one fraction of the polypeptide, A step of generating a fourth composition, c. Extruding or spinning a fourth composition through a plurality of coaxial orifices together with a bore solution containing a coagulation bath solution to produce a plurality of hollow fibers; d. Treating a plurality of hollow fibers with a polycarboxylic acid crosslinking reagent to form covalent crosslinks of esters, thioesters, or amides derived from polycarboxylic acids between and / or within polypeptides in at least one fraction of the polypeptides within the hollow fibers to produce a plurality of covalently crosslinked semipermeable porous hollow fibers; e. i. Treating the covalently crosslinked semipermeable porous hollow fibers with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptide; ii. Annealing the covalently crosslinked semipermeable porous hollow fibers; iii. Treating the covalently crosslinked semipermeable porous hollow fibers with a solvent to remove void-containing elements in the hollow fibers; iv. Washing the covalently crosslinked semipermeable porous hollow fibers with one or more acids, alkalis, and / or buffers to reduce at least one of the Young's modulus, ultimate tensile strength, and / or ultimate tensile strain of the hollow fibers; v. Coating the covalently crosslinked semipermeable porous hollow fibers; vi. Modifying the surface topography of the covalently crosslinked semipermeable porous hollow fibers to assist cell attachment and / or cell alignment; Treating the covalently crosslinked semipermeable porous hollow fibers by at least one post-generation modification process selected from the group consisting of to produce a plurality of processed covalently crosslinked semipermeable porous hollow fibers; f. Drying the processed covalently crosslinked semipermeable porous hollow fibers to produce dried, processed covalently crosslinked semipermeable porous hollow fibers.

[0042] One object of this disclosure is to provide a process for generating Prokitein, which is a. i. A first composition comprising a polypeptide and ii. A step of producing a third composition by combining a solvent and a second composition comprising one or more denaturing agents and / or reducing agents, b. A step of incubating the third composition to produce a fourth composition, c. A step of treating the fourth composition with a polycarboxylic acid crosslinking reagent to produce a polypeptide crosslinked by covalent bonds, d. A step of treating the covalently crosslinked polypeptide with an organic solvent, e. A step of annealing a polypeptide crosslinked by covalent bonds, f. Optional, i. Treating a covalently crosslinked polypeptide with a solvent to remove void-containing elements, and ii. Drying the polypeptide that has been crosslinked by covalent bonds. The process includes the step of processing a covalently crosslinked polypeptide by one or more post-production modification processes selected from the group consisting of the following to produce Prokitein. [Modes for carrying out the invention]

[0043] 1.5 Detailed Description of the Invention The terms "cultured meat," "cultivated meat," "cellular agriculture," "cell-based meat," "laboratory meat," "clean meat," and "cultured animal cell food" refer to the production of meat by culturing animal cells in vitro and are used interchangeably herein. Cultured meat offers three major advantages: sustainability, public health, and animal welfare.

[0044] As stated by the Intergovernmental Panel on Climate Change (IPCC), reducing the consumption of conventional animal products is necessary to mitigate the worst impacts of climate change. However, most consumers are unwilling to do so. Current technologies for cultured meat production are energy-intensive, and sustainable energy sources are needed to ensure the aforementioned benefits. The adoption of next-generation bioreactor platforms for cultured meat production will play a crucial role in this endeavor. It is currently estimated that up to 70% of arable land is used for the production of animal feed, and global meat demand is expected to increase by another 70% by 2050. This indicates that the Earth's resources are currently insufficient to meet this future demand (1).

[0045] A bioreactor, also called a "fermenter" or "culture vessel," is a device or container that provides an environment for the growth and cultivation of cells, microorganisms, or biomolecules. Bioreactors are commonly used in biotechnology, pharmaceuticals, and biochemical engineering for the production of biological products such as cells, vaccines, enzymes, and antibodies.

[0046] Traditional farm-based animal husbandry methods for meat production and subsequent processing pose a risk of introducing potentially deadly infectious diseases such as Salmonella and Listeria to the public. Furthermore, the overuse of antibiotics is common in traditional animal husbandry, contributing to the development of antibiotic-resistant bacteria. In contrast, processes for producing cultured meat can guarantee the absence of these contaminants and do not require the use of antibiotics (1).

[0047] The Sentience Institute (SI) estimates that 99% of animals raised for food are viewed more as industrial products of factory farms than as sentient beings. Reducing the number of such animals is highly desirable (1).

[0048] Conventional bioreactors, culture vessels, or fermenters used interchangeably in this specification and typically used for producing cultured cells usually take the form of a continuous stirred tank reactor (CSTR). A CSTR may be fitted with various control systems for controlling a range of environmental conditions, such as temperature, pH, stirring speed, and the concentration of soluble metabolites (e.g., oxygen, carbon dioxide, glucose, lactic acid, etc.). Typically, a CSTR is fitted with an energy source and a mixing system as means of controlling environmental conditions.

[0049] Many of the current methods used in cellular agriculture have several drawbacks, including: (i) low cell concentrations in the surrounding medium, making subsequent dehydration and recovery steps expensive; (ii) inefficient use of nutrients and growth media by the cells; and / or (iii) difficulty in easily forming tasty and appealing foods from the resulting cell slurry suspension.

[0050] The high cost of cell culture media is recognized as one of the major cost factors in cultured meat production. Cell culture media typically contain fetal bovine serum (FBS), which contains hundreds of different proteins and metabolites. Currently, fully defined substitutes for FBS are generally more expensive and less effective. The high cost of both growth media and FBS may be due to the required growth factor components, which are also typically unstable and have a short shelf life (1).

[0051] CSTRs used to culture cells in suspension or on microcarriers are typically 10 5 ~10 6 Cells can be generated at a density of cells / mL (2;3).

[0052] Semipermeable hollow fibers made of petrochemical polymers have been used to create bioreactors that can achieve and maintain cell densities up to 35 times higher than those achievable with conventional tissue culture techniques (4). Furthermore, it has been demonstrated that the concentration of growth factors required to differentiate stem cells into their terminal state is 1 / 2 to 1 / 8 of that in ceramic semipermeable hollow fiber bioreactors compared to conventional adhesion methods (5;6).

[0053] Scaffolds are of paramount importance in the production of cultured meat because they provide the structural foundation for thick tissue products (7). However, due to limited oxygen and nutrient transport, cell proliferation for cultured meat on conventional porous scaffolds is generally not commercially viable (8). Furthermore, cell slurry suspensions produced from CSTRs are usually not easily palatable, necessitating an additional cell seeding step onto the scaffold. Thus, a typical workflow employed in the production of cultured meat is (i) isolation of stem cells, (ii) culture in suspension to the desired cell concentration, and (iii) seeding onto the scaffold(s). Dehydration of the cell slurry between steps (ii) and (iii) is time- and energy-intensive. Moreover, the final seeding step is complicated by the fact that scaffold design has a significant impact on cell seeding efficiency and the palatability of the final product (3).

[0054] Hollow fibers, such as those derived from petrochemical polymers, ceramics, and cellulose acetate, are used as substrates for culturing cells. While the development and commercial use of such hollow fibers have been successful, challenges remain, including the difficulty of separating cultured cells from the hollow fiber substrate after culturing.

[0055] Hollow fibers can be produced using polypeptides solubilized with acids or alkalis, but this process can disrupt the primary protein structure, resulting in shorter chain lengths, less chain entanglement, and a more brittle material when crosslinked. Furthermore, edible materials such as polypeptides, even when crosslinked, are water-soluble and can swell considerably in aqueous solutions. This swelling can significantly impair the fluid and nutrient transport properties of these materials and may be undesirable, especially in bioreactor applications.

[0056] Certain embodiments of the compositions and methods provided herein attempt to address and overcome some or all of these challenges.

[0057] In certain aspects and embodiments, the Disclosure provides a process for producing a plurality of semipermeable porous hollow fibers comprising covalent esters, thioesters, and / or amide-crosslinked polypeptides.

[0058] Step (a) is, (i) A first composition comprising a polypeptide and (ii) A second composition comprising a solvent and one or more denaturing agents and / or reducing agents This includes combining the elements to produce a third composition.

[0059] The first composition may comprise one or more polypeptides.

[0060] The terms “polypeptide” and “protein” are used interchangeably herein. In some embodiments, the polypeptides of the hollow fibers and / or methods of the present disclosure are obtained from or derived from plants, animals, bacteria, algae, archaea, and / or fungi.

[0061] In some preferred embodiments, the polypeptides of the hollow fibers and / or methods of the present disclosure are derived from one or more extracts and / or isolates obtained from plants, animals, bacteria, algae, archaea, and / or fungi.

[0062] In some embodiments, the polypeptide is a plant polypeptide, where the term “plant” includes, but is not limited to, legumes, nuts, seeds, cereals, and tubers. Examples of such plant sources include, but are not limited to: Legumes: chickpeas, lentils, kidney beans, black beans, white beans, lima beans, pinto beans, soybeans, mung beans, adzuki beans, broad beans, cowpeas, string beans, snow peas, snap peas, and / or split peas; • Nuts: almonds, walnuts, pistachios, cashews, Brazil nuts, hazelnuts, macadamia nuts, pecans, pine nuts, peanuts, chestnuts, and / or coconuts; Seeds: sunflower, pumpkin, chia, flax, sesame, hemp, poppy, quinoa, cumin, fennel, coriander, mustard, caraway, cardamom, fenugreek, ajwain, anise, and / or nigella; Cereals: wheat, rice, corn (maize), oats, barley, rye, millet, sorghum, quinoa, buckwheat, spelt, triticale, amaranth, teff, farro, kamut, freekeh, emmer, and / or fonio; and • Tubers: Potatoes, sweet potatoes, yams, cassava, taro, jicama, Jerusalem artichokes, water chestnuts, Chinese yams, American taro, arrowroot, ginger, turmeric, turnips, and / or beets.

[0063] In some embodiments, the polypeptides are derived from leguminous plants, and these plants include, but are not limited to, Anasazi beans, Canelini beans, Cocoa beans, Coffee beans, Cranberry beans, Edamame beans, Flageoles beans, French green beans, Gigades beans, Great Northern beans, Long beans, Marrowfat peas, Fava beans (broad beans), and Red kidney beans.

[0064] In some embodiments, the polypeptide is an animal polypeptide, where the term “animal” includes, but is not limited to, mammals, marsupials, birds, fish, cephalopods, crustaceans, and insects. Examples of such animal sources include, but are not limited to: Mammals: cattle, sheep, pigs, horses, goats, deer, reindeer, bison, moose, elk, camels, wild boars, wildebeest, and / or guinea pigs; Marsupials: kangaroos, koalas, wombats; Birds: chickens, turkeys, ducks, geese, quail, pheasants, guinea fowl, ostriches, emus, pigeons, partridges, ptarmigans, sandpipers, and / or woodcocks; Fish: salmon, tuna, cod, trout, sardines, haddock, tilapia, catfish, mackerel, swordfish, halibut, mahi-mahi, grouper, crucian carp, sea bass, anchovies, carp, perch, pike, flounder, sole, eel, herring, whitefish, and / or spiny lobster; • Cephalopods: squid, octopus, cuttlefish, and / or nautilus; Crustaceans: shrimp, crabs, lobsters, spiny lobsters, prawns, krill, crayfish, mussels, oysters, bivalves, scallops, and / or cockles; • Reptiles: snakes, turtles, alligators, crocodiles, iguanas, and / or lizards; and Insects: grasshoppers, crickets, mealworms, beetles, ants, termites, cicadas, caterpillars, silkworms, grasshoppers, waxworms, hawk moth larvae, bamboo worms, scorpions, and / or centipedes.

[0065] Examples of bacteria, though not limited to them, include Escherichia coli, Bacillus subtilis, and Pseudomonas fluorescens.

[0066] In some embodiments, the polypeptide is an algal polypeptide, where the term “algae” includes, but is not limited to, Euglenophyta, Chrysophyta, Pyrrophyta, Chlorophyta, Rhodophyta, Paeophyta, and Xanthophyta.

[0067] 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.

[0068] In some embodiments, the polypeptide is an archaeal polypeptide, where the term “archaea” includes, but is not limited to, euryacherchota, crenarchacherchota, and colarchacherchota.

[0069] Examples of archaeal 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.

[0070] In some embodiments, the polypeptide is a fungal polypeptide, where the term “fungus” includes, but is not limited to, Ascomycota, Basidiomycota, Zygomycota, Chytridiomycota, Glomeromycota, and Deuteromycota.

[0071] Examples of fungal sources of polypeptides include, but are not limited to, mushrooms, truffles, yeast, Penicillium, Aspergillus, ergot, chanterelles, morels, blanket fungus, broom fungus, stinkhorn, puffball, tea moss, and colloidal fungi. Other examples include Saccharomyces cerevisiae and Pichia pastoris.

[0072] The extract or isolate may be, for example, an aqueous extract from a plant, animal, bacteria, algae, archaea, or fungus. 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 alkaline solution, optionally with a surfactant, optionally with appropriate mixing and / or homogenization, and then isolating the aqueous extract or isolate from the undissolved material.

[0073] Solid proteins can be recovered from the isolated sample by precipitation, by adjusting the pH of the protein aqueous solution to the isoelectric point of the extracted protein through the addition of an acid or alkali. The solid protein precipitate can then be recovered by centrifugation, followed by washing with water and heating to sterilize the protein extract. Finally, the extracted protein may be spray-dried (9).

[0074] protein In some embodiments, the extract or isolate from a plant, animal, bacterium, algae, archaea, or fungus is a protein extract or isolate.

[0075] Protein extracts can be obtained, in particular, during the production of defatted beans, seeds, or nut flakes. Suitable plant material is first subjected to the processes of washing, drying, conditioning, grinding, dehulling, solvent oil extraction, and flash solvent removal, and the resulting protein is then purified by alcohol washing, acid leaching and / or water leaching (9).

[0076] Furthermore, bacteria, algae, archaea, or fungi can be genetically modified to produce or excrete recombinant proteins. Recombinant proteins can be purified by precipitation at their isoelectric point. Examples of proteins produced by this method 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. (10)

[0077] Examples of proteins that may be used in the compositions and / or methods provided herein include, but are not limited to, the following: Soy proteins such as glycinin and beta-conglycinin, Wheat proteins such as gliadin and glutenin, mung bean proteins such as bignin, phaseolin, and globulin. Corn proteins such as zein, Milk proteins such as whey, Egg proteins such as albumin, Epidermal proteins such as keratin, gelatin, and collagen, Also, insect proteins such as resilin.

[0078] Examples of bacterial polypeptides include, but are not limited to, casein, lactoglobulin, and lactalbumin extracted from recombinant proteins derived from Escherichia coli. For example, chicken or cattle genes encoding casein, lactoglobulin, or lactalbumin polypeptides can be expressed in E. coli (10).

[0079] Examples of yeast-derived proteins include, but are not limited to, casein, lactoglobulin, and lactalbumin extracted from recombinant proteins derived from Saccharomyces cerevisiae and P. pastoris. For example, chicken or cow genes encoding casein, lactoglobulin, or lactalbumin polypeptides can be expressed in Saccharomyces cerevisiae or P. pastoris (10).

[0080] Other examples of fungal proteins include, but are not limited to, casein, lactoglobulin, and lactalbumin extracted from recombinant proteins derived from the filamentous fungus Rhizopus. For example, chicken or cow genes encoding casein, lactoglobulin, or lactalbumin polypeptides can be expressed in Rhizopus (10).

[0081] In some embodiments, the polypeptide is used in a purified form or in combination with one or more other polypeptides and / or other materials.

[0082] polysaccharide The first composition may further contain one or more polysaccharides.

[0083] In some embodiments, the first composition further comprises one or more polysaccharides.

[0084] In some embodiments, the first composition does not contain polysaccharides.

[0085] In some embodiments, the first composition may additionally contain one or more polysaccharides. In certain other embodiments, the first composition may not contain any polysaccharides.

[0086] In some embodiments, extracts or isolates from plants, animals, bacteria, algae, archaea, and / or fungi may additionally contain one or more polysaccharides.

[0087] Polysaccharides are polymers of monosaccharides (simple sugars) linked by glycosidic bonds.

[0088] Numerous methods exist for extracting or isolating polysaccharides from biological materials that may be used in compositions and / or methods provided herein. In one such example, the polysaccharide extract may be obtained during the production of defatted beans, seeds, or nut flakes. A suitable plant material is first subjected to the processes of washing, drying, conditioning, grinding, dehulling, solvent oil extraction, and flash solvent removal. The polysaccharide is then obtained by removing proteins by alcohol washing, acid leaching, and / or water leaching (9).

[0089] Examples of polysaccharides that can be used in addition to the polypeptide in the first composition include, but are not limited to, chitin, chitosan, starch derived from wheat, rice, potato or corn, alginates, agar, hyaluronic acid, dextran, chondroitin sulfate, carrageenan, carrageenan-kappa, carrageenan-iota, pullulan, xanthan gum, gellan gum, and / or pectin.

[0090] In some embodiments, the first composition may additionally include one or more salt derivatives of polysaccharides. Examples of polysaccharide salt derivatives that can be added to the polypeptide in the first composition include, but are not limited to, sodium alginate, potassium alginate, sodium carrageenan, and / or potassium carrageenan.

[0091] Polysaccharides may be used in a purified form or in combination with one or more other polypeptides, polysaccharides, lipids, and / or other materials.

[0092] Lipids The first composition may additionally contain one or more lipids.

[0093] In some embodiments, the first composition further comprises one or more lipids.

[0094] In some embodiments, the first composition does not contain lipids.

[0095] In some embodiments, the first composition may additionally contain one or more lipids.

[0096] In some embodiments, extracts or isolates from plants, animals, bacteria, algae, archaea, and / or fungi may additionally contain one or more lipids.

[0097] Lipids are a class of molecules with diverse structures and functions. Typically, lipids are composed of fatty acids, glycerol, and other hydrocarbon chains.

[0098] Examples of lipids that may be used in addition to polypeptides include, but are not limited to, the following: • Vegetable 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 • Animal fats (including milk, butter, lard, and animal fat): Cattle, sheep, pigs, horses, goats, deer, reindeer, bison, moose, elk, camels, wild boars, guinea pigs, kangaroos, chickens, turkeys, ducks, geese, quail, pheasants, guinea fowl, ostriches, emus, pigeons, partridges, ptarmigans, sandpipers, woodcocks, salmon, tuna, cod, trout, sardines, haddocks, tilapia, catfish, horse mackerel, kelp, halibut, mahi-mahi, grouper, snapper, sea bass, anchovies, carp, perch, pike, flounder, sole, eels, herring, whitefish, and / or spiny lobsters. • Fatty 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). • Salts of fatty acids • Phospholipids, Omega-3 oil, ·Algae oil.

[0099] Lipids may be used in a purified form or in combination with one or more other polypeptides, polysaccharides, lipids, and / or other materials.

[0100] polyol The first composition may further contain one or more polyols and / or polyol polymers.

[0101] In some embodiments, the first composition further comprises one or more polyols and / or polyol polymers.

[0102] In some embodiments, the first composition does not contain polyols and / or polymers of polyols.

[0103] Polyols are organic compounds characterized by having multiple hydroxyl (-OH) groups, usually bonded to carbon atoms, in their molecular structure.

[0104] Examples of polyols include, but are not limited to, ethylene glycol, glycerol, erythritol, treitol, arabitol, xylitol, ribitolmannitol, sorbitol, galactitol, fusitol, iditol, inositol, boremitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, monoacylglycerol, diacylglycerol, and / or triacylglycerol.

[0105] In some embodiments, the first composition further comprises, but is not limited to, a polyol polymer such as polyethylene glycol.

[0106] In some embodiments, the first composition does not contain a polyol polymer such as polyethylene glycol, but is not limited to this.

[0107] In some preferred embodiments, commercially available protein extracts or isolates, polypeptides, polysaccharides, lipids, and / or polyols are used in the production of semipermeable porous hollow fibers, for example, to avoid additional costs. However, the extracts or isolates may also be obtained from the previous processing steps.

[0108] In another preferred embodiment, soy protein isolate (SPI) obtained from soybeans is used for the production of semipermeable porous hollow fibers. SPI is the most highly purified of the commercially available soy protein products, some of which contain more than 90% (by mass) protein. The soy protein extract is produced from defatted soybean flakes, in which most of the fat, sugars, and fiber are removed, leaving protein as a significant residual component. In some embodiments, SPI can be produced by extracting the protein in a weakly alkaline solution. The extract is then isolated by centrifugation and subsequently acidified to produce a protein curd. The curd is washed to remove soluble sugars, neutralized, and finally spray-dried (9). Other plant protein isolates may be produced and used in a similar manner.

[0109] Therefore, in one particularly preferred embodiment, the polypeptide of the composition and / or method provided herein comprises a soy polypeptide. In a particular embodiment, the polypeptide essentially comprises a soy polypeptide.

[0110] In some embodiments, the first composition comprises or essentially comprises a polypeptide, wherein at least 60%, preferably at least 75%, of the polypeptide is a soybean polypeptide (e.g., extracted from soybeans).

[0111] 1.6 Step (A) Dissolution in the first solvent In step (a), a first composition comprising a polypeptide is combined with a second composition comprising a solvent and one or more denaturing agents and / or reducing agents to produce a third composition.

[0112] The first composition may further contain one or more polysaccharides, lipids, polyols, polymers of polyols, and / or any combination thereof.

[0113] In some embodiments, the first composition further comprises one or more polysaccharides.

[0114] In some embodiments, the first composition further comprises one or more lipids.

[0115] In some embodiments, the first composition further comprises one or more polyols and / or polyol polymers.

[0116] In some embodiments, the first composition further comprises one or more polysaccharides and lipids.

[0117] In some embodiments, the first composition further comprises one or more lipids, as well as polyols and / or polymers of polyols.

[0118] In some embodiments, 5% to 35% (by weight per volume) of the first composition is added to the second composition.

[0119] In some embodiments, the first composition is added to the second composition until the final concentration is 5% to 35% (w / v of the second composition).

[0120] In some embodiments, the percentage of polypeptide in the third composition is in the range of 5% to 10%, 10% to 20%, or 20% to 35% (by weight per volume).

[0121] In some embodiments, the first composition is added to the second composition until the final concentration is 5% to 60% (w / v of the second composition).

[0122] In some embodiments, the percentage of polypeptide in the third composition is in the range of 35% to 45%, 45% to 55%, or 55% to 60% (by weight per volume).

[0123] In some embodiments, polysaccharides(s) are added to the first composition in a ratio ranging from 1% to 10000% (by weight of polypeptide).

[0124] In some embodiments, lipids(s) are added to the first composition in a ratio that is (i) between 1% and 10000% (weight per unit weight of polypeptide).

[0125] In some embodiments, polyols and / or polymers of polyols are added to the first composition in a ratio that is (i) between 1% and 10000% (by weight of polypeptide).

[0126] The second composition comprises one or more solvents and one or more denaturing agents.

[0127] In some embodiments, the second composition also comprises one or more reducing agents.

[0128] In some embodiments, the second composition can dissolve some or most of the polypeptide in the first composition.

[0129] In some embodiments, the second composition can dissolve all or substantially all of the polypeptides in the first composition.

[0130] In some embodiments, the second composition can achieve complete dissolution of the polypeptide (and other components, e.g., polysaccharides, lipids, polyols, polymers of polyols, and / or salts) in the first composition.

[0131] An objective of some embodiments is to produce a fourth composition, which is a viscous, extrudeable, or spinnable composition.

[0132] Complete dissolution refers to the process by which a substance, sometimes called a solute, completely dissolves in another substance, sometimes called a solvent.

[0133] Complete dissolution can be experimentally confirmed by visible light spectrophotometric absorbance measurement. By performing absorbance measurements on several samples and ensuring that all measurements are uniform, complete dissolution can be confirmed.

[0134] In this specification, the terms “dissolving” and “solubilising” are used interchangeably.

[0135] Those skilled in the art can easily test the solubility of polypeptides (and other components, if present, such as polysaccharides, lipids, and salts) in various second compositions to determine the suitability of using the second composition.

[0136] In one embodiment, the second composition is capable of disrupting the disulfide bonds and / or hydrogen bonds of at least one polypeptide in the first composition.

[0137] Examples of solvents that may be used in the second composition include aqueous solutions and / or organic solutions.

[0138] Examples of aqueous solutions include acidic solutions, alkaline solutions, and / or salt solutions.

[0139] Examples of organic solutions, though not limited to them, include alcohols.

[0140] In some embodiments, the solvent used in the second composition may include water, ethanol, acetic acid, propanol and / or formaldehyde, and / or mixtures thereof.

[0141] In other embodiments, the solvent used in the second composition is used either alone or in combination with other solvents.

[0142] In some preferred embodiments, the solvent used in the second composition is water.

[0143] In some embodiments, one or more salts are added to the second composition to aid in the dissolution of polypeptides (and other components, if present, such as polysaccharides and lipids) in the first composition. Examples of salts include, but are not limited to, sodium chloride, potassium chloride, zinc chloride, sodium carbonate, potassium carbonate, sodium bicarbonate, and / or zinc sulfate.

[0144] In some embodiments, one or more salts are added to the second composition to aid in the dissolution of polypeptides (and other components, if present, such as polysaccharides and lipids) in the first composition. Examples of salts, but not limited to, include lithium sulfate, sodium malonate, sodium maleate, sodium potassium tartrate, triammonium citrate, trilithium citrate, trisodium citrate, tripotassium citrate, disodium hydrogen phosphate, sodium hypophosphate, monosodium phosphate, dipotassium phosphate, sodium sulfate, sodium bisulfate, potassium sulfate, ammonium sulfate, or sodium sulfite.

[0145] In some embodiments, one or more salts are added to the second composition to aid in the dissolution of polypeptides (and other components, if present, such as polysaccharides and lipids) in the first composition. Examples of salts, but not limited to, include sodium chloride, potassium chloride, zinc chloride, sodium carbonate, potassium carbonate, sodium bicarbonate, zinc sulfate, lithium sulfate, sodium malonate, sodium maleate, sodium potassium tartrate, triammonium citrate, trilithium citrate, trisodium citrate, tripotassium citrate, disodium hydrogen phosphate, sodium hypophosphate, monosodium phosphate, dipotassium phosphate, sodium sulfate, sodium bisulfate, potassium sulfate, ammonium sulfate, or sodium sulfite.

[0146] In some embodiments, one or more hydrated and / or anhydrous salts are added to the second composition.

[0147] In some embodiments, the concentration of each salt added to the second composition is in the range of 0.1% to 70% (by weight relative to the weight of the polypeptide and other components, e.g., polysaccharides, if present), preferably 0.1% to 50%, more preferably 0.5% to 10%, even more preferably 1% to 7%, and most preferably 1% to 2%.

[0148] In some embodiments, the concentration of each salt added to the second composition is in the range of 0.1-1%, 1%-10%, 10%-25%, 25%-50%, or 50%-70%.

[0149] Denaturants and reducing agents The second composition may also contain one or more denaturing agents and / or one or more reducing agents for solubilizing and denaturing the polypeptide.

[0150] In some embodiments, the second composition also comprises one or more modifiers to modify the polypeptide of the first composition.

[0151] In some embodiments, the second composition also comprises one or more reducing agents for reducing the polypeptide of the first composition.

[0152] In some embodiments, the second composition also comprises one or more denaturing agents and / or one or more reducing agents to solubilize and denature the polypeptide of the first composition.

[0153] In some embodiments, the second composition does not contain a reducing agent.

[0154] In some embodiments, the second composition does not contain a denaturant.

[0155] In some embodiments, the second composition does not contain a denaturant or reducing agent.

[0156] The structure of a protein can be described in terms of its primary, secondary, and tertiary structures. The primary structure of a protein is a linear sequence of amino acids linked together by peptide bonds, forming the polypeptide backbone. The secondary structure of a protein refers to the local folding of the polypeptide backbone into specific shapes and patterns stabilized by hydrogen bonds between adjacent amino acids in three-dimensional space. The tertiary structure of a protein refers to the three-dimensional shape of the protein resulting from the interactions of distant amino acids in three-dimensional space (11).

[0157] Protein secondary structures include alpha-helices, beta-sheets, beta-helices, and amorphous structures. Alpha-helices are spiral structures held together by hydrogen bonds between amino acids in the polypeptide backbone. Beta-sheets are flat structures in which hydrogen bonds between adjacent protein chains result in the formation of a folded or accordion-like structure. Beta-helices are helical structures held together by hydrogen bonds between adjacent proteins. Amorphous, disordered, or denatured, as used interchangeably herein, are protein structures that lack a clearly defined or ordered secondary structure (11).

[0158] The primary, secondary, and tertiary structures of polypeptides can be disrupted by applying appropriate agents, thereby denaturing and / or reducing these polypeptides. However, in the production of covalently crosslinked semipermeable porous hollow fibers of this disclosure, in some embodiments, it is desirable to keep the primary structure intact in order to maximize polypeptide chain entanglement. While alkaline and acidic solvents have conventionally been used to solubilize and denature polypeptides, these solvents can also disrupt the primary structure in addition to the secondary and tertiary structures. In contrast, aqueous urea can solubilize polypeptides and, when used in combination with a reducing agent, can denature the secondary and tertiary structures without disrupting the primary structure (12).

[0159] The breakdown of secondary and tertiary structures can enable solvation and stabilization of the polypeptide backbone structure. The linear nature of the unfolded molecular backbone can promote the stacking of molecules in solution, thereby reducing the viscosity of the solution. During extrusion, the linear nature of molecules in solution can ensure chain entanglement and alignment, enabling the realization of appropriate mechanical properties. The degree of polypeptide chain entanglement may be maximized by maximizing the length of the polypeptide chain (12). Therefore, in some embodiments, solubilization of polypeptides with acids and alkalis is undesirable, while solubilization and modification with reducing agents are preferred.

[0160] Polypeptide denaturation and reduction can be achieved by treatment with several chemicals.

[0161] Urea is widely used to denature polypeptides for proteomics and metabolomics studies because it can disrupt the secondary and tertiary structures without destroying the molecular backbone. Urea can solubilize polypeptides through the interaction of hydrophobic motifs on the surface of the polypeptide's tertiary structure, exposing and solvating hydrophilic motifs within the protein structure (13).

[0162] In some embodiments, urea is used in the second composition to solubilize the polypeptide of the first composition at a concentration in the range of 0 mol / L to 8 mol / L, 1 mol / L to 8 mol / L, preferably in the range of 4 mol / L to 8 mol / L or 6 mol / L to 8 mol / L, most preferably at a concentration of about 8 mol / L.

[0163] In some embodiments, the urea concentration is within the range of 0 mol / L to 1 mol / L, 1 mol / L to 2 mol / L, 2 mol / L to 4 mol / L, 4 mol / L to 6 mol / L, or 6 mol / L to 8 mol / L.

[0164] Treatment with an acid or alkali may also be used to denature and solubilize polypeptides, such as those in the solvent of the second composition in step (a). However, the use of acid and alkali chemical treatments may destroy the primary structure of the treated polypeptide (14).

[0165] Examples of acids that can be used as denaturants include, but are not limited to, oxalic acid, malic acid, succinic acid, adipic acid, tartaric acid, citric acid, malonic acid, acetic acid, formic acid, sulfuric acid, nitric acid, and / or hydrochloric acid.

[0166] Examples of alkalis that can be used as denaturing agents include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, and / or magnesium hydroxide.

[0167] Other suitable denaturants that may be used include, but are not limited to, guanidine hydrochloride, sodium dodecyl sulfate (SDS), Triton X-100, ethanol, acetone, dimethyl sulfoxide (DMSO) sophorolipids, chaotropic salts (e.g., ammonium sulfate), and enzymes. Heat may also be used.

