Freeze-dried and sprayable fibroin and modified fibroin forms

Abstract

The present disclosure relates to freeze-dried or sprayable peptide compositions, such as freeze-dried or sprayable fibroin-derived peptide compositions.

Description

Technical Field

[0001] The present disclosure relates to peptide compositions, such as fibroin-derived peptide compositions. Background Art

[0002] Silk is a natural polymer produced by various insects and spiders and comprises a core protein, fibroin, and a gelatinous coating composed of a non-silk protein, sericin. Summary of the Invention

[0003] Embodiments of the present disclosure provide a plurality of substantially solid silk fibroin particles comprising silk fibroin fragments, the particles being characterized by at least one of a bulk density, a surface area, a pore size, a pore volume, an aspect ratio, and / or a Hausner ratio. In some embodiments, the substantially solid silk fibroin particles are annealed. In some embodiments, the substantially solid silk fibroin particles are ground. In some embodiments, the bulk density of the substantially solid silk fibroin particles is less than 0.03 g / ml, less than 0.04 g / ml, less than 0.05 g / ml, less than 0.06 g / ml, less than 0.07 g / ml, less than 0.08 g / ml, less than 0.09 g / ml, less than 0.10 g / ml, less than 0.11 g / ml, less than 0.12 g / ml, less than 0.13 g / ml, less than 0.14 g / ml, less than 0.15 g / ml, less than 0.16 g / ml, less than 0.17 g / ml, less than 0.18 g / ml, less than 0.19 g / ml, less than 0.20 g / ml, less than 0.21 g / ml, less than 0.22 g / ml, less than 0.23 g / ml, less than 0.24 g / ml, less than 0.25 g / ml, less than 0.26 g / ml, less than 0.27 g / ml, less than 0.28 g / ml, less than 0.29 g / ml, less than 0.30 g / ml, less than 0.31 g / ml, less than 0.32 g / ml, less than 0.33 g / ml, less than 0.34 g / ml, less than 0.35 g / ml, less than 0.36 g / ml, less than 0.37 g / ml, less than 0.38 g / ml, .18g / ml, less than 0.19g / ml, less than 0.20g / ml, less than 0.21g / ml, less than 0.22g / ml, less than 0.23g / ml, less than 0.24g / ml, or less than 0.25g / ml, less than 0.26g / ml, less than 0.27g / ml, less than 0.28g / ml, less than 0.29g / ml, less than 0.30g / ml, less than 0.31g / ml, less than 0.32g / ml, less than 0.33g / ml, less than 0.34g / ml, or less than 0.35g / ml. In some embodiments, the substantially solid fibroin particles have an average bulk density of about 0.03 g / ml, about 0.04 g / ml, about 0.05 g / ml, ...6 g / ml, about 0.07 g / ml, about 0.08 g / ml, about 0.09 g / ml, about 0.10 g / ml, about 0.11 g / ml, about 0.12 g / ml, about 0.13 g / ml, about 0.14 g / ml, about 0.15 g / ml, about 0.16 g / ml, about 0.17 g / ml, about 0.18 g / ml, about 0.19 g / ml, about 10 g / ml, about 11 g / ml, about 12 g / ml, about 13 g / ml, about 14 g / ml, about 15 g / ml, about 16 g / ml, about 17 g / ml, about 18 g / ml, about 19 g / ml, about 20 g / ml, about 21 g / ml, about 22 g / ml, about 23 g / ml, about 24 g / ml g / ml, about 0.17 g / ml, about 0.18 g / ml, about 0.19 g / ml, about 0.20 g / ml, about 0.21 g / ml, about 0.22 g / ml, about 0.23 g / ml, about 0.24 g / ml, about 0.25 g / ml, about 0.26 g / ml, about 0.27 g / ml, about 0.28 g / ml, about 0.29 g / ml, about 0.30 g / ml, about 0.31 g / ml, about 0.32 g / ml, about 0.33 g / ml, about 0.34 g / ml or about 0.35 g / ml.In some embodiments, the Hausner ratio of the substantially solid silk fibroin particles is 1.00 to 1.11, 1.12 to 1.18, 1.19 to 1.25, 1.26 to 1.34 or 1.35 to 1.45. In some embodiments, the average diameter of the substantially solid silk fibroin particles is about 3 mm to about 10 mm. In some embodiments, the average diameter of the substantially solid silk fibroin particles is about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm or about 20 mm. In some embodiments, the aspect ratio of the substantially solid silk fibroin particles is 1 to about 1.45. In some embodiments, the substantially solid fibroin particle has an aspect ratio of 1, about 1.10, about 1.15, about 1.20, about 1.25, about 1.30, about 1.35, about 1.40, or about 1.45. In some embodiments, the substantially solid fibroin particle has an aspect ratio of 1, about 1.10, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.20, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, In some embodiments, the substantially solid fibroin particles are substantially spherical. In some embodiments, the substantially solid fibroin particles are mesoporous. In some embodiments, the substantially solid fibroin particles have a BET (Brunauer–Emmett–Teller) surface area of ​​about 2.50 m2. 2 / g to about 6.50m 2 In some embodiments, the substantially solid fibroin particles have a BET (Brunauer–Emmett–Teller) surface area of ​​about 2.50 m 2 / g to about 3.00m 2 / g, about 3.00m 2 / g to about 3.50m 2 / g, about 3.50m 2 / g to about 4.00m 2 / g, about 4.00m 2 / g to about 4.50m 2 / g, about 4.50m 2 / g to about 5.00m 2 / g, about 5.00m 2 / g to about 5.50m 2 / g, about 5.50m 2 / g to about 6.00m 2 / g, or about 6.00m 2 / g to about 6.50m 2 / g. In some embodiments, the average pore size of the substantially solid silk fibroin particles is about to about In some embodiments, the substantially solid silk fibroin particles have an average pore size of about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about or about to about In some embodiments, the substantially solid fibroin particles comprise a plurality of radially oriented microchannels. In some embodiments, the substantially solid fibroin particles comprise an emulsifier, a surfactant, a buffer, an amino acid or a sugar. In some embodiments, the substantially solid fibroin particles comprise a polysaccharide, polysorbate, a glycoside, PBS, arginine, trehalose, glucose or sucrose. In some embodiments, the substantially solid fibroin particles comprise a surfactant selected from sucrose esters, cetearyl glucoside, capryloyl / capryloyl glucoside, sucrose laurate, sucrose palmitate, sucrose stearate, sucrose cocoate, sorbitan monostearate and a combination thereof. In some embodiments, the substantially solid fibroin particles comprise another protein or peptide, C12-C24 fatty alcohol, glycolipid or lipid. In some embodiments, the substantially solid fibroin particles comprise a protein, peptide, sugar surfactant, biosurfactant, lipid or a combination. In some embodiments, the substantially solid silk fibroin particles comprise sugar fatty acid esters, sugar fatty acid monoesters, sugar fat diesters, sugar fat triesters, or sugar fats and polyesters. In some embodiments, the substantially solid silk fibroin particles comprise sucrose fatty acid esters, sorbitan fatty acid esters or sorbitol fatty acid esters, alkyl glucosides, alkyl polyglucosides, or combinations thereof. In some embodiments, the substantially solid silk fibroin particles comprise KCl, NaCl, MgCl2, CaCl2, PBS, Tris, polysorbate 20, polysorbate 80, decanoyl glucoside, sucrose, histidine, glycine, or arginine. In some embodiments, the stabilizer is selected from polysaccharides (such as but not limited to maltodextrin, dextran), surfactants (such as but not limited to polysorbate (20, 60, 80), decanoyl glucoside), buffers (such as but not limited to PBS, triacetate, sodium phosphate), amino acids (such as but not limited to arginine, glycine, cysteine, histidine, lysine, serine) and / or sugars (such as but not limited to trehalose, glucose, sucrose, maltose, fucose).

[0004] Embodiments of the present disclosure provide a silk fibroin nanoclay composite or film comprising a silk fibroin fragment and clay, wherein the % (w / w) of clay in the composite is from about 1% to about 99%. In some embodiments, the clay is bentonite. In some embodiments, the concentration of clay in the composite or film is from about 20% (w / w) to about 33% (w / w), from about 33% (w / w) to about 50% (w / w), or from about 50% (w / w) to about 67% (w / w). In some embodiments, the water vapor permeability (WVP) of the composite is inversely proportional to the concentration of clay in the composite. In some embodiments, the water vapor permeability (WVP, g / m 2 *Pa*24h) is about 0.20 to about 0.30, about 0.30 to about 0.35, about 0.35 to about 0.40, about 0.40 to about 0.45, about 0.45 to about 0.50, about 0.50 to about 0.55, about 0.55 to about 0.60, about 0.60 to about 0.65, about 0.65 to about 0.70, about 0.70 to about 0.75, about 0.75 to about 0.80, about 0.80 to about 0.85.

[0005] Embodiments of the present disclosure provide a stable silk fibroin solution comprising silk fibroin fragments and a stabilizer, wherein the z-average value of the solution is lower than that of a substantially similar silk fibroin solution comprising the silk fibroin fragments but not comprising the stabilizer, and / or the z-average plateau value of the solution is lower than that of a substantially similar silk fibroin solution comprising the silk fibroin fragments but not comprising the stabilizer. In some embodiments, the z-average value is measured after the silk fibroin fragments and the stabilizer are co-formulated for a period of time, wherein the period of time ranges from 1 hour to 250 hours, 1 hour to 350 hours, 1 hour to 450 hours, 1 hour to 550 hours, 1 hour to 650 hours, or 1 hour to 1000 hours. In some embodiments, the z-average value is measured after the silk fibroin fragments and the stabilizer are co-formulated for a period of time, wherein the period of time ranges from 1 minute to 10 minutes, 1 minute to 20 minutes, 1 minute to 30 minutes, 1 minute to 40 minutes, 1 minute to 50 minutes, 1 minute to 60 minutes, 1 minute to 70 minutes, or 1 minute to 80 minutes. In some embodiments, the stabilizer is an emulsifier, a surfactant, a buffer, an amino acid or a sugar. In some embodiments, the stabilizer is a polysaccharide, a polysorbate, a glycoside, PBS, arginine, trehalose, glucose or sucrose. In some embodiments, the stabilizer is a surfactant selected from sucrose ester, cetearyl glucoside, capryloyl / capryl glucoside, sucrose laurate, sucrose palmitate, sucrose stearate, sucrose cocoate, sorbitan monostearate and their combination. In some embodiments, the stabilizer is another protein or peptide, C12-C24 fatty alcohol, glycolipid or lipid. In some embodiments, the stabilizer is a protein, peptide, sugar surfactant, biosurfactant, lipid or combination. In some embodiments, the stabilizer is sugar fatty acid ester, sugar fatty acid monoester, sugar fat diester, sugar fat triester or sugar fat and polyester. In some embodiments, the stabilizer is a sucrose fatty acid ester, a sorbitan fatty acid ester or a sorbitol fatty acid ester, an alkyl glucoside, an alkyl polyglucoside or a combination thereof. In some embodiments, the stabilizer is KCl, NaCl, MgCl2, CaCl2, PBS, Tris, polysorbate 20, polysorbate 80, decanoyl glucoside, sucrose, histidine, glycine or arginine. In some embodiments, the stabilizer is selected from polysaccharides (such as but not limited to maltodextrin, dextran), surfactants (such as but not limited to polysorbate (20, 60, 80), decanoyl glucoside), buffers (such as but not limited to PBS, triacetate, sodium phosphate), amino acids (such as but not limited to arginine, glycine, cysteine, histidine, lysine, serine) and / or sugars (such as but not limited to trehalose, glucose, sucrose, maltose, fucose).

[0006] In some embodiments, the solution is sprayable. Embodiments of the present disclosure provide a liquid suspension in air comprising a plurality of droplets comprising a stable silk fibroin solution as described above, wherein the droplets are sufficiently stable after spraying for a period of time required to reach a surface. In some embodiments, the droplets or droplets are sufficiently stable after formation for a period of time required to reach a surface.

[0007] In some embodiments, the fibroin fragments in any of the embodiments of the plurality of substantially solid fibroin particles, fibroin nanoclay composites or films, stable fibroin solutions, liquid suspensions in air, or plurality of droplets or droplets described herein have a particle size selected from the group consisting of about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 14 kDa to about 30 kDa, about 15 kDa to about 20 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about The invention also provides a weight average molecular weight of about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, about 55 kDa to about 60 kDa, about 60 kDa to about 100 kDa, about 80 kDa to about 144 kDa, about 144 kDa to about 250 kDa, or about 250 kDa to about 350 kDa, and a polydispersity of 1 to about 5. In some embodiments, any of the above embodiments has a polydispersity of 1 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, or about 4.5 to about 5.0. Any of the above embodiments may further comprise from about 0.001% (w / w) to about 10% (w / w) sericin relative to the silk fibroin fragments. In any of the above embodiments, the silk fibroin fragments do not spontaneously or gradually gel and do not significantly change in color or turbidity in aqueous solution for at least 10 days before being formulated into substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, or stabilized silk fibroin solutions.

[0008] In any implementation of the embodiments of a plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets described herein, the silk fibroin fragment may comprise a plurality of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P, and W, wherein at least one of these amino acids is modified, substituted, or replaced. In any of the above embodiments, the silk fibroin is a silk fibroin heavy chain, a silk fibroin light chain, or a silk fibroin hexamer. In any of the above embodiments, the silk fibroin fragment comprises from about 2 to about 100 amino acids. In any of the above embodiments, the silk fibroin fragment comprises from 1 to 5 modifications, substitutions, and / or replacements. In any of the above embodiments, the modifications, substitutions, and / or replacements are selected from asparagine to aspartic acid modifications, substitutions, and / or replacements, glutamine to glutamic acid modifications, substitutions, and / or replacements, and methionine to methionine oxide modifications, substitutions, and / or replacements. In any of the above embodiments, the fibroin is a fibroin heavy chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the fibroin heavy chain. In any of the above embodiments, the modification, substitution and / or replacement is at Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 and / or N5262. In any of the above embodiments, the fibroin is a fibroin light chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 262 of the fibroin light chain. In any of the above embodiments, the modification, substitution and / or replacement is at N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 and / or Q255. In any of the above embodiments, the fibroin is a fibroin hexamer (p25), and the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 220 of the fibroin hexamer (p25). In any of the above embodiments, the modification, substitution and / or replacement is at Q62, N93, M120, N149, N172, N174 and / or N202. In any of the above embodiments, each modification, substitution and / or replacement is independently in the range of about 1% to about 99% in the silk fibroin fragment portion of the substantially solid silk fibroin particle, silk fibroin nanoclay composite or film, or stabilized silk fibroin solution composition.In any of the above embodiments, the % modification, % substitution and / or % replacement is defined as (the number of peptides or protein fragments comprising a modification, substitution and / or replacement at a particular position divided by the total number of peptides or protein fragments comprising the particular position, whether or not comprising a modification, substitution and / or replacement) x 100.

[0009] In any of the plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films described herein, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets, the silk fibroin fragments are contained in one or more fractions, each fraction independently comprising a plurality of silk fibroin heavy chain fragments, a plurality of silk fibroin light chain fragments, and / or a plurality of silk fibroin hexamer (p25) fragments. In any of these embodiments, the silk fibroin fragments have a weight average molecular weight (MW) selected from about 1 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, or about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 250 kDa. w ) and a polydispersity of 1 to about 1.7. In any of the above embodiments, the fibroin fragments have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, or about 160 kDa to about 180 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.2. In any of the above embodiments, the fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.2. In any of the above embodiments, the fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 120 kDa. w) and a polydispersity of 1 to about 1.1. In any of the above embodiments, the fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.2. In any of the above embodiments, the fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. w ) and a polydispersity of 1 to about 1.1. In any of the above embodiments, the fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 20 kDa to about 40 kDa, or about 40 kDa to about 60 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.2. In any of the above embodiments, the one or more fractions are selected from AS77, AS78, AS79, AS80, and AS81. In any of the above embodiments, the one or more fractions are selected from AS82, AS83, AS84, AS85, AS86, AS87, AS88, and AS89. In any of the above embodiments, the one or more fractions are selected from AS90, AS91, AS92, AS93, and AS94. In any of the above embodiments, the one or more fractions are selected from AS95, AS96, AS97, AS98, AS99, and AS100. In any of the above embodiments, the fibroin fragments have a weight average molecular weight (MW) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 220 kDa. w) and a polydispersity of 1 to about 1.7. In any of the above embodiments, the fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. w ) and a polydispersity of 1 to about 1.2 or 1 to about 1.3. In any of the above embodiments, the fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.2. In any of the above embodiments, the fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. w) and a polydispersity of 1 to about 1.2 or 1 to about 1.3. In any of the above embodiments, the one or more fractions are selected from AS101, AS102, AS103, AS104 and AS105. In any of the above embodiments, the one or more fractions are selected from AS106, AS107, AS108, AS109, AS110 and AS111. In any of the above embodiments, the fibroin fragments comprise one or more amino acid modifications, substitutions or replacements of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P and W. In any of the above embodiments, wherein the fibroin fragments comprise about 2 to about 100 amino acids. In any of the above embodiments, the fibroin fragments comprise 1 to 5 modifications, substitutions and / or replacements. In any of the above embodiments, the fibroin is a fibroin heavy chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the fibroin heavy chain. In any of the above embodiments, the fibroin is a fibroin light chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 262 of the fibroin light chain. In any of the above embodiments, the fibroin is a fibroin hexamer (p25) chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 220 of the fibroin hexamer (p25) chain. In any of the above embodiments, the modification, substitution and / or replacement is selected from asparagine to aspartic acid modification, substitution and / or replacement, glutamine to glutamic acid modification, substitution and / or replacement, and methionine to methionine oxide modification, substitution and / or replacement. In any of the above embodiments, the modification, substitution and / or replacement is at a fibroin heavy chain position selected from Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 and / or N5262. In any of the above embodiments, the modification, substitution and / or replacement is at a fibroin light chain position selected from N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 and / or Q255. In any of the above embodiments, the modification, substitution and / or replacement is at a position of the fibroin hexamer (p25) selected from Q62, N93, M120, N149, N172, N174 and / or N202.In any of the above embodiments, each modification, substitution and / or replacement is independently in the range of about 1% to about 99% in the composition. In any of the above embodiments, the % modification, % substitution and / or % replacement is defined as (the number of peptides or protein fragments comprising a modification, substitution and / or replacement at a particular position divided by the total number of peptides or protein fragments comprising a particular position, whether or not comprising a modification, substitution and / or replacement) x 100.

[0010] In any of the above embodiments, the molecular weight is determined by MALS.

