Hollow fibres

Abstract

The present disclosure provides extruded or spun, semi-permeable, porous hollow fibres, comprising covalent ester, thioester and / or amide crosslinked polypeptides as well as processes for their production. The hollow fibres may be produced from protein, 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 fibres may be designed to be used in the production of cartridges that are compatible with existing and / or novel bioreactor platforms, for harbouring cell cultures in cultured meat production.

Description

This disclosure provides semi-permeable porous hollow fibers, including extruded or spun polypeptides comprising covalent esters, thioesters, and / or amide crosslinks, and methods for producing the same. These hollow fibers can be produced from proteins, protein extracts, and / or protein isolates derived from plants, animals, bacteria, algae, archaea, and / or fungi, and in some embodiments are designed to be suitable for human and / or animal ingestion. In some embodiments, these hollow fibers can be designed for the production of tubes compatible with existing and / or novel bioreactor platforms for containing cell cultures in cultured meat production. Abstract

Claims

CLAIMS1 . An edible, semi-permeable, porous hollow fibre, comprising one more polycarboxylic acid derived covalent ester, thioester and / or amide bond crosslinked polypeptides wherein:(a) the external diameter of the hollow fibre is 50 - 6600 pM;(b) the wall thickness of the hollow fibre is 20 - 800 pM;(c) the lumen diameter of hollow fibre is 20 - 5000 pM; and(d) the pore volume of the hollow fibre is 1 - 95 %.

2. A semi-permeable, porous hollow fibre, comprising one more polycarboxylic acid derived covalent ester, thioester and / or amide bond crosslinked polypeptides wherein:(a) the external diameter of the hollow fibre is 50 - 6600 pM;(b) the wall thickness of the hollow fibre is 20 - 800 pM;(c) the lumen diameter of hollow fibre is 20 - 5000 pM; and(d) the pore volume of the hollow fibre is 1- 95 %.

3. An edible, semi-permeable, porous hollow fibre, comprising one more polycarboxylic acid derived covalent ester, thioester and / or amide bond crosslinked polypeptides wherein:(a) the external diameter of the hollow fibre is 50 - 6600 pm;(b) the wall thickness of the hollow fibre is 20 - 800 pm;(c) the lumen diameter of hollow fibre is 20 - 5000 pm; and(d) the porosity of the hollow fibre is 1- 95 %.

4. A semi-permeable, porous hollow fibre, comprising one more polycarboxylic acid derived covalent ester, thioester and / or amide bond crosslinked polypeptides wherein:(a) the external diameter of the hollow fibre is 50 - 6600 pm;(b) the wall thickness of the hollow fibre is 20 - 800 pm;(c) the lumen diameter of hollow fibre is 20 - 5000 pm; and(d) the porosity of the hollow fibre is 1- 95 %.

5. A process for the production of semi-permeable, porous hollow fibres, comprising covalently-crosslinked polypeptides, the process comprising the steps: a. combining: i. a first composition comprising polypeptides, with ii. a second composition comprising a solvent and one or more denaturing agents and / or reducing agents, to produce a third composition; b. incubating the third composition under conditions sufficient to solubilise, denature and / or reduce at least a fraction of the polypeptides, and to produce a fourth composition; c. extruding or spinning the fourth composition, together with a bore solution, through a plurality of co-axial orifices to produce hollow fibres; d. treating the hollow fibres with a polycarboxylic acid crosslinking reagent in order to form inter-polypeptide and / or intra-polypeptide polycarboxylic acid derived ester, thioester or amide covalent bond crosslinks within at least a fraction of the polypeptides in the hollow fibres to produce covalently-crosslinked, semi- permeable, porous hollow fibres hollow fibres; e. treating the covalently-crosslinked, semi-permeable, porous hollow fibres with one of more post-production modification processes selected from the group consisting of: i. treating the covalently-crosslinked, semi-permeable, porous hollow fibres with an organic solvent to increase the relative abundance of beta-sheets in the secondary structure of the polypeptides; and ii. annealing the covalently-crosslinked, semi-permeable, porous hollow fibres; iii. treating the covalently-crosslinked semi-permeable, porous hollow fibres with a solvent to remove void inclusion elements in the hollow fibres; iv. washing the covalently-crosslinked semi-permeable, porous hollow fibres with one or more acid(s), alkali(s) and / or buffer solution(s) todecrease at least one of, the Young’s modulus, ultimate tensile strength and / or ultimate tensile strain of the hollow fibres.; v. coating the covalently-crosslinked semi-permeable, porous hollow fibres; vi. modifying the surface topography of the covalently-crosslinked, semi- permeable, porous hollow fibres to aid cell attachment and / or cell alignment; to produce a plurality of treated covalently-crosslinked, semi-permeable, porous hollow fibres hollow fibres; and f. drying the treated covalently-crosslinked, semi-permeable, porous hollow fibres, to produce dried, treated covalently-crosslinked, semi-permeable, porous hollow fibres.