[0168] The reducing agent can prevent the rearrangement of the polypeptide of the first composition by disrupting the disulfide bonds and inhibiting their oxidation, thereby assisting in the disruption of the secondary and tertiary structures and the formation of a linear molecular skeleton.

[0169] Examples of suitable reducing agents that 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 sulfite, sodium bisulfite, sodium pyrosulfite, sodium hypophosphate, sodium dithionite, mercaptoethanol, and dithiothreitol.

[0170] In some embodiments, one or more denaturing agents, reducing agents, and / or combinations thereof are included in the second composition together with a solvent to assist in the solvation of the polypeptide of the first composition to form a third composition.

[0171] In some embodiments, the concentration of each denaturing agent and / or reducing agent is in the range of 0.01 to 50% (by mass) of the polypeptide in the first composition and is included together with the solvent of the second composition.

[0172] In some embodiments, the concentration of the denaturant and / or reducing agent in the second composition is in the range of 0.01-0.1%, 0.1-1%, 1%-10%, 10%-25%, or 25%-50% (by mass) of the polypeptide in the first composition.

[0173] In some embodiments, the concentration of each denaturing agent and / or reducing agent is in the range of 0.01 to 1500% (by mass) of the polypeptide in the first composition and is included together with the solvent of the second composition.

[0174] In some embodiments, the concentration of the denaturant and / or reducing agent in the second composition is within the range of 50-75%, 75-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or 1000-1500% (by mass) of the polypeptide in the first composition.

[0175] In some embodiments, the pH of the second composition is in the range of 5.01 to 9.99, preferably in the range of 6.0 to 9.0, and most preferably in the range of 6.5 to 8.5.

[0176] In some embodiments, the pH of the second composition is in the range of 5.01 to 6.0, 6.0 to 7.0, 7.0 to 8.0, 8.0 to 9.0, or 9.0 to 9.99.

[0177] In some embodiments, the pH of the second composition is in the range of 9.0 to 9.99, 9.1 to 9.8, 9.2 to 9.7, or 9.3 to 9.4.

[0178] In some embodiments, in step (a), the first composition comprises, essentially consists of, or includes a soybean polypeptide.

[0179] In some embodiments, in step (a), the solvent in the second composition is water.

[0180] In some embodiments, in step (a), the denaturing agent and / or reducing agent in the second composition comprises urea, N-acetylcysteine, and / or sodium sulfite.

[0181] In some embodiments, the pH of the second composition is in the range of 8.0 to 8.5.

[0182] In some embodiments, the pH of the second composition is in the range of 9.0 to 9.5.

[0183] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of a soybean polypeptide, (ii) A second composition comprising water and one or more denaturing agents and / or reducing agents selected from urea, N-acetylcysteine, and sodium sulfite, at a pH in the range of 8.0 to 8.5 This includes combining them to produce a third composition.

[0184] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of a soybean polypeptide, (ii) A second composition comprising water and one or more denaturing agents and / or reducing agents selected from urea, N-acetylcysteine, and sodium sulfite, at a pH in the range of 8.5 to 9.5 This includes combining them to produce a third composition.

[0185] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of a soybean polypeptide, (ii) A second composition comprising water and one or more salts selected from urea, sodium bicarbonate, N-acetylcysteine, and sodium sulfite, a denaturant and / or a reducing agent, at a pH in the range of 8.0 to 8.5 This includes combining them to produce a third composition.

[0186] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of a soybean polypeptide, (ii) A second composition comprising water and one or more salts selected from urea, sodium bicarbonate, N-acetylcysteine, and sodium sulfite, a denaturant and / or a reducing agent, at a pH in the range of 8.5 to 9.5 This includes combining them to produce a third composition.

[0187] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of mung bean polypeptide, (ii) A second composition comprising water and one or more salts selected from urea, sodium bicarbonate, N-acetylcysteine, and sodium sulfite, a denaturant and / or a reducing agent, at a pH in the range of 8.0 to 8.5 This includes combining them to produce a third composition.

[0188] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of mung bean polypeptide, (ii) A second composition comprising water and one or more salts selected from urea, sodium bicarbonate, N-acetylcysteine, and sodium sulfite, a denaturant and / or a reducing agent, at a pH in the range of 8.5 to 9.5 This includes combining them to produce a third composition.

[0189] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of chickpea polypeptide (ii) A second composition comprising water and one or more salts selected from urea, sodium bicarbonate, N-acetylcysteine, and sodium sulfite, a denaturant and / or a reducing agent, at a pH in the range of 8.0 to 8.5 This includes combining them to produce a third composition.

[0190] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of chickpea polypeptide (ii) A second composition comprising water and one or more salts selected from urea, sodium bicarbonate, N-acetylcysteine, and sodium sulfite, a denaturant and / or a reducing agent, at a pH in the range of 8.5 to 9.5 This includes combining them to produce a third composition.

[0191] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of sunflower seed polypeptide (ii) A second composition comprising water and one or more salts selected from urea, sodium bicarbonate, N-acetylcysteine, and sodium sulfite, a denaturant and / or a reducing agent, at a pH in the range of 8.0 to 8.5 This includes combining things.

[0192] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of sunflower seed polypeptide (ii) A second composition comprising water and one or more salts selected from urea, sodium bicarbonate, N-acetylcysteine, and sodium sulfite, a denaturant and / or a reducing agent, at a pH in the range of 8.5 to 9.5 This includes combining things.

[0193] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of a broad bean polypeptide, (ii) A second composition comprising water and one or more salts selected from urea, sodium bicarbonate, N-acetylcysteine, and sodium sulfite, a denaturant and / or a reducing agent, at a pH in the range of 8.0 to 8.5 This includes combining things.

[0194] In some embodiments, step (a) is: (i) A first composition comprising, essentially, or consisting of a broad bean polypeptide, (ii) A second composition comprising water and one or more salts selected from urea, sodium bicarbonate, N-acetylcysteine, and sodium sulfite, a denaturant and / or a reducing agent, at a pH in the range of 8.5 to 9.5 This includes combining things.

[0195] In some embodiments, the first composition of any embodiment of the present disclosure can be used in the production of the Prokitein and / or hollow fibers of the present disclosure.

[0196] In some embodiments, a second composition of any embodiment of the present disclosure can be used in the production of Prokitein and / or hollow fibers of the present disclosure.

[0197] In some embodiments, a third composition of any embodiment of the present disclosure can be used in the production of Prokitein and / or hollow fibers of the present disclosure.

[0198] 1.7 Step (B) Incubation / Aging of the Third Composition In step (b), the third composition is incubated under conditions that may be sufficient to solubilize, denaturate, and / or reduce at least one fraction of the polypeptide of the first composition to form the fourth composition. Doing so may reduce the viscosity of the third composition and disrupt the secondary and tertiary structures of the polypeptide of the first composition.

[0199] In some embodiments, step (b) involves incubating the third composition under conditions sufficient to solubilize, denaturate, and / or reduce at least one fraction of the polypeptide of the first composition to form the fourth composition.

[0200] In some embodiments, the third composition is incubated for a period ranging from 0 minutes to 7 days, preferably 10 minutes to 4 days, more preferably 15 minutes to 1 day, and most preferably 20 minutes to 6 hours.

[0201] In some embodiments, the third composition is incubated for a period of time ranging from 0 to 5 minutes, 5 to 10 minutes, 10 to 20 minutes, 20 minutes to 1 hour, 1 to 3 hours, 3 to 6 hours, 6 to 12 hours, 12 hours to 1 day, 1 to 2 days, 2 to 3 days, 3 to 4 days, 4 to 5 days, 5 to 6 days, or 6 to 7 days.

[0202] In some embodiments, the third composition is incubated at a temperature in the range of -25°C to 150°C, or in the range of 15°C to 90°C, or in the range of 20°C to 90°C.

[0203] In some embodiments, the third composition is incubated at a temperature 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.

[0204] In some embodiments, the third composition is incubated at room temperature.

[0205] In some embodiments, the pH of the third composition is in the range of 5.01 to 9.99, preferably in the range of 6.0 to 9.0, and most preferably in the range of 6.5 to 8.5.

[0206] In some embodiments, the pH of the third composition is in the range of 5.01 to 6.0, 6.0 to 7.0, 7.0 to 8.0, 8.0 to 9.0, or 9.0 to 9.99.

[0207] In this specification, mixing is defined as a mechanical process used to combine different substances or components, typically in a liquid or homogeneous mixture. Mixing involves completely blending two or more substances to create a homogeneous composition, ensuring that the individual components are uniformly distributed throughout the mixture.

[0208] During the incubation step, a third composition may be further mixed.

[0209] During the incubation step, the third composition may be further mixed with a helical ribbon blade.

[0210] In some embodiments, the third composition is mixed during the incubation step.

[0211] In some embodiments, the third composition is mixed with helical ribbon blades during the incubation step.

[0212] In some embodiments, the third composition is not mixed during the incubation step.

[0213] In some embodiments, the third composition is mixed for a period of time within the range of 0 to 5 minutes, 5 to 10 minutes, 10 to 20 minutes, 20 minutes to 1 hour, 1 to 3 hours, 3 to 6 hours, 6 to 12 hours, 12 hours to 1 day, 1 to 2 days, 2 to 3 days, 3 to 4 days, 4 to 5 days, 5 to 6 days, or 6 to 7 days.

[0214] In some other embodiments, the third composition is mixed throughout part, most, or all of the incubation step.

[0215] In some embodiments, the third composition is mixed at a speed 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.

[0216] In some other embodiments, the third composition is 0rpm~5rpm, 5rpm~50rpm, 50rpm~100rpm, 100rpm~150rpm, 150rpm~200rpm, 200rpm~300rpm, 300rpm~400rpm, 400rpm~500rpm, 500rpm~600rpm, 600rpm~700rpm, 700rpm~800rpm, 800rpm~900rpm, 900rpm~1,000rpm, 1,000rpm~1,100rpm, 1,100rpm~1,200rpm, 1,200rpm~1,300rpm pm, 1,300rpm~1,400rpm, 1,400rpm~1,500rpm, 1,500rpm~1,600rpm, 1,600rpm~1,700rpm, 1,700rpm~1,800rpm, 1,800rpm~1,900rpm, 1,900rpm~2,000 rpm, 2,000rpm~2,100rpm, 2,100rpm~2,200rpm, 2,200rpm~2,300rpm, 2,300rpm~2,400rpm, 2,400rpm~2,500rpm, 2,500rpm~2,600rpm, 2,600rpm~2,70 0rpm, 2,700rpm~2,800rpm, 2,800rpm~2,900rpm, 2,900rpm~3,000rpm, 3,000rpm~3,100rpm, 3,100rpm~3,200rpm, 3,200rpm~3,300rpm, 3,300rpm~3, 400rpm, 3,400rpm~3,500rpm, 3,500rpm~3,600rpm, 3,600rpm~3,700rpm, 3,700rpm~3,800rpm, 3,800rpm~3,900rpm, 3,900rpm~4,000rpm, 4,000rpm~4 ,500rpm, 4,500rpm~5,000rpm, 5,000rpm~5,500rpm, 5,500rpm~6,000rpm, 6,000rpm~6,500rpm, 6,500rpm~7,000rpm, 7,000rpm~7,500rpm, 7,500rpm~ 8,000rpm, 8,000rpm~8,500rpm, 8,500rpm~9,000rpm, 9,000rpm~9,500rpm, 9,500rpm~10,000rpm, 10,000rpm~10,500rpm, 10,500rpm~11,000rpm, 11,000rpm~11,500rpm, 11,500rpm~12,000rpm, 12,000rpm~12,500rpm, 12,500rpm~13,000rpm, 13,000rpm~13,500 rpm, 13,500rpm~14,000rpm, 14,000rpm~14,500rpm, 14,500rpm~15,000rpm, 15,000rpm~15,500rpm, 15,500rpm~ The mixture is mixed at speeds within the range of 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 19,000 rpm, 19,000 rpm to 19,500 rpm, or 19,500 rpm to 20,000 rpm.

[0217] In some preferred embodiments, during incubation, the third composition is mixed at a speed in the range of 100 rpm to 400 rpm.

[0218] Room temperature can be within the range of 15°C to 30°C.

[0219] In some embodiments, the room temperature is in 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.

[0220] In some other preferred embodiments, the third composition is mixed with a helical ribbon blade at a speed within the range of 400 rpm for 20 minutes during incubation at room temperature.

[0221] In some embodiments, the viscosity of the fourth composition (i.e., after the incubation step) is 2s at 25°C. -1At the shear rate, 100 cP to 200,000 cP, preferably 100 cP to 1,000 cP, 1,000 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, 20 0,000cP~300,000cP, 300,000cP~400,000cP, 400,000cP~500,000cP, 500,000cP~ 600,000cP, 600,000cP~700,000cP, 700,000cP~800,000cP, 800,000cP~900,000c P, 900,000cP~1,000,000cP, 1,000,000cP~1,100,000cP, 1,100,000cP~1,200,0 00cP, 1,200,000cP~1,300,000cP, 1,300,000cP~1,400,000cP, 1,400,000cP~1,5 The ranges are 00,000cP, 1,500,000cP to 1,600,000cP, 1,600,000cP to 1,700,000cP, 1,700,000cP to 1,800,000cP, 1,800,000cP to 1,900,000cP, and 1,900,000cP to 2,000,000cP.

[0222] The Brookfield rotational viscometer is the most common instrument used to evaluate the viscosity of a sample. The Brookfield rotational viscometer measures the torque required to rotate a spindle in a fluid. For Newtonian fluids, the required torque increases linearly as the spindle rotation speed or the surface area of ​​the spindle used increases. A characteristic apparent viscosity can be measured by evaluating the viscosity of the fluid at a given shear rate and temperature. The rheological properties of the fluid are determined by how the relationship between viscosity and shear rate evolves. A fluid can be a Newtonian fluid where the strain decreases if the viscosity is linearly proportional to the shear rate and the apparent viscosity decreases with increasing shear rate, and increases if the apparent viscosity increases with increasing shear rate; a Bingham plastic fluid requiring a finite yield stress before flowing but then exhibiting a linear shear rate and apparent viscosity profile; or a Bingham pseudoplastic fluid requiring a finite yield stress before flowing but exhibiting a nonlinear shear rate and apparent viscosity profile (15).

[0223] Viscosity may be evaluated using a rotational viscometer manufactured by another manufacturer.

[0224] In some embodiments, a fraction of the polypeptide in the third composition is denatured and / or reduced. Preferably, this fraction is higher than 50%, more preferably higher than 65%, and most preferably higher than 80%.

[0225] In some embodiments, the fraction of the modified and / or reduced polypeptide in the third composition is in the range of 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 99.99%.

[0226] In some embodiments, the fraction of the modified and / or reduced polypeptide in the third composition is in the range of 0% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, or 40% to 50%.

[0227] The denaturation and reduction of polypeptides in the third composition may be quantified using Fourier transform infrared (FTIR) spectroscopy, circular dichroism, wide-angle X-ray scattering (WAXS), or Raman spectroscopy. The degree of reduction may be assessed by recording representative spectra of the starting organic species and using changes in band intensity related to disulfide bonds. Similarly, changes in the secondary structure of the protein may be determined using changes in band intensity related to alpha-helices and beta-sheets.

[0228] Other additives Void fraction components One or more void fractions may be introduced into the first, second, third, or fourth composition to assist in the controlled formation of pores in the extruded or spun hollow fibers. The void fractions can impart porosity by acting as pore templates. This can occur when the doping liquid undergoes phase inversion and solidifies around the particles of the void fractions. Upon removal of the void fractions, the extruded or spun hollow fibers may leave a microstructure consisting of pore morphology that is a negative cast of the original particles of the void fractions. The void-containing elements may be in solid, liquid, and / or gaseous form.

[0229] The pore volume of a material may refer to the total volume of open space or pores within the material. The porosity of a material may refer to the portion of the total material volume occupied by open space or pores, and may be characterized using methods such as mercury porosimetry.

[0230] The pore volume and / or porosity of a material may also be determined by image analysis. Images that may be used to determine the porosity of a material include those generated by a scanning electron microscope. A person skilled in the art may determine porosity by considering the ratio of black pixels to white pixels in a binarized image.

[0231] In some embodiments, one or more void fraction components are introduced into the first, second, third, or fourth composition to assist in the controlled formation of pores in extruded or spun hollow fibers.

[0232] In some embodiments, the void-containing element includes, but is not limited to, powdered or pulverized calcium carbonate.

[0233] In some embodiments, the void-containing element includes crushed or shredded ice.

[0234] In some embodiments, the void-containing element includes, but is not limited to, gases such as air, nitrogen, oxygen, carbon dioxide, helium, neon, argon, xenon, and / or mixtures thereof.

[0235] In some embodiments, the void-containing element includes, but is not limited to, salts such as sodium chloride and potassium chloride.

[0236] In some embodiments, the void-containing elements include lipids such as vegetable 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).

[0237] In some embodiments, when lipids are used as void-filling elements, the lipids have secondary process functions, including, but not limited to, acting as flavor enhancers and plasticizers.

[0238] In some embodiments, the void-containing element comprises one or more polyols and / or polymers of polyols.

[0239] In some embodiments, the void-containing element includes, but is not limited to, a water-soluble polymer such as polyvinyl alcohol.

[0240] For example, solid powdered calcium carbonate may be introduced into the second composition and / or the third composition and / or the fourth composition. The fourth composition may then be extruded into a coagulation bath or spun. Hydrogen ions in the coagulation bath provided by a soluble acidic species (e.g., citric acid) may then react with the calcium carbonate to form a calcium salt and carbon dioxide. After the release of carbon dioxide and the dissolution of the salt, voids may remain where the calcium carbonate particles previously were.

[0241] In another example, a salt such as sodium chloride may be introduced into the second composition and / or the third composition and / or the fourth composition. The fourth composition may then be extruded into an aqueous solidification bath. The excess aqueous solvent in the solidification bath may then dissolve the salt, leaving voids in the place of the salt particles.

[0242] In yet another example, a gas such as air may be introduced into the third composition either before or after incubation by proper mixing, mechanical stirring, and / or injection of the third composition. The fourth composition may then be extruded into an aqueous solidification bath. During the solidification process, void spaces and pores may be formed by air bubbles trapped in the third composition.

[0243] In further examples, pores may be formed via a process called "emulsion molding," where lipids such as sunflower oil may be introduced into the first composition and / or the second composition and / or the third composition and / or the fourth composition. By appropriately mixing or mechanically stirring the third and / or fourth compositions, an oil-in-water emulsion or mixture may be formed, where the oil droplets are dispersed in the aqueous phase. The polypeptide of the first composition may act as a surfactant to stabilize the emulsion. The fourth composition may then be extruded into a coagulation bath or spun. By removing the oil droplets from the resulting extruded or spun hollow fibers, void spaces may be formed, which may function as pores.

[0244] In some embodiments, the void fraction is added to the first composition in step (a).

[0245] In other embodiments, the void fraction is added to the second composition in step (a).

[0246] In other embodiments, the void fraction is added to the third composition in step (b) either before or during the incubation step.

[0247] In other embodiments, the void fraction is added to the third composition after the incubation step in step (b), and then mixed.

[0248] In other embodiments, the void fraction is added to the fourth composition in step (b).

[0249] In a preferred embodiment, the void fraction is added to the fourth composition in step (b).

[0250] In one embodiment, the void-containing elements are added to the composition in step (a) or step (b) such that they constitute 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.

[0251] In some embodiments, void-containing elements are added to the composition in step (a) or step (b) such that the void-containing elements constitute 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.

[0252] plasticizer In some embodiments, one or more other components are added to the composition in step (a) or step (b) to improve the flexibility and ductility of the semipermeable porous hollow fiber.

[0253] In some preferred embodiments, one or more plasticizers are added to the composition in step (a) and / or step (b) to improve the flexibility and ductility of the semipermeable porous hollow fibers.

[0254] Examples of plasticizers include lipids, polyols, and / or polymers of polyols.

[0255] In some embodiments, one or more lipids, polyols, and / or polymers of polyols are added to the composition as plasticizers in step (a) and / or step (b).

[0256] In some embodiments, one or more lipids are added to the composition as plasticizers in step (a) and / or step (b).

[0257] In some embodiments, one or more polyols and / or polymers of polyols are added to the composition as plasticizers in step (a) and / or step (b).

[0258] In one embodiment, one or more plasticizers are added to either the composition at any stage in step (a) or step (b).

[0259] In a preferred embodiment, one or more plasticizers are added to the fourth composition in step (b).

[0260] In some embodiments, no plasticizer is added to the composition in step (a) and / or step (b).

[0261] In some embodiments, the plasticizer has secondary process functions, including, but not limited to, acting as a flavor enhancer and as a void fractionation component.

[0262] In one embodiment, one or more plasticizers are added to the composition in step (a) and / or step (b) such that each constitutes 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.

[0263] In some embodiments, one or more plasticizers are added to the composition in step (a) or step (b) such that each constitutes 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.

[0264] Flavoring One or more other components may be added to the composition of step (a) and / or step (b) as flavorings, which may improve the flavor of the extruded or spun semipermeable porous hollow fibers, including covalent esters, thioesters and / or amide-crosslinked polypeptides.

[0265] In some embodiments, one or more other components are added to the composition as flavorings in step (a) and / or step (b) to improve the flavor of extruded or spun semipermeable porous hollow fibers comprising covalent esters, thioesters and / or amide-crosslinked polypeptides.

[0266] In some embodiments, the flavoring(s) added to the composition in step (a) and / or step (b) may include, but are not limited to, one or more lipids, polyols, and / or polymers of polyols.

[0267] In some other embodiments, the flavorings (plural) added to the composition in step (a) and / or step (b) include, but are not limited to, those enumerated by the European Commission in (EC) No. 1334 / 2008 established in 2012 under Regulation EU 872 / 2012, and non-food flavorings added in accordance with the amendments in accordance with Regulation EU 2018 / 1259.

[0268] In some embodiments, flavorings are not added to the composition in step (a) and / or step (b).

[0269] In some embodiments, the composition of step (a) and / or step (b) is intended to be mixed after the addition of at least one additive, which includes, but is not limited to, void-containing elements, plasticizers, and / or flavorings.

[0270] Degassing In some embodiments, the composition(s) of step (a) and / or step (b) is degassed.

[0271] In this specification, degassing, also known as deflation, is defined as the partial or complete removal of bubbles from any of the compositions.

[0272] Controlled degassing can enable the removal of uncontrolled void spaces in the dope solution. This can ensure that the pore size of the hollow fibers is controlled and uniform, which can then allow for the separation of the luminal and extracapillary spaces of hollow fiber bioreactors by cell size for use in long-term cell culture.

[0273] In some embodiments, the third composition is degassed.

[0274] In some embodiments, the fourth composition is degassed.

[0275] Degassing can be achieved by one or more methods, but are not limited to, reduced pressure, thermal control, membrane degassing, ultrasonic degassing, freeze-pump thaw cycles, and / or centrifugation.

[0276] In some embodiments, degassing is achieved by centrifugation.

[0277] In some embodiments, degassing is achieved by reducing the pressure under vacuum.

[0278] In some embodiments, degassing is achieved by ultrasonic degassing.

[0279] In some embodiments, in step (b), the third composition is incubated for 2 to 4 days.

[0280] In some embodiments, in step (b), the third composition is incubated at a temperature of 18°C ​​to 25°C.

[0281] In some embodiments, in step (b), the third composition is incubated at room temperature.

[0282] In some embodiments, in step (b), the third composition is incubated at a temperature of 18°C ​​to 25°C for 2 to 4 days.

[0283] In some embodiments, step (b) additionally includes introducing one or more void fraction components, preferably powdered calcium carbonate, into the fourth composition.

[0284] In some preferred embodiments, step (b) additionally includes introducing one or more void fraction components, preferably lipids, into the fourth composition.

[0285] In some embodiments, step (b) includes incubating the third composition at a temperature of 18°C ​​to 25°C for 3 days to produce a fourth composition, and introducing lipids into the fourth composition.

[0286] In some embodiments, but not limited to, low-density undissolved components in the third composition may be removed before step (c) by using methods such as centrifugal density-based separation (density gradient centrifugation) carried out via rotary centrifugation.

[0287] In another embodiment, by using centrifugal density-based separation, low-density gases and undissolved components in the third composition can be brought to the surface of the third composition and then mechanically removed.

[0288] In some preferred embodiments, the fourth composition is mixed.

[0289] In some preferred embodiments, the fourth composition is incubated and mixed for 20 minutes.

[0290] In some preferred embodiments, the fourth composition is degassed.

[0291] In some preferred embodiments, one or more lipids are added to the third composition to act as void-containing elements. In a subsequent aging process, the third composition is mixed at 400 rpm at room temperature for 20 minutes in an overhead mixer equipped with helical blades to form a fourth composition. The fourth composition is then degassed by centrifugation.

[0292] In some preferred embodiments, one or more lipids are added to the third composition to act as void-containing elements. In a subsequent aging process, the third composition is mixed at 1000 rpm at room temperature for 20 minutes in an overhead mixer equipped with helical blades to form a fourth composition. The fourth composition is then degassed by centrifugation.

[0293] In some preferred embodiments, one or more lipids are added to the third composition to act as void-containing elements. In a subsequent aging process, the third composition is mixed at 2000 rpm at room temperature for 20 minutes in an overhead mixer equipped with helical blades to form a fourth composition. The fourth composition is then degassed by centrifugation.

[0294] In some embodiments, a fourth composition of any embodiment of the present disclosure can be used in the production of Prokitein and / or hollow fibers of the present disclosure.

[0295] 1.8 Step (C) Extrusion / Spinning Step (c) includes extruding or spinning the fourth composition together with the bore solution through a plurality of coaxial orifices to produce a plurality of hollow fibers.

[0296] In this specification, bore solution is defined as a solution that fills the lumen of an extruded hollow fiber.

[0297] Bore solutions may include aqueous solutions.

[0298] A die, sometimes called a spinneret, is used interchangeably herein and comprises one or more concentric cylinders supplied by a corresponding number of inlet flows.

[0299] The dice can be in the form of a spinneret or multiple coaxial orifices.

[0300] Extrusion may be used to create extruded tubular cylindrical products, which can then be processed to extrude a fourth composition together with a bore solution through multiple coaxial orifices (e.g., dies) of a desired cross-section to produce hollow fibers with a specific cross-sectional profile.

[0301] As used herein, the bore solution is a solution that is extruded into the cavity of the polymer solution, thereby filling and forming the lumen of the hollow fiber of the present disclosure.

[0302] The bore solution may be extruded together with the fourth composition to maintain the channel(s) within the hollow fiber.

[0303] In some embodiments, the bore solution is extruded together with the fourth composition to maintain the channel(s) within the hollow fiber.

[0304] Similar to coagulation bath solutions, bore solutions can be formulated to promote covalent crosslinking-mediated gelation of polycarboxylic acid-derived esters, thioesters, or amides, precipitation of covalently crosslinked hollow fibers, and the formation of extruded or spun hollow fibers containing covalently crosslinked polypeptides.

[0305] In some embodiments, the bore solution is a solution that fills the lumen of a hollow fiber.

[0306] In some further embodiments, the bore solution is a solution that fills the lumen of the hollow fiber and is formulated to promote covalent crosslinking-mediated gelation of polycarboxylic acid-derived esters, thioesters, or amides, precipitation of covalently crosslinked hollow fibers, and to promote the formation of extruded or spun hollow fibers containing covalently crosslinked polypeptides.

[0307] In some embodiments, the bore solution has the same composition as the coagulation bath solution.

[0308] In some embodiments, the bore solution is drawn from the coagulation bath solution and has the same composition as the coagulation bath solution.

[0309] In some other embodiments, the bore solution does not have the same composition as the coagulation bath solution.

[0310] In some other embodiments, the bore solution includes thickeners and / or viscosity modifiers to facilitate the maintenance of channels in the hollow fibers.

[0311] In some embodiments, the thickeners and / or viscosity modifiers in the bore solution are polysaccharides, including chitin, chitosan, starches derived from wheat, rice, potato or maize, alginates, agar, hyaluronic acid, dextran, chondroitin sulfate, carrageenan, carrageenan-kappa, carrageenan-iota, pullulan, xanthan gum, gellan gum, and / or pectin.

[0312] In some embodiments, the bore solution is removed after step (d).

[0313] In some embodiments, step (c) includes extruding or spinning the fourth composition together with the bore solution through a die containing a plurality of coaxial orifices to produce a plurality of hollow fibers.

[0314] The hollow fibers may be in the shape of a tube and / or cylinder.

[0315] In some embodiments, the hollow fibers are tubular and / or cylindrical in shape.

[0316] In some embodiments, the dimensions of the die are selected to produce hollow fibers having the dimensions specified herein.

[0317] In some embodiments, the fourth composition is extruded through a die at a rate of 0.1 mL / hour to 10 mL / hour, more preferably 0.5 mL / hour to 5 mL / hour, and most preferably 1 mL / hour.

[0318] In some embodiments, the fourth composition is extruded through a die at speeds ranging from 0.1 mL / hour to 0.5 mL / hour, 0.5 mL / hour to 1 mL / hour, 1 mL / hour to 5 mL / hour, or 5 mL / hour to 10 mL / hour.

[0319] In some embodiments, the fourth composition is extruded through a die at a rate of 0.1 mL / hour to 100 mL / hour, preferably 5 mL / hour to 75 mL / hour, more preferably 10 mL / hour to 50 mL / hour, and most preferably 12 mL / hour.

[0320] In some embodiments, the fourth composition is extruded through a die at speeds ranging from 0.1 mL / hour to 10 mL / hour, 10 mL / hour to 20 mL / hour, 20 mL / hour to 50 mL / hour, 50 mL / hour to 75 mL / hour, or from 75 mL / hour to 100 mL / hour.

[0321] In some embodiments, the bore solution is extruded through the die at a rate of 0.001 mL / hour to 10 mL / hour, more preferably 0.05 mL / hour to 5 mL / hour, and most preferably 1 mL / hour.

[0322] In some embodiments, the bore solution is extruded through the die at a rate of 10 mL / hour to 30 mL / hour, more preferably 10 mL / hour to 20 mL / hour, and most preferably 12 mL / hour.

[0323] In some embodiments, the bore solution is extruded through the die at speeds ranging from 0.001 mL / hour to 0.05 mL / hour, 0.05 mL / hour to 0.1 mL / hour, 0.1 mL / hour to 0.5 mL / hour, 0.5 mL / hour to 1 mL / hour, 1 mL / hour to 5 mL / hour, or 5 mL / hour to 10 mL / hour.

[0324] In some embodiments, the bore solution is extruded through the die at speeds ranging from 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.

[0325] In some embodiments, the multiple coaxial orifices include 2 to 4 orifices, preferably 2 to 3 orifices, and most preferably 2 orifices.

[0326] In some embodiments, the multiple coaxial orifices include two orifices, three orifices, or four orifices.

[0327] Each of the multiple coaxial orifices may have a profile that is circular, square, triangular, pentagonal, hexagonal, and / or other polygonal in shape.

[0328] In some embodiments, each of the multiple coaxial orifices has a profile that is circular, square, triangular, pentagonal, hexagonal, and / or other polygonal in shape.

[0329] Depending on the composition of the spinning solution forming the fourth composition, dry or wet spinning systems may also be used.