[0011] Embodiments of the present disclosure provide a pouch or laundry bead comprising a plurality of substantially solid silk fibroin particles of any of the above-described embodiments. In some embodiments, the plurality of substantially solid silk fibroin particles are compressed into a multi-particle disc. In some embodiments, the laundry bead further comprises a dissolvable shell comprising polyvinyl alcohol (PVA) or a PVA derivative. In some embodiments, the laundry pod further comprises a shell comprising one or more of nylon, polyglycolide (PGA), polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), polycaprolactone (PCL), poly(butylene succinate) (PBS), polybutylene succinate adipate, poly(p-dioxanone) (PPDO), poly(butylene adipate-co-terephthalate) (PBAT), copolyesters of terephthalic acid and lactic acid, copolyesters of terephthalic acid and glycolic acid, copolyesters of terephthalic acid and succinic acid, poly(hydroxybutyrate), poly(hydroxyvalerate), polyhydroxyhexanoate, poly(hydroxyalkanoate) (PHA), polymethylene adipate / terephthalate.

[0012] Embodiments of the present disclosure provide a method for preparing a plurality of substantially solid silk fibroin particles of any of the above-described embodiments, the method comprising dropping a solution comprising a plurality of silk fibroin fragments into liquid nitrogen. In some embodiments, the method further comprises a freeze-drying step. In some embodiments, the concentration of the silk fibroin fragments in the solution is from about 3% (w / w) to about 50% (w / w). In some embodiments, the concentration of the silk fibroin fragments in the solution is from about 6% (w / w) to about 25% (w / w). In some embodiments, the concentration of the silk fibroin fragments in the solution is from about 6% (w / w) to about 20% (w / w). In some embodiments, the concentration of the silk fibroin fragments in the solution is about 3% (w / w), about 4% (w / w), about 5% (w / w), about 6% (w / w), about 7% (w / w), about 8% (w / w), about 9% (w / w), about 10% (w / w), about 11% (w / w), about 12% (w / w), about 13% (w / w), about 14% (w / w), about 15% (w / w), about 16% (w / w), about 17% (w / w), about 18% (w / w), about 19% (w / w), about 20% (w / w), about 21% (w / w), about 22% (w / w), about 23% (w / w), about 24% (w / w) or about 25% (w / w). In some embodiments, the fibroin fragments comprise one or more of a molecular weight, polydispersity, and / or modifications, substitutions, and / or replacements at specific amino acid positions as defined in any of the above embodiments. In some embodiments, the solution is stable as defined in any of the above embodiments.

[0013] In any of the above embodiments, the particles have a reconstitution yield greater than 90%. In any of the above embodiments, the particles have a reconstitution yield greater than 90% in deionized water.

[0014] In some embodiments, at least 90% of the reconstitution rate is maintained after a stability test comprising simulated aging of the plurality of substantially solid fibroin particles of any one of claims 1 to 92, the aging comprising storage at a temperature of about 40° C. to about 60° C. for a period of time in a range from about 400 days to about 650 days. In some embodiments, the simulated aging range is from about 4 years to about 15 years. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The presently disclosed embodiments will be further explained with reference to the accompanying drawings. The drawings shown are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the presently disclosed embodiments.

[0016] Figure 1Ion exchange sorting scheme for separating populations comprising low-slip and moderate-slip silk / modified polypeptide compositions. Low-slip and moderate-slip silk / modified polypeptide compositions contain negatively charged, positively charged, or neutral silk / modified polypeptides. These populations were separated using Q anion exchange chromatography (A).

[0017] Figure 2 .Chromatogram of a low-slip silk / modified peptide composition loaded on a Q-Sepharose HP column (Cytiva). The effluent contains silk / modified peptides that cannot be captured in the column and are depleted of negatively charged amino acids. After loading the column with a low-slip or medium-slip silk / modified peptide composition and collecting the effluent, the column is washed until the UV-280 absorbance becomes less than 200 AU. The captured negatively charged silk / modified peptides are eluted with a high salt concentration (1 M NaCl) and constitute AS11 and AS22. Chromatography was performed in a buffer containing Tris, but the effluent and Q-eluate were finally dialyzed in water.

[0018] Figure 3 Analytical size exclusion chromatography of low-slip, medium-slip / modified silk compositions and their component AS compositions. Average molecular weight in kDa and polydispersity measurements are shown.

[0019] Figures 4A-4B Analytical size exclusion chromatography of low- and medium-slippery silk / modified peptide compositions and their components (see Table 1 for more details). Figure 4A , the molecular weights of the various new active silk compositions described in this study. Figure 4B , the polydispersity (PDI) of the various novel active silk compositions described in this study. AS24 reconstituted the average molecular weight and polydispersity of the low-slip / modified peptide composition and was composed of 50% AS12 and 50% AS22 (see Table 1 for details). AS6 reconstituted the average molecular weight and polydispersity of the medium-slip / modified peptide composition and was composed of 50% AS1 and 50% AS11 (see Table 1 for details).

[0020] Figure 5 Isoelectric focusing electrophoresis of low-slippery silk / modified peptide compositions. Lanes 2 and 7: Low-slippery silk, different loading amounts. Lanes 3, 5, 8, and 10: AS12 silk, different preparations with different loading amounts. Lanes 4, 6, 9, and 11: AS22 silk, different preparations with different loading amounts.

[0021] Figures 6A-6BSelf-assembly reactions of low- and medium-slip silk / modified peptide compositions and their components (see Table 1 for further details). Both figures depict the kinetic parameters of gel formation during silk self-assembly. In panel A, the calculations of three self-assembly kinetic parameters, t0.5, Amax, and SARF, are shown. See the text for further details.

[0022] Figures 7A-7C Self-assembly kinetics of low- and medium-slippery silk / modified peptide compositions and their components (see Table 1 for more details). Figure 7A , the self-assembly rate factor shows how fast the self-assembly reaction proceeds once it is initiated and the self-assembling cores are organized. Figure 7B , the maximum gel yield shows the density of the silk gel after self-assembly is completed. Figure 7C , the time required for the self-assembly reaction to produce half the maximum amount of gel.

[0023] Figure 8 Low-slippery and medium-slippery silk / modified peptide compositions and their components (see Table 1 for more details). The self-assembly factor reflects the average propensity of silk to self-assemble and form a gel.

[0024] Although the above-mentioned drawings illustrate the presently disclosed embodiments, as noted in the discussion, other embodiments are also contemplated. The present disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art that fall within the scope and spirit of the principles of the presently disclosed embodiments.

[0025] Figure 9 is a graph of the weight average molecular weight average (i.e., mean molecular weight average or mean MW) of silk fibroin dissolved in 9.3 M LiBr as a function of time at 100°C-103°C (i.e., a temperature gradient of 3 degrees Celsius between 100°C and 103°C) as plotted using size exclusion chromatography with a refractive index detector (SEC-RI).

[0026] Figure 10 is a graph of the weight average molecular weight average (i.e., average molecular weight average or average MW) of silk fibroin dissolved in 9.3M LiBr at 122°C-125°C (i.e., a temperature gradient of 3 degrees Celsius between 122°C and 125°C) as a function of time, which was plotted using size exclusion chromatography with a refractive index detector (SEC-RI).

[0027] Figure 11 is a graph showing the percentage of amino acid modifications in silk.

[0028] Figures 12A-12C is a graph showing the percentage of amino acid modifications in low-sliding and medium-sliding silk. Figure 12A Heavy chain modifications are shown, Figure 12BLight chain modifications are shown, and Figure 12C A modified silk fibroin hexamer is shown. N is an asparagine converted to aspartic acid, and Q is a deamidated glutamine. N corresponds to an oxidized methionine. The number following each amino acid indicates its position along the amino acid chain from the corresponding protein.

[0029] Figures 13A-13B is a graph showing the percentage of amino acid modifications in produced and lyophilized low- and medium-smooth silk. Figure 13A Heavy chain modifications are shown, and Figure 13B Light chain modifications are shown. N is asparagine converted to aspartic acid, and Q is deamidated glutamine. N corresponds to oxidized methionine. The number following each amino acid indicates its position along the amino acid chain from the corresponding protein.

[0030] Figures 14A-14B is a graph showing the percentage of amino acid modifications in low-slip silk produced at Walpole and Medford using the slippage process with different process parameters and variable levels. Figure 14A Heavy chain modifications are shown, and Figure 14B Light chain modifications are shown. N is asparagine converted to aspartic acid, and Q is deamidated glutamine. N corresponds to oxidized methionine. The number following each amino acid indicates its position along the amino acid chain from the corresponding protein.

[0031] Figures 15A-15D This graph shows the percentage of amino acid modifications in low- and medium-strength silk produced using the slide and benchtop processes. N represents asparagine converted to aspartic acid, and Q represents deamidated glutamine. N corresponds to oxidized methionine. The number following each amino acid indicates its position along the amino acid chain from the corresponding protein.

[0032] Figure 16 is an explanation of the method used to calculate the percentage of modified amino acids at specific positions along each peptide sequence.

[0033] Figure 17 Anion exchange chromatography and size exclusion chromatography schemes for separating low-slippery silk / modified peptide compositions are shown. Low-slippery silk / modified peptide compositions consist of diverse peptide populations with a wide range of sizes and charges. Different populations of low-slippery silk / modified peptide compositions were isolated using Q-Sepharose anion exchange chromatography as the first step and HiLoad Superdex 200 size exclusion chromatography as the second purification step. The Q-Sepharose eluate was loaded onto a HiLoad Superdex 200 size exclusion chromatograph, which resulted in size sorting of negatively charged silk compositions / modified peptides.

[0034] Figure 18A and 18B is a chromatogram of anion exchange chromatography and subsequent size exclusion chromatography of the eluate (Q-eluate) of the low-slipid / modified polypeptide composition. Figure 18A : Anion exchange chromatography was performed using a Q-Sepharose column (Cytiva). Anion exchange chromatography separated the low-slip silk / modified peptide composition into an uncharged peptide population (flowthrough - light blue background) and eluted negatively charged silk composition (eluate - light pink background). The light yellow background indicates that the column was washed with 50 mM Tris pH = 8.0 before eluting the charged peptide population. Figure 18B The negatively charged eluate was loaded onto a Superdex 200 column and passed through a column containing 50 mM Tris, 200 mM CaCl2, pH = 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the low-slip silk / modified peptide compositions by size. The relative elution volumes of silk compositions AS77 and AS81 are indicated on the chromatogram.

[0035] Figure 19A and 19B Shown are analytical size exclusion chromatograms of the low slippage silk / modified silk composition and its component AS composition. Figure 19A The average molecular weight in kDa of low-slip silk (LS) and AS77-AS81 is shown. Figure 19B Polydispersity (PDI) measurements are shown. Numerical data are presented in Table 7.

[0036] Figure 20 This is an SDS-polyacrylamide gel electrophoresis of low-slippery silk / modified polypeptide compositions. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, with their respective silk compositions being: fraction 6 is AS77, fraction 7 is AS78, fraction 8 is AS79, fraction 9 is AS80, and fraction 10 is AS81.

[0037] Figure 21A and 21B Graph showing the self-assembly reaction of a low-sliding / modified peptide composition. A medium-sliding reaction was used as a positive control. Figure 21A The kinetic parameters of gel formation during silk self-assembly are shown. The self-assembly parameters of the medium-sliding silk are: Amax is 0.6780 (Abs), SARF is 8.676, T0.5 is 3.668 h, and FSAF is 3.08 (Abs / min). Figure 21B This is a snapshot of the same self-assembly assay at a later time point, 12 days after setting up the assay. None of the fractions tested self-assembled over time.

[0038] Figure 22A and 22BCharacterization of low-slip silk compositions by dynamic light scattering is shown. Low-slip silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro to estimate the diameter particle size of each silk composition. Figure 22A . Shown are the intensity diameter particle size distributions measured for silk compositions AS77, AS78, AS79, AS80 and AS81. Figure 22B Correlogram functions for silk compositions AS77, AS78, AS79, AS80, AS81 are shown.

[0039] Figure 23 A size exclusion chromatogram showing the separation of a low-slippery / modified peptide composition. The low-slippery / modified peptide composition consists of a variety of peptide populations with a wide range of sizes. Different populations of low-slippery / modified peptide compositions were separated using HiLoad Superdex 200 size exclusion chromatography.

[0040] Figure 24 This is a chromatogram of a low-slippery / modified peptide composition loaded onto a Superdex 200 gel filtration column. The low-slippery / modified peptide composition was loaded onto a Superdex 200 column and passed through a column containing 50 mM Tris, 200 mM CaCl2, pH 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the low-slippery / modified peptide composition by size. The relative elution volumes of silk compositions AS82, AS86, and AS87 are indicated on the chromatogram.

[0041] Figure 25A and 25B Shown are analytical size exclusion chromatograms of the low slippage silk / modified silk composition and its component AS composition. Figure 25A The average molecular weight in kDa of low-slip silk (LS) and AS82-AS89 is shown. Figure 25B Polydispersity (PDI) measurements are shown. Numerical data are presented in Table 9.

[0042] Figure 26 This is an SDS-polyacrylamide gel electrophoresis of low-slippery silk / modified polypeptide compositions. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, with their respective silk compositions being: fraction 6 is AS82, fraction 7 is AS83, fraction 8 is AS84, fraction 9 is AS85, and fraction 10 is AS86.

[0043] Figure 27A and 27B Graph showing the self-assembly reaction of a low-sliding / modified peptide composition. A medium-sliding reaction was used as a positive control. Figure 27AThe kinetic parameters of gel formation during silk self-assembly are shown. The self-assembly parameters of the medium-sliding silk are: Amax is 0.6978 (Abs), SARF is 8.591, T0.5 is 3.361 h, and FSAF is 3.46 (Abs / min). Figure 27B This is a snapshot of the same self-assembly assay at a later time point, 18 days after assay setup. AS87, AS88, and AS89 show gel formation at this time point, which was already observed five days after the assay (LS, low sliding; MS, medium sliding).

[0044] Figures 28A-28C is a graph showing the characterization of low-slip silk compositions by dynamic light scattering. Low-slip silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro to estimate the particle size of each silk composition. Figure 28A . Shown are the intensity particle size distributions measured for silk compositions AS82, AS83, AS84, AS85, AS86, AS87, AS88, and AS89. Figure 28B Shown are the intensity size distributions measured for silk composition AS82, low slippery silk / modified peptide composition (LS), and medium slippery silk / modified peptide composition (MS). Figure 28C . Shown are the correlation plot functions of silk compositions AS82, AS83, AS84, AS85, AS86, AS87, AS88, AS89, low-sliding silk / modified peptide composition (LS), and medium-sliding silk / modified peptide composition (MS).

[0045] Figure 29Anion exchange chromatography (Q), hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) schemes for separating low-slippery / modified peptide compositions are shown. The low-slippery / modified peptide compositions are composed of a variety of peptide populations with a wide range of sizes and charges. Different low-slippery / modified peptide composition populations are isolated using Q-Sepharose anion exchange chromatography as the first step, Butyl ImpRes hydrophobic interaction resin as the second step, and HiLoad Superdex 200 size exclusion chromatography as the third purification step. The Q-Sepharose eluate is loaded onto a Butyl ImpRes (HIC) column, and the HIC-eluate is loaded onto a HiLoad Superdex 200 size exclusion chromatography, which results in the sorting of negatively charged silk compositions / modified peptides with hydrophobic characteristics sorted by size. The Q-Sepharose eluate contains negatively charged peptides of various sizes. Resolving these peptides by Butyl ImpRes results in the elution of high molecular weight, negatively charged, somewhat hydrophobic silk compositions / modified peptides. Smaller negatively charged peptides are washed as the flow-through and do not bind to the Butyl ImpRes column. The Q-HIC (eluate) is loaded onto a Superdex 200 and separated by size.

[0046] Figures 30A-30E Chromatograms of anion exchange chromatography, hydrophobic interaction chromatography, and subsequent size exclusion chromatography of a low-slipping / modified peptide composition. Figure 30A Anion exchange chromatography using a Q-Sepharose column is shown. Anion exchange chromatography separates the low-slip silk / modified peptide composition into an uncharged peptide population (flowthrough - light blue background) and elutes the negatively charged silk composition (eluate - light pink background). The light yellow background indicates that the column was washed with 50 mM Tris pH = 8.0 before eluting the charged peptide population. Figure 30B . Shown is the negatively charged eluent (Q-eluent) loaded onto a Butyl ImpRes column in the presence of 300 mM ammonium sulfate [(NH4)2SO4] to expose the hydrophobic domain of the silk peptide, which allows binding to the column. The highly charged peptide population does not bind to the column (effluent), highlighted in light blue. The column is washed until the OD280 decreases to approximately 100 units (light yellow). The bound silk peptide (Q-HIC (eluent)) is then eluted using 50 mM Tris, pH = 8.0 (light pink) without ammonium sulfate. Figure 30CThe Q-HIC (eluate) was further fractionated by size exclusion chromatography (SEC) using a Superdex 200 gel filtration column. The Q-HIC (eluate) fraction was passed through a column containing 50 mM Tris, 200 mM CaCl2, pH = 8.0. When the UV-280 absorbance began to increase, fractions were collected to separate the low-slip silk / modified peptide compositions by size. The relative elution volumes of silk compositions AS90 and AS94 are indicated on the chromatogram. Figure 30D The Q-HIC (flow-through) fraction was further fractionated by SEC using a Superdex 200 column following the same procedure as in (VC). The relative elution volumes of silk compositions AS95 and AS100 are indicated on the chromatogram. Figure 30E An overlay of chromatograms (VC) and (VD) is shown. The Q-HIC (eluate) fraction has a higher molecular weight range compared to the Q-HIC (flow-through) fraction, which elutes later in SEC and has a lower molecular weight range.

[0047] Figures 31A-31B is a graph showing analytical size exclusion chromatograms of a low slippery silk / modified silk composition and its component AS composition. Figure 31A The average molecular weight in kDa of low-slip silk (LS) and AS90-AS100 is shown. Figure 31B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 11.

[0048] Figures 32A-32B SDS polyacrylamide gel electrophoresis of low-slip / modified peptide compositions. Figure 32A .Q-HIC (eluate) SEC fractions. Figure 32B .Q-HIC (flow-through) SEC fractions. Lanes are indicated by fraction numbers in the order of elution from the Superdex 200 column, and their respective silk compositions are: Figure 32A In the , fraction 6 is AS90, fraction 7 is AS91, fraction 8 is AS92, fraction 9 is AS93, and fraction 10 is AS94. Figure 32B Of the 100, fraction 8 was AS95, fraction 9 was AS96, fraction 10 was AS97, fraction 11 was AS98, fraction 12 was AS99, and fraction 13 was AS100.