6. The process of claim 5, further providing that the first composition comprises polypeptides derived from plants.

7. The process of claims 5 to 6, further providing that polysaccharide is supplemented to the first composition of claim 5 Step (a) in a ratio that is within the range of 1 % to 10000 % (weight per weight of polypeptide).

8. The process of any one of claims 5 to 7, further providing that lipid is supplemented to the first composition of claim 5 Step (a) in a ratio that is within the range of 1 % to 10000 % (weight per weight of polypeptide).

9. The process any one of claims 5 to 8, further providing that polyol(s) and or polymer(s) of polyols are supplemented to the first composition of claim 5 Step (a) in a ratio that is within the range of 1 % to 10000 % (weight per weight of polypeptide).

10. The process of any one of claims 5 to 9, wherein said the second composition consists of one or more solvent(s) consisting, 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 of any one of claims 5 to 10, wherein said the second composition comprises one or more solvent(s) comprises, 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 of any one of claims 5 to 11 , wherein said the second composition comprises of one or more denaturing agent(s) of at least one of, 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 of any one of claims 5 to 12, wherein said the second composition consists of one or more reducing agent(s) including, N-acetyl-cysteine, L-cysteine, glutathione, ascorbic acid, citric acid, tartaric acid, malic acid, sodium borohydride, sodium sulphite, sodium bisulphite, sodium metabisulphite, sodium hypophosphite, sodium hydrosulphite, mercaptoethanol, and / or dithiothreitol.

14. The process of any one of claims 5 to 13, wherein said the second composition comprises one or more reducing agent(s) including, N-acetyl-cysteine, L-cysteine, glutathione, ascorbic acid, citric acid, tartaric acid, malic acid, sodium borohydride, sodium sulphite, sodium bisulphite, sodium metabisulphite, sodium hypophosphite, sodium hydrosulphite, mercaptoethanol, and / or dithiothreitol.

15. The process of any one of claims 5 to 14, wherein the third composition of Step (b) is incubated for a total duration between 5 minutes and 1 week.

16. The process of any one of claims 5 to 15, wherein the third composition or fourth composition of Step (b) is incubated at temperatures between 0 °C minutes and 150 °C.

17. The process of any one of claims 5 to 16, wherein said the fourth composition comprises one or more void element(s) including calcium carbonate, ice, air, nitrogen, carbon dioxide, argon, sodium chloride, or potassium chloride.

18. The process of any one of claims 5 to 17, further providing that, void inclusion elements are added to the compositions in Step (a) or Step (b), such that the void inclusion elements comprise 1 % to 80 % of the fourth composition (iv).

19. The process of any one of claims 5 to 18, wherein said the coagulation bath solution comprises one or more polycarboxylic acid salts including sodium citrate, sodium malate, potassium malate, and / or potassium citrate.