[0330] Spinning, wet spinning, and / or extrusion are used interchangeably herein and refer to the production of hollow fibers by a process in which a polymer solution can be passed through a die into a solidification bath together with a bore solution which may contain a solidification bath solution, thereby solidifying the polymer solution into hollow fibers. This process may also be called “wet” spinning because the polymer solution is extruded into a liquid bath. Thus, the terms spinning, wet spinning, and / or extrusion refer to a process including (i) preparation of the polymer solution, (ii) assembly of the die / spinneret, (iii) extrusion of the polymer solution with the bore solution, (iv) solidification of the extruded polymer solution, (v) drawing out to stretch the extruded and solidified hollow fibers, (vi) washing to remove residual solvent, and (vii) drying, which involves drying the resulting hollow fibers and winding them onto a spool for storage and future use.

[0331] In some embodiments, the fourth composition is extruded or spun together with the bore solution into an air and / or gaseous atmosphere to produce hollow fibers. The extruded or spun hollow fibers are then sent into a coagulation bath or into an atmosphere that coagulates, precipitates, or dries the hollow fibers.

[0332] In some embodiments, the fourth composition is extruded or spun together with the bore solution in an atmosphere, and then in a coagulation bath.

[0333] In one preferred embodiment, the fourth composition, together with the bore solution, is extruded directly into the solidification bath through a die.

[0334] In some embodiments, step (c) includes extruding or spinning the fourth composition together with a bore solution containing a coagulation bath solution through a plurality of coaxial orifices to directly form one or more hollow fibers having a tubular cylindrical geometry in the coagulation bath.

[0335] In some embodiments, the fourth composition is extruded through the outer orifice of a die having two concentric cylindrical orifices, and the bore solution is extruded through the inner orifice to form hollow fibers having a tubular cylindrical geometric structure.

[0336] In some other embodiments, both the bore solution and the coagulation bath solution compositions include one or more crosslinking reagents, such as sodium citrate, and one or more salts, such as sodium hypophosphite.

[0337] In some further embodiments, both the bore solution and the coagulation bath solution compositions include one or more crosslinking agents, such as sodium malate, and one or more salts, such as sodium hypophosphite.

[0338] In some further embodiments, both the bore solution and the coagulation bath solution compositions include one or more crosslinking reagents, such as sodium citrate.

[0339] In some and further embodiments, both the bore solution and the coagulation bath solution compositions include one or more crosslinking reagents, such as sodium malate.

[0340] In some embodiments, the pH of both the bore solution and the coagulation bath solution is in the range of 7.01 to 9.99, preferably in the range of 8.0 to 9.0, and most preferably in the range of 8.0 to 8.5.

[0341] In some other embodiments, the pH of both the bore solution and the coagulation bath solution is in the range of 7.01–8.0, 8.0–9.0, or 9.0–9.99.

[0342] In some embodiments, step (c) includes extruding the fourth composition together with a bore solution having the same components as the coagulation bath solution through a coaxial orifice comprising two orifices of a circular profile. The fourth composition is extruded through the outer orifice, and the bore solution is extruded through the inner orifice. Both the fourth composition and the bore solution are extruded directly into a coagulation bath filled with the coagulation bath solution. Both the coagulation bath solution and the bore solution contain sodium citrate and sodium hypophosphite in aqueous solutions with a pH of 8 to 8.5.

[0343] In some embodiments, step (c) includes extruding the fourth composition together with a bore solution having the same components as the coagulation bath solution through a coaxial orifice comprising two orifices of a circular profile. The fourth composition is extruded through the outer orifice, and the bore solution is extruded through the inner orifice. Both the fourth composition and the bore solution are extruded directly into a coagulation bath filled with the coagulation bath solution. Both the coagulation bath solution and the bore solution contain sodium malate in an aqueous solution with a pH of 8 to 8.5.

[0344] 1.9 Step (D) Solidification and Crosslinking Step (d) involves treating the plurality of hollow fibers produced in step (c) with a polycarboxylic acid crosslinking reagent capable of forming covalent crosslinks between polypeptides and / or within polypeptides between polycarboxylic acid-derived esters, thioesters, or amides, in at least one fraction of the polypeptide containing the hollow fibers, to produce a plurality of covalently crosslinked semipermeable porous hollow fibers.

[0345] In some embodiments, step (d) includes treating a plurality of hollow fibers with a polycarboxylic acid crosslinking reagent to form covalent crosslinks between and / or within the polypeptides of polycarboxylic acid-derived esters, thioesters, or amides in at least one fraction of the polypeptide containing the hollow fibers, thereby producing a plurality of covalently crosslinked semipermeable porous hollow fibers.

[0346] Step (d) may be carried out in a coagulation bath containing a coagulation bath solution as defined herein.

[0347] In some embodiments, step (d) is carried out in a coagulation bath containing a coagulation bath solution.

[0348] Step (d) may be carried out using multiple and / or consecutive coagulation baths containing coagulation bath solutions of the same and / or different compositions.

[0349] In some embodiments, step (d) is carried out using multiple and / or consecutive coagulation baths containing coagulation bath solutions of the same and / or different compositions.

[0350] During precipitation and solidification, thermodynamically stable solutions of polypeptides can transition from a liquid state to a solid state in a controlled manner.

[0351] A mixture can be formed that begins to separate into different phases by immersing a polypeptide-rich solution (non-solvent), such as the fourth composition extruded or spun from step (c), in a polypeptide-poor solution (solvent), such as a coagulation bath solution. The polypeptide-rich phase may begin to solidify through processes such as gelation, crosslinking, vitrification, and / or crystallization. Separation may involve several methods, including (i) immersion precipitation, where the precipitation of polypeptides may result from liquid-liquid interactions between the solvent and the non-solvent; (ii) controlled evaporation of the solvent, potentially for the isolation of polypeptides; and (iii) freezing, which can be introduced during liquid-liquid transfer between the solvent and the non-solvent, by applying either a crosslinking agent and optionally a catalyst, or by altering the isoelectric point of a salt (16), or by salting out with Hofmeister series salts (17), thereby enabling bond formation between polypeptide molecules.

[0352] Covalent crosslinking of polypeptides with esters, thioesters, or amide bonds derived from polycarboxylic acids can lead to the formation of larger polypeptide aggregates. When larger polypeptide aggregates are formed in extruded or spun hollow fibers, both the water solubility and elasticity of the hollow fibers may decrease, while both the material stiffness and tensile strength may improve (9). Thus, the structure and structural integrity of the hollow fibers may be modified in step (d) to potentially enable the long-term use of the hollow fibers as components in bioreactors, as may be required for culturing cells to produce cultured meat.

[0353] Precipitation of the solid phase in a solidification bath can form porous structures within the solid phase. The main parameters that can determine the porosity of the precipitate are the concentration of the polymer in the polymer-rich phase (i.e., the concentration of the polypeptide in the fourth composition) and the rate at which the precipitate is formed. In a process that may be called non-solvent precipitation, spinodal precipitation can occur by rapidly demixing the binary solvent and solute system under preferred thermodynamic conditions upon the addition of a miscible tertiary non-solvent solution, which may form pores. Typically, rapid demixing and precipitation of the solute into the solid phase results in more porous precipitates. Such occurrences may be characterized by a rapid color change or onset of turbidity in the solute-rich phase (16)(18).

[0354] Furthermore, in a process sometimes called “pore template formation,” void-containing elements may be added to the polymer solution before extrusion and subsequently removed from the precipitate to potentially increase the void fraction of the precipitate (19). When the void-containing elements are lipids such as sunflower oil and are used with the polymer solution to form an oil-in-water emulsion to create a controlled porous structure, this process is sometimes called “emulsion template formation.”

[0355] Covalent crosslinking is carried out by the reaction of polycarboxylic acids with reactive groups on the polypeptide skeleton, including amine (-NH2) groups, hydroxide (-OH) groups, carboxyl (-COOH) groups, and thiol (-SH) groups, potentially forming covalent crosslinks of esters, thioesters, or amides.

[0356] Covalent crosslinking is carried out by the reaction of polycarboxylic acids with reactive groups of polypeptides and polysaccharides, including amine (-NH2) groups, hydroxide (-OH) groups, carboxyl (-COOH) groups, and thiol (-SH) groups, potentially forming covalent crosslinks of polypeptide-polypeptide, polypeptide-polysaccharide, polysaccharide-polysaccharide ester, thioester, and / or amide.

[0357] As used herein, the term “covalent ester, thioester, and / or amide-crosslinked polypeptide” means a polypeptide having at least one ester, thioester, and / or amide crosslink. Generally, any such ester, thioester, and / or amide crosslink is understood to be derived from a polycarboxylic acid. Therefore, the term “polycarboxylic acid-derived, covalent ester, thioester, and / or amide-crosslinked polypeptide” encompasses any covalent ester, thioester, and / or amide-crosslinked polypeptide without the need to demonstrate that it is actually derived from a polycarboxylic acid. Thus, the terms “covalent ester, thioester, and / or amide-crosslinked polypeptide” and “polycarboxylic acid-derived, covalent ester, thioester, and / or amide-crosslinked polypeptide” are used interchangeably herein.

[0358] In some preferred embodiments, the covalent crosslinking of polypeptides in the hollow fibers produced in step (c) is carried out via nonspecific esterification, thioesterification, or amidation reactions, with the use of polycarboxylate salts as non-irritating chemical crosslinking agents.

[0359] In some other embodiments, the covalent crosslinking of polypeptides and polysaccharides in the hollow fibers produced in step (c) is carried out via nonspecific esterification, thioesterification, or amidation reactions, with the use of polycarboxylate salts as chemical crosslinking agents.

[0360] In some embodiments, the term “non-irritating chemical” refers to a chemical designated as GRAS (Generally Recognized as Safe).

[0361] In some other embodiments, the covalent crosslinking of polypeptides and polysaccharides in the hollow fibers produced in step (c) is carried out via nonspecific esterification, thioesterification, or amidation reactions, with the use of polycarboxylate salts as non-irritating chemical crosslinking agents.

[0362] In some embodiments, the polycarboxylate salt used to form covalent crosslinks in the hollow fibers produced in step (c) is not limited to sodium oxalate, potassium oxalate, sodium malate, potassium malate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, sodium tartrate, potassium tartrate, potassium citrate, sodium malonate, potassium malonate, and / or combinations thereof, and preferably sodium citrate.

[0363] In some other embodiments, covalent crosslinking with polycarboxylates having multiple carboxyl groups is carried out under alkaline conditions, resulting in the formation of nonspecific ester, thioester, or amide bond covalent crosslinks (20).

[0364] The use of alkaline conditions may enable covalent crosslinking of polypeptides at low temperatures, thereby improving the energy efficiency of the production process and potentially negating the yellowing of the material that may occur when operated at higher temperatures (20).

[0365] In some further embodiments, polycarboxylate salts having multiple carboxyl groups form crosslinks in a solution containing other salts, but not limited to sodium hypophosphite (22), where sodium hypophosphite acts as a catalyst or is incorporated into the crosslinks (22; 23).

[0366] One or more polycarboxylate crosslinking agents may be used.

[0367] In some embodiments, a single polycarboxylate crosslinking agent is used.

[0368] In other embodiments, one or more polycarboxylate crosslinking agents are used.

[0369] In further embodiments, multiple polycarboxylate crosslinking agents are used in combination.

[0370] In some embodiments, the concentration of each polycarboxylate crosslinking agent in the coagulation bath solution is in the range of 0.01%(w / v) to 50%(w / v), preferably in the range of 0.05%(w / v) to 35%(w / v), more preferably in the range of 0.1%(w / v) to 30%(w / v), even more preferably in the range of 1%(w / v) to 25%(w / v), and most preferably in the range of 5%(w / v) to 15%(w / v), relative to the volume of the solution.

[0371] In some other embodiments, the concentration of each polycarboxylate crosslinking agent in the coagulation bath solution is within the range of 0.01%(w / v)~1%(w / v), 1%(w / v)~5%(w / v), 5%(w / v)~10%(w / v), 10%(w / v)~15%(w / v), 15%(w / v)~20%(w / v), 20%(w / v)~25%(w / v), 25%(w / v)~30%(w / v), 30%(w / v)~35%(w / v), 35%(w / v)~40%(w / v), 40%(w / v)~45%(w / v), or 45%(w / v)~50%(w / v) relative to the volume of the solution.

[0372] In some embodiments, the temperature used in the covalent crosslinking step is either room temperature, or a temperature higher or lower than room temperature, preferably in the range of 5°C to 95°C, more preferably in the range of 10°C to 75°C, even more preferably in the range of 15°C to 65°C, and even more preferably in the range of 20°C to 60°C, and most preferably in the range of 30°C to 55°C.

[0373] In some other embodiments, the temperature used in the covalent crosslinking step 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.

[0374] In some embodiments, the pressure used in the covalent bridging step may be atmospheric pressure, or a pressure 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.

[0375] In some other embodiments, the pressure used in the covalent bridging step 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.

[0376] In some embodiments, the pH used in the covalent crosslinking step may be in the range of 7.01 to 9.99, preferably in the range of 8.0 to 9.0, and most preferably in the range of 8.0 to 8.5.

[0377] In some other embodiments, the pH used in the covalent crosslinking step is within the ranges of 7.01–7.5, 7.5–8.0, 8.0–8.5, 8.5–9.0, 9.0–9.5, and 9.5–9.99.

[0378] In some other embodiments, the duration of the covalent crosslinking step 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.

[0379] In some other embodiments, the duration of the covalent crosslinking step is within the ranges of 30 to 60 seconds, 1 to 30 minutes, 30 to 60 minutes, 1 to 2 hours, 2 to 3 hours, 3 to 4 hours, 4 to 5 hours, and 5 to 6 hours.

[0380] In some other embodiments, the duration of the covalent crosslinking step is within the range of 6 hours and 7 days, preferably within the range of 6 hours and 72 hours, and more preferably within the range of 12 hours and 24 hours.

[0381] In some other embodiments, the duration of the covalent crosslinking step 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, or 96 hours and / or 7 days.

[0382] coagulation bath The coagulation bath solution in step (d) may contain at least one polycarboxylate crosslinking agent dissolved in an aqueous solvent or a mixture of multiple solvents.

[0383] In some embodiments, the coagulation bath solution of step (d) comprises at least one polycarboxylate crosslinking agent dissolved in an aqueous solvent or a mixture of multiple solvents.

[0384] In some embodiments, the coagulation bath solution in step (d) contains an aqueous solvent such as water.

[0385] In some embodiments, the coagulation bath solution of step (d) further comprises at least one alkali, acid, alcohol, catalyst, organic solvent, salt, and / or any combination thereof.

[0386] In some embodiments, the coagulation bath solution of step (d) contains one or more polycarboxylate crosslinking agents, 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.

[0387] In some embodiments, the concentration of each polycarboxylate crosslinking agent in the coagulation bath solution of step (d) is in the range of 0.1% (w / v) to 40% (w / v), preferably in the range of 10% (w / v) to 30% (w / v), more preferably in the range of 20% (w / v) to 28% (w / v), and most preferably 25% (w / v), relative to the volume of the solution.

[0388] In some other embodiments, the concentration of each polycarboxylate crosslinking agent in the coagulation bath solution of step (d) is in the range of 0.1%(w / v) to 1%(w / v), 1%(w / v) to 5%(w / v), 5%(w / v) to 10%(w / v), 10%(w / v) to 20%(w / v), and / or %(w / v) to 40%(w / v) relative to the volume of the solution.

[0389] In some embodiments, the coagulation bath solution in step (d) contains a catalyst comprising zinc sulfate, sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium hypophosphite, and / or ammonium sulfate.

[0390] In some other embodiments, the concentration of each catalyst in the coagulation bath solution in step (d) is in the range of 0.1%(w / v) to 40%(w / v), preferably in the range of 5%(w / v) to 15%(w / v), more preferably in the range of 10%(w / v) to 14%(w / v), and most preferably 12.5%(w / v) relative to the volume of the solution.

[0391] In some embodiments, the concentration of each catalyst in the coagulation bath solution of step (d) is within the range of 0.1%(w / v) to 1%(w / v), 1%(w / v) to 5%(w / v), 5%(w / v) to 10%(w / v), 10%(w / v) to 20%(w / v), and / or %(w / v) to 40%(w / v) relative to the volume of the solution.

[0392] In some embodiments, the coagulation bath solution in step (d) contains an alkali, but is not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide and / or sodium carbonate.

[0393] In some embodiments, the coagulation bath solution in step (d) contains an alkali comprising, but not limited to, ammonium hydroxide, potassium carbonate, and / or sodium bicarbonate.

[0394] In some embodiments, the coagulation bath solution of step (d) contains, but is not limited to, an acid including ascorbic acid, acetic acid, adipic acid, citric acid, formic acid, hydrochloric acid, lactic acid, malic acid, malonic acid, oxalic acid, succinic acid, sulfuric acid, and / or tartaric acid.

[0395] In some embodiments, the pH of the coagulation bath solution in step (d) is in the range of 7.01 to 9.99, preferably in the range of 8.0 to 9.0, and most preferably in the range of 8.0 to 8.5.

[0396] In some other embodiments, the pH of the coagulation bath solution in step (d) is in the range of 7.01-7.5, 7.5-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, and 9.5-9.99.

[0397] In some embodiments, the coagulation bath solution of step (d) contains one or more alcohols, but is not limited to methanol, ethanol, propanol, isopropanol, butanol, and / or isobutanol.

[0398] In some other embodiments, the coagulation bath solution of step (d) contains alcohol having a concentration of 0.5% to 5% (v / v), 5% to 10% (v / v), 10% to 30% (v / v), 20% to 30% (v / v), 30% to 40% (v / v), 40% to 50% (v / v), 50% to 60% (v / v), 60% to 70% (v / v), 70% to 80% (v / v), 80% to 90% (v / v), or 90% to 99.99% (v / v) relative to the total volume of the coagulation bath solution.

[0399] In some embodiments, the coagulation bath solution of step (d) contains, but is not limited to, one or more salts comprising ammonium sulfate, disodium hydrogen phosphate, dipotassium phosphate, lithium sulfate, monosodium phosphate, potassium carbonate, potassium chloride, potassium sulfate, sodium bicarbonate, sodium bisulfate, sodium carbonate, sodium chloride, sodium hypophosphite, sodium malonate, sodium maleate, sodium pyrosulfite, sodium sulfate, sodium sulfite, trilithium citrate, triammonium citrate, trisodium citrate, tripotassium citrate, zinc chloride, and / or zinc sulfate.

[0400] In some embodiments, the coagulation bath solution of step (d) contains one or more salts, but is not limited to, disodium malate.

[0401] In this specification, sodium malate and disodium malate are used interchangeably.

[0402] In some other embodiments, the concentration of each salt in the coagulation bath solution in step (d) is in the range of 0.01%(w / v) to 40%(w / v), preferably in the range of 0.1%(w / v) to 10%(w / v), more preferably in the range of 0.5%(w / v) to 5%(w / v), and most preferably 1%(w / v), relative to the volume of the solution.

[0403] In some other embodiments, the concentration of each salt in the coagulation bath solution of step (d) is within the range of 0.01%(w / v) to 0.1%(w / v), 0.1%(w / v) to 1%(w / v), 1%(w / v) to 5%(w / v), 5%(w / v) to 10%(w / v), 10%(w / v) to 20%(w / v), and / or %(w / v) to 40%(w / v) relative to the volume of the solution.

[0404] In some embodiments, the temperature of the coagulation bath solution in step (d) is either room temperature, or a temperature higher or lower than room temperature, preferably in the range of 5°C to 95°C, more preferably in the range of 10°C to 75°C, even more preferably in the range of 15°C to 65°C, and even more preferably in the range of 20°C to 60°C, and most preferably in the range of 20°C to 35°C.

[0405] In some other embodiments, the temperature of the coagulation bath solution in step (d) is in 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.

[0406] coagulation bath solution In some preferred embodiments, the coagulation bath solution of step (d) comprises water, which also contains 25% (w / v) sodium citrate and 12.5% ​​(w / v) sodium hypophosphite relative to the volume of the coagulation bath solution, and has a pH in the range of 7.01 to 9.99 at room temperature.

[0407] In another preferred embodiment, the coagulation bath solution in step (d) comprises water, which also contains sodium malate, and has a pH in the range of 7.01 to 9.99 at room temperature.

[0408] In some preferred embodiments, the coagulation bath solution of step (d) does not contain more than 5% (w / v) polypeptide before immersion of the extruded or spun fibers.

[0409] In other preferred embodiments, the coagulation bath solution of step (d) does not contain more than 1% (w / v) polypeptide before immersion of the extruded or spun hollow fibers.

[0410] In the case of wet spinning, a suitable coagulation bath solution may comprise one or more solvents along with one or more polycarboxylate crosslinking agents in the mixture, which may also comprise a mixture of catalysts and / or salts, such as sodium hypophosphite. The coagulation bath solution may be formulated to promote covalent crosslinking-mediated gelation of esters, thioesters, or amides derived from polycarboxylic acids, and the precipitation of covalently crosslinked hollow fibers. As a result, the formation of extruded or spun hollow fibers containing covalently crosslinked polypeptides can be promoted.

[0411] In some embodiments, the polypeptide fraction in the covalently crosslinked extruded or spun hollow fiber 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%.

[0412] In some embodiments, the fraction of polypeptides in covalently crosslinked extruded or spun hollow fibers is in the range of 10%-30%, 20%-30%, 30%-40%, 40%-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90%-99.99%.

[0413] The degree to which crosslinking occurs in the polypeptide of extruded or spun hollow fibers can be readily determined by those skilled in the art by methods including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry, Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy.

[0414] To evaluate the degree of covalent crosslinking by SDS-PAGE, a covalently crosslinked sample is passed through an SDS-PAGE gel in parallel with channels containing an uncrosslinked sample and a protein of known mass. The theoretical masses of the expression bands in the crosslinked and uncrosslinked sample channels may be calculated by comparing the distance of the bands along the channel with the distance of the bands in the channel containing the protein of known mass. The degree of crosslinking associated with each band may also be calculated by evaluating the relative masses of the bands in the crosslinked and uncrosslinked sample channels.

[0415] Structural changes associated with crosslinking may be evaluated by changes in band intensity related to various functional groups, such as those determined by FTIR or Raman spectroscopy.

[0416] In some preferred embodiments, step (d) includes treating the hollow fiber polypeptide produced in step (c) in a coagulation bath filled with an aqueous coagulation bath solution containing sodium citrate at pH 8.0 to 8.5, room temperature, and a pressure of 100825 Pa to 101825 Pa for 1 hour.

[0417] In some other preferred embodiments, step (d) includes treating the hollow fiber polypeptide produced in step (c) in a coagulation bath filled with an aqueous coagulation bath solution containing sodium succinate and sodium hypophosphite for 1 hour and 30 minutes at pH 8.0 to 8.5, room temperature, and a pressure of 100825 Pa to 101825 Pa.

[0418] In some more preferred embodiments, step (d) includes treating the hollow fiber polypeptide produced in step (c) in a coagulation bath filled with an aqueous coagulation bath solution containing sodium malate and sodium hypophosphite for 30 minutes at pH 8.0 to 8.5, room temperature, and a pressure of 100825 Pa to 101825 Pa.

[0419] In some more preferred embodiments, step (d) includes treating the hollow fiber polypeptide produced in step (c) in a coagulation bath filled with an aqueous coagulation bath solution containing sodium malate, at a pH of 8.0 to 8.5, room temperature, and a pressure of 100825 Pa to 101825 Pa for 30 minutes.

[0420] In some particularly preferred embodiments, step (d) includes treating the hollow fiber polypeptide produced in step (c) in a coagulation bath filled with an aqueous coagulation bath solution containing sodium citrate and sodium hypophosphite for 1 hour at pH 8.0 to 8.5, room temperature, and a pressure of 100825 Pa to 101825 Pa.

[0421] In some embodiments, the coagulation bath of any embodiment of the present disclosure can be used for the production of Prokitein and / or hollow fibers of the present disclosure.

[0422] In some embodiments, the polycarboxylic acid crosslinking agents of any embodiment of the present disclosure can be used in the production of Prokitein and / or hollow fibers of the present disclosure.

[0423] In some embodiments, the covalently crosslinked polypeptides of any embodiment of the present disclosure can be used in the production of Prokitein and / or hollow fibers of the present disclosure.

[0424] 1.10 Step (E) Post-generation modification Step (e) may include treating the covalently crosslinked semipermeable porous hollow fibers generated in step (d) with at least one post-production modification process.

[0425] In some embodiments, step (e) includes treating the covalently crosslinked semipermeable porous hollow fibers produced in step (d) with at least one post-production modification process.

[0426] Covalently crosslinked semipermeable porous hollow fibers can be further processed, either as part of their creation or as a post-creation modification, to potentially enhance one or more of the mechanical properties, fluid transport, permeability, and water stability of the hollow fibers.

[0427] In some embodiments, covalently crosslinked semipermeable porous hollow fibers are further processed, either as part of the production process or as a post-production modification, to enhance one or more of the mechanical properties, fluid transport, permeability, and water stability of the hollow fibers.

[0428] 1.11 Step (Ei) Modification of the secondary structure of polymers Step (ei) may include treating covalently crosslinked semipermeable porous hollow fibers with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptide.

[0429] In some embodiments, step (ei) includes treating covalently crosslinked semipermeable porous hollow fibers with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptide.

[0430] In particular, covalently crosslinked semipermeable porous hollow fibers can be treated with organic solvents to dehydrate them and modify the secondary polymer structure of their constituent polypeptides.

[0431] In some embodiments, covalently crosslinked semipermeable porous hollow fibers are treated with an organic solvent to dehydrate them and modify the secondary polymer structure of the constituent polypeptides.

[0432] The objective of step (ei) is to potentially modify the secondary polymer structure of polypeptides in covalently crosslinked semipermeable porous hollow fibers in order to increase 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 increased water stability of the hollow fibers, and as a result, the mechanical properties of the hollow fibers may remain relatively unchanged after continuous immersion in aqueous solution for more than three days (24)(25)(27;28).

[0433] In some embodiments, modification of the secondary polymer structure of the polypeptide of covalently crosslinked semipermeable porous hollow fibers is achieved by organic solvents, as well as by either solution washing and / or vapor exposure.

[0434] In some embodiments, modification of the secondary polymer structure of a covalently crosslinked semipermeable porous hollow fiber polypeptide is achieved in aqueous solution using an organic solvent.

[0435] In some embodiments, modification of the secondary polymer structure of a polypeptide of a covalently crosslinked semipermeable porous hollow fiber is achieved by supercritical CO2.

[0436] In some embodiments, covalently crosslinked semipermeable porous hollow fibers are treated with one or more organic solvents, but not limited to methanol, ethanol, propanol, isopropanol, butanol, isobutanol, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, and / or mixtures thereof.

[0437] In some embodiments, covalently crosslinked semipermeable porous hollow fibers are treated with one or more organic solvents, including acetone.

[0438] In some embodiments, covalently crosslinked semipermeable porous hollow fibers are treated with one or more polyols and / or polymers of polyols.

[0439] In some embodiments, modification of the secondary polymer structure of a covalently crosslinked, semipermeable porous hollow fiber polypeptide is achieved in aqueous solution by polyols and / or polymers of polyols.

[0440] In some embodiments, modification of the secondary polymer structure of a covalently crosslinked semipermeable porous hollow fiber polypeptide is achieved by a solution comprising water, an organic solvent, polyols and / or polyol polymers.

[0441] In some embodiments, covalently crosslinked semipermeable porous hollow fibers are treated with one or more fluids, but not limited to supercritical CO2.

[0442] In this specification, supercritical CO2 is considered to behave similarly to organic solvents once it exceeds the critical point of CO2, and is referred to in this manner.

[0443] Similarly, the term "organic solvent" is thought to include supercritical CO2.

[0444] In some embodiments, the concentration of any single component in the solvent mixture used for forming beta-sheets in covalently crosslinked semipermeable porous hollow fibers is in the range of 0%(w / v) to 100%(w / v), preferably in the range of 10%(w / v) to 75%(w / v), and most preferably in the range of 20%(w / v) to 60%(w / v), relative to the total volume of the solvent mixture.

[0445] In some other embodiments, the concentration of any single component in the solvent mixture used in the formation of beta-sheets in covalently crosslinked semipermeable porous hollow fibers is 0%(w / v)~5%(w / v), 5%(w / v)~10%(w / v), 10%(w / v)~15%(w / v), 15%(w / v)~20%(w / v), 20%(w / v)~25%(w / v), 25%(w / v)~30%(w / v), 30%(w / v)~35%(w / v), 35%(w / v)~40%(w / v) relative to the total volume of the solvent mixture. ), 40%(w / v)~45%(w / v), 45%(w / v)~50%(w / v), 50%(w / v)~55%(w / v), 55%(w / v)~60%(w / v), 60%(w / v)~65%(w / v), 65%(w / v)~70%(w / v), 70%( w / v)~75%(w / v), 75%(w / v)~80%(w / v), 80%(w / v)~85%(w / v), 85%(w / v)~90%(w / v), 90%(w / v)~95%(w / v), or 95%(w / v)~100%(w / v).

[0446] In some embodiments, the treatment of covalently crosslinked semipermeable porous hollow fibers with one or more organic solvents is carried out at a temperature in the range of 0°C to 90°C, preferably in the range of 10°C to 80°C, more preferably in the range of 20°C to 70°C, and even more preferably in the range of 30°C to 60°C, most preferably at room temperature.

[0447] In some other embodiments, the treatment of covalently crosslinked semipermeable porous hollow fibers with one or more organic solvents is carried out at temperatures 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.

[0448] In some other embodiments, the treatment of covalently crosslinked semipermeable porous hollow fibers with one or more solvents is carried out at a temperature in the range of 15 to 300°C, preferably in the range of 100°C to 250°C, more preferably in the range of 130°C to 200°C, even more preferably in the range of 150°C to 180°C, and most preferably in the range of 175°C.

[0449] In some other embodiments, the treatment of covalently crosslinked semipermeable porous hollow fibers with one or more solvents is carried out over a range of temperatures.

[0450] In some embodiments, the treatment of covalently crosslinked semipermeable porous hollow fibers with one or more organic solvents is carried out at atmospheric pressure, or at a pressure 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.

[0451] In some other embodiments, the pressure used in the organic solvent washing step 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.

[0452] In some other embodiments, the treatment of covalently crosslinked semipermeable porous hollow fibers with one or more solvents is carried out at a pressure in 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.

[0453] In some embodiments, the treatment of covalently crosslinked semipermeable porous hollow fibers with one or more solvents is carried out in a pressure vessel.

[0454] In some embodiments, the treatment of covalently crosslinked semipermeable porous hollow fibers with one or more organic solvents is carried out over a period of 1 second to 10 days, preferably within the range of 10 minutes and 3 hours, more preferably within the range of 40 and 90 minutes, and most preferably over a period of 1 hour.

[0455] In some other embodiments, the treatment of covalently crosslinked semipermeable porous hollow fibers with one or more organic solvents is performed for 10 minutes and 30 minutes, 30 minutes and 45 minutes, 45 minutes and 1 hour, 1 hour and 2 hours, 2 hours and 3 hours, 3 hours and 4 hours, 4 hours and 5 hours, 5 hours and 6 hours, 6 hours and 7 hours, 7 hours and 8 hours, 8 hours and 9 hours, 9 hours and 10 hours, 10 hours and 11 hours, 11 hours and 12 hours, 12 hours and 13 hours, 13 hours and 14 hours, 14 hours and 15 hours, 15 hours and 16 hours, 16 hours and 17 hours, 17 hours and 18 hours, 18 hours and 19 hours, 19 hours and 20 hours, 20 hours and 21 hours, 21 hours and 22 hours, 22 hours and 23 hours, 23 hours and 24 hours, 24 hours and The work is carried out over a period of time within the range of 25 hours, 25 and 26 hours, 26 and 27 hours, 27 and 28 hours, 28 and 29 hours, 29 and 30 hours, 30 and 31 hours, 31 and 32 hours, 32 and 33 hours, 33 and 34 hours, 34 and 35 hours, 35 and 36 hours, 36 and 37 hours, 37 and 38 hours, 38 and 39 hours, 39 and 40 hours, 40 and 41 hours, 41 and 42 hours, 42 and 43 hours, 43 and 44 hours, 44 and 45 hours, 45 and 46 hours, 46 and 47 hours, 47 and 48 hours, 48 ​​hours and 3 days, 3 days and 4 days, 4 days and 5 days, 5 days and 7 days, or 7 days and 10 days.