[0049] Figure 33The self-assembly reaction of a low-slipping silk / modified peptide combination is shown. The medium-slipping silk reaction was used as a positive control. Kinetic parameters of gel formation during silk self-assembly. Q-HIC (eluate) is the eluate fraction eluted from the Butyl ImpRes column before SEC purification; LS, low-slipping silk; MS, medium-slipping silk. Self-assembly parameters for medium-slipping silk: Amax of 0.6974 (Abs), SARF of 8.661, T0.5 of 3.834 h, and FSAF of 3.03 (Abs / min).

[0050] Figures 34A-34F Characterization of the low-slip silk composition by dynamic light scattering is shown. The low-slip silk and medium-slip silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro to estimate the diameter particle size of each silk composition. Figure 34A Intensity diameter particle size distribution measured for silk composition AS90, Q-HIC (eluate) fraction (before sorting by SEC), low slip silk (LS) and medium slip silk (MS). Figure 34B .Correlation plot function of the silk composition presented in (34A). Figure 34C Intensity diameter particle size distribution measured for silk compositions AS90-AS94 derived from the Q-HIC (eluent)-SEC sorting process. Figure 34D .Correlation plot function of the silk composition presented in (34C). Figure 34E Intensity diameter particle size distribution measured for silk compositions AS95-AS100 derived from the Q-HIC (effluent)-SEC sorting process. Figure 34F .Correlation plot function of the silk composition presented in (34E).

[0051] Figure 35 A size exclusion chromatogram showing the separation of a mid-slippery / modified peptide composition is shown. Mid-slippery / modified peptide compositions consist of diverse peptide populations with a wide range of sizes. Using HiLoad Superdex 200 size exclusion chromatography, distinct mid-slippery / modified peptide composition populations can be separated.

[0052] Figure 36 The figure shows a chromatogram of a medium-smooth silk / modified peptide composition loaded on a Superdex 200 gel filtration column. The medium-smooth silk / modified peptide composition was loaded onto a Superdex 200 column and passed through a column containing 50 mM Tris, 200 mM CaCl2, pH 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the medium-smooth silk / modified peptide composition by size. The relative elution volumes of silk compositions AS107 and AS111 are indicated on the chromatogram.

[0053] Figures 37A-37B . Shown are analytical size exclusion chromatograms of the medium-slip silk / modified silk composition and its constituent AS compositions. Figure 37A The average molecular weight in kDa of medium silk (MS) and AS106-AS111 is shown. Figure 37B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 14.

[0054] Figure 38 Figure 2 is an SDS-polyacrylamide gel electrophoresis of the smooth silk / modified polypeptide compositions. Lanes are indicated by fraction numbers in the order of elution from a Superdex 200 column, with their respective silk compositions being: fraction 6 is AS107, fraction 7 is AS108, fraction 8 is AS109, fraction 9 is AS110, and fraction 10 is AS111.

[0055] Figure 39 The self-assembly reaction of a medium-sliding silk / modified peptide composition is shown. Kinetic parameters for gel formation during silk self-assembly are shown. The red dashed line shows how the self-assembly parameters Amax, SARF, and T0.5 were calculated for unsorted medium-sliding silk (MS). These numerically calculated parameters for silk compositions AS106-AS111 can be found in Table 16. Low-sliding silk (LS) served as a negative control. LS, low-sliding silk; MS, medium-sliding silk.

[0056] Figures 40A-40B Figure 4 shows the characterization of the medium-sliding silk compositions by dynamic light scattering. The medium-sliding silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro to estimate the particle size of each silk composition. Figure 40A . Intensity particle size distribution measured for silk compositions AS106, AS107, AS108, AS109, AS110, AS111 and medium-slip silk (MS). Figure 50B .Correlation functions for the silk compositions presented in (40A).

[0057] Figure 41 .Anion exchange chromatography and size exclusion chromatography schemes for separating mid-slide silk / modified peptide compositions are shown. Mid-slide silk / modified peptide compositions consist of diverse peptide populations with a wide range of sizes and charges. Different mid-slide silk / modified peptide composition populations were separated using Q-Sepharose anion exchange chromatography as the first step and HiLoad Superdex 200 size exclusion chromatography as the second purification step. The Q-Sepharose eluate was loaded onto a HiLoad Superdex 200 size exclusion chromatograph, which resulted in size sorting of negatively charged silk compositions / modified peptides.

[0058] Figures 42A-42B. is a chromatogram of anion exchange chromatography and subsequent size exclusion chromatography of the eluate (Q-eluate) of the mid-slippery silk / modified polypeptide composition. Figure 42A Anion exchange chromatography was performed using a Q-Sepharose column (Cytiva). Anion exchange chromatography separated the mid-slip silk / modified peptide composition into an uncharged peptide population (flowthrough - light blue background) and eluted negatively charged silk composition (eluate - light pink background). The light yellow background indicates that the column was washed with 50 mM Tris pH = 8.0 before eluting the charged peptide population. Figure 42B The negatively charged eluate (Q-eluate) was loaded onto a Superdex 200 column and passed through a column containing 50 mM Tris, 200 mM CaCl2, pH = 8.0. When UV-280 absorbance began to increase, fractions were collected to separate the mid-slip silk / modified peptide compositions by size. The relative elution volumes of silk compositions AS101 and AS105 are indicated on the chromatogram.

[0059] Figures 43A-43B . Shown are analytical size exclusion chromatograms of the medium-slip silk / modified silk composition and its constituent AS compositions. Figure 43A The average molecular weight in kDa of medium silk (MS) and AS101-AS105 is shown. Figure 43B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 16.

[0060] Figure 44A Figure 2 is an SDS-polyacrylamide gel electrophoresis of the smooth silk / modified polypeptide compositions. Lanes are indicated by fraction numbers, in the order of elution from a Superdex 200 column, with their respective silk compositions being: fraction 6 is AS101, fraction 7 is AS102, fraction 8 is AS103, fraction 9 is AS104, and fraction 10 is AS105.

[0061] Figure 44B The self-assembly reaction of the medium-slip silk / modified peptide composition is shown. The low-slip silk reaction is used as a negative control. The kinetic parameters of gel formation during silk self-assembly are shown. The red dotted line is shown to illustrate the calculation of the Amax, SARF (self-assembly rate factor) and T0.5 parameters in Table 17.

[0062] Figure 45A 、 Figure 45B and Figure 45C . is a graph showing the characterization of the medium-sliding silk compositions by dynamic light scattering. The medium-sliding silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro (Malvern) to estimate the diameter particle size of each silk composition. Figure 45A. Intensity diameter particle size distribution determined by intensity measurements for silk compositions AS101, AS102, AS103, AS104 and AS105. Figure 45B Intensity diameter particle size distribution determined by intensity measurements for silk compositions AS101, AS105 and medium-slip silk (MS) to emphasize the particle size difference between AS101 and AS105. Figure 45C . Correlation plot functions of silk compositions AS101, AS102, AS103, AS104, AS105 and medium sliding silk (MS).

[0063] Figure 46 is a graphical illustration of the values ​​of the three molar mass moments (Mn, Mw, and Mz) as they relate to molar mass and the number of molecules per molar mass. This example applies to polydisperse samples; for monodisperse samples, Mn = Mw = Mz.

[0064] Figures 47A-47B It is an analytical SEC-MALS for low molecular weight silk, medium molecular weight silk and high molecular weight silk. Figure 47A . Weight average molecular weight in kDa of low molecular weight silk, medium molecular weight silk and high molecular weight silk. Figure 47B . Shows the polydispersity index (PDI) measurements for low molecular weight silk, medium molecular weight silk, and high molecular weight silk.

[0065] Figures 48A-48B Analytical SEC-MALS of low-, medium-, and high-molecular-weight silk produced with different process parameters and levels. Individual data points are shown, and the mean is represented by the height of the box. The bars cover one standard deviation. Figure 48A .The weight average molecular weight range of low molecular weight silk, medium molecular weight silk and high molecular weight silk. Figure 48B .PDI range of low molecular weight silk, medium molecular weight silk and high molecular weight silk.

[0066] Figures 49A-49B Analytical SEC-MALS of low slippery / modified silk compositions and component AS compositions separated by Q-SEC (Q-eluate). Figure 49A The average molecular weight in kDa of low-slip silk (LS) and AS77-AS81 is shown. Figure 49B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 24.

[0067] Figures 50A-50B Analytical SEC-MALS of low-slip silk / modified silk compositions and component AS compositions separated by SEC. Figure 50A The average molecular weight in kDa of low-slip silk (LS) and AS82-AS89 is shown. Figure 50BThe polydispersity (PDI) measurements are shown. The numerical data are presented in Table 25.

[0068] Figures 51A-51B Analytical SEC-MALS of low slippery / modified silk compositions and component AS compositions separated by Q-HIC-SEC (Q-HIC-eluate). Figure 51A The average molecular weight in kDa of low-slip silk (LS) and AS90-AS94 is shown. Figure 51B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 26.

[0069] Figures 52A-52B Analytical SEC-MALS of low slip / modified silk composition and component AS composition separated by Q-HIC-SEC (Q-HIC-flowthrough). Figure 52A The average molecular weight in kDa of low-slip silk (LS) and AS95-AS100 is shown. Figure 52B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 26.

[0070] Figures 53A-53B Analytical SEC-MALS of the mid-slip silk / modified silk composition and the component AS composition separated by Q-SEC (Q-flowthrough). Figure 53A The average molecular weight in kDa of medium silk (MS) and AS101-AS105 is shown. Figure 53B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 27.

[0071] Figures 54A-54B is the analysis of the mid-slip silk / modified silk composition and the component AS composition separated by SEC-MALS. Figure 54A The average molecular weight in kDa of medium silk (MS) and AS106-AS111 is shown. Figure 54B The polydispersity (PDI) measurements are shown. The numerical data are presented in Table 28.

[0072] Figure 55A 、 Figure 55B and Figure 55C A sequence listing of the fibroin heavy chain is shown.

[0073] Figure 56 A sequence listing of the fibroin light chain is shown.

[0074] Figure 57 A sequence listing of a fibroin hexamer is shown.

[0075] Figure 58 Three chromatographic principles of silk sorting are shown.

[0076] Figure 59 Anion exchange chromatography followed by size exclusion chromatography of silk sorting is shown.

[0077] Figure 60 Anion exchange chromatography followed by hydrophobic interaction chromatography and size exclusion chromatography is shown.

[0078] Figure 61 is a diagram including the assay used to characterize the silk fraction.

[0079] Figure 62 Shown are graphs presenting the data of Table 44 and Table 45. Data are shown with standard deviation. "Nanoclay" refers to Elementis Bentone Hydroclay.

[0080] Figure 63 is an illustration of a typical bentonite structure.

[0081] Figure 64 Shown is an SEM image of a cross-section of a membrane cast from Elementis Bentone Hydroclay 2001. Highly ordered stacking of clay layers is visible.

[0082] Figure 65 is an SEM image of a cross section of a membrane cast with pure RSF.

[0083] Figure 66 Figure 2 is a SEM image of a cross section of a 1:1 RSF / 2001 membrane cast under neutral (pH 7.0) conditions. The layered structure of the clay is preserved.

[0084] Figure 67 Figure 2 is a SEM image of a cross section of a 1:1 RSF / 2001 film cast under acidic conditions (pH 3.5). Note the ribbon-like structure.

[0085] Figure 68 The enhanced diffusion pathways created by the RSF / nanoclay composite are shown.

[0086] Figure 69 This is an FTIR scan of the amide I region of an RSF / 2001 membrane cast under neutral conditions. The nanoclay concentration was varied from 0% (red) to 70% (yellow). As the nanoclay content increased, the amide I peak shifted to the left, away from the β-sheet region.

[0087] Figure 70 is a flow chart showing various embodiments for producing pure fibroin-based protein fragments (SPF) of the present disclosure.

[0088] Figure 71is a flow chart showing various parameters that may be modified during the process of producing the SPF of the present disclosure during the extraction and solubilization steps.

[0089] Figure 72 and Figure 73 is a graph showing the effect of extraction volume on % mass loss.

[0090] Figure 74 Graph summarizing the effect of extraction time on molecular weight of silk processed under conditions of 100°C extraction temperature, 100°C LiBr, and 100°C oven dissolution (oven / dissolution time variation).

[0091] Figure 75 is a graph summarizing the effect of extraction time on the molecular weight of silk processed under conditions of 100°C extraction temperature, boiling LiBr, and 60°C oven dissolution (oven / dissolution time variation).

[0092] Figure 76 Graph summarizing the effect of extraction time on molecular weight of silk processed under conditions of 100°C extraction temperature, 60°C LiBr, and 60°C oven dissolution (oven / dissolution time variation).

[0093] Figure 77 Graph summarizing the effect of extraction time on the molecular weight of silk processed under conditions of 100°C extraction temperature, 80°C LiBr, and 80°C oven dissolution (oven / dissolution time variation).

[0094] Figure 78 Graph summarizing the effect of extraction time on molecular weight of silk processed under conditions of 100°C extraction temperature, 80°C LiBr, and 60°C oven dissolution (oven / dissolution time variation).

[0095] Figure 79 Graph summarizing the effect of extraction time on molecular weight of silk processed under conditions of 100°C extraction temperature, 100°C LiBr, and 60°C oven dissolution (oven / dissolution time variation).

[0096] Figure 80 Graph summarizing the effect of extraction time on molecular weight of silk processed under conditions of 100°C extraction temperature, 140°C LiBr, and 140°C oven dissolution (oven / dissolution time variation).

[0097] Figure 81 Graph summarizing the effect of extraction temperature on molecular weight of silk processed under conditions of 60 min extraction time, 100°C LiBr, and 100°C oven dissolution (oven / dissolution time variation).

[0098] Figure 82is a graph summarizing the effect of LiBr temperature on the molecular weight of silk processed under conditions of 60 min extraction time, 100°C extraction temperature, and 60°C oven dissolution (oven / dissolution time variation).

[0099] Figure 83 is a graph summarizing the effect of LiBr temperature on the molecular weight of silk processed under conditions of 30 minute extraction time, 100°C extraction temperature, and 60°C oven dissolution (oven / dissolution time variation).

[0100] Figure 84 Graph summarizing the effect of oven / dissolution temperature on the molecular weight of silk processed under conditions of 100°C extraction temperature, 30 minute extraction time, and 100°C lithium bromide (oven / dissolution time variation).

[0101] Figure 85 This graph summarizes the effects of oven / dissolution temperature on the molecular weight of silk processed under the conditions of 100°C extraction temperature, 60 minute extraction time, and 100°C lithium bromide.

[0102] Figure 86 Graph summarizing the effect of oven / dissolve temperature on the molecular weight of silk processed under conditions of 100°C extraction temperature, 60 minute extraction time, and 140°C lithium bromide (oven / dissolve time variation).

[0103] Figure 87 Graph summarizing the effect of oven / dissolve temperature on the molecular weight of silk processed under conditions of 100°C extraction temperature, 30 minute extraction time, and 140°C lithium bromide (oven / dissolve time variation).

[0104] Figure 88 Graph summarizing the effect of oven / dissolution temperature on the molecular weight of silk processed under conditions of 100°C extraction temperature, 60 minute extraction time, and 80°C lithium bromide (oven / dissolution time variation).

[0105] Figure 89 is a graph summarizing the molecular weight of silk processed under different conditions, including extraction time, extraction temperature, lithium bromide (LiBr) temperature, oven temperature for dissolution, and oven time for dissolution.

[0106] Figure 90 is a graph summarizing the molecular weight of silk processed at an oven / melting temperature equal to the LiBr temperature.

[0107] Figures 91A-91C Shown are low MW silk solids obtained from lyophilization as described herein at different stages of grinding. Figure 91A Shown are coarse particles of low MW silk solids immediately after removal from the lyophilization vial. Figure 91B Particles are shown reduced in size midway through the milling process. Figure 91C Fine particles with a uniform particle size distribution are shown upon completion of the grinding.

[0108] Figure 92 Solid particles of medium MW silk solids are shown.

[0109] Figure 93 Shown are examples of two different sizes of solid filament particles formed in the thin film evaporation process described herein.

[0110] Figure 94A and Figure 94B Shown are examples of microparticles prepared by the solution precipitation process described herein.

[0111] Figure 95 Shown is the ground silk powder used for the uses described herein.

[0112] Figures 96A-96B Shown is a pouch containing a solid formulation comprising the silk fibroin fragments described herein. Figure 96A Shown are pockets containing loose fibroin fragments as described herein. Figure 96B Shown is a pouch containing a disc of cryogenically pelleted fibroin fragments as described herein.

[0113] Figure 97A Lyophilized silk fibroin pellets are shown. Figure 97B is a graph showing the reconstitution yield of activated fibroin.

[0114] Figure 98A Lyophilized silk is shown. Figure 98B is a graph showing freeze-drying temperature.

[0115] Figure 99A Low temperature pelletized filaments are shown. Figure 99B is a graph showing freeze-drying temperature.

[0116] Figure 100 Different concentrations of lyophilized beads are shown.

[0117] Figures 101A-101G are SEM images of freeze-dried silk under different processing conditions. Figure 101A 6% lyophilized silk is shown without annealing. Figure 101B Shown are 6% lyophilized silk in the annealed condition. Figure 101C 6% lyophilized silk is shown without annealing. Figure 101D Shown are 6% lyophilized silk in the annealed condition. Figure 101E 16% of the silk is shown without annealing. Figure 101F 16% silk is shown with 4 hours annealing. Figure 101G16% silk is shown with 20 hours annealing.

[0118] Figure 102A-102B is an image of powdered lyophilized filaments made from dense lyophilized pellets. Figure 102A Shown is 10% medium Mw Activated Silk ground from dense pellets TM . Figure 102B Shown is 16% low Mw Activated Silk ground from dense pellets TM .

[0119] Figure 103 Figure 2 is an image showing the effect of particle size and reconstitution method on reconstitution yield. Compared to coarse grinding, finely ground filament powder remains above the liquid level and does not wet out when reconstituted using the static method.

[0120] Figures 104A-104C Is to show Activated Silk TM Image of spray capability. Figure 104A A close-up of the aerosol nozzle during spraying. Figure 104B The spray stream is shown. Figure 104C Shown is the spray pattern using an aerosol can at 6" above the surface.

[0121] Figure 105A is an image showing the bulk material "lifting" from the tray during freeze drying.

[0122] Figure 105B is an image showing meltback.

[0123] Figure 106 is a diagram showing an overview of an example of bulk freeze-drying.

[0124] Figure 107 is an image of a bulk freeze-dried product.

[0125] Figure 108 It is an image of the product being melted back.

[0126] Figure 109 is an image showing the color change of the final product.

[0127] Figure 110 is a graph showing the results of Profile 5.

[0128] Figures 111A-111C is an image of the freeze-dried pellets. Figure 111A It is 6% freeze-dried pellets. Figure 111B It is 10% freeze-dried pellets. Figure 111C It is 17% freeze-dried pellets.

[0129] Figure 112 It is a graph showing a BET isotherm linear graph.