20. The process of any one of claims 5 to 19, further providing that a salt is added to the composition of the coagulation bath solution zinc sulphate, sodium sulphite, sodium bisulphite, sodium meta-bisulphite, sodium hypophosphite and / or ammonium sulphate.21 . The process of any one of claims 5 to 20, wherein said the coagulation bath solution comprises one or more polycarboxylic acid salts including sodium oxalate, potassium oxalate, sodium malate, potassium malate, sodium succinate, potassium succinate, sodium adipate, potassium adipate, sodium tartrate, potassium tartrate, sodium malonate, and / or potassium malonate and is supplemented with a salt including zinc sulphate, sodium sulphite, sodium bisulphite, sodium meta-bisulphite, sodium hypophosphite and / or ammonium sulphate.

22. The process of any one of claims 5 to 21 , wherein the pH of the coagulation bath solution is above 7.0 and less than 10.0.

23. The process of any one claims 5 to 22, wherein the temperature of the coagulation bath solution is between 0 °C and 90 °C.

24. The process of any one of claims 5 to 23, where in the hollow fibre is washed in an excess of aqueous solvent with a pH between 7.0 and 8.0 after step (d).

25. The process of any one of claims 5 to 24 wherein the organic solvent used in step (e.i) includes at least one of methanol, ethanol, propanol, iso-propanol, butanol, iso-butanol, dimethyl sulfoxide, dimethylformamide, and / or dimethylacetamide.

26. The process of any one of claims 5 to 25, wherein the organic solvent used in step (e.i) includes at least one of polyol(s) and / or polymer(s) of polyols.

27. The process of any one of claims 5 to 26, wherein the annealing in step (e(ii)) is carried out at 75 °C to 180 °C.

28. The process of any one of claims 5 to 27, wherein the annealing in step (e.ii) is carried out at 75 °C to 180 °C over a total duration that ranges between 10 minutes and 6 hours.

29. The process of 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 of 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, iso-propanol, butanol, iso-butanol, dimethyl sulfoxide, dimethylformamide, dimethylacetamide.31 . The process of any one of claims 5 to 30, wherein the solvent used in the void removal step (e(iii)) comprises polyol(s) and / polymer(s) of polyols.

32. The process of any one of claims 5 to 31 , where in the solvent used to lower, at least one of, the Young’s modulus, ultimate tensile strength and / or ultimate tensile strain of the hollow fibres in step (e(iv)) comprise 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, sulphuric acid, tartaric acid sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or sodium carbonate.

33. The process of any one of claims 5 to 32, wherein the coating in Step (e(v)) comprises at least one of, collagen, gelatine, fibrinogen, fibronectin or laminin.

34. The process of any one of claims 5 to 33, wherein the coating in Step (e(v)) comprises at least one short peptide sequence embodying a cell attachment peptide sequenceselected from, AELDVP (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: 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), PRARI (SEQ ID NO: 33), REDV (SEQ ID NO: 34), RGD, SIGFRGDGQTC (SEQ ID NO: 35), SIKVAV (SEQ ID NO: 36), 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), YIGSR (SEQ ID NO: 45).

35. The process of any one of claims 5 to 34, wherein the surface is modified in Step (e(vi)) by a process of plasma treatment, physical vapour deposition, ultrasonification or mechanical etching.

36. A hollow fibre obtained or obtainable by a process of any one of claims 1 - 35.

37. The hollow fibre of claim 1 , 2, 3, 4, or 36, wherein the porosity is 1 % to 95 %.

38. The hollow fibre of claim 1 , 2, 3, 4, or 37, wherein the porosity is 45 % to 85 %.

39. The hollow fibre of claim 38, wherein the hollow fibre is water stable for 3 or more days.

40. A semi-permeable porous hollow fibre, comprising one more polypeptides crosslinked with polycarboxylic acid derived ester, thioester or amide covalent bonds wherein the materials of the hollow fibres are GRAS.41 . A hollow fibre cartridge wherein the materials include products of any one of claims 5 - 40.

42. A hollow fibre bioreactor wherein the materials include products of any one of claims 5 - 41.

43. A process to produce a food product that comprises the hollow fibres produced by the processes any one of claims 5-42 and a plurality of cells 44. A food product which comprises polypeptides crosslinked with polycarboxylic acid derived ester, thioester or amide covalent bonds.

45. A food product produced by the processes any one of claims 5-44, which comprises covalently-crosslinked polypeptide hollow fibre.

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