[0456] Changes in the secondary polymer structure of polypeptides in covalently crosslinked hollow fibers can be determined by evaluating changes in the intensity of peaks associated with amorphous random coils, alpha-helices, beta-helices, beta-sheets, and disordered structures. This type of analysis can be performed by those skilled in the art using methods including Fourier transform infrared (FTIR) spectroscopy, circular dichroism, wide-angle X-ray scattering (WAXS), and Raman spectroscopy.

[0457] In some particularly preferred embodiments, step (ei) includes treating the covalently crosslinked semipermeable porous hollow fibers generated in step (d) in a liquid solvent bath containing at least 40% ethanol at room temperature and atmospheric pressure for 1 hour.

[0458] In some possible embodiments, the coagulation bath solution in step (d) contains an organic solvent so that step (d) is effectively combined with step (ei). In this embodiment, beta-sheet formation occurs simultaneously with the coagulation, solidification, and crosslinking of the polypeptide.

[0459] In some embodiments, the organic solvent used in any embodiment of the present disclosure can be used in the production of the Prokitein and / or hollow fibers of the present disclosure.

[0460] 1.12 Step (E.ii) Annealing Step (e.ii) may include annealing the covalently crosslinked semipermeable porous hollow fibers.

[0461] The annealing process typically allows for the rearrangement of the crystalline structure into a more stable form, which may be used to reduce the internal stress of covalently crosslinked semipermeable porous hollow fibers. More specifically, the annealing process may allow the beta-sheets formed in step (ei) to become a stable microstructure state of the hollow fibers. As a result, the microstructure of the beta-sheets remains intact during rehydration of the hollow fibers, preventing hydrolysis of the ester crosslinks formed in step (d). In summary, the successive covalent ester crosslinks, beta-sheet formation, and annealing steps can impart long-term water stability to the hollow fibers, enabling their use in long-term cell cultures required for the production of cultured meat products.

[0462] In some embodiments, the annealing process allows the crystalline structure to be rearranged into a more stable form, reducing the internal stress of the covalently crosslinked semipermeable porous hollow fibers. More specifically, the annealing process results in the beta-sheet formed in step (ei) becoming a stable microstructure of the hollow fiber. As a result, the microstructure of the beta-sheet remains intact during rehydration of the hollow fiber, preventing hydrolysis of the ester crosslinks formed in step (d). In summary, the successive covalent ester crosslinking, beta-sheet formation, and annealing steps impart long-term water stability to the hollow fiber, enabling its use in long-term cell cultures required for the production of cultured meat products.

[0463] The treated covalently crosslinked semipermeable porous hollow fibers may be partially or completely dried in a process referred to herein as “pre-drying” prior to annealing.

[0464] In some embodiments, the treated covalently crosslinked semipermeable porous hollow fibers are partially dried before annealing, resulting in some moisture remaining within the microstructure of the hollow fibers.

[0465] In some embodiments, the treated covalently crosslinked semipermeable porous hollow fibers are completely dried before annealing, so that, in theory, all moisture is removed from the microstructure of the hollow fibers.

[0466] In some embodiments, the treated covalently crosslinked semipermeable porous hollow fibers are not dried before annealing.

[0467] In some embodiments, the pre-drying process is carried out at a temperature in the range of 5 to 90°C, preferably in the range of 15 to 80°C, more preferably in the range of 40 to 70°C, and most preferably in the range of 50 to 70°C.

[0468] In some other embodiments, the pre-drying process is carried out at temperatures in the range of 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.

[0469] In some embodiments, the treated covalently crosslinked semipermeable porous hollow fibers are dried by freeze-drying before annealing.

[0470] In some embodiments, the treated covalently crosslinked semipermeable porous hollow fibers are not dried by freeze-drying before annealing.

[0471] In some embodiments, the pre-drying process is carried out at a temperature in the range of 5 to -90°C, preferably in the range of -15°C to -90°C, more preferably in the range of -40°C to -90°C, even more preferably in the range of -60°C to -90°C, and most preferably in the range of -80°C to -86°C.

[0472] In some other embodiments, the pre-drying process is carried out at temperatures in the range of 5°C to 0°C, 0°C to -10°C, -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, -80°C to -86°C, or -80°C to -90°C.

[0473] In some embodiments, the treated covalently crosslinked semipermeable porous hollow fibers are dried under conditions below the triple point of water.

[0474] In some embodiments, the pre-drying process is carried out over a period ranging from 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 2 hours to 1 day.

[0475] In some other embodiments, the pre-drying process is carried out over a period of time ranging from 0 to 5 minutes, 5 to 10 minutes, 10 to 15 minutes, 15 to 20 minutes, 20 to 25 minutes, 25 to 30 minutes, 30 to 35 minutes, 35 to 40 minutes, 40 to 45 minutes, 45 to 50 minutes, 50 to 55 minutes, 55 minutes to 1 hour, 1 to 2 hours, 2 to 3 hours, 3 to 4 hours, 4 to 5 hours, 5 to 6 hours, 6 to 12 hours, 12 to 18 hours, 18 to 24 hours, 24 to 30 hours, 30 to 36 hours, 36 to 42 hours, 42 to 48 hours, 48 ​​to 54 hours, 54 to 60 hours, 60 to 66 hours, 66 hours to 3 days, 3 to 4 days, 4 to 5 days, 5 to 6 days, or 6 to 7 days.

[0476] The pre-drying process may be carried out by convection, infrared heat source, and / or on a heated surface.

[0477] In some embodiments, the pre-drying process is carried out by convection, radiation, electromagnetic waves, infrared heat sources, and / or on a heated surface.

[0478] The annealing step may be carried out in different atmospheres, liquids, and / or mixtures thereof.

[0479] In some embodiments, the annealing step is performed in different atmospheres, liquids, and / or mixtures thereof.

[0480] The atmosphere used in the annealing step may include, but is not limited to, air, carbon dioxide, water vapor, inert gases (such as nitrogen, helium, neon, argon, and xenon), vapors (such as ethanol, vegetable oil, and water), and / or mixtures thereof.

[0481] In some embodiments, the atmosphere used in the annealing step may include, but is not limited to, air, carbon dioxide, water vapor, inert gases (such as nitrogen, helium, neon, argon, and xenon), vapors (such as ethanol, vegetable oil, water), and / or mixtures thereof.

[0482] The liquids used in the annealing step may include, but are not limited to, ethanol, vegetable oils, and / or mixtures thereof.

[0483] The fluid used in the annealing step may include, but is not limited to, alcohols such as methanol, ethanol, propanol, isopropanol, butanol, and / or mixtures thereof.

[0484] The fluid used in the annealing step is not limited, but may include supercritical CO2.

[0485] In some embodiments, the liquid used in the annealing step includes, but is not limited to, ethanol, vegetable oil, and / or mixtures thereof.

[0486] In some embodiments, the fluid used in the annealing step includes, but is not limited to, alcohols such as methanol, ethanol, propanol, isopropanol, butanol, and / or mixtures thereof.

[0487] In some embodiments, the fluid used in the annealing step includes, but is not limited to, supercritical CO2.

[0488] In some embodiments, the liquid used in the annealing step does not contain polyols.

[0489] In some embodiments, the hollow fibers are annealed by baking them in a convection oven, steaming them in an autoclave or pressure cooker, and / or immersing them in a hot oil bath.

[0490] In some preferred embodiments, the annealing step is performed in an air atmosphere.

[0491] In some embodiments, the annealing step is carried out at a temperature in the range of 50°C to 180°C, preferably in the range of 75°C to 160°C, more preferably in the range of 110°C to 140°C, and most preferably in the range of 120°C to 130°C.

[0492] In some other embodiments, the annealing step is performed at temperatures in the range of 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.

[0493] In some embodiments, the annealing step is carried out at a temperature in the range of 180°C to 300°C, preferably in the range of 200°C to 280°C, more preferably in the range of 220°C to 260°C, and most preferably in the range of 240°C to 250°C.

[0494] In some other embodiments, the annealing step is performed at temperatures in the range of 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.

[0495] In some embodiments, the annealing step is performed over a period of time ranging from 30 minutes to 6 hours, preferably within the range of 10 minutes and 4 hours, more preferably within the range of 30 minutes and 3 hours, even more preferably within the range of 45 hours and 2 hours, and most preferably within the range of 1 hour and 1.5 hours.

[0496] In some other embodiments, the annealing step can be 5 to 15 minutes, 15 to 30 minutes, 30 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 The process is carried out over a period of time within the range of 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.

[0497] Semipermeable porous hollow fibers crosslinked by annealed covalent bonds can be rehydrated in water and / or aqueous solutions in a process referred to herein as rehydration.

[0498] In some embodiments, the annealed, covalently crosslinked, semipermeable porous hollow fibers are rehydrated in water and / or aqueous solution.

[0499] In some embodiments, the semipermeable porous hollow fibers crosslinked by annealed covalent bonds are not rehydrated in water and / or aqueous solutions.

[0500] In some embodiments, the components in the aqueous solution for rehydration include, but are not limited to, water, alcohol, polyol, acid, alkali, salt, and / or combination thereof.

[0501] In some preferred embodiments, the rehydrated aqueous solution contains a polyol.

[0502] In some preferred embodiments, step (e.ii) includes annealing the covalently crosslinked semipermeable porous hollow fibers in air at 130°C for 2 hours.

[0503] In some particularly preferred embodiments, step (e.ii) includes drying the covalently crosslinked semipermeable porous hollow fibers by convection at 60°C for 2 hours, followed by annealing them in air at 130°C for 1 hour, and then rehydrating them in an aqueous solution containing 20% ​​glycerol (v / v).

[0504] In some embodiments, the pre-drying conditions used in any of the embodiments of the present disclosure can be used for the production of Prokitein and / or hollow fibers of the present disclosure.

[0505] In some embodiments, the annealing conditions used in any of the embodiments of this disclosure can be used for the production of Prokitein and / or hollow fibers of this disclosure.

[0506] In some embodiments, the process by which Prokitein is produced involves protein annealing.

[0507] In some embodiments, Prokitein is annealed to modify and / or adapt its material properties.

[0508] In some other embodiments, Prokitein is annealed to modify and / or adapt its Young's modulus.

[0509] In some other embodiments, Prokitein is annealed to modify and / or adapt its ultimate tensile stress.

[0510] In some other embodiments, Prokitein is annealed to modify and / or adapt its ultimate tensile strain.

[0511] 1.13 Step (E.iii) Removal of void-containing elements Step (e.iii) may include treating the covalently crosslinked semipermeable porous hollow fibers with one or more solvents to remove void-containing elements in the hollow fibers and to impart porosity.

[0512] In some embodiments, step (e.iii) includes treating covalently crosslinked semipermeable porous hollow fibers with one or more solvents to remove void-containing elements in the hollow fibers, thereby imparting porosity.

[0513] In some embodiments, the solvent used to remove void-containing elements includes aqueous solutions and / or organic solutions.

[0514] In some embodiments, a supercritical fluid is used to remove void-containing elements.

[0515] In some embodiments, supercritical CO2 is used to remove void-containing elements.

[0516] In some embodiments, the aqueous solution includes water, an acid, an alkali, and / or a salt solution.

[0517] In some other embodiments, one of the solvents is water.

[0518] In some embodiments, one or more of the solvents are aqueous solutions containing at least one of oxalic acid, malic acid, succinic acid, adipic acid, tartaric acid, citric acid, malonic acid, acetic acid, or formic acid, preferably an acid containing citric acid and / or malic acid.

[0519] In some embodiments where the void-containing element is powdered calcium carbonate, semipermeable porous hollow fibers crosslinked by multiple covalent bonds are treated with an acidic aqueous solution such as citric acid. Hydrogen ions react with the calcium carbonate embedded in the walls of the hollow fibers to form calcium salts and carbon dioxide. As carbon dioxide is released and the salt dissolves, voids are formed in the walls of the hollow fibers in the spaces previously occupied by calcium carbonate, thereby increasing the void fraction of the hollow fibers.

[0520] In some embodiments, the organic solution comprises at least one of ethanol, propanol, isopropanol, butanol, isobutanol, dimethyl sulfoxide, dimethylformamide, and / or dimethylacetamide, preferably ethanol.

[0521] In some embodiments, the organic solution includes acetone.

[0522] In some embodiments, the organic solution comprises polyols and / or polymers of polyols.

[0523] In some embodiments, a combination of aqueous solution and organic solution may be used to form a solvent mixture.

[0524] In some embodiments where the void-containing elements are lipids, multiple hollow fibers are exposed to an organic solvent such as ethanol. As the oil droplets dissolve in the organic phase and are subsequently removed, voids are formed in the walls of the hollow fibers in the spaces previously occupied by the oil droplets, thereby imparting porosity to the hollow fibers.

[0525] In some embodiments, the concentration of any single component in the solvent mixture used to remove void-containing elements from covalently crosslinked semipermeable porous hollow fibers is in the range of 0%(w / v) to 100%(w / v), preferably in the range of 10%(w / v) to 75%(w / v), and most preferably in the range of 20%(w / v) to 60%(w / v), relative to the total volume of the solvent mixture.

[0526] In some other embodiments, the concentration of any single component in the solvent mixture used to remove void-containing elements from covalently crosslinked semipermeable porous hollow fibers is 0%(w / v)~5%(w / v), 5%(w / v)~10%(w / v), 10%(w / v)~15%(w / v), 15%(w / v)~20%(w / v), 20%(w / v)~25%(w / v), 25%(w / v)~30%(w / v), 30%(w / v)~35%(w / v), and 35%(w / v)~40% relative to the total volume of the solvent mixture. (w / v), 40%(w / v)~45%(w / v), 45%(w / v)~50%(w / v), 50%(w / v)~55%(w / v), 55%(w / v)~60%(w / v), 60%(w / v)~65%(w / v), 65%(w / v)~70%(w / v), 70%(w / v)~75%(w / v), 75%(w / v)~80%(w / v), 80%(w / v)~85%(w / v), 85%(w / v)~90%(w / v), 90%(w / v)~95%(w / v), or 95%(w / v)~100%(w / v).

[0527] In some embodiments, step (e.iii) does not involve treating the covalently crosslinked semipermeable porous hollow fibers with any solvent.

[0528] In some embodiments that do not use solvents, step (e.iii) includes subjecting a covalently crosslinked semipermeable porous hollow fiber to pyrolysis-induced pore formation. In this specification, the term “pyrolysis-induced pore formation” refers to a process of pyrolysis of one or more void-containing elements within the wall of a hollow fiber to remove such void-containing elements and thereby impart porosity.

[0529] In some particularly preferred embodiments, step (e.iii) includes treating covalently crosslinked semipermeable porous hollow fibers, comprising powdered calcium carbonate, with 25% (w / v) citric acid in an aqueous solvent at room temperature for 30 minutes.

[0530] In some other particularly preferred embodiments, step (e.iii) includes treating a covalently crosslinked semipermeable porous hollow fiber containing lipids with a 40% ethanol solution at room temperature for 30 minutes.

[0531] In some other particularly preferred embodiments, step (e.iii) includes treating a covalently crosslinked, semipermeable porous hollow fiber containing lipids with isopropanol at room temperature for 30 minutes.

[0532] In some other particularly preferred embodiments, step (e.iii) includes treating a covalently crosslinked, semipermeable porous hollow fiber containing lipids with supercritical CO2.

[0533] One or more void-containing elements may be removed from the hollow fiber in a processing step other than step (e.iii), including, but not limited to, step (d), step (ei), and / or step (e.iv). For example, water-soluble void-containing elements may dissolve in the coagulation bath solution of step (d). In another example, alcohol-soluble void-containing elements may dissolve in the coagulation bath solution of step (d), which contains an aqueous solvent and one or more alcohols.

[0534] In some possible embodiments, one or more void-containing elements are removed from the hollow fiber in a processing step other than step (e.iii), which includes, but is not limited to, step (d), step (ei), and / or step (e.iv).

[0535] In some embodiments, the solvent used to remove void-containing elements in any of the embodiments of the present disclosure can be used in the production of Prokitein and / or hollow fibers of the present disclosure.

[0536] 1.14 Step (E.iv) Buffer Wash Step (e.iv) may include washing the covalently crosslinked semipermeable porous hollow fibers with one or more acids, alkalis, and / or buffers to potentially reduce at least one of the Young's modulus, ultimate tensile strength, and / or ultimate tensile strain of the hollow fibers.

[0537] In some embodiments, step (e.iv) includes washing the covalently crosslinked semipermeable porous hollow fibers with one or more acids, alkalis, and / or buffers to potentially reduce at least one of the Young's modulus, ultimate tensile strength, and / or ultimate tensile strain of the hollow fibers.

[0538] In some embodiments, the acid used in step (e.iv) includes, but is not limited to, ascorbic acid, acetic acid, adipic acid, citric acid, formic acid, hydrochloric acid, lactic acid, malic acid, malonic acid, oxalic acid, succinic acid, sulfuric acid, and / or tartaric acid.

[0539] In some embodiments, the alkali used in step (e.iv) includes, but is not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and / or sodium carbonate.

[0540] In some embodiments, covalently crosslinked semipermeable porous hollow fibers are washed in step (e.iv) with a buffered solution with a pH of 10 or higher.

[0541] In some other embodiments, covalently crosslinked semipermeable porous hollow fibers are washed in step (e.iv) with a sodium carbonate buffer buffered to pH 10 or higher.

[0542] In some embodiments, step (e.iv) is continued for a period of time ranging from 10 minutes to 3 hours, preferably 15 minutes and 2 hours, and most preferably 30 minutes to 1 hour.

[0543] In some other embodiments, step (e.iv) is continued for a period of time within the range of 10 minutes and 15 minutes, 15 minutes and 30 minutes, 30 minutes and 45 minutes, 45 minutes and 1 hour, 1 hour and 2 hours, or 2 hours and 3 hours.

[0544] In some embodiments, step (e.iv) is carried out at a temperature in the range of 10°C to 80°C, preferably in the range of 20°C to 60°C, more preferably in the range of 30°C to 50°C, and most preferably at room temperature.

[0545] In some other embodiments, step (e.iv) is carried out at a temperature 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.

[0546] In some particularly preferred embodiments, step (e.iv) includes washing the covalently crosslinked semipermeable porous hollow fibers with an aqueous solution of sodium carbonate buffer at pH 11.0 at 37°C for 60 minutes.

[0547] 1.15 Step (Ev) Coating Step (ev) may include coating a semipermeable porous hollow fiber that is crosslinked by covalent bonds.

[0548] In some embodiments, step (ev) includes coating a covalently crosslinked semipermeable porous hollow fiber.

[0549] Coatings are widely used in combination with conventional cell culture techniques to aid cell adhesion and / or proliferation. Possible coatings include, but are not limited to, protein complexes, cell adhesion peptide sequences (CAPs), and / or growth factors (30;31).

[0550] Protein complexes can be used to enhance cell adhesion to covalently crosslinked, semipermeable porous hollow fibers. These include, but are not limited to, collagen, gelatin, fibrinogen, fibronectin, and laminin.

[0551] In some embodiments, protein complexes are used to enhance cell adhesion to covalently crosslinked semipermeable porous hollow fibers.

[0552] In some embodiments, protein complexes are not used to enhance cell adhesion to covalently crosslinked semipermeable porous hollow fibers.

[0553] CAP is a short sequence of amino acids that embodies the minimum motifs necessary for cell adhesion (30).

[0554] CAPs that can be used to enhance cell adhesion to covalently crosslinked semipermeable porous hollow fibers include, but are not limited to, AELDPPF (SEQ ID NO: 1), CGGNGEPRGDTYRAY (SEQ ID NO: 2), CFALRGDNP (SEQ ID NO: 3), CKKQRFRHRNRKG (SEQ ID NO: 4), CNYYSNS (SEQ ID NO: 5), CSVTCG (SEQ ID NO: 6), DGEA (SEQ ID NO: 7), ELVTDFPTDLPAT (SEQ ID NO: 8), FHRRIKA (SEQ ID NO: 8), Number 9), FQGVLQNVRFVF (Sequence ID 10), GACRGDCLGA (Circular) (Sequence ID 11), GFOGER (Sequence ID 12), GFRGDGQ (Sequence ID 13), GRGDS (Sequence ID 14), GRGDAC (Sequence ID 15), GTFALRGDNGQ (Sequence ID 16), IDAPS (Sequence ID 17), IKLLI (Sequence ID 18), IKVAV (Sequence ID 19), IWKHKGRDVILKKDVRFYC (Sequence ID 20), KAFDI TYVRLKF (SEQ ID NO: 21), KLDAPT (FN5) (SEQ ID NO: 22), KQAGDV (SEQ ID NO: 23), KRSR (SEQ ID NO: 24), LIGRKK (SEQ ID NO: 25), LGTIPG (SEQ ID NO: 26), LRE, LRGDN (SEQ ID NO: 27), MNYYSNS (SEQ ID NO: 28), NPWHSIYITRFG (SEQ ID NO: 29), PDGSR (SEQ ID NO: 30), PHRSN (SEQ ID NO: 31), PKRGDL (SEQ ID NO: 32), PRARI (SEQ ID NO: 3 3) This includes REDV (sequence code 34), RGD, SIGFRGDGQTC (sequence code 35), SIKVAV (sequence code 36), SINNNR (sequence code 37), SPPRRARV (sequence code 38), SVVYGLR (sequence code 39), TWYKIAFQRNRK (sequence code 40), VALDEP (sequence code 41), VGVAPG (sequence code 42), VPGIG (sequence code 43), WQPPRARI (sequence code 44), and YIGSR (sequence code 45).

[0555] In some embodiments, CAP is used to enhance cell adhesion to covalently crosslinked semipermeable porous hollow fibers.

[0556] In some embodiments, the CAP used to enhance cell adhesion to covalently crosslinked semipermeable porous hollow fibers includes, but is not limited to, AELDPPF (SEQ ID NO: 1), CGGNGEPRGDTYRAY (SEQ ID NO: 2), CFALRGDNP (SEQ ID NO: 3), CKKQRFRHRNRKG (SEQ ID NO: 4), CNYYSNS (SEQ ID NO: 5), CSVTCG (SEQ ID NO: 6), DGEA (SEQ ID NO: 7), ELVTDFPTDLPAT (SEQ ID NO: 8), FH RRIKA (SEQ ID NO: 9), FQGVLQNVRFVF (SEQ ID NO: 10), GACRGDCLGA (cyclic) (SEQ ID NO: 11), GFOGER (SEQ ID NO: 12), GFRGDGQ (SEQ ID NO: 13), GRGDS (SEQ ID NO: 14), GRGDAC (SEQ ID NO: 15), GTFALRGDNGQ (SEQ ID NO: 16), IDAPS (SEQ ID NO: 17), IKLLI (SEQ ID NO: 18), IKVAV (SEQ ID NO: 19), IWKHKGRDVILKKDVRFYC (SEQ ID NO: 20) KAFDITYVRLKF (SEQ ID NO: 21), KLDAPT (FN5) (SEQ ID NO: 22), KQAGDV (SEQ ID NO: 23), KRSR (SEQ ID NO: 24), LIGRKK (SEQ ID NO: 25), LGTIPG (SEQ ID NO: 26), LRE, LRGDN (SEQ ID NO: 27), MNYYSNS (SEQ ID NO: 28), NPWHSIYITRFG (SEQ ID NO: 29), PDGSR (SEQ ID NO: 30), PHRSN (SEQ ID NO: 31), PKRGDL (SEQ ID NO: 32), PRARI32), This includes numbers 33), REDV (sequence number 34), RGD, SIGFRGDGQTC (sequence number 35), SIKVAV (sequence number 36), SINNNR (sequence number 37), SPPRRARV (sequence number 38), SVVYGLR (sequence number 39), TWYKIAFQRNRK (sequence number 40), VALDEP (sequence number 41), VGVAPG (sequence number 42), VPGIG (sequence number 43), WQPPRARI (sequence number 44), and YIGSR (sequence number 45).

[0557] In some embodiments, CAP is not used to enhance cell adhesion to covalently crosslinked semipermeable porous hollow fibers.

[0558] Growth factors may be used to enhance cell adhesion to covalently crosslinked, semipermeable porous hollow fibers. These 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 / or vascular endothelial growth factor (VEGF).

[0559] In some embodiments, growth factors are used to enhance cell adhesion to covalently crosslinked semipermeable porous hollow fibers.

[0560] In some embodiments, growth factors are used to enhance cell adhesion to covalently crosslinked, semipermeable porous hollow fibers. These 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 / or vascular endothelial growth factor (VEGF).

[0561] In some embodiments, growth factors are not used to enhance cell adhesion to covalently crosslinked semipermeable porous hollow fibers.

[0562] In some embodiments, protein complexes, CAPs, and / or growth factors are used to enhance cell adhesion to covalently crosslinked semipermeable porous hollow fibers.

[0563] Chemical methods such as grafting may be used to enhance cell adhesion to covalently crosslinked, semipermeable porous hollow fibers by coating the surface with protein complexes, CAPs, and / or growth factors.

[0564] In some embodiments, chemical methods are used to enhance cell adhesion to covalently crosslinked semipermeable porous hollow fibers by coating the surface with protein complexes, CAPs, and / or growth factors.

[0565] In some embodiments, no chemical methods are used to enhance cell adhesion to the hollow fibers by coating the surface of covalently crosslinked semipermeable porous hollow fibers with protein complexes, CAPs, and / or growth factors.

[0566] In one particularly preferred embodiment, step (ev) includes leaving the covalently crosslinked semipermeable porous hollow fibers uncoated.

[0567] 1.16 Step (E.vi) Surface Topography Correction Step (e.vi) may include correcting the surface topography of covalently crosslinked semipermeable porous hollow fibers to aid in cell adhesion and / or cell alignment.

[0568] In some embodiments, step (e.vi) includes correcting the surface topography of covalently crosslinked semipermeable porous hollow fibers to aid in cell adhesion and / or cell alignment.

[0569] Step (e.vi) may include stretching a semipermeable porous hollow fiber crosslinked by covalent bonds to induce tearing in the microstructure and form a porous structure.

[0570] In some embodiments, step (e.vi) includes stretching covalently crosslinked semipermeable porous hollow fibers to induce tearing in the microstructure and form a porous structure.

[0571] The surface topography of the outer wall of covalently crosslinked semipermeable porous hollow fibers can be modified.

[0572] In some embodiments, the surface topography of the outer wall of covalently crosslinked semipermeable porous hollow fibers is modified.

[0573] In some embodiments, the surface topography of the outer wall of the covalently crosslinked semipermeable porous hollow fibers is not modified.

[0574] Methods that may be used to modify a surface include, but are not limited to, physical methods such as plasma treatment, physical vapor deposition, ultrasonic treatment, and mechanical etching.

[0575] In some embodiments, the methods used to modify the surface include, but are not limited to, physical methods such as plasma treatment, physical vapor deposition, ultrasonic treatment, and mechanical etching.

[0576] Mechanical etching may be used to impart grooves along the surface of covalently crosslinked semipermeable porous hollow fibers.

[0577] In some embodiments, mechanical etching is used to impart grooves along the surface of covalently crosslinked semipermeable porous hollow fibers.

[0578] The shape of the die or spinaret orifice used in step (c) may be used to provide grooves along the surface of the hollow fibers in order to assist in the alignment of cells along the longitudinal axis of the covalently crosslinked semipermeable porous hollow fibers.

[0579] In some embodiments, the shape of the die or spinaret orifice used in step (c) is used to impart grooves along the surface of the hollow fibers in order to assist in the alignment of cells along the longitudinal axis of the covalently crosslinked semipermeable porous hollow fibers.

[0580] In one particularly preferred embodiment, step (e.vi) includes leaving the surface topography of the covalently crosslinked semipermeable porous hollow fibers uncorrected.

[0581] The post-generation modification processes described herein in steps (ei) to (e.vi) may be carried out in any combination and in any order.

[0582] In some embodiments, step (ei) is performed.

[0583] In some embodiments, step (ei) is not performed.

[0584] In some embodiments, step (e.ii) is performed.

[0585] In some embodiments, step (e.ii) is not performed.

[0586] In some embodiments, step (e.iii) is performed.

[0587] In some embodiments, step (e.iii) is not performed.

[0588] In some embodiments, step (e.iv) is performed.

[0589] In some embodiments, step (e.iv) is omitted.

[0590] In some embodiments, step (ev) is performed.

[0591] In some embodiments, step (ev) is not performed.

[0592] In some embodiments, step (e.vi) is performed.

[0593] In some embodiments, step (e.vi) is omitted.

[0594] In some embodiments, steps (ei) and (e.ii) are performed sequentially in a specified order.

[0595] In some embodiments, steps (ei), (e.ii), and (e.iii) are performed sequentially in a specified order.

[0596] In some embodiments, steps (ei), (e.iii), and (e.ii) are performed sequentially in a specified order.

[0597] In some embodiments, one or more of steps (ei) to (e.vi) are performed sequentially in a specified order.

[0598] In some embodiments, steps (ei), (e.ii), (e.iii), and (e.iv) are performed sequentially in a specified order.

[0599] In some embodiments, steps (ei), (e.ii), (e.iii), (e.iv), and (ev) are performed sequentially in a specified order.

[0600] In some embodiments, steps (ei), (e.ii), (e.iii), (e.iv), (ev), and (e.vi) are performed sequentially in a specified order.

[0601] In some embodiments, steps (ei), (e.ii), (e.iv), (ev), and (e.vi) are performed sequentially in a specified order.

[0602] In some embodiments, steps (ei), (e.ii), (e.iii), (ev), and (e.vi) are performed sequentially in a specified order.

[0603] In some embodiments, steps (ei), (e.ii), (e.iii), (e.iv), and (e.vi) are performed sequentially in a specified order.

[0604] In some embodiments, steps (ei), (e.ii), (e.iii), (e.iv), and (ev) are performed sequentially in a specified order.

[0605] In some embodiments, steps (ei), (e.ii), and (e.iii) are performed sequentially in a specified order.

[0606] In some embodiments, steps (ei), (e.ii), and (e.iv) are performed sequentially in a specified order.

[0607] In some embodiments, steps (ei), (e.ii), and (ev) are performed sequentially in a specified order.

[0608] In some embodiments, steps (ei), (e.ii), and (e.vi) are performed sequentially in a specified order.

[0609] In some embodiments, steps (ei), (e.ii), and (e.iii) are performed within a single process step.

[0610] 1.17 Step (F) Storage and Drying Step (f) may include drying the treated covalently crosslinked semipermeable porous hollow fibers to produce dried, treated covalently crosslinked semipermeable porous hollow fibers, and then storing them in a low-humidity atmosphere.

[0611] In some embodiments, step (f) includes drying the treated covalently crosslinked semipermeable porous hollow fibers to produce dried, treated covalently crosslinked semipermeable porous hollow fibers, and then storing them in a low-humidity atmosphere.

[0612] In some embodiments, the treated covalently crosslinked semipermeable porous hollow fibers are partially dried, and preferably completely dried.