[0130] Figure 113 It is freeze-dried Activated Silk prepared by low-temperature granulation method TM SEM image of .

[0131] Figure 114 is a graph showing the "z-average" particle size.

[0132] Figure 115 are images showing silk solution samples.

[0133] Figure 116 This is a graph showing sample output for intensity and Z-average particle size. Over time, the particle size distribution shifts toward higher size regimes, with the intensity peak in this region increasing (left). This can be summarized by the increase in Z-average value (right). The Python program allows for quick comparison of data across time points and samples.

[0134] Figure 117 This image shows the workflow for dynamic light scattering and data processing in Python and JMP.

[0135] Figure 118 Graph showing that in a filament-only control, 27P is, as expected, several orders of magnitude more stable than 33B across a range of temperatures and concentrations (lower left, upper right). However, 33B aggregates more rapidly, making it a useful model system for studying the effects of excipients on aggregation rate (lower right). (Unless otherwise noted, n = 3 replicates).

[0136] Figure 118 Figures 2 and 3 show the baseline system aggregation of both 27P and 33B. These figures show that 27P is several orders of magnitude more stable than 33B over a range of temperatures and concentrations.

[0137] Figure 119A is a graph showing the baseline aggregation profile of 27P.

[0138] Figure 119B is a graph showing the main effect of the baseline system on the aggregation rate.

[0139] Figure 120 is a graph showing the z-average values ​​of various silk solutions.

[0140] Figure 121 is a graph showing the z-average values ​​of various silk solutions.

[0141] Figure 122 is a graph showing the z-average values ​​of various silk solutions.

[0142] Figure 123 Images showing soluble silk, gel silk, and microparticles in solution.

[0143] Figure 124 is a graph showing the z-average values ​​of various silk solutions.

[0144] Figure 125 is a graph showing the z-average values ​​of various silk solutions.

[0145] Figure 126 is a graph showing the z-average values ​​of various silk solutions.

[0146] Figures 127A-127C is a graph showing the tested stability of concentrated 33B solutions. Figure 127A The DLS test results of the 33B sample are shown. Figure 127B Shown are the z-average values ​​of various silk solutions at 4°C. Figure 127C Shown are incubation curves at 70° C. These figures highlight the dramatic difference in aggregation profiles with temperature and further demonstrate the impressive performance of PBS buffer in slowing aggregation.

[0147] Figures 128A-128B 40C test results of the excipients are shown. Figure 128A Arginine HCl, MgCl2>NaCl, KCl>PBS>CaCl2. Figure 128B The two best performers (ArgHCl, MgCl2) showed no pH change and no aggregation after 1085 hours (45 days). After 10 days at this temperature, the equivalent of 33B / deionized water was completely gelled.

[0148] Figures 129A-129B is a scan of a solution stored at 40°C over a period of approximately 45 days ( Figure 129A ), showing a similar aggregation profile to the solution scanned continuously at 70 °C during 1 h ( Figure 129B ).

[0149] Figures 130A-130C is a graph showing that the normalized relative aggregation rate appears to be temperature dependent.

[0150] Figures 131A-131B is a graph showing that the excipient that performs best at 40°C also shows significant improvement at 70°C compared to deionized water.

[0151] Figure 132 Shows the formula used to model a 70°C polymerization in Prism.

[0152] Figures 133A-133C The 70°C curves are shown and were analyzed via a single correlation model in Prism software to extract better quantitative data from the samples.

[0153] Figure 134is a graph showing the results of a preservative screening, specifically the serial z-average measurements at 70°C.

[0154] Figure 135 is a bar graph showing the effect of various preservatives on aggregation.

[0155] Figure 136 is a graph showing the potential of salts to stabilize aggregation-prone solutions.

[0156] Figure 137 is a bar graph showing the potential of salts to stabilize aggregation-prone solutions. DETAILED DESCRIPTION

[0157] The present disclosure provides lyophilized or sprayable peptides or protein fragments comprising a plurality of amino acids selected from the group consisting of M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P, and W, wherein at least one of the amino acids is modified, substituted, or replaced as disclosed herein.

[0158] The present disclosure provides a pouch comprising a plurality of apertures, wherein the pouch encapsulates a substantially solid formulation comprising silk fibroin fragments. The present disclosure also provides a method for reconstituting the substantially solid formulation comprising silk fibroin fragments in a solvent. The present disclosure also provides a method for preparing a pouch encapsulating a substantially solid formulation comprising silk fibroin fragments.

[0159] Silk is a natural polymer produced by various insects and spiders. Silk produced by the silkworm Bombyx mori (Bombyx mori) consists of a core protein, fibroin, and a gelatinous coating composed of the non-silk protein, sericin. Fibroin is an FDA-approved, edible, non-toxic, and relatively inexpensive protein derived from silkworm cocoons. The structure and content of amino acids in fibroin closely resemble those in human tissue.

[0160] Methods of preparing fibroin fragments or fibroin-based protein fragments are known and described, for example, in US Patents 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177, all of which are incorporated herein by reference in their entirety.

[0161] definition

[0162] As used in the foregoing part of this specification and throughout the rest of this specification, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

[0163] Unless otherwise specified, all percentages, parts, and ratios are based upon the total weight of the eye care compositions of the present disclosure. All such weights as they relate to listed ingredients are based on the active level and, therefore, do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified. The term "weight percent" may be expressed herein as "wt %" or % w / w.

[0164] As used herein, the terms "a," "an," or "the" are generally construed to cover both the singular and the plural.

[0165] As used herein, the term "about" generally refers to a particular value that includes variations and an acceptable range of error as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean zero variation, as well as a range of ±20 percent, ±10 percent, or ±5 percent of a given value.

[0166] For example, as used herein, the term "dermatologically acceptable carrier" means a carrier that is suitable for use in contact with mammalian keratinous tissue without causing any adverse reactions (such as excessive toxicity, incompatibility, instability, allergic reactions). Dermatologically acceptable carriers may include, but are not limited to, water, liquid or solid emollients, humectants, solvents, and the like.

[0167] As used herein, the term "hydrophile-lipophile balance" (HLB) of a surfactant is a measure of its degree of hydrophilicity or hydrophobicity, as determined by calculating the values ​​for different regions of the molecule, as determined by the Griffin method: HLB = 20*M h / M, where M h Where HLB is the molecular weight of the hydrophilic portion of the surfactant, and M is the molecular weight of the entire surfactant. The results range from 0 to 20. An HLB value of 0 corresponds to a completely lipophilic molecule, while an HLB value of 20 corresponds to a completely hydrophilic molecule. The HLB value can be used to predict the surfactant properties of a molecule: HLB < 10: fat-soluble (water-insoluble), HLB > 10: water-soluble (fat-insoluble), HLB = 1-3: defoamers, 3-6: W / O (water-in-oil) emulsifiers, 7-9: wetting and spreading agents, 8-16: O / W (oil-in-water) emulsifiers, 13-16: detergents, and 16-18: solubilizers or hydrotropes.

[0168] As used herein, "average weight average molecular weight" refers to the average of two or more values ​​of weight average molecular weight of fibroin or fragments thereof of the same composition, the two or more values ​​being determined by two or more separate experimental readings.

[0169] As used herein, the term polymer "polydispersity (PD)" is generally used as a measure of the breadth of the molecular weight distribution of a polymer and is given by the formula: definition.

[0170] As used herein, the term "substantially uniform" may refer to fibroin-based protein fragments that are distributed in a normal distribution about an identified molecular weight. As used herein, the term "substantially uniform" may refer to a uniform distribution of components or additives, such as fibroin fragments, dermatologically acceptable carriers, etc., throughout the composition of the present disclosure.

[0171] As used herein, the terms "silk fibroin peptide," "silk fibroin protein fragment," and "silk fibroin fragment" are used interchangeably. Where molecular size is an important parameter, the molecular weight or the number of amino acid units is defined.

[0172] As used herein, the term "fast-dissolving solid form" refers to fast-dissolving solid forms including lyophilized forms (filter cakes, flakes, films) and compressed tablets.

[0173] As used herein, the term "peptide" or "protein" refers to a chain of amino acids linked together by peptide bonds (also called amide bonds). The fundamental differentiating factors between proteins and peptides are size and structure. Peptides are smaller than proteins. Traditionally, peptides are defined as molecules consisting of 2 to 50 amino acids, while proteins are composed of 50 or more amino acids. Additionally, the structure of peptides is often less well-defined than that of proteins, which can adopt complex conformations known as secondary, tertiary, and quaternary structures.

[0174] As used herein, the term "fibroin" or "silk protein" refers to a class of structural proteins produced by certain silk-producing spider and insect species (see the definition provided in WIPO Pearl - WIPO's Multilingual Terminology Portal database https: / / wipopearl.wipo.int / en / linguistic). Fibroin can include silkworm fibroin, insect or spider silk proteins (e.g., spidroin), recombinant spider proteins, silk proteins present in other spider silk types, such as tubular silk proteins (TuSP), flagellar silk proteins, minor ampullate silk proteins, alveolar silk proteins, piriform silk proteins, polymeric sericin), silkworm fibroin produced by genetically modified silkworms, or recombinant silkworm fibroin.

[0175] As used herein, the term "fibroin" refers to silkworm fibroin, silk fibroin produced by genetically modified silkworms, or recombinant silkworm fibroin (see (1) Narayan, ed., Encyclopedia of Biomedical Engineering, Vol. 2, Elsevier, 2019; (2) Kobayashi et al., ed., Encyclopedia of Polymeric Nanomaterials, Springer, 2014, https: / / link.springer.com / referenceworkentry / 10.1007%2F978-3-642-36199-9_323-1). In one embodiment, the silk fibroin is obtained from silkworms.

[0176] As used herein, the term "solid solution" refers to an active agent that is molecularly dissolved in a solid excipient matrix, such as a hydrophobic polymer, wherein the active agent is miscible with the polymer matrix excipient.

[0177] As used herein, the term "solid dispersion" refers to an active agent dispersed as crystalline or amorphous particles, wherein the active agent is dispersed in an amorphous polymer and randomly distributed among the polymer matrix excipients.

[0178] As used herein, the term "substantially uniform" may refer to a silk fibroin-based protein fragment that is distributed in a normal distribution with respect to an identified molecular weight. As used herein, the term "substantially homogeneous" may also refer to a uniform distribution of components or additives (e.g., silk fibroin-based protein fragments, dermatologically acceptable carriers, etc.) throughout the silk composition or formulation.

[0179] As used herein, the term "surface tension" refers to the tendency of a fluid's surface to contract to the smallest possible surface area. At a liquid-air interface, surface tension arises because the liquid's molecules are more attracted to each other (due to cohesion) than the molecules in air are to each other (due to adhesion). The net effect is an inward force on the liquid's surface, which causes the liquid to behave as if its surface were covered by a stretched elastic film. Water has a higher surface tension (72.8 mN / m at 20°C) than most other liquids due to the relatively high attraction between its molecules through a network of hydrogen bonds.

[0180] SPF definition and properties

[0181] As used herein, "silk protein fragment" (SPF) includes, but is not limited to, one or more of the following: "fibroin fragment" as defined herein; "recombinant silk fragment" as defined herein; "spider silk fragment" as defined herein; "fibroin-like protein fragment" as defined herein; "chemically modified silk fragment" as defined herein; and / or "sericin or sericin fragment" as defined herein. The SPF may have any molecular weight value or range described herein, and any polydispersity value or range described herein. As used herein, in some embodiments, the term "silk protein fragment" also refers to a silk protein comprising or consisting of at least two identical repeating units, each of which is independently selected from a naturally occurring silk polypeptide or a variant thereof, an amino acid sequence of a naturally occurring silk polypeptide, or a combination of both.

[0182] SPF molecular weight and polydispersity

[0183] In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 1 kDa to about 5 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 5 kDa to about 10 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 10 kDa to about 15 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 15 kDa to about 20 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 14 kDa to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 20 kDa to about 25 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 25 kDa to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 30 kDa to about 35 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 35 kDa to about 40 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 39 kDa to about 54 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 40 kDa to about 45 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 45 kDa to about 50 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 50 kDa to about 55 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 55 kDa to about 60 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 60 kDa to about 65 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 65 kDa to about 70 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 70 kDa to about 75 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 75 kDa to about 80 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 80 kDa to about 85 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 85 kDa to about 90 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 90 kDa to about 95 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 95 kDa to about 100 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 100 kDa to about 105 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 105 kDa to about 110 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 110 kDa to about 115 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 115 kDa to about 120 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 120 kDa to about 125 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 125 kDa to about 130 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 130 kDa to about 135 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 135 kDa to about 140 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 140 kDa to about 145 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 145 kDa to about 150 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 150 kDa to about 155 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 155 kDa to about 160 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 160 kDa to about 165 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 165 kDa to about 170 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 170 kDa to about 175 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 175 kDa to about 180 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 180 kDa to about 185 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 185 kDa to about 190 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 190 kDa to about 195 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 195 kDa to about 200 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 200 kDa to about 205 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 205 kDa to about 210 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 210 kDa to about 215 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 215 kDa to about 220 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 220 kDa to about 225 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 225 kDa to about 230 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 230 kDa to about 235 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 235 kDa to about 240 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 240 kDa to about 245 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 245 kDa to about 250 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 250 kDa to about 255 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 255 kDa to about 260 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 260 kDa to about 265 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 265 kDa to about 270 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 270 kDa to about 275 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 275 kDa to about 280 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 280 kDa to about 285 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 285 kDa to about 290 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 290 kDa to about 295 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 295 kDa to about 300 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 300 kDa to about 305 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 305 kDa to about 310 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 310 kDa to about 315 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 315 kDa to about 320 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 320 kDa to about 325 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 325 kDa to about 330 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 330 kDa to about 335 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 335 kDa to about 340 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 340 kDa to about 345 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 345 kDa to about 350 kDa.

[0184] In some embodiments, the compositions of the present disclosure include SPF compositions selected from compositions #1001 to #3500 having a weight average molecular weight selected from about 1 kDa to about 250 kDa, and a polydispersity selected from 1 to about 5 (including but not limited to a polydispersity of 1), 1 to about 1.5 (including but not limited to a polydispersity of 1), about 1.5 to about 2, about 1.5 to about 3, about 2 to about 2.5, about 2.5 to about 3, about 3 to about 3.5, about 3.5 to about 4, about 4 to about 4.5, and about 4.5 to about 5:

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] As used herein, "low molecular weight," "low MW," or "low-MW" SPF can include SPF having a weight average molecular weight or average weight average molecular weight selected from about 5 kDa to about 38 kDa, about 14 kDa to about 30 kDa, or about 6 kDa to about 17 kDa. In some embodiments, the target low molecular weight for certain SPFs can be a weight average molecular weight of about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, or about 38 kDa.

[0195] As used herein, "medium molecular weight," "medium MW," or "mid-MW" SPFs may include SPFs having a weight average molecular weight or average weight average molecular weight selected from about 31 kDa to about 55 kDa or about 39 kDa to about 54 kDa. In some embodiments, the target medium molecular weight for certain SPFs may be a weight average molecular weight of about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 kDa, about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa, about 50 kDa, about 51 kDa, about 52 kDa, about 53 kDa, about 54 kDa, or about 55 kDa.

[0196] As used herein, "high molecular weight," "high MW," or "high-MW" SPFs can include SPFs having a weight average molecular weight or average weight average molecular weight selected from about 55 kDa to about 150 kDa. In some embodiments, the target high molecular weight for certain SPFs can be about 55 kDa, about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa, about 60 kDa, about 61 kDa, about 62 kDa, about 63 kDa, about 64 kDa, about 65 kDa, about 66 kDa, about 67 kDa, about 68 kDa, about 69 kDa, about 70 kDa, about 71 kDa, about 72 kDa, about 73 kDa, about 74 kDa, about 75 kDa, about 76 kDa, about 77 kDa, about 78 kDa, about 79 kDa, or about 80 kDa.

[0197] In some embodiments, the molecular weights described herein (e.g., low molecular weight, medium molecular weight, high molecular weight) can be converted to the approximate number of amino acids contained in the corresponding SPF, as understood by those of ordinary skill in the art. For example, the average weight of amino acids can be about 110 Daltons (i.e., 110 g / mol). Therefore, in some embodiments, the molecular weight of a linear protein divided by 110 Daltons can be used to approximate the number of amino acid residues contained therein.

[0198] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from 1 to about 5.0, including but not limited to a polydispersity of 1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 1.5 to about 3.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from 1 to about 1.5, including but not limited to a polydispersity of 1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 1.5 to about 2.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 2.0 to about 2.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 2.5 to about 3.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 3.0 to about 3.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 3.5 to about 4.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 4.0 to about 4.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 4.5 to about 5.0.

[0199] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of 1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.9.In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 5.0.

[0200] In some embodiments, in compositions described herein having a combination of low, medium, and / or high molecular weight SPFs, such low, medium, and / or high molecular weight SPFs can have the same or different polydispersities.

[0201] Silk fibroin fragments

[0202] Methods for preparing silk fibroin or silk fibroin fragments and their use in various fields are known and described, for example, in U.S. Patents 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369 and 10,166,177, 10,287,728 and 10,301,768, all of which are incorporated herein by reference in their entirety. Raw silk from the silkworm (Bombyx mori) consists of two main proteins: fibroin (about 75%) and sericin (about 25%). Fiber fibroin is a fibrous protein with a semi-crystalline structure that provides stiffness and strength. The term "fibroin" as used herein refers to fibers from the cocoon of the silkworm Bombyx mori having a weight-average molecular weight of about 370,000 Da. Crude silkworm fiber consists of double strands of fibroin. The adhesive material that binds these twin fibers together is sericin. Silk fibroin is composed of heavy chains (H chains) with a weight-average molecular weight of approximately 350,000 Da and light chains (L chains) with a weight-average molecular weight of approximately 25,000 Da. Silk fibroin is an amphiphilic polymer with a large hydrophobic domain (of high molecular weight) that constitutes the majority of the polymer. The hydrophobic region is interrupted by small hydrophilic spacers, and the N- and C-termini of the chain are also highly hydrophilic. The hydrophobic domain of the H chain contains a repeating hexapeptide sequence of Gly-Ala-Gly-Ala-Gly-Ser and repeats of the Gly-Ala / Ser / Tyr dipeptide, which can form stable anti-parallel-sheet crystallites. The amino acid sequence of the L chain is non-repeating, making it more hydrophilic and relatively elastic. The alternating arrangement of hydrophilic (Tyr, Ser) and hydrophobic (Gly, Ala) segments in the silk fibroin molecule allows for self-assembly of the fibroin molecule.

[0203] Provided herein are methods for producing pure and highly scalable solutions of silk fibroin fragment mixtures that can be used in a variety of applications across multiple industries. Without wishing to be bound by any particular theory, it is believed that these methods are equally applicable to the fragmentation of any SPF described herein, including but not limited to recombinant silk proteins, and fragmentation of silk-like or silk-like proteins.