[0613] In some embodiments, the semipermeable porous hollow fibers crosslinked by the treated covalent bonds are not dried.

[0614] In some embodiments, the drying process is carried out by convection, radiation, freeze-drying, electromagnetic wave sources, infrared heat sources, and / or on a heated surface.

[0615] Freeze-drying may aid in the formation of pores in covalently crosslinked semipermeable porous hollow fibers and / or modify their microstructure.

[0616] In some embodiments, freeze-drying is used to modify the porosity of covalently crosslinked semipermeable porous hollow fibers.

[0617] In some embodiments, freeze-drying is additionally used to modify the porosity of covalently crosslinked semipermeable porous hollow fibers.

[0618] In some embodiments, freeze-drying is not used to correct the porosity of covalently crosslinked semipermeable porous hollow fibers.

[0619] In some embodiments, freeze-drying is used, preferably additionally, and most preferably not used, to correct the porosity of covalently crosslinked semipermeable porous hollow fibers.

[0620] In some embodiments, the covalently crosslinked semipermeable porous hollow fibers are partially or completely dried at a temperature in the range of 5 to 90°C, preferably in the range of 15 to 80°C, more preferably in the range of 40 to 70°C, and most preferably in the range of 50 to 70°C.

[0621] In some other embodiments, covalently crosslinked semipermeable porous hollow fibers are partially or completely dried at temperatures in the range of 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.

[0622] In some embodiments, the covalently crosslinked semipermeable porous hollow fibers are dried using conditions applicable to the pre-drying step (e.ii).

[0623] Dry, processed, covalently crosslinked, semi-permeable porous hollow fibers can be stored in air, in inert gases (such as nitrogen, helium, neon, argon, and xenon), in CO2, under vacuum, and / or in PBS buffer solution, water, aqueous solutions, organic solutions, and / or mixtures thereof.

[0624] In some embodiments, dried, treated, covalently crosslinked, semipermeable porous hollow fibers are stored under vacuum.

[0625] In some embodiments, dried, treated, covalently crosslinked, semipermeable porous hollow fibers are stored in PBS buffer.

[0626] In some embodiments, dried, treated, covalently crosslinked, semipermeable porous hollow fibers are stored in water and / or aqueous solutions.

[0627] Suitable components present in an aqueous solution may include, but are not limited to, water, alcohol, polyol, acid, alkali, salt, and / or combinations thereof.

[0628] In some preferred embodiments, the aqueous solution used to store the dried, treated, covalently crosslinked semipermeable porous hollow fibers contains a polyol.

[0629] In some embodiments, dried, treated, covalently crosslinked, semipermeable porous hollow fibers can be stored in an organic solvent.

[0630] In other embodiments, dried, treated, covalently crosslinked semipermeable porous hollow fibers may be stored in a mixture of an organic solvent and an aqueous solvent, such as water and ethanol, but are not limited to these.

[0631] In some embodiments, dried, treated, covalently crosslinked, semipermeable porous hollow fibers can be stored refrigerated at temperatures of 8–0°C.

[0632] In some embodiments, dried, treated, covalently crosslinked, semipermeable porous hollow fibers can be stored frozen at temperatures of 0 to -90°C.

[0633] In some preferred embodiments, step (f) includes partially drying the treated covalently crosslinked semipermeable porous hollow fibers by exposure to heat to produce partially dried, treated covalently crosslinked semipermeable porous hollow fibers, and then storing them in air.

[0634] In some preferred embodiments, step (f) includes partially drying the treated covalently crosslinked semipermeable porous hollow fibers by exposure to heat to produce partially dried, treated covalently crosslinked semipermeable porous hollow fibers, and then storing them in a 40% ethanol (v / v) aqueous solution.

[0635] In some preferred embodiments, step (f) includes partially drying the treated covalently crosslinked semipermeable porous hollow fibers by exposure to heat to produce partially dried treated covalently crosslinked semipermeable porous hollow fibers, then rehydrating them in a 20% aqueous glycerol solution, and then storing them in the air.

[0636] In some preferred embodiments, step (f) includes partially drying the treated covalently crosslinked semipermeable porous hollow fibers by exposure to heat to produce partially dried, treated covalently crosslinked semipermeable porous hollow fibers, and then storing them in a 20% glycerol (v / v) aqueous solution.

[0637] In a preferred embodiment, the process steps are carried out in the specified order (a) to (f) (sequentially).

[0638] In some particularly preferred embodiments, a process for producing a plurality of semipermeable porous hollow fibers comprising covalent esters, thioesters, and / or amide-linked polypeptides includes sequentially carrying out steps (a), (b), (c), (d), (ei), (e.ii), and (e.iii) in a specified order.

[0639] In some particularly preferred embodiments, a process for producing a plurality of semipermeable porous hollow fibers comprising covalent esters, thioesters, and / or amide-linked polypeptides includes sequentially carrying out steps (a), (b), (c), (d), (ei), (e.iii), and (e.ii) in the specified order.

[0640] The applicant also provides a number of semipermeable porous hollow fibers, comprising covalent esters, thioesters, and / or amide-crosslinked polypeptides, which are obtained or can be obtained by the process of the present application.

[0641] In some embodiments, the drying conditions used in any of the embodiments of this disclosure can be used for the production of Prokitein and / or hollow fibers of this disclosure.

[0642] 1.18 Physical structure of porous, semi-permeable hollow fibers crosslinked by covalent bonds In further embodiments, semipermeable porous hollow fibers are provided, the semipermeable porous hollow fibers comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, wherein, (a) The outer diameter of the hollow fiber is 50 to 6600 μM. (b) The wall thickness of the hollow fiber is 20 to 800 μM. (c) The inner diameter of the hollow fiber is 20 to 5000 μM. (d) The pore volume of the hollow fiber is 1-95%.

[0643] In further embodiments, semipermeable porous hollow fibers are provided, the semipermeable porous hollow fibers comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, wherein, (a) The outer diameter of the hollow fiber is 50 to 6600 μm. (b) The wall thickness of the hollow fiber is 20 to 800 μm. (c) The inner diameter of the hollow fiber is 20 to 5000 μm. (d) The porosity of the hollow fibers is 1-95%.

[0644] The hollow fibers of the present disclosure may comprise covalent esters, thioesters, and / or amide-crosslinked polypeptides, each generally comprising a cylindrical tubular structure (e.g., a tube) having a wall, an outer surface, an inner surface, and a lumen. A schematic diagram illustrating an example of this geometry may be shown in Figure 3. In Figure 3, the lumen, inner wall, and outer wall are labeled 3006, 3005, and 3004, respectively.

[0645] In some embodiments, the hollow fibers of the present disclosure comprise covalent esters, thioesters, and / or amide-crosslinked polypeptides, each comprising a cylindrical tubular structure (e.g., a tube) having a wall, an outer surface, an inner surface, and a lumen.

[0646] Each hollow fiber in this disclosure may have a cylindrical tubular structure, such as the one labeled 3003 in Figure 3.

[0647] In some embodiments, each hollow fiber cylindrical tubular structure of the present disclosure has a major axis, such as the one labeled 3003 in Figure 3.

[0648] The cross-section of each fiber in this disclosure may consist of a single tube, preferably circular, but other shapes such as elliptical, square, and rectangular may be used, but are not limited to these. A schematic diagram of an example of multiple fibers in a bundle may be shown in Figure 4. An example of a single fiber having a circular cylindrical tubular structure is labeled 4001 in Figure 4.

[0649] In some embodiments, the cross-section of each fiber in the present disclosure consists of a single tube, preferably circular, but other shapes such as elliptical, square, and rectangular may be used, but are not limited to these.

[0650] The cross-sections of each fiber in this disclosure may include, but are not limited to, concentric shapes such as circular, elliptical, square, and rectangular, or combinations thereof.

[0651] In some embodiments, the cross-section of each fiber of the present disclosure includes, but is not limited to, concentric shapes such as circular, elliptical, square, and rectangular, or combinations thereof.

[0652] The hollow fibers of this disclosure may be in the shape of a tubular cylinder, preferably a circular tubular cylinder.

[0653] In some embodiments, the hollow fibers of the present disclosure are in the shape of a tubular cylinder, preferably a circular tubular cylinder.

[0654] Each hollow fiber in this disclosure may have a lumen (i.e., an orifice) extending along its long axis, so that a fluid can flow through the lumen.

[0655] In some embodiments, each hollow fiber of the present disclosure has a lumen (i.e., an orifice) extending along its long axis, so that a fluid can flow through the lumen.

[0656] In some embodiments, the lumen of each fiber of this application is open at one or both ends.

[0657] In a preferred embodiment, the lumen of each fiber of this application is open at both ends.

[0658] The hollow fibers in this disclosure may be of any desired length.

[0659] In some embodiments, the hollow fibers of the present disclosure are of any desired length.

[0660] In some embodiments, the length of the hollow fiber is not limited, but is within the range of 2 mm and 2 m, preferably within the range of 1 cm and 1 m, and most preferably within the range of 2.5 cm and 10 cm.

[0661] In some embodiments, the length of the hollow fiber is not limited, but is within the range of 2 mm and 5 m, preferably within the range of 1 cm and 1 m, and most preferably within the range of 2.5 cm and 10 cm.

[0662] In some other embodiments of the Disclosure, the length of the hollow fiber is within the range of 2 mm and 1 cm, 1 cm and 5 cm, 5 cm and 10 cm, 10 cm and 50 cm, 50 cm and 1 m, or 1 m and 2 m.

[0663] In some other embodiments of the Disclosure, the length of the hollow fiber is within the range of 2 mm and 1 cm, 1 cm and 5 cm, 5 cm and 10 cm, 10 cm and 50 cm, 50 cm and 1 m, 1 m and 2 m, 2 m and 3 m, 3 m and 4 m, or 4 m and 5 m.

[0664] The hollow fibers of the present disclosure can be classified, based on their cross-sectional structure, into either (i) symmetrical (isotropic) (constant cross-sectional structure) or (ii) heterogeneous (asymmetrical) (anisotropic) (non-constant cross-sectional structure).

[0665] In some embodiments, the hollow fibers of the present disclosure are symmetrical.

[0666] In some embodiments, the hollow fibers of the present disclosure are symmetrical, non-uniform, asymmetrical, or any combination thereof.

[0667] Each non-uniform fiber in this disclosure may include either (i) a composite of two materials (non-uniform composite) or (ii) a composite of the same material (non-uniform integrated coating).

[0668] In one embodiment, the hollow fibers of the present disclosure consist solely of an uneven composite, where each fiber comprises a composite of two materials.

[0669] In further embodiments, all hollow fibers in the present disclosure are non-uniform, integrated coatings, and each fiber is made of the same material.

[0670] In some embodiments, a plurality of fibers of the present disclosure are provided, and such plurality of fibers are, (i) Multiple symmetrical fibers, (ii) Multiple fibers that are an uneven composite, and / or (iii) Multiple fibers that are unevenly and integrally coated to form a bundle of fibers, A mixture of these, or containing at least one of them.

[0671] In some embodiments, the outer diameter of the hollow fibers of the present disclosure is in the range of 50 μm to 6600 μm, preferably in the range of 100 μm to 1500 μm, and most preferably in the range of 200 μm to 900 μm.

[0672] In some other embodiments, the outer diameter of the hollow fiber of the present disclosure is 50μm~100μm, 100μm~200μm, 200μm~300μm, 300μm~400μm, 400μm~500μm, 500μm~600μm, 600μm~700μm, 700μm~800μm, 800μm~900μm, 900μm~1000μm, 1000μm~1100μm, 1100μm~1200μm, 1200μm~1300μm, 1300μm~1400μm, 1400μm~1500μm, 1500μm~1600μm, 1600 μm~1700μm, 1700μm~1800μm, 1800μm~1900μm, 1900μm~2000μm, 2000μm ~2100μm, 2100μm~2200μm, 2200μm~2300μm, 2300μm~2400μm, 2400μm~25 00μm, 2500μm~2600μm, 2600μm~2700μm, 2700μm~2800μm, 2800μm~2900μm m, 2900μm~3000μm, 3000μm~3100μm, 3100μm~3200μm, 3200μm~3300μm, 3 300μm~3400μm, 3400μm~3500μm, 3500μm~3600μm, 3600μm~3700μm, 370 0μm~3800μm, 3800μm~3900μm, 3900μm~4000μm, 4000μm~4100μm, 4100μm ~4200μm, 4200μm~4300μm, 4300μm~4400μm, 4400μm~4500μm, 4500μm~46 00μm, 4600μm~4700μm, 4700μm~4800μm, 4800μm~4900μm, 4900μm~5000μm m is within the range of 5000μm~5100μm, 5100μm~5200μm, 5200μm~5300μm, 5300μm~5400μm, 5400μm~5500μm, 5500μm~5600μm, 5600μm~5700μm, 5700μm~5800μm, 5800μm~5900μm, 5900μm~6000μm, 6000μm~6100μm, 6100μm~6200μm, 6200μm~6300μm, 6300μm~6400μm, 6400μm~6500μm, or 6500μm~6600μm.

[0673] In some embodiments, the wall thickness of each fiber of this application is in the range of 1 μm to 800 μm, preferably in the range of 50 μm to 800 μm, more preferably in the range of 70 μm to 500 μm, and most preferably in the range of 75 μm to 150 μm.

[0674] In some embodiments, the wall thickness of each fiber of this application is in the range of 20 μm to 50 μm, 50 μm to 100 μm, 100 μm to 200 μm, 200 μm to 500 μm, or 500 μm to 800 μm.

[0675] In some embodiments, the lumen diameter of the hollow fiber of the present disclosure is in the range of 20 μm to 5000 μm, preferably in the range of 50 μm to 1000 μm, more preferably in the range of 100 μm to 850 μm, and most preferably in the range of 150 μm to 500 μm.

[0676] In some other embodiments, the lumen diameter of the hollow fiber of the present disclosure is in the range of 20 μm to 50 μm, 50 μm to 100 μm, 100 μm to 200 μm, 200 μm to 500 μm, 500 μm to 1000 μm, 1000 μm to 2500 μm, or 2500 μm to 5000 μm.

[0677] A scanning electron microscope (SEM) may be used to scan the surface of the material with an electron beam, generating images of the material at a wide range of magnifications from 10x to over 500,000x. SEM image analysis can be used by those skilled in the art to quantify various structural properties of the hollow fibers of the present disclosure, including lumen diameter, wall thickness, surface pore diameter, and internal structure (32;33).

[0678] A key feature of this application is that the hollow fibers disclosed herein are semi-permeable.

[0679] In some embodiments, the hollow fibers of the present disclosure are semipermeable, and as a result, fluids containing nutrients and some solutes can pass through, but not limited to, soluble growth factors, proteins, and cells cannot.

[0680] In some embodiments, the hollow fibers of the present disclosure are semipermeable, so that fluids and some solutes, including but not limited to nutrients, soluble growth factors, and proteins, can pass through, but cells cannot.

[0681] In some embodiments, the hollow fibers of the present disclosure are semipermeable, thereby allowing fluids, nutrients, soluble growth factors, proteins, and cells to pass through.

[0682] Soluble growth factors that are 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) (34).

[0683] Soluble growth factors can vary in size. For example, bFGF ranges from 18 to 34 kDa depending on its form. HGF is cleaved to form two active polypeptide chains of sizes 69 kDa and 34 kDa. On the other hand, IGF-1 is 7.6 kDa, EGF is 6 kDa, and VEGF has 17 isoforms ranging in size from 16 to 45 kDa.

[0684] In some embodiments, the hollow fibers of the present disclosure are semi-permeable, so that fluids and some solutes smaller than 7 kDa can pass through, but some solutes larger than 7 kDa cannot.

[0685] In some embodiments, the hollow fibers of the present disclosure are porous and semipermeable, and as a result, liquids and solutes in the range of 7kDa-12kDa, 12kDa-17kDa, 17kDa-22kDa, 22kDa-27kDa, 27kDa-32kDa, 32kDa-37kDa, 37kDa-42kDa, 42kDa-47kDa, 47kDa-52kDa, 52kDa-57kDa, 57kDa-62kDa, 62kDa-67kDa, 67kDa-72kDa, 72kDa-77kDa, or greater than 77kDa can pass through.

[0686] In some embodiments, the hollow fibers of the present disclosure are semipermeable, and as a result, liquids and solutes in the range of 7kDa-12kDa, 12kDa-17kDa, 17kDa-22kDa, 22kDa-27kDa, 27kDa-32kDa, 32kDa-37kDa, 37kDa-42kDa, 42kDa-47kDa, 47kDa-52kDa, 52kDa-57kDa, 57kDa-62kDa, 62kDa-67kDa, 67kDa-72kDa, 72kDa-77kDa, or larger than 77kDa can pass through.

[0687] Materials can be classified as macroporous (>50 nm), mesoporous (2-50 nm), or microporous (<2 nm) depending on the range of pore sizes within the material. Microporous materials are sometimes called nanoporous. Materials are classified by the largest pore size present. For example, porous materials containing pores smaller than 50 nm and pores larger than 50 nm are classified as macroporous.

[0688] In some embodiments, the hollow fibers of the Disclosure are macroporous, mesoporous, or microporous.

[0689] In some embodiments, the pore diameter of the pores in the wall of each hollow fiber of the present disclosure is in the range of 1 nm to 100 μm, preferably in the range of 5 nm to 15 μm, more preferably in the range of 10 nm to 5 μm, even more preferably in the range of 50 nm to 3 μm, and most preferably in the range of 100 nm to 2 μm.

[0690] In some other embodiments, the pore diameter of each hollow fiber of the present disclosure is in 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 μm, 1 μm to 5 μm, 5 μm to 10 μm, 10 μm to 50 μm, and / or 50 μm to 100 μm.

[0691] In some embodiments, combinations of hollow fibers of the present disclosure are provided, and such combinations are (i) Multiple fibers having pore sizes in the range of 1 μm to 15 μm, and (ii) comprising a plurality of fibers having pore sizes in the range of 1 nm to 10 nm.

[0692] In some embodiments, the porosity of the wall of each hollow fiber in the present disclosure is in the range of 1% to 95%, preferably in the range of 50% to 90%, and most preferably in the range of 60% to 80%.

[0693] In some other embodiments, the porosity of the walls of each hollow fiber in the present disclosure is in the range of 1% to 20%, 20% to 40%, 40% to 60%, 60% to 80%, or 80% to 90%.

[0694] In some other embodiments, the porosity of the wall of each hollow fiber in the present disclosure is less than 1%.

[0695] Mercury porosimetry is the gold standard for measuring pore volume, porosity, and pore size of semipermeable porous hollow fibers. Based on the capillary method for small pores, porosity can be determined by the total permeable volume of non-wet fluid per unit volume of the sample. Pore size may be calculated from Washburn's equation. Modern mercury porosimetry can measure pore diameters in the range of 900 μm to 0.005 μm (35). Furthermore, surface pore diameters may be measured by SEM image analysis (32;33). Such analytical methods can be applied to the hollow fibers of the present disclosure by those skilled in the art.

[0696] The molecular weight cutoff (MWCO) is defined as the lowest molecular weight at which more than 90% of a solute with a known molecular weight is retained by the hollow fiber. Typically, polyethylene glycol, poly(ethylene oxide), or bovine serum albumin are used as marker molecules to determine the MWCO (36). Changes in the abundance of these marker molecules in the feed and permeate flows can be measured using refractive index signal intensities separated by size exclusion chromatography using a high-performance liquid chromatography analyzer. This method may be used by those skilled in the art to determine the molecular weight cutoff of the hollow fibers of the present disclosure.

[0697] In some embodiments, the MWCO of each hollow fiber of the present disclosure is 5kDa to 100kDa, preferably 5kDa to 7kDa, more preferably 7kDa and 10kDa, even more preferably 10kDa and 20kDa, and most preferably in the range of 20kDa and 30kDa.

[0698] In some other embodiments, the MWCO of each hollow fiber of the present disclosure is in the range of 5kDa to 10kDa, 10kDa to 15kDa, 15kDa to 20kDa, 20kDa to 25kDa, 25kDa to 30kDa, 30kDa to 35kDa, 35kDa to 40kDa, 40kDa to 45kDa, 45kDa to 50kDa, 50kDa to 55kDa, 55kDa to 60kDa, 60kDa to 65kDa, 65kDa to 70kDa, 70kDa to 75kDa, 75kDa to 80kDa, 80kDa to 85kDa, 85kDa to 90kDa, 90kDa to 95kDa, or 95kDa to 100kDa.

[0699] In some embodiments, the pure water filtration coefficient (PWP) of the transformer wall of the hollow fiber of the present disclosure is 10 L / (m³). 2 h Bar) and 1,000 L / (m 2 h bar), preferably 10 L / (m 2 h bar) ~200L / (m 2 h bar), most preferably 0.5 L / (m 2 h bar) ~30L / (m2 is within the range of (h bar).

[0700] The PWP of the hollow fibers of the present disclosure is defined as the volume (L) of pure water passing through the fiber wall per unit area (m 2 ) of the hollow fiber, per unit time (h), and per transmembrane pressure (bar).

[0701] One of ordinary skill in the art may be able to determine the PWP of the hollow fibers of the present disclosure using a hydraulic filtration system as shown in FIG. 7. In such an apparatus, pressure gauges located at the inlet and outlet of the cartridge continuously measure the upstream and downstream pressures. Pure water is pumped from a reservoir through a positive displacement pump through the lumen of the hollow fibers within the cartridge. Alternatively, a gear pump can also be used. The pure water can penetrate through the wall of the hollow fiber into an additional extra-capillary space on the shell side or can flow through the lumen. The outlet on the shell side of the hollow fiber transfers the liquid to a permeate reservoir. The mass of the feed water and the shell side permeate reservoir is continuously recorded.

[0702] The PWP can be determined by measuring the change in the mass of water collected on the permeate side of the wall throughout the operation (37;36).

[0703] For the hollow fibers of the present disclosure to be suitable for use in a bioreactor, it is preferred that the PWP remains within an appropriate range over a typical culture period. Such a period can last at least three days.

[0704] The particle rejection size (PRS) is defined as the smallest particle size at which more than 90% of the particles in a standard test slurry of a given uniform particle size are retained by a separation device such as the hollow fibers of the present disclosure (36).

[0705] In some embodiments, the PRS exclusion size of each fiber of the present disclosure is in the range of 0.1 to 100 μm, preferably at least 100 μm, more preferably at least 50 μm, even more preferably at least 25 μm, even more preferably at least 10 μm, even more preferably at least 5 μm, even more preferably at least 2.5 μm, even more preferably at least 1 μm, even more preferably at least 0.5 μm, and most preferably at least 0.1 μm.

[0706] In some other embodiments, the PRS of each fiber in the present disclosure is within the range of 0.1μm-1μm, 1μm-5μm, 5μm-10μm, 10μm-15μm, 15μm-20μm, 20μm-25μm, 25μm-30μm, 30μm-35μm, 35μm-40μm, 40μm-45μm, 45μm-50μm, 50μm-55μm, 55μm-60μm, 60μm-65μm, 65μm-70μm, 70μm-75μm, 75μm-80μm, 80μm-85μm, 85μm-90μm, 90μm-95μm, or 95μm-100μm.

[0707] Those skilled in the art may determine the PRS of the hollow fibers of the present disclosure using a multisizer that evaluates the particle sizes in a slurry containing a suspension of polystyrene balls of different sizes. Once the test slurry has passed through the hollow fibers, the PRS of the hollow fibers of the present disclosure may then be calculated using the difference in the abundance of polystyrene ball sizes in the test slurry and the permeate (6).

[0708] 1.19 Composition of porous, semi-permeable hollow fibers crosslinked by covalent bonds In certain embodiments and examples, the hollow fibers of the Disclosure comprise one or more covalent esters, thioesters, and / or amide-crosslinked polypeptides.

[0709] In some embodiments, Prokitein is used to produce hollow fibers.

[0710] In some embodiments, the hollow fiber contains Prokitein.

[0711] In one embodiment, the covalent ester, thioester, and / or amide-crosslinked polypeptide may be a polymer, and as a result, comprises multiple chains of polypeptide.

[0712] In some embodiments, the polypeptides in the hollow fibers of the Disclosure are partially covalently crosslinked, substantially covalently crosslinked, or fully covalently crosslinked.

[0713] In some embodiments, the hollow fibers of the present disclosure contain at least 50% (by weight) of covalently crosslinked polypeptides.

[0714] In some embodiments, the hollow fibers of the present disclosure contain at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.99% (by weight) of covalently crosslinked polypeptides.

[0715] In other embodiments, the hollow fibers of the present disclosure contain 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% (by weight) of covalently crosslinked polypeptides.

[0716] In some embodiments, the hollow fibers of the present disclosure further contain polysaccharides (each present in a mass ratio ranging from 1% to 10,000% (weight per unit weight of polypeptide)).

[0717] In some other embodiments, the hollow fibers of the present disclosure further contain lipids (each present in a mass ratio ranging from 1% to 10,000% (weight per unit weight of polypeptide)).

[0718] In some other embodiments, the hollow fibers of the present disclosure further comprise polyols (each present in a mass ratio ranging from 1% to 10,000% (weight per unit weight of polypeptide)).

[0719] In some further embodiments, the hollow fibers of the present disclosure further comprise at least one of polysaccharides and lipids (each present in a mass ratio ranging from 1% to 10,000% (weight per unit weight of polypeptide)).

[0720] In some further embodiments, the hollow fibers of the present disclosure further comprise at least one of polysaccharides, lipids, and polyols (each present in a mass ratio ranging from 1% to 10,000% (weight per unit weight of polypeptide)).

[0721] In some embodiments, polypeptides in the hollow fibers of the Disclosure are crosslinked by a combination of intramolecular (within individual polypeptides) and intermolecular (between multiple polypeptides) covalent esters, thioesters, or amide bonds.

[0722] In other embodiments, polypeptides and polysaccharides in the hollow fibers of the Disclosure are crosslinked by a combination of intramolecular and intermolecular bonds to form polypeptide-polypeptide, polypeptide-polysaccharide, polysaccharide-polysaccharide ester, thioester and / or amide covalent crosslinks.

[0723] In some other embodiments of the present disclosure, in which the hollow fiber comprises only one type of polypeptide, all or substantially all of the polypeptides of the hollow fiber may be covalently crosslinked.

[0724] In further embodiments of the present disclosure, in which the hollow fiber comprises multiple types of polypeptides, there may be covalent crosslinks between all or substantially all of the types of polypeptides of the hollow fiber.

[0725] In some other embodiments of the present disclosure, in which the hollow fibers further contain one or more polysaccharides, all or substantially all of the polypeptides and / or polysaccharides of the hollow fibers may be covalently crosslinked.

[0726] In some embodiments, the polypeptide in the hollow fiber of the Disclosure is crosslinked by covalent bonds by at least 20% (by weight of the polypeptide), preferably at least 50% to 95%, more preferably at least 55% to 90%, even more preferably at least 65% to 85%, and most preferably at least 70% to 80%.

[0727] In other embodiments, the polypeptides within the hollow fibers of the present disclosure are crosslinked by covalent bonds by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.99% (by weight of the polypeptides).

[0728] In some other embodiments of the present disclosure, the range of covalently crosslinked polypeptides in the hollow fibers of the present disclosure is 20% and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90%, or 90% and 99.99% (by weight of polypeptides).

[0729] In some embodiments, Prokitein comprises a polypeptide obtained from a single source (e.g., soybeans or broad beans).

[0730] This disclosure is also at least in part based on the recognition that Prokitein comprising polypeptides derived from multiple sources (e.g., soybeans and broad beans) may have different properties than Prokitein comprising polypeptides derived from a single source.

[0731] As used herein, “Prokitein mixture” refers to Prokitein comprising polypeptides derived from multiple different sources.

[0732] In some embodiments, Prokitein, Prokitein mixtures, and / or polypeptides of the methods of the present disclosure are obtained from or derived from plants, animals, bacteria, algae, archaea, and / or fungi.

[0733] The prokitein mixture may contain polypeptides derived from two, three, four, five, six, or more than six sources.

[0734] In some embodiments, the Prokitein mixture comprises polypeptides derived from two, three, four, five, six, or more than six sources.

[0735] In some other embodiments, Prokitein comprises a Prokitein mixture.

[0736] Prokitein may additionally contain polysaccharides, lipids, polyols, and / or any combination thereof.

[0737] In some embodiments, Prokitein contains a polysaccharide.

[0738] In some embodiments, Prokitein contains lipids.

[0739] In some embodiments, Prokitein includes a polyol.

[0740] In some embodiments, Prokitein comprises polysaccharides and lipids.

[0741] In some embodiments, Prokitein comprises lipids and polyols.

[0742] In some embodiments, Prokitein comprises polysaccharides and polyols.

[0743] In some embodiments, Prokitein comprises polysaccharides, lipids, and polyols.

[0744] Methods for producing esters, thioesters, and / or amide-crosslinked polypeptides are discussed herein.

[0745] Polypeptides in hollow fibers may be crosslinked through esterification, thioesterification, and / or amidation reactions with polycarboxylic acids.

[0746] Polypeptides within hollow fibers may be crosslinked through eration, thioesterification, and / or amidation reactions with polycarboxylates containing, but not limited to, 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, and / or potassium malonate, and preferably sodium citrate and / or sodium malate.

[0747] Polycarboxylic acids having multiple carboxyl groups may be used to form covalent ester, thioester, or amide crosslinks between polypeptides (i.e., between two polypeptide chains) and / or within a polypeptide (i.e., between two locations along one polypeptide chain) (20).

[0748] In some embodiments, polycarboxylic acids having multiple carboxyl groups are used to form covalent esters, thioesters, or amide crosslinks between and / or within polypeptides.

[0749] Using polycarboxylic acids having multiple carboxyl groups, covalent ester crosslinks between and / or within polypeptides may be formed in the presence of a secondary catalyst such as sodium hypophosphite. In such cases, two carboxyl groups may first form a cyclic anhydride at high temperature (38;39). 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 form a polypeptide ester, thioester, or amide. A secondary addition reaction between the polycarboxyl derivative and sodium hypophosphite may then react with a second polypeptide ester, thioester, or amide to form an intermediate that may potentially give rise to crosslinking. Other reaction pathways using bifunctional polycarboxylic acids may be possible, and catalysts may also be used (23;40).

[0750] In some embodiments, polycarboxylic acids having multiple carboxyl groups are used to form covalent ester crosslinks between and / or within polypeptides in the presence of a secondary catalyst such as sodium hypophosphite.

[0751] Polycarboxylic acids having three or more carboxyl groups may be used to form covalent ester crosslinks between and / or within polypeptides. In such cases, two of the carboxyl groups on the polycarboxylic acid may first 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 form a polypeptide ester, thioester, or amide having two or more carboxyl groups. Two of the carboxyl groups on the polypeptide ester, thioester, or amide may then be dehydrated again to form a cyclic anhydride. Finally, the polypeptide ester having a cyclic anhydride may then react with a functional group on a second polypeptide chain to form an ester, thioester, or amide-crosslinked polypeptide derived from the polycarboxylic acid (20;41).

[0752] In some embodiments, polycarboxylate salts having three or more carboxyl groups are used to form covalent esters, thioesters, or amide crosslinks between and / or within polypeptides.

[0753] In some embodiments, polycarboxylate salts having three or more carboxyl groups are used to form covalent esters, thioesters, or amide crosslinks between and / or within polypeptides in the presence of a catalyst.