[0204] The term "fibroin" as used herein includes silk fibroin and insect or spider silk proteins. In one embodiment, the silk fibroin is obtained from the silkworm, Bombyx mori. Raw silk from the silkworm is composed of two major proteins: fibroin (about 75%) and sericin (about 25%). Fibroin is a fibrous protein with a semi-crystalline structure that provides stiffness and strength. The term "fibroin" as used herein refers to fibers from the cocoon of the silkworm, Bombyx mori, having a weight average molecular weight of about 370,000 Da. Converting these insoluble fibroin fibrils into water-soluble fibroin fragments requires the addition of concentrated neutral salts (e.g., 8-10 M lithium bromide), which interfere with the intermolecular and intramolecular ionic and hydrogen bonding that originally makes the silk fibroin insoluble in water. Methods of preparing fibroin fragments and / or compositions thereof are known and described, for example, in US Patents 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177.

[0205] Raw silk cocoons from silkworms are cut into fragments. The silk cocoon fragments are processed in an aqueous solution of Na2CO3 at about 100°C for about 60 minutes to remove sericin (degumming). The volume of water used is equal to about 0.4 times the weight of the raw silk, and the amount of Na2CO3 is about 0.848 times the weight of the raw silk cocoon fragments. The resulting degummed silk cocoon fragments are rinsed three times with deionized water at about 60°C (each rinse lasting 20 minutes). The volume of rinse water for each cycle is 0.2L x the weight of the raw silk cocoon fragments. Excess water is removed from the degummed silk cocoon fragments. After the deionized water washing step, the wet degummed silk cocoon fragments are dried at room temperature. The degummed silk cocoon fragments are mixed with a LiBr solution, and the mixture is heated to about 100°C. The heated mixture is placed in a drying oven and heated at about 100°C for about 60 minutes to achieve complete dissolution of the natural silk protein. The resulting fibroin solution is filtered and dialyzed for 72 hours using tangential flow filtration (TFF) and a 10kDa membrane through deionized water. The resulting aqueous silk fibroin solution has a concentration of approximately 8.5% by weight. This 8.5% silk solution is then diluted with water to produce a 1.0% w / v silk solution. TFF can then be used to further concentrate the pure silk solution to a concentration of 20.0% w / w silk / water.

[0206] Silk dialysis through a series of water changes is a manual and time-intensive process that can be accelerated by changing certain parameters, such as diluting the silk solution before dialysis. The dialysis process can be scaled up using semi-automated equipment, such as a tangential flow filtration system.

[0207] In some embodiments, silk solutions were prepared under various preparation conditions, such as 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, 9.3 M LiBr was prepared and allowed to stand at room temperature for at least 30 minutes. 5 mL of LiBr solution was added to 1.25 g of silk and placed in a 60°C oven. Samples were removed from each group at 4, 6, 8, 12, 24, 168, and 192 hours.

[0208] In some embodiments, silk solutions were prepared under various preparation conditions, such as 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, a 9.3 M LiBr solution was heated to one of four temperatures: 60°C, 80°C, 100°C, or boiling. 5 mL of the hot LiBr solution was added to 1.25 g of silk and placed in a 60°C oven. Samples were removed from each group at 1, 4, and 6 hours.

[0209] In some embodiments, silk solutions are prepared under various preparation condition parameters, such as using four different silk extraction combinations: 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, a 9.3M LiBr solution is heated to one of four temperatures: 60°C, 80°C, 100°C, or boiling. 5 mL of hot LiBr solution is added to 1.25 g of silk and placed in an oven at the same temperature as the LiBr. Samples are removed from each group at 1, 4, and 6 hours. 1 mL of each sample is added to 7.5 mL of 9.3M LiBr and refrigerated for viscosity testing.

[0210] In some embodiments, SPF is obtained by dissolving raw undegummed, partially degummed, or degummed silkworm fiber with a neutral lithium bromide salt. The raw silk is processed at a temperature and other conditions selected to remove any sericin and achieve a desired weight average molecular weight (MW) of the fragment mixture. W ) and polydispersity (PD). The selection of process parameters can be varied to achieve different final silk protein fragment properties depending on the intended use. The resulting final fragment solution is a silk core protein fragment and water with process contaminants ranging from parts per million (ppm) to undetectable levels, which is an acceptable level in the pharmaceutical, medical, and consumer eye care markets. The concentration, size, and polydispersity of the SPF can be further varied depending on the desired use and performance requirements.

[0211] Methods for preparing silk protein fragments for use in the compositions of the present disclosure are described in U.S. Patent Application Publication Nos. 2015 / 00933340, 2015 / 0094269, 2016 / 0193130, 2016 / 0022560, 2016 / 0022561, 2016 / 0022562, 2016 / 0022563, and 2016 / 0222579, 2016 / 0281294, and U.S. Patent Nos. 9,187,538, 9,522,107, 9,517,191, 9,522,108, 9,511,012, and 9,545,369, which are incorporated herein by reference in their entireties. However, exemplary methods are described in Figure 70 , which is a flow chart showing various embodiments for producing pure silk fibroin-based protein fragments (SPF) of the present disclosure. It should be understood that not all of the illustrated steps are necessary to prepare all silk solutions of the present disclosure. Figure 70 As shown in step A, cocoons (heat treated or non-heat treated), silk fibers, silk powder or spider silk can be used as the silk source. If starting with raw silk cocoons from silkworms, the cocoons can be cut into small pieces, such as pieces of approximately equal size, step B1. Then in step C1a, the raw silk is extracted and rinsed to remove any sericin. This produces raw silk that is essentially free of sericin. In one embodiment, water is heated to a temperature of 84°C to 100°C (ideally boiling) and then Na2CO3 (sodium carbonate) is added to the boiling water until the Na2CO3 is completely dissolved. The raw silk is added to the boiling water / Na2CO3 (100°C) and immersed for about 15-90 minutes, where the longer boiling time produces smaller silk protein fragments. In one embodiment, the water volume is equal to about 0.4x the weight of the raw silk, and the Na2CO3 volume is equal to about 0.848x the weight of the raw silk. In one embodiment, the water volume is equal to 0.1x the weight of the raw silk and the Na2CO3 volume is maintained at 2.12g / L. This is Figure 72 and Figure 73 This was demonstrated in the figures: varying the silk mass (x-axis) in the same volume of extraction solution (i.e., same volume of water and same concentration of Na2CO3) achieved sericin removal (essentially free of sericin), as demonstrated by a total silk mass loss (y-axis) of 26%-31%.

[0212] Subsequently, the water-dissolved Na2CO3 solution is drained and excess water / Na2CO3 is removed from the fibroin fibers (e.g., by manually looping the fibroin extract, using a rotary cycle of a machine, etc.). The resulting fibroin extract is rinsed with warm to hot water, typically at a temperature ranging from about 40°C to about 80°C, to remove any residual adsorbed sericin or contaminants, with the volume of water being changed at least once (repeated as needed). The resulting fibroin extract is fibroin substantially free of sericin. In one embodiment, the resulting fibroin extract is rinsed with water at a temperature of about 60°C. In one embodiment, the volume of rinse water for each cycle is equal to 0.1 L to 0.2 L x the weight of the raw silk. It may be advantageous to stir, tumble, or circulate the rinse water to maximize the rinsing effect. After rinsing, excess water is removed from the extracted fibroin fibers (e.g., by manually or by machine squeezing the fibroin extract). Alternatively, methods known to those skilled in the art, such as pressure, temperature, or other agents, or combinations thereof, may be used for sericin extraction. Alternatively, the silk glands can be directly removed from the worm (100% sericin-free silk protein). This will yield sericin-free liquid silk protein without any changes in the protein structure.

[0213] The extracted silk fibroin fibers are then completely dried. Once dried, the extracted silk fibroin is dissolved using a solvent added to the silk fibroin at a temperature between ambient temperature and boiling point, step C1b. In one embodiment, the solvent is a lithium bromide (LiBr) solution (LiBr has a boiling point of 140°C). Alternatively, the extracted silk fibroin fibers are not dried, but rather wet and placed in the solvent; the solvent concentration can then be varied to achieve a concentration similar to that achieved when the dried silk is added to the solvent. The final concentration of the LiBr solvent can range from 0.1M to 9.3M. Table D summarizes the molecular weight of silk dissolved by different concentrations of lithium bromide (LiBr) and different extraction and dissolution sizes. Complete dissolution of the extracted silk fibroin fibers can be achieved by varying the treatment time and temperature, as well as the concentration of the dissolution solvent. Other solvents can be used, including but not limited to phosphate phosphate, calcium nitrate, calcium chloride solution, or other concentrated inorganic salt aqueous solutions. To ensure complete dissolution, the silk fibers should be completely immersed in the heated solvent solution and then maintained at a temperature of about 60°C to about 140°C for 1-168 hours. In one embodiment, the silk fibers should be completely immersed in the solvent solution and then placed in a drying oven at a temperature of about 100°C for about 1 hour.

[0214] Table D: Molecular weight of silk dissolved by different concentrations of LiBr and by different extraction and dissolution sizes

[0215]

[0216] The temperature at which the silk fibroin extract is added to the LiBr solution (or vice versa) has an impact on the time required to completely dissolve the silk fibroin and the resulting molecular weight and polydispersity of the final SPF mixture solution. In one embodiment, the silk solvent solution concentration is less than or equal to 20% w / v, and in addition, stirring during the introduction or dissolution process can be used to promote dissolution at different temperatures and concentrations. The temperature of the LiBr solution provides control over the molecular weight and polydispersity of the resulting silk protein fragment mixture. In one embodiment, higher temperatures dissolve the silk faster to provide enhanced process scalability and large-scale production of silk solutions. In one embodiment, using a LiBr solution heated to a temperature of 80°C-140°C reduces the time required in an oven to achieve complete dissolution. Changing the time and dissolving the solvent at a temperature of 60°C or above will change and control the MW and polydispersity of the SPF mixture solution formed from native silk fibroin of original molecular weight.

[0217] Alternatively, extraction can be bypassed by placing the entire cocoon directly into a solvent, such as LiBr (step B2). This requires subsequently filtering the silkworm particles from the silk and solvent solution and removing the sericin using methods known in the art for separating hydrophobic and hydrophilic proteins (e.g., column separation and / or chromatography, ion exchange, chemical precipitation using salt and / or pH, and / or enzymatic digestion and filtration or extraction), all of which are common examples of standard protein isolation methods and are not limiting (step C2). Alternatively, extraction can be bypassed by placing the unheated cocoon, from which the silkworms have been removed, into a solvent, such as LiBr. This method can be used for sericin isolation, with the advantage that unheated cocoons contain significantly less insect debris.

[0218] Dialysis can be used to remove the dissolving solvent from the resulting dissolved silk fibroin fragment solution by dialyzing the solution against a certain volume of water, step E1. Pre-filtration before dialysis helps to remove any debris (i.e., silkworm residue) from the silk and LiBr solution, step D. In one example, a 0.1% to 1.0% silk-LiBr solution is filtered using a 3 μm or 5 μm filter at a flow rate of 200-300 mL / min before dialysis and possible concentration as needed. The method disclosed herein as described above utilizes time and / or temperature to reduce the concentration from 9.3M LiBr to a range of 0.1M to 9.3M to facilitate filtration and downstream dialysis, especially when considering establishing a scalable process. Alternatively, without using additional time or temperature, the 9.3M LiBr-silk protein fragment solution can be diluted with water to facilitate debris filtration and dialysis. The result of dissolution under filtration at the desired time and temperature is a translucent, particle-free, room temperature storage-stable silk protein fragment-LiBr solution with a known MW and polydispersity. It is advantageous to regularly change the dialysis water until the solvent is removed (e.g., change the water after 1 hour, 4 hours, and then every 12 hours for a total of 6 water changes). The total number of water volume changes can be varied based on the resulting concentration of the solvent used for silk protein dissolution and fragmentation. After dialysis, the final silk solution can be further filtered to remove any remaining debris (i.e., silkworm residues).

[0219] Alternatively, tangential flow filtration (TFF), a rapid and efficient method for separating and purifying biomolecules, can be used to remove the solvent from the resulting dissolved silk fibroin solution, step E2. TFF provides a highly pure aqueous solution of silk protein fragments and ensures that the process can be scaled up to produce large quantities of solution in a controlled and reproducible manner. The silk-LiBr solution can be diluted before TFF (from 20% to 0.1% silk in water or LiBr). Prefiltration as described above before TFF treatment maintains filtration efficiency and may avoid the formation of a silk gel boundary layer on the filter surface due to the presence of debris particles. Prefiltration before TFF also helps to remove any residual debris (i.e., silkworm residues) from the silk and LiBr solution, which may cause spontaneous or long-term gelation of the resulting aqueous-only solution, step D. Recirculating or single-pass TFF can be used to produce water-silk protein fragment solutions ranging from 0.1% silk to 30.0% silk (more preferably, 0.1%-6.0% silk). The desired concentration, molecular weight and polydispersity of the silk protein fragment mixture based on the solution may require the TFF membrane of different cut-off sizes. For example, by changing the length of the extraction boiling time or the silk solution of different molecular weight made by the time and temperature in the dissolving solvent (such as LiBr), the film of 1-100kDa may be required. In one embodiment, TFF 5 or 10kDa membrane is used to purify the silk protein fragment mixture solution and produce the silk-water ratio of final expectation. After removing the dissolving solvent (such as LiBr), single-pass TFF, TFF and other methods as known in the art can also be used, such as falling film evaporator to concentrate the solution (the desired concentration obtained is 0.1% to 30% silk). This can be used as a substitute for the standard HFIP concentration method for preparing water-based solutions known in the art. It is also possible to use a larger-pore membrane to filter out small silk protein fragments and produce a solution with and / or without the higher molecular weight silk of narrower polydispersity value.

[0220] Table C summarizes the molecular weights of some embodiments of the silk protein solutions disclosed herein. The silk protein solutions were processed under the following conditions: extraction at 100°C for 20 minutes, rinsing at room temperature, and LiBr in a 60°C oven for 4-6 hours. The water-soluble membranes were processed under the following TFF conditions: extraction at 100°C for 60 minutes, rinsing at 60°C, and LiBr in a 100°C oven for 60 minutes. Figures 93 to 10 4 further shows manipulation of extraction time, LiBr dissolution conditions, and TFF processing and the resulting exemplary molecular weights and polydispersities. These examples are not intended to be limiting, but rather to demonstrate the potential of specifying parameters for specific molecular weight silk fragment solutions.

[0221] Table C: Molecular weight of silk protein solutions disclosed herein

[0222]

[0223] The assay method for detecting LiBr and Na2CO3 is carried out using an HPLC system equipped with an evaporative light scattering detector (ELSD). Calculated by linear regression of the obtained peak area of ​​the analyte drawn relative to concentration. More than one sample of many preparations disclosed herein is used for sample preparation and analysis. Typically, four samples of different preparations are directly weighed into a 10mL volumetric flask. The sample is suspended in 5mL of 20mM ammonium formate (pH 3.0) and kept at 2 to 8°C for 2 hours, occasionally shaken to extract analytes from the film. After 2 hours, the solution is diluted with 20mM ammonium formate (pH 3.0). The sample solution from the volumetric flask is transferred to an HPLC vial and injected into the HPLC-ELSD system to estimate sodium carbonate and lithium bromide.

[0224] The analytical method developed for the quantification of Na2CO3 and LiBr in silk fibroin preparations was found to be linear over the range of 10-165 μg / mL, with RSDs of 2% for injection precision, 1% for area, and 0.38% and 0.19% for retention time of sodium carbonate and lithium bromide, respectively. This analytical method can be used for the quantitative determination of sodium carbonate and lithium bromide in silk fibroin preparations.

[0225] The final silk protein fragment solution is pure silk protein fragments and water, containing particulate debris and / or processing contaminants, including LiBr and Na2CO3, at PPM to undetectable levels. Tables A and B are tables summarizing the concentrations of LiBr and Na2CO3 in the solutions of the present disclosure. In Table A, the processing conditions include extraction at 100°C for 60 minutes, rinse at 60°C, and LiBr at 100°C in a 100°C oven for 60 minutes. The TFF conditions were varied, including the pressure difference and the number of diafiltration volumes. In Table B, the processing conditions include boiling at 100°C for 60 minutes, rinse at 60°C, and LiBr in a 60°C oven for 4-6 hours.

[0226] Table A: Concentrations of lithium bromide and sodium carbonate in silk protein solutions

[0227]

[0228] Table B: Contents of lithium bromide and sodium carbonate in silk protein solution

[0229]

[0230]

[0231] *ND = Not Detected

[0232] The silk fragment-water solution, freeze-dried silk protein fragment mixture, or any other composition comprising SPF can be sterilized according to standard methods in the art, including but not limited to filtration, heating, radiation, or electron beam. It is expected that the silk protein fragment mixture will withstand sterilization better than intact silk protein solutions described in the art due to its shorter protein polymer length. In addition, silk products produced from the SPF mixture described herein can be sterilized as needed for the application.

[0233] Figure 71 Flowchart showing various parameters that can be modified during the extraction and solubilization steps in the process of producing the silk protein fragment solutions of the present disclosure. Selected process parameters can be varied to achieve different final solution properties, such as molecular weight and polydispersity, depending on the intended use. It should be understood that not all illustrated steps are required to prepare all silk solutions of the present disclosure.

[0234] In one embodiment, a method for producing a silk protein fragment solution of the present disclosure includes forming a sheet of silk cocoons from a Bombyx mori silkworm; extracting the sheet in a solution of water and Na2CO3 at about 100°C for about 60 minutes, wherein the volume of the water is equal to about 0.4 times the weight of the raw silk and the amount of Na2CO3 is about 0.848 times the weight of the sheet to form a fibroin extract; rinsing the fibroin extract in a certain volume of rinse water at about 60°C for three times, each rinse for about 20 minutes, wherein the rinse water in each cycle is equal to about 0.2 L x the weight of the fragment; extracting the fibroin extract from the silk fibroin extract; Excess water is removed from the white extract; the silk fibroin extract is dried; the dried silk fibroin extract is dissolved in a LiBr solution, wherein the LiBr solution is first heated to about 100°C to produce a silk-LiBr solution and maintained; the silk-LiBr solution is placed in a drying oven at about 100°C for about 60 minutes to achieve complete dissolution of the natural silk protein structure and further fragmentation into a mixture with the desired molecular weight and polydispersity; the solution is filtered to remove any residual debris from the silkworm; the solution is diluted with water to obtain a 1% silk solution; and tangential flow filtration (TFF) is used to remove the solvent from the solution. In one embodiment, a 10kDa membrane is used to purify the silk solution and produce the final desired silk-water ratio. TFF can then be used to further concentrate the pure silk solution to a silk-to-water concentration of 2%.