[0754] Using a polycarboxylic acid having three or more carboxyl groups, covalent ester crosslinks between and / or within polypeptides may be formed in the presence of a secondary catalyst such as sodium hypophosphite. In such cases, two carboxyl groups may be dehydrated first to form a cyclic anhydride. Subsequently, acylation between the anhydride derived from the polycarboxylic acid and sodium hypophosphite may lead to the formation of an intermediate. Then, a nucleophilic substitution reaction between the anhydride having one of the amine (-NH2), hydroxide (-OH), or thiol (-SH) functional groups present on the polypeptide chain and the sodium hypophosphite intermediate may produce a polypeptide ester, thioester, or amide having two or more carboxyl groups. Then, two of the carboxyl groups on the ester, thioester, or amide derived from the polypeptide polycarboxylic acid may be dehydrated again to form a polypeptide ester, thioester, or amide cyclic anhydride intermediate. When this intermediate is acylated with sodium hypophosphite, another intermediate may be formed, which may undergo nucleophilic substitution by another functional group present on the polypeptide chain to form a polycarboxylic acid-derived polypeptide ester, thioester, or amide crosslink and a rearrangement of sodium hypophosphite, which acts as a catalyst and is not consumed (22).

[0755] In some embodiments, catalysts used to catalytically influence the formation of covalent esters, thioesters, or amide crosslinks between and / or within polycarboxylic acid-derived polypeptides include, but are not limited to, sodium sulfite, sodium bisulfite, sodium pyrosulfite, and / or sodium hypophosphite.

[0756] Those skilled in the art may be able to determine the chemical composition and structure of polypeptides in the hollow fibers of the present disclosure, as well as polypeptides from which they are derived. Methods that may be used include mass spectrometry (MS), SDS-PAGE, Fourier transform infrared (FTIR) spectroscopy, circular dichroism, wide-angle X-ray scattering (WAXS), and Raman spectroscopy.

[0757] Those skilled in the art may determine the chemical composition and structure of polypeptides in Prokitein and polypeptides from which they are derived using the same methods applicable to hollow fibers in the present disclosure. Methods that may be used include mass spectrometry (MS), SDS-PAGE, Fourier transform infrared (FTIR) spectroscopy, circular dichroism, wide-angle X-ray scattering (WAXS), and Raman spectroscopy.

[0758] Mass spectrometry is commonly used to determine primary and higher-order protein structures. MS analysis is generally performed on protein samples that are either completely intact (top-down) or digested into fragments first (bottom-up). In a typical form of this type of analysis, both top-down and bottom-up, the sample is first converted into an ionized gas by methods such as electrospray ionization or matrix-assisted laser desorption / ionization (MADLI). The mass(s) of the ionized sample or sample fragment may then be determined using a time-of-flight (TOF) detector. The peptide sequence can then be determined by comparing the recorded sample fragment with the predicted peptide sequence mass. To avoid evaluating complex mixtures of proteins at once, the protein mixture may be separated before analysis using methods such as SDS-PAGE, high-performance liquid chromatography (HPLC), or gas chromatography (GC) (42).

[0759] Proteins may be separated based on molecular weight using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by molecular sieving provided by the variable pore size in the PAGE gel. SDS denatures and unravels most protein structures, eliminating differences in the secondary and tertiary structures of polypeptides and minimizing the effects of their surface charge differences. As a result, since the 1970s, SDS-PAGE has become one of the most widely used techniques for protein separation and characterization. The mass of unknown bands may be estimated by using standards of known mass. These estimated masses can then be cross-referenced with MS or Western blot data from the literature to identify bands isolated in the sample (43). By comparing the mass of expressed bands from a cross-linked polypeptide sample with the mass of bands from known polypeptides, the degree of cross-linking in a cross-linked polypeptide sample can be estimated by the multiplicity of the masses of known bands related to the bands in the known polypeptide sample.

[0760] For example, SDS-PAGE is used to identify polypeptides present in soy protein, which can be separated into four main groups called 2S, 7S, 11S, and 15S. Of the proteins identified during SPI, 7S globulin (beta-conglycinin) and 11S globulin (globulin) are the two most abundant. Beta-conglycinin, with a molecular weight of approximately 180 kDa, is a trimer glycoprotein consisting of three subunits: alpha (approximately 67 kDa), alpha' (approximately 71 kDa), and beta (approximately 50 kDa). Glycinin (approximately 350 kDa) is a hexamer consisting of five main subunits, two of which are basic (approximately 19.6 kDa) or acidic (approximately 34.8 kDa) (44;45). The degree of crosslinking in a soybean-derived crosslinked polypeptide sample can be estimated by comparing the estimated masses of bands identified in the sample with the multiplicity of these masses. Such a method may also be used to analyze polypeptides of other origins.

[0761] Representative samples of the material used to produce semipermeable hollow fibers may be taken, and the masses of the identified bands may be estimated by evaluating the samples using SDS-PAGE. These bands can then be identified by comparison with known masses from the literature to determine their components in the starting organic material. Bands with masses that do not correlate with those in the literature may be identified by mass spectrometry.

[0762] Representative samples of prokitein may be taken, and the masses of the identified bands may be estimated by evaluating the samples using SDS-PAGE. These bands can then be identified by comparison with known masses from the literature to determine their components in the starting organic material. Bands with masses that do not correlate with those in the literature may be identified by mass spectrometry.

[0763] Fourier transform infrared (FTIR) spectroscopy is one of the most commonly used methods for determining the chemical structure of molecules due to its low associated equipment costs and wide molecular size applicability. The chemical structure of a molecule is largely determined by the vibrational frequencies recorded in response to the strength of vibrational couplings and the mass of the vibrational atoms. However, while chemical structure cannot be determined from FTIR data, changes in molecular structure can. One of the most common applications of FTIR is the analysis of the secondary structure of proteins, which is largely determined through the evaluation of amide I, II, and III bands as well as the near-infrared region. The secondary structure is generally determined from these bands by one of two methods: (1) fitting the component bands to the amide I band, or (2) decomposing the amide I band into a ground spectrum determined from a calibration protein (46). For example, FTIR has been used to evaluate changes in the secondary structure of silk proteins upon exposure to organic solvents. The components of the secondary structure of silk proteins are determined at assigned wavelengths: beta-sheet (1616–1637 cm⁻¹). -1 and 1697~1703cm -1 ), random coil (1638~1655cm -1 ), Alpha Helix (1656~1662cm) -1 ) and Beta Turn (1663~1696cm) -1 ), quantified from the peak area (27;28). Therefore, FTIR may be used to evaluate the change in the secondary structure of polypeptides in the hollow fibers of the present disclosure.

[0764] Circular dichroism (CD) is an inexpensive and rapid method for evaluating the secondary structure of proteins based on the adsorption difference between left-handed and right-handed polarized light. Various structural elements of the polypeptide backbone interact with light, resulting in characteristic CD spectra in specific conformations. Specifically, alpha-helix proteins have negative bands at 222 nm and 208 nm, and a positive band at 193 nm. Proteins with beta-helices have a negative band at 218 nm and a positive band at 195 nm, while disordered polypeptides have very low ellipticity above 210 nm and a negative band around 195 nm. By evaluating the change in relative band intensity, changes in the prevalence of each of these secondary protein structures can generally be determined (47). Therefore, CD may be used to evaluate changes in the secondary structure of polypeptides of hollow fibers in this disclosure.

[0765] X-ray diffraction (XRD) is a well-established technique used to characterize the crystal structure of materials containing polypeptides (48). Wide-angle X-ray scattering can be used to determine the secondary structure of proteins, as the association of 9° and 20° peaks is associated with changes in the alpha-helix and beta-sheet, respectively (49). Therefore, XRD may be used to evaluate changes in the secondary structure of polypeptides in hollow fibers of the present disclosure.

[0766] Raman spectroscopy may be used to analyze polypeptide structures by utilizing several vibrational modes. The conformations of the secondary structures of different polypeptides may be identified by considering vibrational bands associated with amides A, B, and I-VII. The relative abundances of alpha-helices and beta-sheets in a polypeptide sample can be determined from the positions of amides I and III. Specifically, for amide I and amide III modes, the average wavelengths of the alpha-helices are 1662-1655 and 1272-1264 cm, respectively. -1 The average wavelengths of the beta-sheet structure are 1674–1672 and 1242–1227 cm. -1(50) Therefore, Raman spectroscopy may be used to evaluate the changes in the secondary structure of the polypeptide of the hollow fiber of the present disclosure.

[0767] FTIR, CD, XRD, and Raman spectroscopy may also be used to evaluate changes in the secondary structure of the Prokitein polypeptide.

[0768] Differential scanning calorimetry (DSC) can be used to characterize the thermal and conformational stability of proteins and other biomacromolecules. DSC measures heat capacity as a function of temperature to create a thermograph. The thermogram has three characteristic regions: (1) the annealing peak (enthalpy of crystallization, ΔH c (1) Determined from, (2) Melting peak (Enthalpy of melting, ΔH f (3) Melting point (T m ) may contain.

[0769] Crystal formation in the Prokitein and hollow fibers of this disclosure may be induced using annealing. During annealing, the sample is raised to a temperature between the glass transition temperature and the melting point, which may allow the molecular chains to be reconfigured into a more stable form with reduced internal stress. Upon subsequent cooling, the molecular chains may be frozen in place.

[0770] When annealed samples are analyzed by DSC, they may show an increase in melting temperature, an increase in crystallization temperature, a sharper and more prominent melting peak, and / or a decrease in the number of melting peaks. Depending on the conditions used, re-annealing an already annealed sample may result in only limited changes to the thermal properties of the sample, since the crystalline structure is already somewhat stable (i.e., internal stresses have decreased). Therefore, those skilled in the art may be able to use DSC to assess whether polymers such as Prokitein or hollow fibers of this disclosure have been annealed.

[0771] Those skilled in the art may also use XRD to assess the crystallinity of the Prokitein and / or hollow fibers of this disclosure. In XRD, crystalline regions typically produce sharp peaks, while amorphous regions usually contribute to a broad, diffused background. Crystallinity may also be assessed by integrating the relative magnitudes of the peaks in the XRD spectrum. Semicrystalline polymers such as Prokitein may yield XRD spectra that include peaks associated with crystalline and amorphous regions, respectively, and a broad, diffused background. The internal stress of the Prokitein and hollow fibers of this disclosure can be assessed using Bragg's law, peak shift analysis, and sine wave analysis related to the tilt angle. 2 The characteristics may also be evaluated by analysis of the XRD spectrum using the ψ method and / or a method including fitting of the entire powder pattern. Furthermore, the uniform distribution of grain size and shape can be determined by those skilled in the art through analysis of peaks in the XRD spectrum.

[0772] In some embodiments, the XRD spectrum of Prokitein includes sharp peaks and a broad diffuse background.

[0773] In some embodiments, Prokitein is almost perfectly crystalline.

[0774] In some embodiments, Prokitein is almost completely amorphous.

[0775] In some embodiments, the crystallinity of Prokitein is greater than 0%.

[0776] In some embodiments, the crystallinity of Prokitein is higher than 5%.

[0777] In some embodiments, the crystallinity of Prokitein is higher than 10%.

[0778] In some embodiments, the crystallinity of Prokitein is higher than 20%.

[0779] In some embodiments, the crystallinity of Prokitein is higher than 30%.

[0780] In some embodiments, the crystallinity of Prokitein is higher than 40%.

[0781] In some embodiments, the crystallinity of Prokitein is higher than 50%.

[0782] In some embodiments, the crystallinity of Prokitein is higher than 60%.

[0783] In some embodiments, the crystallinity of Prokitein is higher than 70%.

[0784] In some embodiments, the crystallinity of Prokitein is higher than 80%.

[0785] In some embodiments, the crystallinity of Prokitein is higher than 90%.

[0786] In some embodiments, the crystallinity of Prokitein is higher than 95%.

[0787] In some embodiments, the degree of crystallinity of Prokitein is in 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%, or 99.99%.

[0788] The ratio of crystalline regions to amorphous regions within Prokitein may be modified to adapt its material properties. Crystalline materials are typically hard, robust, and brittle, while amorphous materials are flexible. The ratio of crystalline regions to amorphous regions within Prokitein can be adapted by the annealing temperature and time.

[0789] In some embodiments, the ratio of crystalline regions to amorphous regions within Prokitein is modified and / or adapted by protein annealing.

[0790] Functional properties of covalently crosslinked porous semipermeable hollow fibers A characteristic feature of certain aspects and embodiments of the present disclosure is that the hollow fibers of the present disclosure remain mechanically stable while immersed in an aqueous solution for at least one day, preferably at least three days.

[0791] A characteristic feature of certain aspects and embodiments of the Disclosure is that one or more of the described process steps, particularly steps (d) and (ei), facilitate the formation of a beta-sheet structure within the polypeptide of the hollow fiber of the Disclosure. The formation of a beta-sheet in the secondary structure of the polypeptide imparts a certain degree of water stability to the hollow fiber produced in the Disclosure.

[0792] A characteristic feature of certain aspects and embodiments of the present disclosure is that the described process steps, in particular one or more of steps (d), (ei), and (e.ii), are performed sequentially in the specified order. Step (ei) may promote the formation of beta-sheets within the secondary protein structure of the crosslinked polypeptide of the hollow fibers of the present disclosure, thereby conferring some degree of water stability. Step (e.ii) may anneal the covalently crosslinked hollow fibers, thereby relieving internal stress in the crosslinked material, which may allow the beta-sheet structure to remain intact when immersed in an aqueous solvent, thereby preventing hydrolysis of the covalent ester crosslinks.

[0793] The hollow fibers of this disclosure can maintain good mechanical properties after being continuously immersed in an aqueous solution.

[0794] In some embodiments, the hollow fibers of the present disclosure maintain good mechanical properties after continuous immersion in an aqueous solution. In particular, at least one of the Young's modulus, ultimate tensile strength, and / or ultimate tensile strain of the hollow fibers of the present disclosure does not decrease by more than 60% after immersion in an aqueous solution such as PBS at a temperature of 18°C ​​to 38°C and pH 7.0 to 8.0 for 3 days, and is measured immediately after removal from the aqueous solution, preferably after 5 days, more preferably after 10 days, even more preferably after 15 days, and most preferably after 30 days.

[0795] In another embodiment, the hollow fibers of the present disclosure maintain good mechanical properties after continuous immersion in an aqueous solution. In particular, when measured by SEM and / or mercury porosimetry, the wall thickness, pore diameter, and pore void fraction of the hollow fibers produced in the present disclosure do not change by more than 30% after immersion in an aqueous solution such as PBS at a temperature of 18°C ​​to 38°C and pH 7.0 to 8.0 for 3 days, preferably after 5 days, more preferably after 10 days, even more preferably after 15 days, and most preferably after 30 days.

[0796] When evaluating the functional mechanical properties of a material by applying tensile force to a sample, the resulting displacement can be recorded as a function of the force applied until the sample fractures, using a universal uniaxial tensile test. The mechanical properties of the material can be calculated from the measured relationship between the tensile force applied to the sample and the resulting displacement. Properties that can be calculated by knowing the cross-sectional area of ​​the test material when measured by SEM include the yield point, ultimate tensile strength, ultimate tensile strain, and Young's modulus.

[0797] Ultimate tensile strength is the maximum force applied to a material before it fractures. Ultimate tensile strain is the elongation of the material at the fracture point. Young's modulus is the ratio of the change in stress to the change in strain before the yield point. The yield point is the point at which the relationship between stress and strain is no longer directly proportional. By evaluating these properties, the mechanical profile of the hollow fibers of this disclosure can be determined (51).

[0798] The yield point, ultimate tensile strength, ultimate tensile strain, and Young's modulus of prokitein can also be evaluated by universal uniaxial tensile testing.

[0799] In yet another embodiment, the hollow fibers of the present disclosure maintain good aqueous fluid transport properties after being immersed in an aqueous solution. In particular, the PWP of the hollow fibers produced in the present disclosure does not decrease by more than 60% and does not increase by more than 20% after being immersed in an aqueous solution such as phosphate buffer (PBS) at a temperature of 18°C ​​to 38°C and a pH of 7.0 to 8.0 for 3 days, preferably after 5 days, more preferably after 10 days, even more preferably after 15 days, and most preferably after 30 days.

[0800] In yet another further embodiment, the hollow fibers of the present disclosure maintain good mass transport properties after immersion in an aqueous solution. In particular, the molecular weight cutoff and particle retention size of the hollow fibers produced in the present disclosure do not decrease by more than 60% and do not increase by more than 20% after immersion in an aqueous solution such as phosphate buffer (PBS) at a temperature of 18°C ​​to 38°C and pH 7.0 to 8.0 for 3 days, preferably after 5 days, more preferably after 10 days, even more preferably after 15 days, and most preferably after 30 days.

[0801] Treatment of hollow fibers to modify their mechanical properties may be performed either before and / or after culturing cells on the hollow fibers.

[0802] In some embodiments, treatment of the hollow fibers to modify their mechanical properties is carried out before culturing cells on the hollow fibers.

[0803] In some embodiments, the treatment of hollow fibers to modify their mechanical properties is carried out after culturing cells on the hollow fibers.

[0804] In some embodiments, no treatment of the hollow fibers is performed to modify their mechanical properties.

[0805] In some embodiments, the hollow fibers of the present disclosure remain stable while immersed in an aqueous solution such as phosphate buffer (PBS) at a temperature of 18°C ​​to 38°C and a pH of 7.0 to 8.0 for 3 days, and as a result, the hollow fibers maintain good mechanical properties, structural characteristics, aqueous fluid transport properties, and mass transport properties for at least 3 days, preferably 5 days, more preferably 10 days, even more preferably 15 days, and most preferably 30 days.

[0806] In some embodiments, Prokitein remains mechanically stable while immersed in an aqueous solution for at least 1 day, preferably at least 3 days, more preferably at least 5 days, even more preferably at least 10 days, and even more preferably at least 15 days, most preferably at least 30 days.

[0807] In some embodiments, Prokitein remains mechanically stable while immersed in an aqueous solution for at least one month, preferably at least three months, and most preferably at least six months.

[0808] In some embodiments, Prokitein maintains good mechanical properties after continuous immersion in an aqueous solution. In particular, at least one of Prokitein's Young's modulus, ultimate tensile strength, and / or ultimate tensile strain does not decrease by more than 60% after immersion in an aqueous solution such as PBS at a temperature of 18°C ​​to 38°C and pH 7.0 to 8.0 for 3 days, and is measured immediately after removal from the aqueous solution, preferably after 5 days, more preferably after 10 days, even more preferably after 15 days, and most preferably after 30 days.

[0809] In one embodiment, the hollow fibers of the present disclosure are edible.

[0810] In one embodiment, the Prokitein of this disclosure is edible.

[0811] In some embodiments, the hollow fibers of the present disclosure are suitable for human and / or animal ingestion.

[0812] In some embodiments, the Prokitein of this disclosure is suitable for human and / or animal administration.

[0813] In some embodiments, the hollow fibers of the present disclosure are GRAS (Generally Recognized as Safe).

[0814] In some embodiments, the hollow fibers of the present disclosure are produced solely from components that are GRAS (Generally Recognized as Safe).

[0815] In some embodiments, the Prokitein of this disclosure is generated solely from GRAS components.

[0816] In some embodiments, the hollow fibers of the present disclosure are partially generated from a component that is GRAS (Generally Recognized as Safe).

[0817] In some embodiments, the Prokitein of this disclosure is partially derived from a component that is GRAS (Generally Recognized as Safe).

[0818] In some embodiments, the hollow fibers of the present disclosure have a good taste.

[0819] In some embodiments, the Prokitein of this disclosure has a good taste.

[0820] In some embodiments, the hollow fibers of the present disclosure constitute food.

[0821] In some embodiments, the Prokitein of this disclosure constitutes a food product.

[0822] In some embodiments, the hollow fibers of the present disclosure are food products.

[0823] In some embodiments, the Prokitein of this disclosure is a food product.

[0824] In some embodiments, the hollow fibers of the present disclosure are components of food.

[0825] In some embodiments, the Prokitein of this disclosure is a food ingredient.

[0826] In some embodiments, the hollow fibers of the present disclosure are biodegradable.

[0827] In some embodiments, the Prokitein of this disclosure is biodegradable.

[0828] In some embodiments, the term “edible” is defined as material suitable for consumption by humans and / or animals.

[0829] In some other embodiments, the term “edible” refers to material that is suitable for consumption by humans and / or animals and can be digested as food.

[0830] While it's impossible to guarantee that a particular food product poses absolutely no risk to consumers, various risk management frameworks exist to mitigate potential risks. These are embodied in pre-market approval decisions by food safety authorities.

[0831] In the European Union, this is replaced by a pre-market approval decision, as described by the European Food Safety Authority (EFSA) Novel Food Regulation (EU) 2015 / 2283. Certain foods are considered “new” if they fall into several defined categories and have not been significantly consumed in the EU prior to May 15, 1997. Defined categories include foods with new or intentionally modified molecular structures that have not been used in food or as food in the EU prior to May 15, 1997. In some embodiments, the term “edible” refers to materials that are qualified as food by the European Food Safety Authority. In some embodiments, the term “edible” refers to materials that are qualified as food by the European Food Safety Authority as of January 1, 2023.

[0832] Similarly, the United States Food and Drug Administration (FDA) designates foods or food additives as “generally recognized as safe” (GRAS) for consumption, based on consensus among qualified experts. This designation can be obtained in two ways: (i) long-term, sustained use (the food has been widely consumed since before 1958 and there have been no reports of adverse effects), or (ii) a series of scientific safety studies and subsequent review by the FDA of the data obtained. As used herein, the terms “generally recognized as safe” and “GRAS” are interchangeable and refer to materials suitable for consumption by humans or animals. In some embodiments, “GRAS” refers to materials that qualify as GRAS in accordance with the United States Food and Drug Administration. In some embodiments, “GRAS” refers to materials that qualify as GRAS in accordance with the United States Food and Drug Administration as of January 1, 2023. In some embodiments, the term “edible” refers to materials that are GRAS.

[0833] In some embodiments, "edible" refers to ingredients that are rated as food by the European Food Safety Authority and are also GRAS (Generally Recognized as Safe).

[0834] The EFSA has published guidance on food safety assessments under Novel Food Regulation (EU) 2015 / 2283. These regulations mandate scientific data on the kinetics and toxicity of proposed novel foods, including an enumeration of the novel food's identity, a detailed description of the production process, compositional information, stability data, absorption, distribution, metabolism and excretion (ADME) data, as well as nutritional and toxicological information. Toxicological data should include details of expected use, kinetic data, toxin data for constituent materials, human trial data, and available relevant information.

[0835] Petrochemical polymers and ceramic hollow fibers have not been widely consumed in the EU since before 1997, nor are they currently consumed in the US since before 1958. They are not specifically designated as novel foods by the EFSA or as GRAS by the FDA. These materials may not be safe to consume.

[0836] In some preferred embodiments, the hollow fibers of the present disclosure are non-cytotoxic.

[0837] The edible extruded or spun fibers of this disclosure may comprise covalent esters, thioesters, and / or amide-crosslinked polypeptides and may be used as substrates for cell growth in cell culture or in bioreactors. To ensure that the edible hollow fibers produced in this disclosure are suitable for cell culture, they are non-cytotoxic in certain embodiments.

[0838] The International Organization for Standardization (ISO) standard ISO 10993-1:2018 (Biological evaluation of medical devices) is accredited for the selection of biological materials by specifying test procedures for demonstrating carcinogenicity, genotoxicity, hematological compatibility, cytotoxicity, transplantability, sensitization, irritation, and acute, subacute, subchronic, and chronic toxicity. Cytotoxic effects can be determined by either qualitative or quantitative methods. However, quantitative methods are preferred, and qualitative methods should be used only for screening purposes. Accordingly, the materials produced in this disclosure may be classified as non-cytotoxic because, in some embodiments, they are quantified by one of the methods outlined in supplementary documents A to D of ISO 10993-5 (Biological evaluation of medical devices - Part 5: In vitro cytotoxicity testing).

[0839] In some preferred embodiments, the outer or inner surface of the hollow fibers of the present disclosure can act as a substrate for cell adhesion and proliferation.

[0840] In some embodiments, the hollow fibers of the present disclosure can sustain cell culture for at least 1 day, preferably at least 3 days, more preferably at least 5 days, even more preferably at least 10 days, even more preferably at least 15 days, and most preferably at least 30 days.

[0841] In some other embodiments, the hollow fibers of the present disclosure are used for 1 second to 1 minute, 1 minute to 1 hour, 1 hour to 6 hours, 6 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, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 11 days, 11 days to 12 days, 12 days to 13 days, 13 days to 14 days, 14 days to 15 days, and 15 days. Cell culture can be sustained for a period of time within the range of 1 to 16 days, 16 to 17 days, 17 to 18 days, 18 to 19 days, 19 to 20 days, 20 to 21 days, 21 to 22 days, 22 to 23 days, 23 to 24 days, 24 to 25 days, 25 to 26 days, 26 to 27 days, 27 to 28 days, 28 to 29 days, 29 to 30 days, or more than 30 days.

[0842] Since the substrates are characterized in relation to their cytotoxicity, they may be suitable for cell proliferation. ISO 10993 certifies standardized methods for determining the cytotoxicity of materials and means by which materials should be tested according to the structure of the material. A material may be considered non-cytotoxic only if it meets the test protocols indicated by ISO 10993 (52). Those skilled in the art may evaluate the cytotoxicity of extruded or spun fibers of the present disclosure, including covalent esters, thioesters, and / or amide-crosslinked polypeptides, using the methods outlined in ISO 10993.

[0843] In some embodiments, the hollow fibers of the present disclosure are non-cytotoxic, as determined by the method described in ISO 10993.

[0844] Cell proliferation is defined as an increase in cell number as a result of cell proliferation and division. The cell number in a given sample can be quantified directly and / or indirectly by those skilled in the art using several online and offline methods. Direct cell counting is the most common method, performed under a microscope, manually or using a hemocytometer. Other direct methods may include flow cytometry and spectrophotometry. Alternatively, indirect methods may be employed, such as recording trends in the concentrations of metabolites (e.g., biomass, glucose, ethanol, lactic acid, acetic acid, succinic acid, and citrate) and dissolved gases (e.g., oxygen, carbon dioxide, and ammonia). Cell number can also be measured indirectly by metabolic assays such as Alamar blue or CellTiter Glo. Furthermore, samples may be frozen to obtain a snapshot of the culture performance at a given point in time. These samples may then be imaged using techniques such as confocal microscopy, scanning electron microscopy, or histology. Such methods may be applied to evaluate the proliferation of cells grown on the hollow fibers of this disclosure.

[0845] Furthermore, cell counts can also be indirectly measured by metabolic assays utilizing solutions containing PrestoBlue, PrestoBlue High Sensitivity, and / or resazurin salts.

[0846] 1.20 Bioreactor A characteristic feature of certain aspects and embodiments of the present disclosure is that the hollow fibers of this disclosure are suitable for use as components in bioreactors intended for use in cell culture.

[0847] In some embodiments, the hollow fibers of the present disclosure are suitable for use as components in multimodal, mechanically dynamic bioreactors intended for use in cell culture.

[0848] In some embodiments, the hollow fibers of the present disclosure are suitable for use as components in hydrostatic cell cultures such as well plates or flasks.

[0849] As used herein, the term “multimodal” refers to providing chemical, electrical, and / or mechanical stimuli to cultured cells. As used herein, the term “mechanically dynamic” refers to the function of a bioreactor that applies controlled mechanical forces to cultured cells. Such conceivable multimodal mechanically dynamic bioreactors may be used under dynamic fluid and / or mechanically dynamic conditions in which the bioreactor platform and / or cell substrate is either stretched or compressed.

[0850] A characteristic feature of certain aspects and embodiments of the disclosure is that the hollow fibers of the disclosure are suitable for consumption alone or in combination with products intended for ingestion by humans and / or animals.

[0851] In some embodiments, the hollow fibers of the present disclosure are non-cytotoxic and can be used to maintain cell proliferation for at least three days.

[0852] In some other embodiments, the hollow fibers of the present disclosure are suitable for use in the production of one or more cultured meat products intended for consumption in humans and / or animals.

[0853] In some embodiments, the fibers of the present disclosure are suitable for use as components in a bioreactor.

[0854] In some other embodiments, one or more of the hollow fibers of the present disclosure are used in a bioreactor.

[0855] In some embodiments, the Disclosure provides a bioreactor comprising one or more of the hollow fibers of the Disclosure.

[0856] In some other embodiments, a bioreactor comprising one or more hollow fibers of the present disclosure is suitable for promoting cell culture.

[0857] In further embodiments, a bioreactor comprising one or more hollow fibers of the present disclosure is suitable for use in the production of one or more cultured meat products intended for human and / or animal consumption.

[0858] 1.21 Cartridge The hollow fibers of the present disclosure are suitable for use as components within bioreactor cartridges, and each cartridge containing one or more of the hollow fibers of the present disclosure is a characteristic feature of certain aspects and embodiments of the present disclosure.

[0859] The cartridges of the present disclosure may include one or more of the hollow fibers of the present disclosure, aligned parallel to the long axis of the hollow fibers and separated by an outer wall having an inlet port and an outlet port. A schematic diagram of a quarter of the cross-section of such a cartridge may be shown in Figure 5. In Figure 5, typical components of the cartridge are labeled, including the inlet to the cartridge (5001), the medium dissipation cut section of the cartridge cap (5002), the cartridge cap (5003), the hollow fiber potting layer (5004), the bundle of hollow fibers (5005), and the outer shell of the cartridge (5006).

[0860] In the cartridge of the present disclosure, the intracapillary (IC) space may be located within the lumen of the hollow fiber of the present disclosure, and the extracapillary (EC) space may be located surrounding the hollow fiber and separated by the outer shell of the cartridge.

[0861] In some embodiments, the cartridge of the present disclosure has an intracapillary (IC) space within the lumen of the hollow fiber of the present disclosure, and an extracapillary (EC) space surrounding the hollow fiber, separated by the outer shell of the cartridge.

[0862] Claw's cylindrical model is commonly used to model the distance between capillaries in vivo before oxygen concentration restricts cell proliferation (53). Claw's cylindrical model suggests that hollow fibers should be bundled so that the distance is less than 100 μm (54).

[0863] In some embodiments, the shortest distance between any two adjacent fibers of the present disclosure in a particular bundle of cartridges is in the range of 0 μm to 150 μm, preferably in the range of 20 μm to 200 μm, more preferably in the range of 50 μm to 130 μm, even more preferably in the range of 80 μm to 120 μm, and most preferably in the range of 50 μm to 130 μm.

[0864] In some embodiments, the shortest distance between any two adjacent fibers of the present disclosure in a particular bundle of cartridges is within the range of 0μm~10μm, 10μm~20μm, 20μm~30μm, 30μm~40μm, 40μm~50μm, 50μm~60μm, 60μm~70μm, 70μm~80μm, 80μm~90μm, 90μm~100μm, 110μm~120μm, 120μm~130μm, 130μm~140μm, 140μm~150μm, 150μm~160μm, 160μm~170μm, 170μm~180μm, 180μm~190μm, and / or 190μm~200μm.

[0865] In some other embodiments, the shortest distance between any two adjacent fibers of the present disclosure in a particular bundle of cartridges is within the range of 200 μm to 250 μm, 250 μm to 300 μm, 300 μm to 350 μm, 350 μm to 400 μm, 400 μm to 450 μm, 450 μm to 500 μm, 500 μm to 550 μm, 550 μm to 600 μm, 600 μm to 650 μm, 650 μm to 700 μm, 700 μm to 750 μm, 750 μm to 800 μm, 800 μm to 850 μm, 850 μm to 900 μm, 900 μm to 950 μm, and / or 950 μm to 1000 μm.