[0235] Each process step, from raw cocoons to dialysis, is scalable to improve manufacturing efficiency. Whole cocoons are currently purchased as raw material, but pre-cleaned cocoons or non-heat-treated cocoons, where the removal of the worms leaves minimal debris, have also been used. Cutting and cleaning the cocoons is a manual process; however, for scalability, this process can be made less labor-intensive by, for example, using automated machines combined with compressed air to remove the worms and any particulates, or by using a cutting grinder to cut the cocoons into smaller pieces. The extraction step, currently performed in small batches, can be completed in larger vessels, such as industrial washing machines, where temperatures of 60°C to 100°C or higher can be maintained. The rinsing step can also be completed in an industrial washing machine, eliminating manual rinse cycles. The dissolution of silk in the LiBr solution can be performed in vessels other than convection ovens, such as stirred tank reactors. Silk dialysis, performed with a series of water changes, is a manual and time-intensive process that can be accelerated by varying certain parameters, such as diluting the silk solution before dialysis. The dialysis process can be scaled up for manufacturing by using semi-automated equipment, such as a tangential flow filtration system.

[0236] Without wishing to be bound by any particular theory, varying the extraction (i.e., time and temperature), LiBr (i.e., the temperature of the LiBr solution when added to the fibroin extract (or vice versa), and dissolution (i.e., time and temperature) parameters resulted in solvent-silk solutions with varying viscosities, uniformities, and colors. While also not wishing to be bound by any particular theory, increasing the extraction temperature, extending the extraction time, using higher temperature LiBr solutions initially and over time when dissolving the silk, and increasing the time at temperature (e.g., in an oven or alternative heat source as shown here) all resulted in solvent-silk solutions with lower viscosities and more uniformity. While almost all parameters resulted in viable silk solutions, methods that achieved complete dissolution in less than 4 to 6 hours were preferred for process scaling.

[0237] In one embodiment, a solution of silk fibroin fragments having a weight average molecular weight selected from about 6 kDa to about 17 kDa is prepared according to the following steps: degumming a silk source by adding the silk source to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes; removing sericin from the solution to produce a silk fibroin extract comprising an undetectable level of sericin content; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a lithium bromide solution having an initial temperature of about 60°C to about 140°C when the silk fibroin extract is placed in the lithium bromide solution; maintaining the silk fibroin-lithium bromide solution in an oven at a temperature of about 140°C for a period of up to 1 hour; removing the lithium bromide from the silk fibroin extract; and preparing an aqueous solution of silk fibroin fragments, the aqueous solution comprising: fragments having a weight average molecular weight selected from about 6 kDa to about 17 kDa and a polydispersity of 1 to about 5 or about 1.5 to about 3.0. The method may further include drying the silk fibroin extract before the dissolving step. The aqueous solution of the fibroin fragments may contain less than 300 ppm of lithium bromide residues as measured using a high performance liquid chromatography lithium bromide assay. The aqueous solution of the fibroin fragments may contain less than 100 ppm of sodium carbonate residues as measured using a high performance liquid chromatography sodium carbonate assay. The aqueous solution of the fibroin fragments may be lyophilized. In some embodiments, the fibroin fragment solution may be further processed into various forms, including gels, powders, and nanofibers.

[0238] In one embodiment, a solution of fibroin fragments having a weight average molecular weight selected from the group consisting of about 17 kDa to about 39 kDa is prepared according to the following steps: adding a silk source to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes to cause degumming; removing sericin from the solution to produce a fibroin extract comprising an undetectable level of sericin content; draining the solution from the fibroin extract; dissolving the fibroin extract in a lithium bromide solution having a starting temperature of about 80°C to about 140°C when the fibroin extract is placed in the lithium bromide solution. temperature; maintaining the fibroin-lithium bromide solution in a drying oven at a temperature of about 60° C. to about 100° C. for a period of up to 1 hour; removing lithium bromide from the fibroin extract; and preparing an aqueous solution of fibroin fragments, wherein the aqueous solution of fibroin fragments comprises from about 10 ppm to about 300 ppm of lithium bromide residues, wherein the aqueous solution of fibroin fragments comprises from about 10 ppm to about 100 ppm of sodium carbonate residues, wherein the aqueous solution of fibroin fragments comprises fragments having a weight average molecular weight selected from about 17 kDa to about 39 kDa and a polydispersity of 1 to about 5 or from about 1.5 to about 3.0. The method may further include drying the fibroin extract prior to the dissolving step. The aqueous solution of fibroin fragments may comprise less than 300 ppm of lithium bromide residues as measured using a high performance liquid chromatography lithium bromide assay. The aqueous solution of fibroin fragments may comprise less than 100 ppm of sodium carbonate residues as measured using a high performance liquid chromatography sodium carbonate assay.

[0239] In some embodiments, a method for preparing an aqueous solution of silk fibroin fragments having an average weight average molecular weight selected from about 6 kDa to about 17 kDa comprises the following steps: degumming a silk source by adding the silk source to a boiling (100° C.) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes; removing sericin from the solution to produce a silk fibroin extract comprising an undetectable level of sericin content; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a lithium bromide solution having an initial temperature of about 60° C. to about 140° C. when the silk fibroin extract is placed in the lithium bromide solution; maintaining the silk fibroin-lithium bromide solution in an oven at a temperature of about 140° C. for at least 1 hour; removing the lithium bromide from the silk fibroin extract; and preparing an aqueous solution of silk fibroin fragments, the aqueous solution comprising: fragments having an average weight average molecular weight selected from about 6 kDa to about 17 kDa and a polydispersity of 1 to about 5 or about 1.5 to about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin fragments may contain less than 300 ppm of residual lithium bromide, as measured using a high-performance liquid chromatography (HPLC) lithium bromide assay. The aqueous solution of pure silk fibroin fragments may contain less than 100 ppm of residual sodium carbonate, as measured using a high-performance liquid chromatography (HPLC) sodium carbonate assay. The method may further include adding a therapeutic agent to the aqueous solution of pure silk fibroin fragments. The method may further include adding a molecule selected from one of an antioxidant and an enzyme to the aqueous solution of pure silk fibroin fragments. The method may further include adding a vitamin to the aqueous solution of pure silk fibroin fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin fragments may be lyophilized. The method may further include adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin fragments. The alpha hydroxy acid may be selected from glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further include adding hyaluronic acid or a salt thereof to the aqueous solution of pure silk fibroin fragments at a concentration of about 0.5% to about 10.0%. The method may further include adding at least one of zinc oxide or titanium dioxide. A film can be prepared from an aqueous solution of pure silk fibroin fragments produced by this method. The film can contain from about 1.0% to about 50.0% by weight of vitamin C or its derivatives. The film can have a water content of from about 2.0% to about 20.0% by weight. The film can contain from about 30.0% to about 99.5% by weight of pure silk fibroin fragments. A gel can be prepared from an aqueous solution of pure silk fibroin fragments produced by this method. The gel can contain from about 0.5% to about 20.0% by weight of vitamin C or its derivatives. The gel can have a silk content of at least 2% and a vitamin content of at least 20%.

[0240] In some embodiments, a method for preparing an aqueous solution of fibroin fragments having an average weight average molecular weight selected from about 17 kDa to about 39 kDa comprises the following steps: adding a silk source to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes to cause degumming; removing sericin from the solution to produce a fibroin extract comprising an undetectable level of sericin content; draining the solution from the fibroin extract; dissolving the fibroin extract in a lithium bromide solution having a temperature of about 80°C to about 140°C; and The method further comprises the steps of: maintaining the silk fibroin extract at a starting temperature of about 60° C. to about 100° C. in a drying oven at a temperature of about 60° C. to about 100° C. for at least 1 hour; removing lithium bromide from the silk fibroin extract; and preparing an aqueous solution of pure silk fibroin fragments, wherein the aqueous solution of pure silk fibroin fragments comprises from about 10 ppm to about 300 ppm of lithium bromide residues, wherein the aqueous solution of silk fibroin fragments comprises from about 10 ppm to about 100 ppm of sodium carbonate residues, wherein the aqueous solution of pure silk fibroin fragments comprises fragments having an average weight average molecular weight selected from about 17 kDa to about 39 kDa and a polydispersity of 1 to about 5 or from about 1.5 to about 3.0. The method may further comprise drying the silk fibroin extract before the dissolving step. The aqueous solution of pure silk fibroin fragments may comprise less than 300 ppm of lithium bromide residues as measured using a high performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin fragments may comprise less than 100 ppm of sodium carbonate residues as measured using a high performance liquid chromatography sodium carbonate assay. The method may further include adding a therapeutic agent to the aqueous solution of pure silk fibroin fragments. The method may further include adding a molecule selected from the group consisting of an antioxidant and an enzyme to the aqueous solution of pure silk fibroin fragments. The method may further include adding a vitamin to the aqueous solution of pure silk fibroin fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin fragments may be lyophilized. The method may further include adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin fragments. The alpha hydroxy acid may be selected from glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further include adding hyaluronic acid or a salt thereof to the aqueous solution of pure silk fibroin fragments at a concentration of about 0.5% to about 10.0%. The method may further include adding at least one of zinc oxide or titanium dioxide. A film may be prepared from the aqueous solution of pure silk fibroin fragments prepared in this manner. The film may comprise about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The film may have a water content of about 2.0% to about 20.0% by weight. The film may comprise from about 30.0% to about 99.5% by weight of pure silk fibroin fragments. A gel may be prepared from an aqueous solution of pure silk fibroin fragments prepared by this method. The gel may comprise from about 0.5% to about 20.0% by weight of vitamin C or a derivative thereof.The gel may have a silk content of at least 2% and a vitamin content of at least 20%.

[0241] In one embodiment, a solution of fibroin fragments having a weight average molecular weight selected from the group consisting of about 39 kDa to about 80 kDa is prepared according to the following steps: adding a silk source to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to cause degumming; removing sericin from the solution to produce a fibroin extract comprising an undetectable level of sericin content; draining the solution from the fibroin extract; dissolving the fibroin extract in a lithium bromide solution having a temperature of about 80°C to about 140°C; and placing the fibroin extract in a solution of lithium bromide having a temperature of about 80°C to about 140°C. The method further comprises the steps of: drying the silk fibroin extract before the dissolving step. The silk fibroin extract comprises a starting temperature when in a lithium bromide solution; maintaining the silk fibroin-lithium bromide solution in a drying oven at a temperature of about 60° C. to about 100° C. for a period of up to 1 hour; removing lithium bromide from the silk fibroin extract; and preparing an aqueous solution of silk fibroin fragments, wherein the aqueous solution of silk fibroin fragments comprises about 10 ppm to about 300 ppm of lithium bromide residues, about 10 ppm to about 100 ppm of sodium carbonate residues, and fragments having a weight average molecular weight selected from about 39 kDa to about 80 kDa and a polydispersity of 1 to about 5 or about 1.5 to about 3.0. The method may further comprise drying the silk fibroin extract before the dissolving step. The aqueous solution of silk fibroin fragments may comprise less than 300 ppm of lithium bromide residues as measured using a high performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin fragments may comprise less than 100 ppm of sodium carbonate residues as measured using a high performance liquid chromatography sodium carbonate assay. In some embodiments, the method may further comprise adding an active agent (e.g., a therapeutic agent) to the aqueous solution of pure silk fibroin fragments. The method may further include adding an active agent selected from an antioxidant or an enzyme to the aqueous solution of pure silk fibroin fragments. The method may further include adding a vitamin to the aqueous solution of pure silk fibroin fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin fragments may be freeze-dried. The method may further include adding an α-hydroxy acid to the aqueous solution of pure silk fibroin fragments. The α-hydroxy acid may be selected from glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further include adding hyaluronic acid or a salt thereof at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin fragments prepared by this method. A film may be prepared from the aqueous solution of pure silk fibroin fragments prepared by this method. The film may contain about 1.0% to about 50.0% vitamin C or a derivative thereof. The film may have a water content of about 2.0% to about 20.0% by weight. The film may contain about 30.0% to about 99.5% pure silk fibroin fragments by weight. A gel may be prepared from the aqueous solution of pure silk fibroin fragments prepared by this method. The gel may comprise from about 0.5 wt % to about 20.0 wt % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2 wt % and a vitamin content of at least 20 wt %.

[0242] The molecular weight of the silk protein fragments can be controlled based on specific parameters used during the extraction step, including extraction time and temperature; specific parameters used during the dissolution step, including the LiBr temperature when the silk is immersed in lithium bromide and the time the solution is held at a specific temperature; and specific parameters used during the filtration step. By controlling the process parameters using the disclosed method, SPF mixture solutions with a polydispersity equal to or less than 2.5 can be produced, having a variety of different molecular weights ranging from 1 kDa to 250 kDa, 5 kDa to 200 kDa, 5 kDa to 150 kDa, 10 kDa to 150 kDa, or 10 kDa to 80 kDa. By varying the process parameters to produce silk solutions with varying molecular weights, a desired range of fragment mixtures with a polydispersity equal to or less than 2.5 can be targeted based on desired performance requirements. For example, a lower molecular weight silk film containing a drug can have a faster release rate than a higher molecular weight SPF formulation. Furthermore, SPF mixture solutions with a polydispersity greater than 2.5 can be obtained. Furthermore, two solutions with different average molecular weights and polydispersities can be mixed to produce a combined solution. Alternatively, liquid silk glands that have been removed directly from insects (100% sericin-free silk protein) can be used in combination with any of the SPF mixture solutions disclosed herein. The molecular weight of the protein fragment composition based on pure fibroin was determined using high pressure liquid chromatography (HPLC) with a refractive index detector (RID). Polydispersity was calculated using Cirrus GPC Online GPC / SEC software version 3.3 (Agilent).

[0243] Differences in processing parameters can produce regenerated silk fibroin with different molecular weights and peptide chain size distributions (polydispersity, PD), which in turn affect the properties of regenerated silk fibroin, including mechanical strength and water solubility.

[0244] Parameters are varied during the processing of raw silk cocoons into silk solution. Varying these parameters affects the MW of the resulting silk solution. The manipulated parameters include (i) extraction time and temperature, (ii) LiBr temperature, (iii) dissolution oven temperature, and (iv) dissolution time. Figures 74 to 90 Molecular weights were determined by mass spectrometry as shown.

[0245] Experiments were performed to determine the effect of varying the extraction time. Figures 74 to 90 is a graph showing these results, and Tables A to G summarize the results. Here is a summary:

[0246] – 30 minutes of sericin extraction time resulted in a higher molecular weight than 60 minutes of sericin extraction time

[0247] –Molecular weight decreases with time in the oven

[0248] –140℃ LiBr and oven treatment resulted in the lower limit of the confidence interval being below the molecular weight of 9500Da

[0249] – 30 min extraction with undigested silk at 1 and 4 h time points

[0250] – A 30-minute extraction resulted in a significantly higher molecular weight at the 1-hour time point, with the lower limit of the confidence interval being 35,000 Da

[0251] - The molecular weights achieved at the upper limit of the confidence interval range from 18000 to 216000 Da (important to provide a solution with the specified upper limit).

[0252]

[0253]

[0254]

[0255] Experiments were performed to determine the effect of varying the extraction temperature. Figure 74 is a graph showing these results, and Table H summarizes the results. Here is a summary:

[0256] – Sericin extraction at 90°C resulted in higher MW than sericin extracted at 100°C

[0257] Both -90°C and 100°C show a decrease in MW with time in the oven.

[0258]

[0259] Experiments were conducted to determine the effect of varying the temperature of lithium bromide (LiBr) when added to silk. Figures 82 to 83 is a graph showing these results, and Table IJ summarizes the results. Here is a summary:

[0260] – No effect on molecular weight or confidence intervals (all CI ~10500-6500 Da)

[0261] – Studies have shown that since most of the material is silk at room temperature, when LiBr is added and begins to dissolve, the temperature at which the LiBr-silk dissolves quickly drops below the original LiBr temperature

[0262]

[0263]

[0264] Experiments were performed to determine the effect of oven / dissolution temperature. Figures 84 to 88is a graph showing these results, and Table KO summarizes the results. Here is a summary:

[0265] - Oven temperature had less effect on the 60-minute extracted silk than on the 30-minute extracted silk. Without wishing to be bound by theory, it is believed that the 30-minute silk degraded less during the extraction process, and therefore the oven temperature had a greater effect on the larger MW, less degraded fraction of the silk.

[0266] - For 60°C vs. 140°C ovens, the 30 minute extracted silk shows a very significant lower MW effect at the higher oven temperature, while the 60 minute extracted silk has a much smaller effect

[0267] The -140°C oven resulted in a lower limit of the confidence interval of ∼6000 Da.

[0268]

[0269]

[0270] In one embodiment, the methods disclosed herein produce solutions with characteristics that can be controlled during manufacturing, including but not limited to: MW - can be changed by varying extraction and / or dissolution time and temperature (e.g., LiBr temperature), pressure, and filtration (e.g., size exclusion chromatography); structure - removal or cleavage of heavy or light chains of the fibroin polymer; purity - hot water rinse temperature for improved sericin removal or filtration capacity for improved particle removal that adversely affects the storage stability of the silk fragment protein mixture solution; color - the color of the solution can be controlled using, for example, LiBr temperature and time; viscosity; clarity; and stability of the solution. The resulting pH of the solution is typically about 7 and can be altered using acid or base depending on storage requirements.

[0271] Raw silk cocoons from silkworms are cut into fragments. The raw silk cocoon fragments are boiled in an aqueous solution of Na2CO3 (about 100°C) for about 30 minutes to about 60 minutes to remove sericin (degumming). The volume of water used is equal to about 0.4x the weight of raw silk, and the amount of Na2CO3 is about 0.848x the weight of the raw silk cocoon fragments. The resulting degummed silk cocoon fragments are rinsed three times with deionized water at about 60°C (each rinse lasting 20 minutes). The volume of the rinse water for each cycle is 0.2L x the weight of the raw silk cocoon fragments. Excess water is removed from the degummed silk cocoon fragments. After the deionized water washing step, the wet degummed silk cocoon fragments are dried at room temperature. The degummed silk cocoon fragments are mixed with a LiBr solution and the mixture is heated to about 100°C. The heated mixture is placed in a drying oven and heated at a temperature of about 60°C to about 140°C for about 60 minutes to achieve complete dissolution of the natural silk protein. The resulting solution was cooled to room temperature and then dialyzed using a 3,500Da MWCO membrane to remove the LiBr salt. Multiple exchanges were performed in deionized water until the solution was as described in Oakton Bromide (Br - Br measured in hydrolyzed silk fibroin solution on a double-junction ion selective electrode - Ions are less than 1ppm.