[0866] In some embodiments, the cartridge of the present disclosure comprises a plurality of hollow fibers of the present disclosure arranged in one or more bundles, with a density of 1 hollow fiber / cm³. 2 ~1000 hollow fibers / cm 2 Preferably, 60 hollow fibers / cm 2 ~500 hollow fibers / cm 2Most preferably, 80 hollow fibers / cm 2 ~300 hollow fibers / cm 2 It is within the range.

[0867] In some other embodiments, the cartridge of the present disclosure comprises a plurality of hollow fibers of the present disclosure arranged in one or more bundles, with a density of 25 hollow fibers / cm³. 2 ~150 hollow fibers / cm 2 Preferably, 50 hollow fibers / cm 2 ~125 hollow fibers / cm 2 , more preferably 75 hollow fibers / cm 2 ~105 hollow fibers / cm 2 Most preferably, 70 hollow fibers / cm 2 ~95 hollow fibers / cm 2 It is within the range.

[0868] In some other embodiments, the cartridge of the Disclosure comprises a plurality of hollow fibers of the Disclosure, wherein these hollow fibers are 40 hollow fibers / cm². 2 ~60 hollow fibers / cm 2 , 60 hollow fibers / cm 2 ~80 hollow fibers / cm 2 , 80 hollow fibers / cm 2 ~100 hollow fibers / cm 2 , 100 hollow fibers / cm 2 ~120 hollow fibers / cm 2 , 120 hollow fibers / cm 2 ~180 hollow fibers / cm 2 , 180 hollow fibers / cm 2 ~220 hollow fibers / cm 2 , 220 hollow fibers / cm 2 ~250 hollow fibers / cm 2 , 250 hollow fibers / cm 2 ~300 hollow fibers / cm 2 300 hollow fibers / cm 2 ~350 hollow fibers / cm 2 350 hollow fibers / cm 2 ~400 hollow fibers / cm 2 , 400 hollow fibers / cm 2 ~450 hollow fibers / cm 2 , 450 hollow fibers / cm2 ~500 hollow fibers / cm 2 , 500 hollow fibers / cm 2 ~600 hollow fibers / cm 2 , 600 hollow fibers / cm 2 ~800 hollow fibers / cm 2 , and / or 800 hollow fibers / cm 2 ~1000 hollow fibers / cm 2 They are arranged in one or more bundles having a density within the range.

[0869] To ensure the integrity of the bundle and to ensure that the fluid entering the cartridge flows through the intercapillary space rather than the extracapillary space of the hollow fiber cartridge, the hollow fibers may be fixed within a non-cytotoxic matrix through a process called potting. Potting agents that may be used to ensure the integrity of the bundle include, but are not limited to, epoxy, plaster, gypsum, silicone rubber, and polytetrafluoroethylene.

[0870] In some embodiments, the potting agent used to ensure bundle integrity comprises a crosslinked polypeptide and / or polysaccharide.

[0871] In some embodiments, the potting agent used to ensure bundle integrity comprises crosslinked polypeptides and / or polysaccharides, including covalent erates, thioerates, and / or amide crosslinks.

[0872] In some embodiments, the cartridge of the present disclosure comprises hollow fibers of the present disclosure that are potted with a potting agent.

[0873] Potting is a process that can seal the ends of hollow fibers with a matrix to create a tight fluid seal between the hollow fibers and the outer wall separating them from the capsule. This process is typically necessary to prevent fluid from bypassing the hollow fibers and flowing into the space outside the capillary, and instead to push the fluid into the space inside the capillary.

[0874] The potting agent is a sealing matrix used in the potting process.

[0875] In some embodiments, the hollow fiber cartridge is assembled by reversibly enclosing one or more bundles of the hollow fibers of the present disclosure within an outer shell and adding appropriate end caps.

[0876] In some other embodiments, the cartridge is easily disassembled.

[0877] In further embodiments, individual, multiple, and / or bundles of the hollow fibers of the present disclosure are removable from the cartridge assembly.

[0878] In further embodiments, the individual, multiple, or bundled hollow fibers of the present disclosure cannot be removed from the cartridge assembly.

[0879] In some embodiments, individual, multiple, or bundles of the hollow fibers of the present disclosure are removable from the cartridge assembly to which the cells are attached.

[0880] In some embodiments, the cultured meat product containing the hollow fibers of the present disclosure is removable from the cartridge assembly.

[0881] In some preferred embodiments, the cultured meat product containing the hollow fibers of the present disclosure may be further processed to improve, preserve, and / or modify the flavor, mouthfeel, taste, and / or texture of the cultured meat product before it is removed from the cartridge assembly.

[0882] The inner diameter of the outer shell of each bioreactor cartridge in this disclosure may be in the range of 1 cm to 5 mm.

[0883] In some embodiments, the inner diameter of the outer shell of each bioreactor cartridge of the present disclosure is in the range of 1 cm to 50 cm, preferably 10 cm, more preferably 3 cm, and most preferably 7.5 cm.

[0884] In other embodiments, the inner diameter of the outer shell of each bioreactor cartridge of the present disclosure is 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, 45 cm, 50 cm, or any integer and decimal value in between.

[0885] In some embodiments, the inner diameter of the outer shell of each bioreactor cartridge of the present disclosure is in the range of 1 cm to 500 cm, preferably 10 cm and 300 cm, more preferably 30 cm and 200 cm, and most preferably 50 cm and 100 cm.

[0886] In some other embodiments, the inner diameter of the outer shell of each bioreactor cartridge of the present disclosure is 1cm-5cm, 5cm-10cm, 10cm-15cm, 15cm-20cm, 20cm-25cm, 25cm-30cm, 30cm-35cm, 35cm-40cm, 40cm-45cm, 45cm-50cm, 50cm-55cm, 55cm-60cm, 60cm-65cm, 65cm-70cm, 70cm-75cm, 75cm-80cm, 80cm-85cm, 85cm-90cm, 90cm-95cm, 95cm-100cm, 100cm-1 05cm, 105cm~110cm, 110cm~115cm, 115cm~120cm, 120cm~125cm, 125cm~130cm, 130cm~135cm, 135cm~140cm, 140cm~145cm, 145cm~150cm, 150cm~155cm, 155cm~160cm, 160cm~165cm, 165cm~170cm, 170cm~175cm, 175cm~180cm, 180cm~185cm, 185cm~190cm, 190cm~195cm, 195cm~200cm, 200cm~205cm, 205cm ~210cm, 210cm~215cm, 215cm~220cm, 220cm~225cm, 225cm~230cm, 230cm~235cm, 235cm~240cm, 240cm~245cm, 245cm~250cm, 250cm~255cm, 255cm~260 cm, 260cm~265cm, 265cm~270cm, 270cm~275cm, 275cm~280cm, 280cm~285cm, 285cm~290cm, 290cm~295cm, 295cm~300cm, 300cm~305cm, 305cm~310cm, 31 0cm~315cm, 315cm~320cm, 320cm~325cm, 325cm~330cm, 330cm~335cm, 335cm~340cm, 340cm~345cm, 345cm~350cm, 350cm~355cm, 355cm~360cm, 360cm~3 65cm, 365cm~370cm, 370cm~375cm, 375cm~380cm, 380cm~385cm, 385cm~390cm, 390cm~395cm, 395cm~400cm, 400cm~405cm, 405cm~410cm, 410cm~415cm,The height range is within the following categories: 415cm~420cm, 420cm~425cm, 425cm~430cm, 430cm~435cm, 435cm~440cm, 440cm~445cm, 445cm~450cm, 450cm~455cm, 455cm~460cm, 460cm~465cm, 465cm~470cm, 470cm~475cm, 475cm~480cm, 480cm~485cm, 485cm~490cm, 490cm~495cm, or 495cm~500cm.

[0887] Cartridges for assemblies of petrochemical polymer hollow fibers are well known in the art, as disclosed in Japanese Patents US6,001,585,A and US2012 / 0308531.

[0888] The hollow fibers or cartridges of the present disclosure are not limited to any particular bioreactor configuration in their use. In a preferred bioreactor configuration, the supply of culture medium and process control are suitable for enabling confluence of cell culture.

[0889] In some embodiments, the hollow fiber cartridges of the present disclosure can be used in existing commercially available reactor platforms, including, but not limited to, systems from Terumo BCT Quantum®, FiberCell® Systems Duet Pump, Sartorius Sartoflow®, and Cell Culture Company (HF Primer®, AlutovaxID®, AcuSyst-Maximizer®, or AcuSyst-Xcellerator®).

[0890] In some embodiments, the hollow fiber cartridges of the present disclosure can be used in a bioreactor platform supporting a single cartridge.

[0891] In some embodiments, the hollow fiber cartridges of the present disclosure can be used in a bioreactor platform supporting multiple cartridges.

[0892] A bioreactor platform capable of supporting cell culture using hollow fibers or cartridges fabricated therefrom may have a housing sized to accommodate one or more cartridges. The platform may regulate environmental conditions within the housing so that the temperature, humidity, and gas composition of the enclosed atmosphere are controlled. The cartridges within the platform may be connected to an inlet feed for culture medium. The inlet feed line may pass through a pump, thereby allowing the rate of the inlet feed to be regulated. The outlet of the cartridge may be connected to a waste reservoir. Alternatively, the outlet of the cartridge may be connected to an inlet so that culture medium recycling is established. The platform may be fitted with sensors for detecting and measuring various culture process parameters, including, but not limited to, temperature, pH, flow rate, system weight, and concentrations of lysed metabolites (e.g., oxygen, carbon dioxide, glucose, lactate, etc.). Furthermore, a control system may be suitable for automating the operation of the bioreactor platform and may be connected to a sensor array. Such control systems can be controlled locally through the use of a human-machine interface (HMI) and / or remotely through installation on a broader control and monitoring network (e.g., SCADA or DeltaV).

[0893] Furthermore, the bioreactor platform may include one or more ports from which materials can be added or removed.

[0894] In some embodiments, a bioreactor platform capable of supporting cell culture using hollow fibers or cartridges fabricated therefrom has a housing sized to accommodate one or more cartridges.

[0895] In some embodiments, the platform adjusts the environmental conditions within the enclosure so that the temperature, humidity, and gas composition of the enclosed air are controlled.

[0896] In some embodiments, each cartridge is connected to one or more inlet feeds of the culture medium. The inlet feed line(s) passes through one or more pumps, thereby allowing the rate of the inlet feed to be adjusted.

[0897] In some embodiments, each cartridge is connected to one or more inlets supplied from one or more fresh culture medium reservoirs.

[0898] In some embodiments, the outlet of each cartridge is connected to one or more waste reservoirs.

[0899] In some embodiments, the outlet of each cartridge is connected to the inlet of the same cartridge so that the recycling of the culture medium is established.

[0900] In some embodiments, the outlet of each cartridge is connected to one or more waste reservoirs and the inlet of the same cartridge so that partial recycling of the culture medium is established.

[0901] In some embodiments, the outlet of each cartridge is connected to one or more waste reservoirs and intermediate reservoirs. The rate of outlet flow from the cartridge to the intermediate reservoir(s) is indicated by the recirculation rate.

[0902] In some embodiments, the cartridge is connected to one or more inlets supplied from one or more intermediate culture medium reservoirs. Each inlet feed is supplied with a mixture of fresh medium and recycled medium, the ratio of which is indicated by one or more recycling rates.

[0903] In some embodiments, the bioreactor platform is fitted with sensors for detecting and measuring temperature.

[0904] In some embodiments, the bioreactor platform is equipped with sensors for detecting and measuring pH.

[0905] In some embodiments, the bioreactor platform is fitted with sensors for detecting and measuring flow rate.

[0906] In some embodiments, the bioreactor platform is fitted with sensors for detecting and measuring the system weight.

[0907] In some embodiments, the bioreactor platform is equipped with sensors for detecting and measuring the concentration(s) of dissolved metabolites(s) (e.g., oxygen, carbon dioxide, glucose, lactate, etc.).

[0908] In some embodiments, a control system is attached to the bioreactor platform.

[0909] In some embodiments, the bioreactor platform is fitted with a control system for partially automating the operation of the bioreactor platform.

[0910] In some embodiments, the bioreactor platform is fitted with a control system for automating the operation of the bioreactor platform.

[0911] In some embodiments, the bioreactor platform is fitted with a control system equipped with a sensor array.

[0912] In some embodiments, the bioreactor platform is fitted with a control system that can be controlled locally using a human-machine interface (HMI).

[0913] In some embodiments, the bioreactor platform is fitted with a control system that can be controlled locally by the use of a human-machine interface (HMI) and / or remotely by installation on a broader control and monitoring network (e.g., SCADA or DeltaV).

[0914] In some embodiments, the bioreactor platform is connected to a cell retention device such as XCell® ATF6, but is not limited to XCell® ATF6.

[0915] In some embodiments, the bioreactor platform includes one or more ports through which material can be added or removed.

[0916] In some embodiments, the inner and / or outer surfaces of the hollow fibers of the present disclosure can act as a substrate for cell adhesion and proliferation, particularly during the production of cultured meat products.

[0917] In some embodiments, the types of cells cultured on the hollow fibers of the Disclosure, either alone or in combination, include, but are not limited to, satellite cells, mesenchymal stem cells, induced pluripotent stem cells, myocytes, fibroblasts, adipocytes, or engineered cells.

[0918] Other cultured cells suitable for use in the production of cultured meat products include, but are not limited to, satellite cells, mesenchymal stem cells, induced pluripotent stem cells, myocytes, fibroblasts, adipocytes, or engineered cells derived from animal sources. Examples of such animal sources include, but are not limited to, the following: Mammals: cattle, sheep, pigs, horses, goats, deer, reindeer, bison, alpacas, llamas, moose, elk, camels, wild boars, buffalo, wildebeest, whales, dolphins, and / or guinea pigs; Marsupials: kangaroos, koalas, wombats; Birds: chickens, turkeys, ducks, geese, quail, peacocks, pheasants, guinea fowl, ostriches, emus, pigeons, partridges, ptarmigans, sandpipers, and / or woodcocks; Fish: salmon, tuna, cod, trout, sardines, haddock, tilapia, catfish, mackerel, swordfish, halibut, mahi-mahi, grouper, crucian carp, sea bass, anchovies, carp, perch, pike, flounder, sole, eel, herring, whitefish, and / or spiny lobster; • Cephalopods: squid, octopus, cuttlefish, and / or nautilus; Crustaceans: shrimp, crabs, lobsters, spiny lobsters, prawns, krill, crayfish, mussels, oysters, bivalves, scallops, and / or cockles; • Reptiles: snakes, turtles, alligators, crocodiles, iguanas, and / or lizards; and Insects: grasshoppers, crickets, mealworms, beetles, ants, termites, cicadas, caterpillars, silkworms, grasshoppers, waxworms, hawk moth larvae, bamboo worms, scorpions, and / or centipedes.

[0919] In some possible embodiments, engineered cells derived from the DNA of extinct animals are cultured on hollow fibers of the present disclosure. Examples of such animals include, but are not limited to, mammuths (Mammuthus) and / or dodos (Raphus).

[0920] In some embodiments, the cells cultured on the hollow fibers of the present disclosure are derived from mammals, including, but not limited to, human, primate, dog, cat, and / or mouse sources.

[0921] In some embodiments, cells cultured on the hollow fibers of this disclosure are not intended for use in the production of cultured meat products.

[0922] In some embodiments, the type of cells cultured on the hollow fibers of this disclosure is the same, thereby constituting a homoculture.

[0923] In some other embodiments of the present disclosure, different types of cells are cultured on the hollow fibers, thereby constituting a co-culture.

[0924] Cell co-culture is realized by simultaneously culturing multiple cell types within a single bioreactor.

[0925] In some embodiments, the cells cultured on the hollow fibers of the present disclosure are derived from multiple animal sources.

[0926] In some embodiments, the cells cultured on the hollow fibers of the present disclosure originate from multiple sources.

[0927] In some embodiments, the combinations of cell types that can be cultured together on the hollow fibers of the present disclosure in co-culture include, but are not limited to, myocytes and fibroblasts, myocytes and engineered cells, adipocytes and fibroblasts, adipocytes and engineered cells, myocytes and fibroblasts and adipocytes, myocytes and fibroblasts and engineered cells, myocytes and adipocytes and engineered cells, fibroblasts and adipocytes and engineered cells, or myocytes and fibroblasts and engineered cells.

[0928] In some embodiments, but not limited to, cells derived from non-animal protein sources such as plants, bacteria, fungi, algae, and / or archaea can be appropriately cultured in a bioreactor comprising hollow fibers and / or cartridges thereof of the present disclosure.

[0929] In some embodiments, the cells cultured on the hollow fibers of the present disclosure are derived from a plurality of sources, including animals, plants, bacteria, fungi, algae, and / or archaea.

[0930] Edible cells, including satellite cells, muscle cells, and adipocytes, can be sensitive to fluid strain stress, i.e., mechanical forces resulting from the friction of fluid flow against the outside of the cell membrane. In CSTRs, high fluid strain stress can ensure efficient mass transfer and uniform mixing. However, hollow fiber bioreactors can maintain very low fluid strain stress in the extracapillary space compared to CSTRs (55).

[0931] Phagocytic cells, including satellite cells, muscle cells, and adipocytes, are typically naturally occurring adherent cell types. During the culture of adherent phagocytic cells in CSTR, the cells can grow on either the surface of the bioreactor or on microcarriers present in the contained suspension. Alternatively, phagocytic cells may be selectively bred to be cultured in a suspension (without microcarriers) or within spherical aggregates. However, selective breeding programs are typically time-consuming and costly. Furthermore, the highest cell concentrations achieved in suspension are still typically lower than those achieved in hollow fiber bioreactors (55)(4)(2;3).

[0932] In some embodiments, the disclosure also provides semipermeable porous hollow fibers comprising covalent esters, thioesters, and / or amide-crosslinked polypeptides to which cells adhere.

[0933] In some embodiments, the disclosure also provides semipermeable porous hollow fibers comprising covalent esters, thioesters, and / or amide-crosslinked polypeptides on which cells adhere and are cultured.

[0934] Food containing porous, semi-permeable hollow fibers crosslinked by covalent bonds This disclosure may also provide food products including: (i) one or more fibers of the present disclosure, and (ii) Multiple cells.

[0935] In some embodiments, the present disclosure provides a food product that includes: (i) one or more fibers of the present disclosure, and (ii) Multiple cells.

[0936] In some embodiments, the Disclosure provides a food product comprising one or more of the fibers of the Disclosure.

[0937] In some embodiments, multiple cells are growing on a single hollow fiber of the present disclosure.

[0938] In some other embodiments, multiple cells are growing on multiple hollow fibers of the present disclosure.

[0939] In some further embodiments, multiple cells are growing in a suspension around multiple hollow fibers of the present disclosure.

[0940] Multiple cells may be dead and / or alive.

[0941] In some embodiments, multiple cells on the hollow fibers of the present disclosure are dead.

[0942] In other embodiments, the multiple cells on the hollow fiber of the present disclosure are living.

[0943] In some other embodiments, the cells cultured on the hollow fibers of the present disclosure include dead cells and living cells.

[0944] In some embodiments, the food is subjected to an optional heat treatment step in a process such as cooking.

[0945] In some embodiments, the food is subjected to one or more food preservation methods to inhibit microbial growth and extend the shelf life of the food. Such food preservation methods include, but are not limited to, canning, chemical preservation, refrigeration, dehydration, fermentation, freeze-drying, freezing, high-pressure treatment, irradiation, processing atmosphere packing, pickling, salting, smoking, sugaring, and vacuum packing. These methods may overlap or be used in combination.

[0946] In some preferred embodiments, the food is a cultured meat product.

[0947] In this disclosure, the term “cultured meat product” includes, but is not limited to, whole, sliced, cut, diced, minced, and / or reshaped pieces of such meat product.

[0948] 1.22 Formation of food containing covalently crosslinked porous semipermeable hollow fibers Providing a process for producing food products containing hollow fibers and multiple cells is a feature of certain aspects and embodiments of the present disclosure.

[0949] This process may involve five steps, including cell augmentation, bioreactor seeding, cell culture, post-production modification, and harvesting.

[0950] In some embodiments, the process includes five steps: cell augmentation, bioreactor seeding, cell culture, post-production modification, and harvesting.

[0951] In some embodiments, the process includes four steps: cell augmentation, bioreactor seeding, cell culture, and harvesting.

[0952] Cells used in the seeding stage may be initially grown (cultured) on an existing bioreactor platform to allow a smaller number of starting cells to grow more, if necessary for seeding. Such existing bioreactor platforms may include, but are not limited to, conventional T-flasks, shaking flasks, or plates, or bioreactors such as CSTRs, wave bag reactors, or microcarrier bioreactors.

[0953] The augmented cell population can then be seeded into a bioreactor containing hollow fibers of the present disclosure. To achieve this, the cells can be arranged in the extracapillary space between the hollow fibers and separated by the inner wall of the cartridge shell. The cells can then be injected as a slurry into each cartridge through the tertiary port of the outer shell. A second tertiary port of the outer shell can facilitate the movement of gases or liquids during this seeding process.

[0954] After seeding, food can be produced through a culture process using a bioreactor containing hollow fibers of the present disclosure. Throughout the cell culture process, a sufficient amount of cell growth medium can be supplied to promote cell proliferation. Process parameters may be monitored to ensure that inhibition of cell proliferation is minimized until a desired cell density is achieved, at which point the culture may be terminated.

[0955] In some embodiments of the bioreactor, the hollow fiber cartridge of the present disclosure is supplied with culture medium into the lumen of each fiber through one port of the cartridge. In this way, nutrients and growth factors necessary for cell proliferation and differentiation are supplied to the cells in the extracapillary space by the movement of clumps and / or fluids.

[0956] In some embodiments of the bioreactor, the hollow fibers or cartridges of the present disclosure are supplied with culture medium before, during, and / or after the cell seeding step.

[0957] In some embodiments of the bioreactor, the culture medium constitutes a co-flow channel through each hollow fiber cartridge of the present disclosure. The fluid flow in this configuration can be understood by examining the simplified shell and tubular hollow fiber bioreactor port diagram shown in Figure 6. In this configuration, the culture medium enters through both a lumen inlet port (6001) that supplies the medium into the lumen of each fiber and a second tertiary port (6002) on the outer shell of the cartridge into the extracapillary space. The fluid flowing into the bioreactor may flow through the lumen of each fiber and exit through a lumen outlet port (6005), permeate through the hollow fiber, or exit through an auxiliary port (6004) on the outer shell at the same end as the lumen outlet.

[0958] In some embodiments of the bioreactor, the culture medium constitutes a backflow channel through each hollow fiber cartridge of the present disclosure. The fluid flow in this configuration can be understood by examining a simplified diagram of the shell and tubular hollow fiber bioreactor port shown in Figure 6. In this configuration, the culture medium enters through both a lumen inlet port (6001) that supplies the culture medium into the lumen of each fiber, and a second tertiary port (6004) on the outer shell of the cartridge, located at the opposite end of the bioreactor cartridge, which flows into the extracapillary space. The fluid flowing into the bioreactor may flow through the lumen of each fiber and exit through a lumen outlet port (6005), permeate through the fiber, or exit through an auxiliary port (6002) on the outer shell at the opposite end of the lumen outlet.

[0959] In other embodiments of the bioreactor, the culture medium constitutes a flow path through each hollow fiber cartridge of the present disclosure. The fluid flow path in this configuration can be understood by examining the simplified shell and tubular hollow fiber bioreactor port diagram shown in Figure 6. In this configuration, the culture medium enters both lumen inlet ports (6001) that supply the medium into the lumen of the hollow fiber. The auxiliary ports (6002 and 6004) of the outer shell are closed, and as a result, the medium exits only through the lumen outlet port (6005).

[0960] In some embodiments, the cell density achieved in a bioreactor including a hollow fiber cartridge of the present disclosure is, but is not limited to, 100,000 cells / cm³. 3 ~100,000,000 cells / cm 3 It is within the range.

[0961] In some embodiments, the cell density achieved in a bioreactor including a hollow fiber cartridge of the present disclosure is, but is not limited to, 100,000 cells / cm³. 3 ~200,000,000 cells / cm 3 It is within the range.

[0962] In some embodiments, the cell density achieved in a bioreactor including a hollow fiber cartridge of the present disclosure is, but is not limited to, 100,000 cells / cm³. 3 ~300,000,000 cells / cm 3 It is within the range.

[0963] In some embodiments, the cell density achieved in a bioreactor including a hollow fiber cartridge of the present disclosure is, but is not limited to, 100,000 cells / cm³. 3 ~150,000 cells / cm 3 , 150,000 cells / cm 3 ~200,000 cells / cm 3 , 200,000 cells / cm 3 ~500,000 cells / cm 3 , 500,000 cells / cm 3 ~1,000,000 cells / cm 3 , 1,000,000 cells / cm 3 ~10,000,000 cells / cm 3 , 10,000,000 cells / cm 3 ~50,000,000 cells / cm 3 ,50,000,000 cells / cm 3 ~75,000,000 cells / cm 3 ,75,000,000 cells / cm 3 ~100,000,000 cells / cm 3 It is within the range.

[0964] In some embodiments, the cell density achieved in the bioreactor comprising the hollow fiber cartridge of the present disclosure is, without limitation, 100,000 cells / cm 3 ~150,000 cells / cm 3 、150,000 cells / cm 3 ~200,000 cells / cm 3 、200,000 cells / cm 3 ~500,000 cells / cm 3 、500,000 cells / cm 3 ~1,000,000 cells / cm 3 、1,000,000 cells / cm 3 ~10,000,000 cells / cm 3 、10,000,000 cells / cm 3 ~50,000,000 cells / cm 3 、50,000,000 cells / cm 3 ~75,000,000 cells / cm 3 、75,000,000 cells / cm 3 ~100,000,000 cells / cm 3 、100,000,000 cells / cm 3 ~125,000,000 cells / cm 3 、125,000,000 cells / cm 3 ~150,000,000 cells / cm 3 、150,000, cells / cm 3 ~175,000,000 cells / cm 3 、175,000,000 cells / cm 3 ~200,000,000 cells / cm 3 、200,000,000 cells / cm 3 ~225,000,000 cells / cm 3 、225,000,000 cells / cm 3 ~250,000,000 cells / cm 3 、250,000,000 cells / cm 3 ~275,000,000 cells / cm 3 、and / or 275,000,000 cells / cm 3~300,000,000 cells / cm 3 It is within the range.

[0965] In some embodiments, cell culture performed in a bioreactor is carried out until the cells reach confluence (percentage of surface covered by adherent cells), which is in the range of 70% to 99%.

[0966] In some embodiments, cell culture performed in a bioreactor is carried out until the cells reach a confluence (percentage of surface covered by adherent cells) which is in the range of 70%–75%, 75%–80%, 80%–85%, 85%–90%, or 90%–99%.

[0967] In some embodiments, cell culture performed in a bioreactor is carried out until the cells reach confluence (percentage of surface covered by adherent cells), which is in the range of 10% to 70%.

[0968] In some embodiments, cell culture performed in a bioreactor is carried out until the cells reach a confluence (percentage of surface covered by adherent cells) which is in the range of 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, or 60%-70%.

[0969] After the cultured cells have reached the desired cell density and / or confluence, the hollow fibers and cultured cell aggregates of this disclosure may be subjected to one or more treatments after the cell culture step to improve flavor, aroma, texture, smell, or appearance.

[0970] In some embodiments of the final harvesting step, the hollow fiber cartridge of the present disclosure is removed from the support bioreactor platform. The edible cell mass and fibers are then removed from the cartridge as a food that can be further processed.

[0971] 1.23 Post-proliferation treatment of cells Any hollow fibers and / or foods derived therefrom of the present disclosure may be subjected to further processing to modify their physical and / or chemical properties for the purpose of improving the flavor of the hollow fibers and / or foods derived therefrom.

[0972] In some embodiments, the hollow fibers and / or any food products derived therefrom of the present disclosure are subjected to further processing to modify their physical and / or chemical properties.

[0973] In other embodiments, the hollow fibers and / or any food products derived therefrom of the present disclosure are not subjected to further processing to modify their physical and / or chemical properties.

[0974] In some other embodiments of the present disclosure, a process for processing or improving the flavor of a composition comprising hollow fibers: Step (A) provides a composition containing the fibers of the present disclosure within a cartridge, (B1) A step of physiologically and chemically treating the hollow fibers in the composition to reduce their mechanical strength, and / or Step (B2) to flush the lumen of the hollow fibers and / or the remaining void space of the hollow fibers in the space outside the capillary of the cartridge. One or more of the following, Step (C) removes the composition containing the hollow fibers of the present disclosure from the cartridge, Step (D1) of mechanically processing a composition containing hollow fibers of the present disclosure, and / or Step (D2) to physically reduce the length of the hollow fibers in the composition. A process is provided that includes one or more of the following:

[0975] In some embodiments, in step (B1), the hollow fibers in the composition are treated with an acid, alkali, or buffer, with or without cells.

[0976] In some embodiments, in step (B1), the hollow fibers in the composition are washed with a sufficient amount of water or buffered aqueous solution, with or without the presence of cells. The buffer may be, for example, a carbonate buffer with a pH of 10-13.

[0977] In some embodiments, in step (B1), the hollow fibers in the composition are treated with an enzyme, with or without the presence of cells. Examples of enzymes include, but are not limited to, bromelain.

[0978] In some embodiments, compositions comprising hollow fibers of the present disclosure are treated with heat or infrared radiation in processes such as cooking.

[0979] In other embodiments, the mechanical strength of a composition containing hollow fibers of the present disclosure is reduced, as determined by a uniaxial tensile test.

[0980] In some embodiments, in step (B2), the lumen of the hollow fibers in the composition, and the extracapillary space of any cartridge containing such composition, are flushed with one or more fluids containing, but not limited to, water, acid, alkali, aqueous buffer, water-oil emulsion, animal fat, vegetable oil, saline solution, flavoring, emulsifier, stabilizer, colorant, thickener and / or gelling agent, or any combination thereof in any order.

[0981] In some embodiments, fats and oils used to flush the lumen of the hollow fibers in the composition and the extracapillary space of the cartridge include, but are not limited to, vegetable oils, rapeseed oil, sunflower oil, linseed oil, sunflower oil, avocado oil, corn oil, lard, coconut oil, palm oil, sesame oil, soybean oil, canola oil, olive oil, peanut oil, nut oil, omega-3 oils, fish-derived oils, animal milk, animal butter, processed animal fats or adipose tissue derived from cellular agriculture, or any combination thereof, in any order.

[0982] In some embodiments, the colorants contained in the solution(s) used to flush the lumen of the hollow fibers in the composition and the extracapillary space of the cartridge may include, but are not limited to, curcumin, riboflavin, riboflavin-5'-phosphate, tartrazine, quinoline yellow, sunset yellow FCF; orange yellow S, cochineal; carmine; carmine, azorbin; carmoisine, amaranth, ponseau 4R; cochineal red A, erythrosine, allura red AC, patent blue V, indigotine; indigocarmine, brilliant blue FCF, chlorophyll and chlorophyllin, copper complexes of chlorophyll and chlorophyllin, green S, plain caramel, sulfite caramel, ammonia caramel, ammonia sulfite caramel, brilliant black BN; black PN, or any combination thereof, in any order.

[0983] In some embodiments, the preservatives contained in the solution(s) used to flush the lumen of the hollow fibers in the composition and the extracapillary space of the cartridge include, but are not limited to, potassium sorbate, benzoic acid, sodium benzoate, potassium benzoate, calcium benzoate, ethyl parahydroxybenzoate, sodium parahydroxybenzoate, methyl parahydroxybenzoate, sodium parahydroxybenzoate, sulfur dioxide, sodium sulfite, sodium bisulfite, sodium pyrosulfite, potassium pyrosulfite, calcium sulfite, calcium bisulfite, potassium bisulfite, nisin, natamycin, hexamethylenetetramine, dimethyl dicarbonate, lauroyl ethyl alginate, potassium nitrite, sodium nitrite, sodium nitrate, potassium nitrate, propionic acid, sodium propionate, calcium propionate, potassium propionate, boric acid, sodium tetraborate, borax, or lysozyme, or any combination thereof.

[0984] In some embodiments, the antioxidants in the solution(s) used to flush the lumen of the hollow fibers in the composition and the extracapillary space of the cartridge include, but are not limited to, ascorbic acid, sodium ascorbate, calcium ascorbate, fatty acid esters of ascorbic acid, tocopherol, alpha-tocopherol, gamma-tocopherol, delta-tocopherol, propyl gallate, erythorbic acid, sodium erythorbate, tert-butylhydroquinone (TBHQ), butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), rosemary extract, or 4-hexylresorcinol, or any combination thereof.

[0985] In some embodiments, the flavorings contained in the solution(s) used to flush the lumen of the hollow fibers in the composition and the extracapillary space of the cartridge include, but are not limited to, those listed in the European Union list of flavorings found in Annex I of Regulation EU1334 / 2008, and non-food-derived flavorings added in accordance with Regulation EU2018 / 1259.

[0986] In some embodiments, other additives in the solution(s) used to flush the lumen of the hollow fibers in the composition and the extracapillary space of the cartridge include, but are not limited to, potassium chloride, calcium chloride, magnesium chloride, monosodium glutamate, monosodium glutamate, monosodium glutamate, sodium citrate, potassium citrate, calcium citrate, sodium phosphate, potassium phosphate, calcium phosphate, magnesium phosphate, sodium carbonate, potassium carbonate, sodium sulfate, potassium sulfate, calcium sulfate, L-leucine, or L-cysteine.

[0987] In some embodiments, the emulsifiers, stabilizers, thickeners, and gelling agents contained in the solution(s) used to flush the lumen of the hollow fibers in the composition and the extracapillary space of the cartridge include, but are not limited to, alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, propane-1,2-diol alginate, agar, carrageenan, processed Eukema algae, locust bean gum; carob gum, guar gum, tragacanth, acacia gum; acacia gum, xanthan gum, karaya gum, tara gum, gellan gum, konjac, soybean hemicellulose, cassia gum, polyoxyethylene sorbitan monolaurate; polysorbate 20, polyoxyethylene sorbitan monooleate; polysorbate 80, polyoxyethylene sorbitan monopalmitate; polysorbate 40, This includes at least one of the following: polyoxyethylene sorbitan monostearate; polysorbate 60, polyoxyethylene sorbitan tristearate; polysorbate 65, pectin, phosphatidoammonium, sucrose acetate isobutyrate, woodrosin glycerin ester, cellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, ethylmethylcellulose, carboxymethylcellulose, cross-linked sodium carboxymethylcellulose, enzymatic hydrolysis of carboxymethylcellulose, sodium stearoyl-2-lactate, calcium stearoyl-2-lactate, stearyl tartrate, sorbitan monostearate, sorbitan tristearate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, or invertase.

[0988] In some embodiments, the additives included in the solution(s) used to flush the lumen of the hollow fibers in the composition and the extracapillary space of the cartridge include, but are not limited to, those listed in EU Regulation (EC) No. 1333 / 2008 and its annexes. These additives include sweeteners, colorants, preservatives, antioxidants, carriers, acids, acidity modifiers, anticaking agents, defoamers, excipients, emulsifiers, emulsifying salts, solidifying agents, flavor enhancers, foaming agents, gelling agents, glazing agents, modified starches, leavening agents, metal encapsulants, stabilizers, and thickeners. These additives may be added in any combination and any order.

[0989] In other embodiments, additives in the solution(s) used to flush the lumen of the hollow fibers in the composition and the extracapillary space of the cartridge may include, but are not limited to, those generally considered safe (GRAS) by the FDA, such as those listed in the GRAS Substances (SCOGS) database. These additives may include sweeteners, colorants, preservatives, antioxidants, carriers, acids, acidity modifiers, anticaking agents, defoamers, excipients, emulsifiers, emulsifying salts, solidifying agents, flavor enhancers, foaming agents, gelling agents, glazing agents, modified starches, leavening agents, metal encapsulants, stabilizers, and thickeners. These additives may be added in any combination and any order.

[0990] In some embodiments, in step (D1), the composition containing hollow fibers is beaten, chopped, macerated, ground, and / or processed by other machine-based food preparation methods, with or without cells.

[0991] In some embodiments, in step (D2), the composition containing hollow fibers is cut to reduce the length of the hollow fibers.

[0992] This disclosure also extends to compositions that can be obtained or obtained by such process.

[0993] 1.24 Food Providing a food product containing a plurality of cells containing polypeptides covalently crosslinked by esters, thioesters, and / or amide bonds derived from hollow fibers of the present disclosure is a feature of certain aspects and embodiments of the present disclosure.

[0994] The cells may be any of the cells disclosed herein.

[0995] In some embodiments, the food is cylindrical and / or has a nearly circular cross-section.

[0996] In some embodiments, the food has a cross-section that is, but is not limited to, circular, near-circular, elliptical, square, rectangular, triangular, pentagonal, hexagonal, or other polygonal in shape.

[0997] In some embodiments, the cross-section of the food contains multiple cross-linked polypeptide rings intertwined with the cell mass.

[0998] In some embodiments, the cross-section of the food includes a plurality of rings derived from the hollow fibers of the Disclosure that are intertwined with the cell mass.

[0999] In some embodiments, the ring is filled with one or more polypeptides, polysaccharides, lipids, polyols, and / or any combination thereof.

[1000] In some embodiments, the cross-section of the food contains multiple fibrous structures derived from the hollow fibers of the present disclosure, intertwined with the cell mass.

[1001] In some embodiments, the muscle is filled with polypeptides, polysaccharides, lipids, polyols, and / or any combination thereof.

[1002] In some embodiments, the food is a mixture of cellular aggregates and hollow fibers of the present disclosure that have been beaten, chopped, and / or ground.

[1003] In some embodiments, the food(s) of the Disclosure are mechanically reformed masses comprising cell aggregates and hollow fibers of the Disclosure.

[1004] In some embodiments, the food(s) of the present disclosure also contain a supplemental polypeptide.

[1005] In some embodiments, the food(s) of the present disclosure also contain polysaccharides.

[1006] In some embodiments, the food(s) of the present disclosure also contain lipids.

[1007] In some embodiments, the food(s) of the present disclosure also contain polyols.

[1008] In some embodiments, the food(s) of the present disclosure also contain animal-derived meat products.

[1009] In some embodiments, the food(s) of the present disclosure also contain proteins derived from non-animal sources, including, but not limited to, plants, bacteria, fungi, algae, and / or archaea.

[1010] In some embodiments, the food(s) of this disclosure may contain additives, including, but are not limited to, those listed in EU Regulation (EC) No. 1333 / 2008 and its annexes. These additives include sweeteners, colorants, preservatives, antioxidants, carriers, acids, acidity modifiers, anticaking agents, defoamers, excipients, emulsifiers, emulsifying salts, solidifying agents, flavor enhancers, foaming agents, gelling agents, glazing agents, water-retaining agents, modified starches, packaging gases, propellants, leavening agents, metal encapsulants, stabilizers, thickeners, and flour treatment agents. These additives may be present in any combination.

[1011] In some embodiments, the food(s) of the Disclosure contain additives, including, but are not limited to, those designated by the FDA as generally recognized as safe (GRAS), such as those listed in the GRAS Substances (SCOGS) database. These additives include sweeteners, colorants, preservatives, antioxidants, carriers, acids, acidity modifiers, anticaking agents, defoamers, excipients, emulsifiers, emulsifying salts, solidifiers, flavor enhancers, foaming agents, gelling agents, glazing agents, modified starches, leavening agents, metal encapsulants, stabilizers, and thickeners. These additives may be present in any combination.

[1012] A person skilled in the art may be able to easily determine whether a food contains hollow fibers of the present disclosure using techniques that may include nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry.

[1013] NMR can be used to determine whether a food product contains polycarboxylic acid-derived esters, thioesters, or polypeptides crosslinked by amide crosslinking covalent bonds. Specifically, 1 The presence of a carboxylic acid derivative may also be determined by the presence of a carbonyl group using 1H proton NMR. 1 The protons on the carbon bonded to the alkoxide oxygen in the ester resonate in the 2.0–3.0 ppm region of the 1H NMR spectrum. On the other hand, the protons on the carbon bonded to the sulfur in the thioester resonate in the 2.0–3.0 ppm region (56). Since these groups are not native to polypeptides, the identification of these groups in food can therefore be used as evidence that the fibers of the present disclosure are used in the preparation of food and thus constitute the products of the processes of the present disclosure.

[1014] Similarly, 13The presence of carboxylic acid derivatives may be determined by using 13C NMR based on the difference in resonance frequencies between the carbonyl carbon in carboxylic acids (160–180 ppm) and the derivative esters (50–90 ppm), aldehydes, and ketones (180–220 ppm). In carboxylic acids, the carbonyl carbon is strongly deprotected by the adjacent highly electronegative double-bonded oxygen. The carboxyl carbon in carboxylic acid derivatives exhibits a variety of resonance frequencies when subjected to different levels of deprotection. Carbons of other functional groups also exhibit different resonance frequencies, such as the nitrogen-bonded carbon in amides (20–65 ppm) and the sulfur-bonded carbon in thioesters (20–45 ppm) (56). Similarly, since these groups are not natural in polypeptides, the identification of these groups in food can therefore be used as evidence that the fibers of the present disclosure are used in the preparation of food and thus constitute the products of the processes of the present disclosure.

[1015] By applying these NMR spectral analysis techniques, it may be possible to identify polypeptides specifically crosslinked by covalent bonds of polycarboxylic acid-derived esters, thioesters, or amides from the NMR spectrum of food samples, distinguishing them from polypeptides crosslinked by other means, such as thermophysical crosslinking. Therefore, NMR analysis may be performed to distinguish food samples containing covalent crosslinks of polycarboxylic acid-derived esters, thioesters, or amides from other food samples, because those food samples are within the scope disclosed above. 1 1H NMR or 13 This is because it can show a significantly higher peak in the 1C NMR spectrum.

[1016] Using mass spectrometry, asylium ions (R-CO) + The presence of a peak related to ) may indicate the presence of a carboxylic acid derivative. However, since the mass of the substance (R-) bound to the asylium ion is variable, the theoretical mass of these ions must be calculated on a case-by-case basis (56).

[1017] Due to the non-standard number of peaks associated with carboxylic acid derivatives measured by mass spectrometry, NMR should be used.

[1018] Once the hollow fibers of the present disclosure have been identified by NMR and / or mass spectrometry, SDS-PAGE may be applied to determine whether the hollow fibers have been subjected to acid, alkali, and / or buffer washing. Such washing disrupts the backbone of the polypeptide molecule, shortens the molecule, and reduces the molecular weight of each fragment. Comparison of the resulting SDS-PAGE bonds may show molecular weight differences caused by hydrolysis compared to an untreated sample. Similarly, mass spectrometry may be used to identify the reduction in fragment size to the same effect.

[1019] In some embodiments, the food products of this disclosure are subjected to an optional heat treatment step in a process such as cooking.

[1020] In some embodiments, the foods of this disclosure are subjected to one or more food preservation methods to inhibit microbial growth and extend the shelf life of the foods. Such food preservation methods include, but are not limited to, canning, chemical preservation, refrigeration, dehydration, fermentation, freeze-drying, freezing, high-pressure treatment, irradiation, processing atmosphere packing, pickling, salting, smoking, sugaring, and vacuum packing. These methods may overlap or be used in combination.

[1021] The disclosures of each reference document included herein are incorporated herein by reference in their entirety.

[1022] 2 Exemplary Embodiments In addition to the embodiments and models disclosed elsewhere in this specification, the following are exemplary embodiments specifically intended by this disclosure.

[1023] 2.1 Embodiments of the Composition 1. An edible semi-permeable porous hollow fiber comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, (a) The outer diameter of the hollow fiber is 50 to 6600 μM, (b) The thickness of the wall of the hollow fiber is 20 to 800 μM, (c) The inner diameter of the hollow fiber is 20 to 5000 μM, (d) The edible semi-permeable porous hollow fiber having a pore volume of 1 to 95%.

[1024] 2. A semipermeable porous hollow fiber comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, (a) The outer diameter of the hollow fiber is 50 to 6600 μM, (b) The thickness of the wall of the hollow fiber is 20 to 800 μM, (c) The inner diameter of the hollow fiber is 20 to 5000 μM, (d) The semipermeable porous hollow fiber having a pore volume of 1 to 95%.

[1025] 3. An edible semi-permeable porous hollow fiber comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, (a) The outer diameter of the hollow fiber is 50 to 6600 μm, (b) The thickness of the wall of the hollow fiber is 20 to 800 μm, (c) The inner diameter of the hollow fiber is 20 to 5000 μm, (d) The edible semi-permeable porous hollow fiber having a porosity of 1 to 95%.

[1026] 4. A semipermeable porous hollow fiber comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, (a) The outer diameter of the hollow fiber is 50 to 6600 μm, (b) The thickness of the wall of the hollow fiber is 20 to 800 μm, (c) The inner diameter of the hollow fiber is 20 to 5000 μm, (d) The semipermeable porous hollow fiber having a porosity of 1 to 95%.

[1027] 5. A semipermeable porous hollow fiber comprising one or more polypeptides crosslinked by polycarboxylic acid-derived esters, thioesters, or amide covalent bonds.

[1028] 6. A semipermeable porous hollow fiber comprising one or more polypeptides crosslinked by polycarboxylic acid-derived esters, thioesters, or amide covalent bonds, wherein the material of the hollow fiber is GRAS.

[1029] 7. A regular semi-crystalline polymer comprising ester, thioester, and / or amide-crosslinked polypeptides having a beta-sheet secondary structure.

[1030] 8. A regular semi-crystalline polymer, Prokitein, comprising ester, thioester, and / or amide-crosslinked polypeptides having a beta-sheet secondary structure.

[1031] 9. The hollow fiber is a hollow fiber from any of the prior embodiments of the composition, wherein the hollow fiber contains Prokitein.

[1032] 10. A hollow fiber from any of the prior embodiments of the composition, wherein the hollow fiber comprises a regular semicrystalline polymer having a beta-sheet secondary structure, comprising an ester, thioester, and / or amide-crosslinked polypeptide.

[1033] 11. A hollow fiber from any of the prior embodiments of the composition, wherein the cross-section of the hollow fiber is circular, elliptical, square, rectangular, or polygonal.

[1034] 12. A hollow fiber from any of the prior embodiments of the composition, wherein the cross-section of the hollow fiber is circular.

[1035] 13. A hollow fiber from any of the prior embodiments of the composition, wherein the cross-section of the hollow fiber is elliptical.

[1036] 14. A hollow fiber from any of the prior embodiments of the composition, wherein the cross-section of the hollow fiber is square.

[1037] 15. A hollow fiber of any of the prior embodiments of the composition, wherein the cross-section of the hollow fiber is rectangular.

[1038] 16. A hollow fiber of any of the prior embodiments of the composition, wherein the cross-section of the hollow fiber is polygonal.

[1039] 17. A hollow fiber in any of the prior embodiments of the composition, wherein the hollow fiber has a tubular or cylindrical shape.

[1040] 18. A hollow fiber of any of the prior embodiments of the composition, wherein the hollow fiber has a lumen.

[1041] 19. A hollow fiber in any of the prior embodiments of the composition, wherein one end of the lumen of the hollow fiber is open.

[1042] 20. A hollow fiber of any of the prior embodiments of the composition, wherein both ends of the lumen of the hollow fiber are open.

[1043] 21. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 15% to 25%.

[1044] 22. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 30% to 45%.

[1045] 23. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 40% to 65%.

[1046] 24. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 70% to 95%.

[1047] 25. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 5% to 10%.

[1048] 26. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 60% to 85%.

[1049] 27. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 30% to 35%.

[1050] 28. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 45% to 70%.

[1051] 29. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 5% to 30%.

[1052] 30. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 5% to 75%.

[1053] 31. A hollow fiber of any of the prior embodiments of the composition, wherein the porosity is in the range of 65% to 70%.

[1054] 32. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 65% to 95%.

[1055] 33. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 30% to 75%.

[1056] 34. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 25% to 85%.

[1057] 35. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 30% to 70%.

[1058] 36. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 60% to 70%.

[1059] 37. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 50% to 75%.

[1060] 38. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 20% to 45%.

[1061] 39. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 10% to 65%.

[1062] 40. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 60% to 75%.

[1063] 41. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 60% to 65%.

[1064] 42. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 55% to 75%.

[1065] 43. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 10% to 85%.

[1066] 44. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 30% to 55%.

[1067] 45. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 35% to 65%.

[1068] 46. ​​A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 40% to 55%.

[1069] 47. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 25% to 40%.

[1070] 48. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 35% to 95%.

[1071] 49. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 25% to 75%.

[1072] 50. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 5% to 90%.

[1073] 51. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 15% to 30%.

[1074] 52. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 25% to 70%.

[1075] 53. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 10% to 80%.

[1076] 54. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 75% to 80%.

[1077] 55. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 45% to 65%.

[1078] 56. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 45% to 90%.

[1079] 57. A hollow fiber of any of the prior embodiments of the composition, wherein the porosity is in the range of 10% to 70%.

[1080] 58. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 55% to 70%.

[1081] 59. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 10% to 60%.

[1082] 60. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 15% to 55%.

[1083] 61. A hollow fiber from any of the prior embodiments of the composition, wherein the porosity is in the range of 50% to 70%.

[1084] 62. A hollow fiber from any of the prior embodiments of the composition, wherein the po...

Claims

1. A semi-permeable porous hollow fiber for food use, comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, (a) The outer diameter of the hollow fiber is 50 to 6600 μM, (b) The thickness of the wall of the hollow fiber is 20 to 800 μM, (c) The inner diameter of the hollow fiber is 20 to 5000 μM, (d) The edible semi-permeable porous hollow fiber having a pore volume of 1 to 95%.

2. A semipermeable porous hollow fiber comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, (a) The outer diameter of the hollow fiber is 50 to 6600 μM, (b) The thickness of the wall of the hollow fiber is 20 to 800 μM, (c) The inner diameter of the hollow fiber is 20 to 5000 μM, (d) The semipermeable porous hollow fiber having a pore volume of 1 to 95%.

3. A semi-permeable porous hollow fiber for food use, comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, (a) The outer diameter of the hollow fiber is 50 to 6600 μm, (b) The thickness of the wall of the hollow fiber is 20 to 800 μm, (c) The inner diameter of the hollow fiber is 20 to 5000 μm, (d) The edible semi-permeable porous hollow fiber having a porosity of 1 to 95%.

4. A semipermeable porous hollow fiber comprising one or more covalent esters, thioesters, and / or amide-linked polypeptides derived from polycarboxylic acids, (a) The outer diameter of the hollow fiber is 50 to 6600 μm, (b) The thickness of the wall of the hollow fiber is 20 to 800 μm, (c) The inner diameter of the hollow fiber is 20 to 5000 μm, (d) The semipermeable porous hollow fiber having a porosity of 1 to 95%.

5. A process for producing semipermeable porous hollow fibers containing covalently crosslinked polypeptides, a. i. A first composition comprising a polypeptide and ii. A second composition comprising a solvent and one or more denaturing agents and / or reducing agents. The steps include: combining these to produce a third composition, b. Incubate the third composition under conditions sufficient to solubilize, denaturate and / or reduce at least one fraction of the polypeptide, A step of generating a fourth composition, c. The fourth composition is extruded or spun together with the bore solution through a plurality of coaxial orifices to produce hollow fibers, d. A step of treating the hollow fiber with a polycarboxylic acid crosslinking reagent to form polycarboxylic acid-derived ester, thioester, or amide covalent crosslinks between polypeptides and / or within polypeptides in at least one fraction of the polypeptide within the hollow fiber, thereby producing a covalently crosslinked semipermeable porous hollow fiber; e. i. To increase the relative abundance of beta-sheets in the secondary structure of the polypeptide by treating the covalently crosslinked semipermeable porous hollow fibers with an organic solvent, and ii. Annealing the semipermeable porous hollow fibers crosslinked by the aforementioned covalent bonds, iii. Treating the semipermeable porous hollow fibers crosslinked by the covalent bonds with a solvent to remove void-containing elements from the hollow fibers, iv. Washing the covalently crosslinked semipermeable porous hollow fibers with one or more acids, alkalis, and / or buffers to reduce at least one of the Young's modulus, ultimate tensile strength, and / or ultimate tensile strain of the hollow fibers. v. Coating the semipermeable porous hollow fibers crosslinked by the aforementioned covalent bonds, vi. To correct the surface topography of the covalently crosslinked semipermeable porous hollow fibers to assist in cell adhesion and / or cell alignment. A step of processing the covalently crosslinked semipermeable porous hollow fibers by one or more post-production modification processes selected from the group consisting of the above, to produce a plurality of processed covalently crosslinked semipermeable porous hollow fibers, f. A step of drying the treated covalently crosslinked semipermeable porous hollow fibers to produce dried, treated covalently crosslinked semipermeable porous hollow fibers, The process including the process described above.

6. The process according to claim 5, further specifying that the first composition comprises a polypeptide derived from a plant.

7. The process according to claims 5 to 6, further specifying that a polysaccharide is supplemented to the first composition of step (a) of claim 5 in a ratio within the range of 1% to 10,000% (weight per unit weight of polypeptide).

8. The process according to any one of claims 5 to 7, further specifying that lipids are supplemented to the first composition of step (a) of claim 5 in a ratio within the range of 1% to 10,000% (weight per unit weight of polypeptide).

9. The process according to any one of claims 5 to 8, further specifying that polyols and / or polymers of polyols are supplemented to the first composition of step (a) of claim 5 in a ratio ranging from 1% to 10,000% (weight per unit weight of polypeptide).

10. The process according to any one of claims 5 to 9, wherein the second composition comprises one or more solvents consisting of water, ethanol, oxalic acid, malic acid, succinic acid, adipic acid, tartaric acid, citric acid, malonic acid, acetic acid, formic acid, sodium hydroxide, and / or potassium hydroxide.

11. The process according to any one of claims 5 to 10, wherein the second composition comprises one or more solvents including water, ethanol, oxalic acid, malic acid, succinic acid, adipic acid, tartaric acid, citric acid, malonic acid, acetic acid, formic acid, sodium hydroxide, and / or potassium hydroxide.

12. The process according to any one of claims 5 to 11, wherein the second composition comprises at least one denaturing agent selected from oxalic acid, malic acid, succinic acid, adipic acid, tartaric acid, citric acid, malonic acid, acetic acid, formic acid, urea, sodium hydroxide, and / or potassium hydroxide.

13. The process according to any one of claims 5 to 12, wherein the second composition comprises one or more reducing agents including N-acetyl-cysteine, L-cysteine, glutathione, ascorbic acid, citric acid, tartaric acid, malic acid, sodium borohydride, sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium hypophosphate, sodium dithionite, mercaptoethanol and / or dithiothreitol.

14. The process according to any one of claims 5 to 13, wherein the second composition comprises one or more reducing agents, including N-acetyl-cysteine, L-cysteine, glutathione, ascorbic acid, citric acid, tartaric acid, malic acid, sodium borohydride, sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium hypophosphate, sodium dithionite, mercaptoethanol and / or dithiothreitol.

15. The process according to any one of claims 5 to 14, wherein the third composition of step (b) is incubated for a total period between 5 minutes and 1 week.

16. The process according to any one of claims 5 to 15, wherein the third or fourth composition of step (b) is incubated at a temperature between 0°C and 150°C.

17. The process according to any one of claims 5 to 16, wherein the fourth composition comprises one or more void elements, each comprising calcium carbonate, ice, air, nitrogen, carbon dioxide, argon, sodium chloride, or potassium chloride.

18. The process according to any one of claims 5 to 17, further specifying that the void-containing element is added to the composition in step (a) or step (b) such that the void-containing element constitutes 1% to 80% of the fourth composition (iv).

19. The process according to any one of claims 5 to 18, wherein the coagulation bath solution comprises one or more polycarboxylates containing sodium citrate, sodium malate, potassium malate and / or potassium citrate.

20. The process according to any one of claims 5 to 19, further specifying that a salt is added to the composition of the coagulation bath solution comprising zinc sulfate, sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium hypophosphite and / or ammonium sulfate.

21. The process according to any one of claims 5 to 20, wherein the coagulation bath solution comprises one or more polycarboxylates including sodium oxalate, potassium oxalate, sodium malate, potassium malate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, sodium tartrate, potassium tartrate, sodium malonate, and / or potassium malonate, and is supplemented with salts including zinc sulfate, sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium hypophosphite, and / or ammonium sulfate.

22. The process according to any one of claims 5 to 21, wherein the pH of the coagulation bath solution is greater than 7.0 and less than 10.

0.

23. The process according to any one of claims 5 to 22, wherein the temperature of the solidification bath solution is between 0°C and 90°C.

24. The process according to any one of claims 5 to 23, wherein the hollow fibers are washed after step (d) in an excess aqueous solvent having a pH between 7.0 and 8.

0.

25. The process according to any one of claims 5 to 24, wherein the organic solvent used in step (e.i) comprises at least one of methanol, ethanol, propanol, isopropanol, butanol, isobutanol, dimethyl sulfoxide, dimethylformamide, and / or dimethylacetamide.

26. The process according to any one of claims 5 to 25, wherein the organic solvent used in step (e.i) comprises at least one of polyols and / or polymers of polyols.

27. The process according to any one of claims 5 to 26, wherein the annealing in step (e(ii)) is carried out at a temperature of 75°C to 180°C.

28. The process according to any one of claims 5 to 27, wherein the annealing in step (e.iii) is carried out at a temperature of 75°C to 180°C for a total period of time ranging from 10 minutes to 6 hours.

29. The process according to any one of claims 5 to 28, wherein the solvent used in the void removal step (e(iii)) comprises at least one of oxalic acid, malic acid, succinic acid, adipic acid, tartaric acid, citric acid, malonic acid, acetic acid, or formic acid.

30. The process according to any one of claims 5 to 29, wherein the solvent used in the void removal step (e(iii)) comprises at least one of water, oxalic acid, malic acid, succinic acid, adipic acid, tartaric acid, citric acid, malonic acid, acetic acid, or formic acid, ethanol, propanol, isopropanol, butanol, isobutanol, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide.

31. The process according to any one of claims 5 to 30, wherein the solvent used in the void removal step (e(iii)) comprises polyols and / or polymers of polyols.

32. The process according to any one of claims 5 to 31, wherein the solvent used in step (e(iv)) to reduce at least one of the Young's modulus, ultimate tensile strength, and / or ultimate tensile strain of the hollow fiber comprises at least one of ascorbic acid, acetic acid, adipic acid, citric acid, formic acid, hydrochloric acid, lactic acid, malic acid, malonic acid, oxalic acid, succinic acid, sulfuric acid, tartaric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or sodium carbonate.

33. The process according to any one of claims 5 to 32, wherein the coating in step (e(v)) comprises at least one of collagen, gelatin, fibrinogen, fibronectin, or laminin.

34. The coating in step (e(v)) is AELDVP (SEQ ID NO: 1), CGGGEPRGDTYRAY (SEQ ID NO: 2), CFALRGDNP (SEQ ID NO: 3), CKKQRRFHRRNRKG (SEQ ID NO: 4), CNYYSNS (SEQ ID NO: 5), CSVTCG (SEQ ID NO: 6), DGEA (SEQ ID NO: 7), ELVTDFPTDLPAT (SEQ ID NO: 8), FHRRIKA (SEQ ID NO: 9), FQGVLQNVRFVF (SEQ ID NO: 10), GACRGDCLGA (cyclic) (SEQ ID NO: 11), GFOGER (SEQ ID NO: 12), GFRGDGQ (SEQ ID NO: 13), GRGDS (SEQ ID NO: 14), GRGDAC (SEQ ID NO: 15), GTFALRGDNGQ (SEQ ID NO: 16), IDAPS (SEQ ID NO: 17), IKLLI (SEQ ID NO: 18), IKVAV (SEQ ID NO: 19), IWKHKGRDVILKKDVRFYC (SEQ ID NO: 20), KAFDITYVRLKF (SEQ ID NO: 21), KLDAPT (FN5) (SEQ ID NO: 22), KQAGDV (SEQ ID NO: 11), GFOGER (SEQ ID NO: 12), GFRGDGQ (SEQ ID NO: 13), GRGDS (SEQ ID NO: 14), GRGDAC (SEQ ID NO: 15), GTFALRGDNGQ (SEQ ID NO: 16), IDAPS (SEQ ID NO: 17), IKLLI 23) KRSR (SEQ ID NO: 24), LIGRKK (SEQ ID NO: 25), LGTIPG (SEQ ID NO: 26), LRE, LRGDN (SEQ ID NO: 27), MNYYSNS (SEQ ID NO: 28), NPWHSIYITRFG (SEQ ID NO: 29), PDGSR (SEQ ID NO: 30), PHRSN (SEQ ID NO: 31), PKRGDL (SEQ ID NO: 32), PRARI (SEQ ID NO: 33), REDV (SEQ ID NO: 34), RGD, SIGFRGDGQTC (SEQ ID NO: 35), SIKVAV (SEQ ID NO: 36), The process according to any one of claims 5 to 33, comprising at least one short-chain peptide sequence that embodies a cell adhesion peptide sequence, selected from SINNNR (SEQ ID NO: 37), SPPRRARV (SEQ ID NO: 38), SVVYGLR (SEQ ID NO: 39), TWYKIAFQRNRK (SEQ ID NO: 40), VALDEP (SEQ ID NO: 41), VGVAPG (SEQ ID NO: 42), VPGIG (SEQ ID NO: 43), WQPPRARI (SEQ ID NO: 44), and YIGSR (SEQ ID NO: 45).

35. The process according to any one of claims 5 to 34, wherein in step (e(vi)), the surface is modified by a process of plasma treatment, physical vapor deposition, ultrasonic treatment, or mechanical etching.

36. A hollow fiber obtained or obtainable by the process described in any one of claims 1 to 35.

37. The hollow fiber according to claim 1, 2, 3, 4, or 36, wherein the porosity is 1% to 95%.

38. The hollow fiber according to claim 1, 2, 3, 4, or 37, wherein the porosity is 45% to 85%.

39. The hollow fiber according to claim 38, wherein the hollow fiber is water-resistant for three days or more.

40. A semipermeable porous hollow fiber comprising one or more polypeptides crosslinked by polycarboxylic acid-derived esters, thioesters, or amide covalent bonds, wherein the material of the hollow fiber is GRAS.

41. A hollow fiber cartridge wherein the material includes the product described in any one of claims 5 to 40.

42. A hollow fiber bioreactor wherein the material includes the product described in any one of claims 5 to 41.

43. A process for producing a food product comprising the hollow fiber produced by the process described in any one of claims 5 to 42 and a plurality of cells.

44. Foods containing polycarboxylic acid-derived esters, thioesters, or polypeptides crosslinked by amide covalent bonds.

45. A food product produced by a process according to any one of claims 5 to 44, comprising a polypeptide hollow fiber crosslinked by covalent bonds.

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