[0272] The resulting aqueous silk fibroin solution has a concentration of about 8.0% w / v, containing pure silk fibroin fragments having an average weight average molecular weight selected from about 6 kDa to about 16 kDa, about 17 kDa to about 39 kDa, and about 39 kDa to about 80 kDa, and a polydispersity of about 1.5 to about 3.0. The 8.0% w / v solution is diluted with deionized water to provide 1.0% w / v, 2.0% w / v, 3.0% w / v, 4.0% w / v, and 5.0% w / v, based on the coating solution.

[0273] Various silk concentration percentages (%) were prepared using tangential flow filtration (TFF). In all cases, a 1% silk solution was used as the input feed. Starting volumes ranged from 750-18,000 mL of 1% silk solution. The solution was diafiltered in TFF to remove lithium bromide. Once below a specified residual LiBr level, the solution was subjected to ultrafiltration to increase the concentration by removing water. See the following examples.

[0274] Six (6) silk solutions were used in the standard silk structure and the results were as follows:

[0275] Solution #1 had a silk concentration of 5.9 wt%, an average molecular weight of 19.8 kDa, and a PDI of 2.2 (prepared by 60 minute boiling extraction and 100°C LiBr dissolution for 1 hour).

[0276] Solution #2 had a silk concentration of 6.4 wt% (prepared by 30 min boiling extraction and 4 h LiBr dissolution at 60°C).

[0277] Solution #3 had a silk concentration of 6.17 wt% (prepared by 30 minute boiling extraction and 100°C LiBr dissolution for 1 hour).

[0278] Solution #4 had a silk concentration of 7.30 wt%: A 7.30% silk solution was generated starting with 30-minute extraction batches of 100 g of silk cocoons per batch. °° The extracted silk fibers were then dissolved in a 100°C oven using 100°C 9.3 M LiBr for 1 hour. 100 g of silk fibers were dissolved per batch to make 20% silk in LiBr. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 15,500 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once the LiBr was removed, the solution was ultrafiltered to a volume of approximately 1300 mL. 1262 mL of 7.30% silk was then collected. Water was added to the feed to aid in removing the remaining solution, and 547 mL of 3.91% silk was then collected.

[0279] Solution #5 had a silk concentration of 6.44 wt%: A 60-minute extraction batch of 25, 33, 50, 75, and 100 g of silk cocoons was used to produce a 6.44 wt% silk solution. °°The extracted silk fibers were then dissolved in a 100°C oven using 100°C 9.3 M LiBr for 1 hour. 35, 42, 50, and 71 g of silk fibers were dissolved in each batch to make 20% silk in LiBr and combined. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 17,000 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once the LiBr was removed, the solution was ultrafiltered to a volume of approximately 3000 mL. 1490 mL of 6.44% silk was then collected. Water was added to the feed to help remove the remaining solution, and 1454 mL of 4.88% silk was then collected.

[0280] Solution #6 had a silk concentration of 2.70 wt%: A 2.70% silk solution was generated starting with a 60-minute extraction batch of 25 g silk cocoons per batch. °° The extracted silk fibers were then dissolved in a 100°C oven using 100°C 9.3 M LiBr for 1 hour. 35.48 g of silk fibers were dissolved per batch to make 20% silk in LiBr. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 1000 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once the LiBr was removed, the solution was ultrafiltered to a volume of approximately 300 mL. 312 mL of 2.7% silk was then collected.

[0281] The preparation of fibroin solutions with higher molecular weight is given in Table O.

[0282] Table O. Preparation and properties of silk fibroin solutions.

[0283]

[0284]

[0285] The silk aqueous coating compositions for application to fabric are given in Tables P and Q below.

[0286]

[0287]

[0288] Three (3) silk solutions were used in the membrane preparation with the following results:

[0289] Solution #1 was 5.9% silk concentration, average MW of 19.8 kDa, and PD of 2.2 (prepared using a 60-minute boiling extraction and 1-hour LiBr dissolution at 100°C).

[0290] o Solution #2 was 6.4% silk concentration (prepared by 30 min boiling extraction and 4 h LiBr dissolution at 60°C).

[0291] o Solution #3 was 6.17% silk concentration (prepared by 30 minute boiling extraction and 1 hour LiBr dissolution at 100°C).

[0292] Membranes were prepared according to Rockwood et al. (Nature Protocols; Vol. 6; No. 10; published online Sept. 22, 2011; doi:10.1038 / nprot.2011.379). 4 mL of a 1% or 2% (wt / vol) silk solution in water was added to a 100 mm Petri dish (the volume of silk can be varied for thicker or thinner membranes and is not important) and left open to dry overnight. The bottom of a vacuum desiccator was filled with water. The dry membrane was placed in a desiccator and vacuum was applied to water anneal the membrane for 4 hours before removal from the dish. The membrane cast from solution #1 did not result in a structurally continuous membrane; the membrane broke into several pieces. Despite the water annealing treatment, these membrane fragments dissolved in water.

[0293] Silk solutions of various molecular weights and / or molecular weight combinations can be optimized for gel applications. An example of this approach is provided below, but is not intended to be limiting in application or formulation. Three (3) silk solutions were used in gel preparation, with the following results:

[0294] Solution #1 was 5.9% silk concentration, average MW of 19.8 kDa, and PD of 2.2 (prepared using 60 min boiling extraction and 100°C LiBr dissolution for 1 hour).

[0295] o Solution #2 was 6.4% silk concentration (prepared by 30 min boiling extraction and 4 h LiBr dissolution at 60°C).

[0296] o Solution #3 was 6.17% silk concentration (prepared by 30 minute boiling extraction and 1 hour LiBr dissolution at 100°C).

[0297] "Egel" is an electrogelation method as described by Rockwood et al. Briefly, 10 ml of a silk solution in water was added to a 50 ml conical tube and a pair of platinum wire electrodes were immersed in the silk solution. A 20 volt potential was applied to the platinum electrodes for 5 minutes, the power was turned off, and the gel was collected. Solution #1 did not form an Egel during the 5-minute application of current.

[0298] Solutions #2 and #3 were gelled according to the published horseradish peroxidase (HRP) procedure. The properties appeared typical of the disclosed solutions.

[0299] Materials and Methods: The following equipment and materials were used in the determination of silk molecular weight: Agilent 1100 with chemstation software; version 10.01; refractive index detector (RID); analytical balance; volumetric flasks (1000 mL, 10 mL, and 5 mL); HPLC grade water; ACS grade sodium chloride; ACS grade sodium phosphate dibasic heptahydrate; phosphoric acid; dextran MW standards - nominal molecular weight 5 kDa, 11.6 kDa, 23.8 kDa, 48.6 kDa, and 148 kDa; 50 mL PET or polypropylene disposable centrifuge tubes; graduated pipettes; amber glass HPLC vials with Teflon caps; and a Phenomenex PolySep GFC P-4000 column (size: 7.8 mm × 300 mm).

[0300] Procedure:

[0301] A) Preparation of 1 L of mobile phase (0.1 M sodium chloride solution in 0.0125 M sodium phosphate buffer)

[0302] Take a clean, dry 250mL beaker, place it on a scale, and tare it. Add approximately 3.3509g of disodium hydrogen phosphate heptahydrate to the beaker. Record the exact weight of the weighed disodium hydrogen phosphate. Dissolve the weighed sodium phosphate by adding 100mL of HPLC water to the beaker. Be careful not to spill any of the contents of the beaker. Carefully transfer the solution to a clean, dry 1000mL volumetric flask. Rinse the beaker and transfer the rinse solution to the volumetric flask. Repeat the rinse 4-5 times. In a separate, clean, dry 250mL beaker, accurately weigh approximately 5.8440g of sodium chloride. Dissolve the weighed sodium chloride in 50mL of water and transfer this solution to the sodium phosphate solution in the volumetric flask. Rinse the beaker and transfer the rinse solution to the volumetric flask. Adjust the pH of the solution to 7.0±0.2 with phosphoric acid. Bring the volume in the volumetric flask to 1000mL with HPLC water and shake vigorously to mix the solution evenly. Filter the solution through a 0.45μm polyamide membrane filter. Transfer the solution to a clean, dry solvent bottle and label the bottle. The volume of this solution can be varied as required by varying the amounts of disodium hydrogen phosphate heptahydrate and sodium chloride accordingly.

[0303] B) Preparation of Dextran Molecular Weight Standard Solution

[0304] At least five different molecular weight standards should be used for each sample run so that the expected values ​​for the test samples are encompassed by the values ​​of the standards used. Label six 20 mL scintillation glass vials as molecular weight standards. Accurately weigh approximately 5 mg of each dextran molecular weight standard and record the weight. Dissolve the dextran molecular weight standard in 5 mL of mobile phase to prepare a 1 mg / mL standard solution.

[0305] C) Preparation of sample solution

[0306] When preparing sample solution, if there is limitation on how many samples can be provided, the preparation can be scaled up, as long as the ratio is maintained. According to the silk protein content in sample type and sample, enough samples are weighed into 1mg / mL sample solution for preparation for analysis in 50mL disposable centrifuge tubes on analytical balance. The sample is dissolved in an equal-volume mobile phase to prepare a 1mg / mL solution. Tightly cover these tubes and mix the sample (in solution). The sample solution was left standstill for 30 minutes at room temperature. Gently mix the sample solution 1 minute and centrifuge for 10 minutes at 4000RPM again.

[0307] D) HPLC analysis of samples

[0308] Transfer 1.0 mL of all standard and sample solutions to separate HPLC vials. Inject the molecular weight standards (one injection each) and each sample in duplicate. Analyze all standard and sample solutions using the following HPLC conditions:

[0309] column PolySepGFCP-4000(7.8×300mm) Column temperature 25℃ detector Refractive index detector (temperature at 35°C) Injection volume 25.0μL Mobile phase 0.1 M sodium chloride solution in 0.0125 M sodium phosphate buffer flow rate 1.0mL / min Runtime 20.0min

[0310] Data Analysis and Calculations - Calculation of Average Molecular Weight Using Cirrus Software

[0311] Upload the chromatographic data files of the standard and analytical samples to the Cirrus SEC data collection and molecular weight analysis software. Calculate the weight average molecular weight (M) of each sample injected. w ), number average molecular weight (M n ), peak average molecular weight (M p ) and polydispersity.

[0312] Spider silk fragments

[0313] Spider silk is a natural polymer composed of three domains: a repetitive central core domain that dominates the protein chain and non-repetitive N- and C-terminal domains. The large core domain is organized in a block copolymer-like arrangement, with two basic sequences—a crystalline polypeptide (poly(A) or poly(GA)) and a less crystalline polypeptide (GGX or GPGXX (SEQ ID NO:6))—alternating within the core domain. Dragline silk is a protein complex composed of major ampullate dragline protein 1 (MaSp1) and major ampullate dragline protein 2 (MaSp2). Both silks are approximately 3500 amino acids long. MaSp1 is found in the fiber core and periphery, while MaSp2 forms clusters in certain core regions. The large central domains of MaSp1 and MaSp2 are organized in a block copolymer-like arrangement, with two basic sequences—a crystalline polypeptide (poly(A) or poly(GA)) and a less crystalline polypeptide (GGX or GPGXX (SEQ ID NO:6))—alternating within the core domain. Specific secondary structures have been assigned to poly(A) / (GA), GGX, and GPGXX (SEQ ID NO:6) motifs, comprising β-sheets, α-helices, and β-helices, respectively. The primary sequence, composition, and secondary structural elements of the repetitive core domain determine the mechanical properties of spider silk, while the non-repetitive N- and C-terminal domains are crucial for storing the liquid silk dope in the lumen and forming fibers in the spinning duct.

[0314] The main difference between MaSp1 and MaSp2 is the presence of proline (P) residues, which make up 15% of the total amino acid content in MaSp2, while MaSp1 contains no prolines. By counting the number of proline residues in dragline silk from the spider N. clavipes, it was possible to estimate the presence of both proteins in the fiber: 81% MaSp1 and 19% MaSp2. Different spiders have different ratios of MaSp1 and MaSp2. For example, dragline silk fibers from the orb weaver Argiope aurantia contain 41% MaSp1 and 59% MaSp2. This variation in the ratio of large ampullate silk can determine the properties of the silk fiber.

[0315] At least seven different types of silk proteins are known for a single species of Theridiidae spider. The silks differ in primary sequence, physical properties and function. For example, dragline silks, used to construct the framework, radii and lifelines, are known for their outstanding mechanical properties, including strength, toughness and elasticity. On an equal weight basis, spider silk is tougher than steel and Kevlar. Flageliform silks, found in capture spirals, have a ductility of up to 500%. Small ampullate silks, found in the auxiliary spirals of orb-webs and prey wrapping, have a high toughness and strength almost similar to large ampullate silks, but do not supercontract in water.

[0316] Spider silks are known for their high tensile strength and toughness. Recombinant silk proteins also impart advantageous properties to cosmetic or dermatological compositions, particularly improved hydration or softening, good film-forming properties, and low surface density. The diverse and unique biomechanical properties, combined with biocompatibility and slow degradation rates, make spider silk an excellent candidate as a biomaterial for tissue engineering, guided tissue repair, and drug delivery, in cosmetic products (e.g., nail and hair strengtheners, skin care products), and in industrial materials (e.g., nanowires, nanofibers, surface coatings).

[0317] In one embodiment, the silk protein may include a polypeptide derived from a natural spider silk protein. The polypeptide is not particularly limited as long as it is derived from a natural spider silk protein, and examples of the polypeptide include natural spider silk proteins and recombinant spider silk proteins, such as variants, analogs, derivatives, etc. of natural spider silk proteins. In terms of excellent toughness, the polypeptide may be derived from the main dragline silk protein produced in the spider's ampulla. Examples of the main dragline silk protein include the major ampulla silk proteins MaSp1 and MaSp2 from the spider's genus Nephilaclavipes and ADF3 and ADF4 from the spider Araneus diadematus. Examples of polypeptides derived from the main dragline silk protein include variants, analogs, derivatives, etc. of the main dragline silk protein. In addition, the polypeptide may be derived from the whip gland silk protein produced in the spider's whip gland. Examples of whip gland silk proteins include whip gland silk proteins derived from the spider's genus Nephilaclavipes, etc.

[0318] Examples of polypeptides derived from the major dragline protein include polypeptides containing two or more units of the amino acid sequence represented by Formula 1: REP1-REP2 (1), preferably polypeptides containing five or more units thereof, and more preferably polypeptides containing ten or more units thereof. Alternatively, the polypeptide derived from the major dragline protein may be a polypeptide containing units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) and having an amino acid sequence represented by any one of SEQ ID NOs: 52 to 54 (also described in U.S. Patent No. 9,051,453, which is incorporated herein by reference in its entirety) at the C-terminus, or an amino acid sequence having 90% or more homology to the amino acid sequence represented by any one of SEQ ID NOs: 52 to 54 (also described in U.S. Patent No. 9,051,453, which is incorporated herein by reference in its entirety). In the polypeptide derived from the major dragline protein, the units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) may be the same as or different from each other. In the case of producing a recombinant protein using a microorganism such as Escherichia coli as a host, the molecular weight of the polypeptide derived from the major dragline protein is 500 kDa or less, or 300 kDa or less, or 200 kDa or less in consideration of productivity.

[0319] In formula (1), REP1 refers to polyalanine. In REP1, the number of consecutively arranged alanine residues is preferably 2 or more, more preferably 3 or more, further preferably 4 or more, and particularly preferably 5 or more. In addition, in REP1, the number of consecutively arranged alanine residues is preferably 20 or less, more preferably 16 or less, further preferably 12 or less, and particularly preferably 10 or less. In formula (1), REP2 is an amino acid sequence consisting of 10 to 200 amino acid residues. The total number of glycine, serine, glutamine and alanine residues contained in the amino acid sequence is 40% or more, preferably 60% or more, and more preferably 70% or more relative to the total number of amino acid residues contained therein.

[0320] In the primary dragline, REP1 corresponds to the crystalline region of the fiber, where crystalline beta sheets form, and REP2 corresponds to the amorphous region of the fiber, where most of the strands lack a regular structure and are more flexible. Furthermore, [REP1-REP2] corresponds to a repeating region (repeating sequence) consisting of crystalline and amorphous regions, which is a characteristic sequence of dragline proteins.

[0321] Recombinant silk fragment

[0322] In some embodiments, the recombinant silk protein refers to a recombinant spider silk polypeptide, a recombinant insect silk polypeptide or a recombinant spidroin polypeptide. In some embodiments, the recombinant silk protein fragments disclosed herein include recombinant spider silk polypeptides of the family Araneidae or Araneoids, or recombinant insect silk polypeptides of the silkworm (Bombyx mori). In some embodiments, the recombinant silk protein fragments disclosed herein include recombinant spider silk polypeptides of the family Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragments disclosed herein include block copolymers having repeating units derived from natural spider silk polypeptides of the family Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragments disclosed herein include block copolymers having synthetic repeating units derived from spider silk polypeptides of the family Araneidae or Araneoids and block copolymers of non-repeating units derived from natural repeating units of spider silk polypeptides of the family Araneidae or Araneoids.

[0323] Recent advances in genetic engineering have provided routes for producing various types of recombinant silk proteins. Recombinant DNA technology has been used to provide a more practical source of silk proteins. As used herein, "recombinant silk protein" refers to a synthetic protein produced heterologously in a prokaryotic or eukaryotic expression system using genetic engineering methods.

[0324] Various methods for synthesizing recombinant silk peptides are known and described by Ausubel et al., Current Protocols in Molecular Biology §8 (John Wiley & Sons 1987, (1990)), which is incorporated herein by reference. The Gram-negative, rod-shaped bacterium Escherichia coli (E. coli) is a recognized host for industrial-scale protein production. Therefore, most recombinant silk has been produced in E. coli. E. coli is easy to manipulate, has a short generation time, is relatively low-cost, and can be scaled up for larger protein production.

[0325] Recombinant silk proteins can be produced by transforming eukaryotic or prokaryotic systems containing cDNA encoding for silk proteins, fragments of such proteins, or analogs of such proteins. The recombinant DNA approach is capable of producing recombinant silk with programmed sequence, secondary structure, architecture, and precise molecular weight. There are four main steps in this method: (i) designing and assembling the synthetic silk-like gene into a gene "cassette," (ii) inserting this fragment into a recombinant DNA vector, (iii) transforming this recombinant DNA molecule into a host cell, and (iv) expression and purification of the selected clone.

[0326] The term "recombinant vector" as used herein includes any vector known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). Said vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain the desired coding sequence and the appropriate DNA sequences necessary for expressing the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast or plant) or in an in vitro expression system. Cloning vectors are generally used for engineering and amplification of specific desired DNA fragments and may lack the functional sequences required for expressing the desired DNA fragments.

[0327] The prokaryotic system includes gram-negative bacteria or gram-positive bacteria. The prokaryotic expression vector may include a host organism recognizable origin of replication, a functional homologous or heterologous promoter in the host, a spidroin, a fragment of this protein, or a DNA sequence encoding a similar protein. Non-limiting examples of prokaryotic expression organisms are Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum, Anabaena, Caulobacter, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus (such as Bacillus subtilis), Brevibacterium, Corynebacterium, Rhizobium (Sinorrhiza), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylbacterium, Propionibacterium, Staphylococcus or Streptomyces cells.

[0328] Eukaryotic systems include yeast and insect, mammalian or plant cells. In this case, the expression vector may include a yeast plasmid origin of replication or an autonomously replicating sequence, a promoter, a DNA sequence encoding a spider silk protein, a fragment or a similar protein, a polyadenylation sequence, a transcription termination site and finally, a selection gene. Non-limiting examples of eukaryotic expression organisms include yeast, such as Saccharomyces cerevisiae, Pichia pastoris, basidiosporogenous yeasts, ascosporogenous yeasts, filamentous fungi, such as Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, Acremonium chrysogenum), Candida, Hansenula, Kluyveromyces, Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces, Pichia (e.g., Pichia pastoris) or Yarrowia cells, etc., mammalian cells, such as HeLa cells, COS cells, CHO cells, etc., insect cells, such as Sf9 cells, MEL cells, etc., “insect host cells”, such as Spodoptera frugiperda or Trichoplusia ni cells, SF9 cells, SF-21 cells or High-Five cells, wherein SF-9 and SF-21 are ovary cells from Spodoptera frugiperda, and High-Five cells are egg cells from Trichoplusia ni, “plant host cells”, such as tobacco, potato or pea cells.

[0329] A variety of heterologous host systems have been developed for the production of different types of recombinant silk. Recombinant partial spider silk proteins and engineered silk have been cloned and expressed in bacteria (Escherichia coli), yeast (Pichia pastoris), insects (Bombyx mori larvae), plants (tobacco, soybean, potato, Arabidopsis), mammalian cell lines (BHT / hamster), and transgenic animals (mouse, goat). Most silk proteins produced have an N-terminal or C-terminal His-tag to facilitate purification and produce sufficient protein.

[0330] In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems may include transgenic animals and plants. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include bacteria, yeast, and mammalian cell lines. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include Escherichia coli. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include transgenic silkworms (B. mori) generated using genome editing technology (e.g., CRISPR).

[0331] The recombinant silk proteins in the present disclosure comprise synthetic proteins based on the repeating units of natural silk proteins. In addition to the synthetic repeating silk protein sequences, these may further comprise one or more natural non-repeating silk protein sequences.

[0332] In some embodiments, "recombinant silk protein" refers to recombinant silk protein or fragments thereof. Recombinant production of fibroin and sericin has been reported. Various hosts have been used for such production, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas, Rhodopseudomonas, Bacillus, and Streptomyces. See EP 0230702, which is incorporated herein by reference in its entirety.

[0333] Also provided herein is the design and biosynthesis of fibroin-like multi-block polymers comprising the GAGAGX (SEQ ID NO: 1) hexapeptide (X is A, Y, V, or S) derived from the repeat domain of the Bombyx mori silk heavy chain (H chain).

[0334] In some embodiments, the present disclosure provides a silk protein-like multi-block polymer derived from a repeating domain of a Bombyx mori silk heavy chain (H chain) comprising a GAGAGS (SEQ ID NO: 2) hexapeptide repeating unit. The GAGAGS (SEQ ID NO: 2) hexapeptide is the core unit of the H chain and plays an important role in the formation of the crystalline domain. The silk protein-like multi-block polymer containing the GAGAGS (SEQ ID NO: 2) hexapeptide repeating unit spontaneously aggregates into a β-pleated structure similar to natural silk fibroin, wherein the silk protein-like multi-block polymer has any weight average molecular weight described herein.

[0335] In some embodiments, the present disclosure provides a silk peptide-like multi-block copolymer composed of a GAGAGS (SEQ ID NO: 2) hexapeptide repeat derived from the H chain of a Bombyx mori silk heavy chain and a mammalian elastin VPGVG (SEQ ID NO: 3) motif produced by Escherichia coli. In some embodiments, the present disclosure provides a fusion silk fibroin composed of a GAGAGS (SEQ ID NO: 2) hexapeptide repeat derived from the H chain of a Bombyx mori silk heavy chain and GVGVP (SEQ ID NO: 4) produced by Escherichia coli, wherein the silk protein-like multi-block polymer has any weight average molecular weight described herein.

[0336] In some embodiments, the present disclosure provides a mixture consisting of (GAGAGS) 16 (SEQ ID NO: 55) silkworm recombinant protein composed of repeated segments. In some embodiments, the present disclosure provides a recombinant protein composed of (GAGAGS) 16 (SEQ ID NO: 55) repeats and non-repeated fragments produced by E. coli (GAGAGS) 16 –F-COOH(SEQ ID NO:56), (GAGAGS) 16 –FF-COOH(SEQ ID NO:57),(GAGAGS) 16 –FFF-COOH(SEQ ID NO:58),(GAGAGS) 16 –FFFF-COOH(SEQ ID NO:59),(GAGAGS) 16 –FFFFFFFF-COOH(SEQ ID NO:60),(GAGAGS) 16 –FFFF–FFFFFFFF-COOH (SEQ ID NO: 61), wherein F has the following amino acid sequence SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG (SEQ ID NO: 5), and wherein the silk protein-like multi-block polymer has any weight average molecular weight described herein.

[0337] In some embodiments, "recombinant silk protein" refers to a recombinant spider silk protein or a fragment thereof. The production of recombinant spider silk proteins based on partial cDNA cloning has been reported. The recombinant spider silk protein thus produced comprises a portion of a repeating sequence derived from the spider rod thread spider silk protein Spidroin 1, which is from the spider Nephila. See Xu et al. (Proc. Natl. Acad. Sci. USA, 87:7120–7124 (1990). The cDNA clone encoding a portion of the repeating sequence of Spidroin 2, the second fibroin of the dragline silk from the Nephila spider and its recombinant synthesis are described in J. Biol. Chem., 1992, volume 267, pp. 19320–19324. The recombinant synthesis of spider silk proteins comprising protein fragments and variants by transformed Escherichia coli is described in U.S. Patents 5,728,810 and 5,989,894. The cDNA cloning encoding the ampullate gland spider silk protein and its expression are described in U.S. Patents 5,733,771 and 5,756,677. The cDNA cloning encoding the whip gland silk protein from the orb-webspinning spider is described in U.S. Patent 5,994,099. U.S. Patent 6,268,169 describes the recombinant synthesis of spider silk-like proteins derived from the repetitive peptide sequence present in the natural spider dragline silk of the genus Nephila by Escherichia coli, Bacillus subtilis and Pichia pastoris recombinant expression systems. WO 03 / 020916 describes a protein having a repetitive peptide sequence derived from the golden orb spider Nephila madagascariensis, Nephila senegalensis, Tetragnathakauaiensis, Tetragnatha versicolor, Argiope aurantia, Argiope trifasciata, Gasteracantha mammosa and the large ampullate glands of Latrodectus geometricus, the whip glands of Argiope trifasciata, the ampullate glands of Dolomedes tenebrosus, the two sets of silk glands of Plectreurys tristis and the silk glands of the mygalomorph Euagrus chisoseus. Each of the above references is incorporated herein by reference in its entirety.

[0338] In some embodiments, the recombinant spidroin protein is a hybrid protein of a spidroin protein and an insect silk protein, a spidroin protein and collagen, a spidroin protein and elastic protein, or a spidroin protein and keratin. The spider silk repeating unit comprises or consists of the amino acid sequence of a region comprising or consisting of at least one peptide motif that is repeated in a naturally occurring large ampullate polypeptide, such as a dragline silk polypeptide, a small ampullate polypeptide, a whip-like polypeptide, an aggregated silk polypeptide, a grape-like silk polypeptide, or a pyriform silk polypeptide.

[0339] In some embodiments, the recombinant spidroin protein of the present disclosure comprises a synthetic spidroin protein comprising repeating units derived from a natural spidroin protein, a consensus sequence, and optionally one or more natural non-repetitive spidroin protein sequences. The repeating units of the natural spidroin polypeptide may comprise a dragline silk polypeptide or a whip gland spidroin polypeptide of the family Araneidae or Araneoids.

[0340] As used herein, a spider silk "repeat unit" comprises or consists of at least one peptide motif that is repeated in a naturally occurring macroampullar gland polypeptide, such as a dragline silk polypeptide, a microampullar gland polypeptide, a whip gland polypeptide, a polymorphic gland silk polypeptide, a grape gland silk polypeptide, or a piriform gland silk polypeptide. A "repeat unit" refers to a region of amino acid sequence that corresponds to or consists of at least one peptide motif (e.g., AAAAAA (SEQ ID NO: 20) or GPGQQ (SEQ ID NO: 15)) that is repeated in a naturally occurring silk polypeptide (e.g., MaSpI, ADF-3, ADF-4, or Flag) (i.e., the same amino acid sequence) or a region that corresponds to an amino acid sequence that is substantially similar thereto (i.e., a variant amino acid sequence). A "repeat unit" having an amino acid sequence that is "substantially similar" to the corresponding amino acid sequence in a naturally occurring silk polypeptide (i.e., a wild-type repeat unit) is also similar in its properties, for example, a silk protein comprising a "substantially similar repeat unit" remains insoluble and maintains its insolubility. A "repeat unit" having an amino acid sequence that is "identical" to the amino acid sequence of a naturally occurring silk polypeptide can, for example, be a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI (SEQ ID NO: 48), MaSpII (SEQ ID NO: 49), ADF-3 (SEQ ID NO: 50) and / or ADF-4 (SEQ ID NO: 51). A "repeat unit" having an amino acid sequence that is "substantially similar" to the amino acid sequence of a naturally occurring silk polypeptide can, for example, be a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI (SEQ ID NO: 48), MaSpII (SEQ ID NO: 49), ADF-3 (SEQ ID NO: 50) and / or ADF-4 (SEQ ID NO: 51) but having one or more amino acid substitutions at specific amino acid positions.

[0341] As used herein, the term "consensus peptide sequence" refers to an amino acid sequence containing an amino acid that frequently occurs at a certain position (e.g., "G") and in which other amino acids that are not further identified are replaced by the placeholder "X." In some embodiments, the consensus sequence is at least one of the following: (i) GPGXX (SEQ ID NO: 6), wherein X is an amino acid selected from A, S, G, Y, P, and Q; (ii) GGX, wherein X is an amino acid selected from Y, P, R, S, A, T, N, and Q, preferably Y, P, and Q; (iii) A x , where x is an integer from 5 to 10.

[0342] The consensus peptide sequence of GPGXX (SEQ ID NO: 6) and GGX, i.e., a glycine-rich motif, provides flexibility to silk polypeptides and therefore to the threads formed by the silk proteins containing the motif. In detail, the iterative GPGXX (SEQ ID NO: 6) motif forms a rotating helical structure that imparts elasticity to the silk polypeptide. Both the major ampullate and whip gland silks have the GPGXX (SEQ ID NO: 6) motif. The iterative GGX motif is associated with a helical structure with three amino acids per turn and is present in most spider silks. The GGX motif can provide additional elasticity to the silk. The iterative polyalanine Ax (peptide) motif forms a crystalline β-pleated structure to provide strength to the silk polypeptide, as described, for example, in WO 03 / 057727.

[0343] In some embodiments, the recombinant spider silk protein of the present disclosure comprises two identical repeating units, each of which comprises at least one, preferably one, amino acid sequence selected from: GGRPSDTYG (SEQ ID NO: 7) and GGRPSSSYG (SEQ ID NO: 8), derived from resilin. Resilin is an elastomeric protein found in most arthropods that provides low stiffness and high strength.

[0344] As used herein, "non-repeating unit" refers to an amino acid sequence that is "substantially similar" to the corresponding non-repeating (carboxyl terminal) amino acid sequence in naturally occurring dragline silk polypeptides (i.e., wild-type non-repeating (carboxyl terminal) units), preferably the amino acid sequences in ADF-3 (SEQ ID NO: 50), ADF-4 (SEQ ID NO: 51), NR3 (SEQ ID NO: 62), and NR4 (SEQ ID NO: 63) of the spider Araneus cruciferus (also described in U.S. Patent No. 9,217,017, which is incorporated herein by reference in its entirety), the 16-repeat C16 peptide (spider silk protein eADF4, molecular weight 47.7 kDa, AMSilk) comprising the sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 9), and the amino acid sequence modified from the native sequence of ADF4 from Araneus cruciferus. Non-repeating ADF-4 and its variants exhibit efficient assembly properties.

[0345] Among synthetic spider silk proteins, the recombinant silk proteins of the present disclosure, in some embodiments, comprise a C16 protein having the polypeptide sequence of SEQ ID NO: 64 (also described in U.S. Patent No. 8,288,512, which is incorporated herein by reference in its entirety). In addition to the polypeptide sequence shown in SEQ ID NO: 64, functional equivalents, functional derivatives, and salts of this sequence are also specifically included.

[0346] As used herein, "functional equivalents" refer to mutants which have an amino acid different from the specifically mentioned amino acid in at least one sequence position of the above-mentioned amino acid sequences.

[0347] In some embodiments, the recombinant spidroin proteins of the present disclosure comprise an effective amount of at least one natural or recombinant silk protein, including spidroin proteins corresponding to Spidroin major 1 described by Xu et al., PNAS, USA, 87, 7120, (1990), Spidroin major 2 described by Hinman and Lewis, J. Biol. Chem., 267, 19320, (1922), recombinant spidroin proteins as described in U.S. Patent Application No. 2016 / 0222174 and U.S. Patent Nos. 9,051,453, 9,617,315, 9,689,089, 8,173,772, 8,642,734, 8,367,8038,097,583, 8,030,024, 7,754,851, 7,148,039, 7,060,260, or patent application No. WO 2016 / 0222174. 95 / 25165. Each of the above-cited references is incorporated herein by reference in its entirety. Additional recombinant spidroins suitable for use in the recombinant RSPF of the present disclosure include ADF3 and ADF4 from the "major ampulla gland" of the spider Araneus crusae.

[0348] Recombinant silk is also described in other patents and patent applications incorporated herein by reference: US2004590196, US7,754,851, US2007654470, US 7,951,908, US2010785960, US 8,034,897, US20090263430, US2008226854, US20090123967, US2005712095, US2007991037, US 20090162896, US200885266, US 8,372,436, US2007989907, US 2009267596, US2010319542, US2009265344, US2012684607, US 2004583227, US 8,030,024, US2006643569, US 7,868,146, US 2007991916, US 8,097,583、US2006643200、US 8,729,238、US 8,877,903, US20190062557, US20160280960, US20110201783, US2008991916, US2011986662, US2012697729, US20150328363, US 9,034,816, US20130172478, US 9,217,017, US20170202995, US 8,721,991, US2008227498, US 9,233,067, US 8,288,512, US2008161364, US 7,148,039、US1999247806、US2001861597、US 2004887100, US 9,481,719, US 8,765,688, US200880705, US 2010809102, US 8,367,803, US2010664902, US 7,569,660, US 1999138833, US2000591632, US20120065126, US20100278882, US2008161352, US20100015070, US2009513709, US 20090194317, US2004559286, US200589551, US2008187824, US 20050266242, US20050227322 and US20044418.

[0349] Recombinant silk is also described in other patents and patent applications incorporated herein by reference: US20190062557, US20150284565, US20130225476, US 20130172478, US20130136779, US20130109762, US20120252294, US20110230911, US20110201783, US20100298877, US10,478,520, US10,253,213, US10,072,152, US 9,233,067, US 9,217,017, US 9,034,816, US 8,877,903, US8,729,238, US 8,721,991, US 8,097,583, US 8,034,897, US 8,030,024, US 7,951,908, US 7,868,146 and US 7,754,851.

[0350] In some embodiments, the recombinant spider silk protein of the present disclosure comprises or consists of 2 to 80 repeating units, each of which is independently selected from GPGXX (SEQ ID NO: 6), GGX and A as defined herein. x .

[0351] In some embodiments, the recombinant spider silk protein of the present disclosure comprises or consists of repeating units, each of which is selected from the group consisting of GPGAS (SEQ ID NO: 10), GPGSG (SEQ ID NO: 11), GPGGY (SEQ ID NO: 12), GPGGP (SEQ ID NO: 13), GPGGA (SEQ ID NO: 14), GPGQQ (SEQ ID NO: 15), GPGGG (SEQ ID NO: 16), GPGQG (SEQ ID NO: 17), GPGGS (SEQ ID NO: 18), GGY, GGP, GGA, GGR, GGS, GGT, GGN, GGQ, AAAAA (SEQ ID NO: 19), AAAAAA (SEQ ID NO: 20), AAAAAAA (SEQ ID NO: 21), AAAAAAAA (SEQ ID NO: 22), AAAAAAAAA (SEQ ID NO: 23), AAAAAAAAAA (SEQ ID NO: 24), GGRPSDTYG (SEQ ID NO: 7), and GGRPSSSYG (SEQ ID NO: 8). ID NO: 8), (i) GPYGPGASAAAAAAGGYGPGSGQQ (SEQ ID NO: 25), (ii) GSSAAAAAAAASGPGGYGPENQGSGPGGYGPGGP (SEQ ID NO: 9), (iii) GPGQQGPGQQGPGQQGPGQQ (SEQ ID NO: 26), (iv) GPGGAGGPYGPGGAGGPYGPGGAGGPY (SEQ ID NO: 9) NO:27), (v)GGTTIIEDLDITIDGADGPITISEELTI(SEQ ID NO:28), (vi)PGSSAAAAAAAAASGPGQGQGQGQGQGGRPSDTYG(SEQ ID NO:29), (vii)SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG(SEQ ID NO:30), (viii)GGAGGAGGAGGSGGAGGS(SEQ ID NO: 31), (ix) GPGGAGPGGYGPGSGPGGYGPGSGPGGY (SEQ ID NO: 32), (x) GPYGPGASAAAAAAGGYGPGCGQQ (SEQ ID NO: 33), (xi) GPYGPGASAAAAAAGGYGPGKGQQ (SEQ IDNO:34), (xii)GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP(SEQNo. 8,877,903, for example, a synthetic spider peptide having a sequence order of GPGAS (SEQ ID NO: 10), GGY, GPGSG (SEQ ID NO: 11) in the peptide chain or a sequence order of AAAAAAAA (SEQ ID NO: 22), GPGGY (SEQ ID NO: 12), GPGGP (SEQ ID NO: 13) in the peptide chain, or a sequence order of AAAAAAAA (SEQ ID NO: 22), GPGQG (SEQ ID NO: 17), GGR in the peptide chain.

[0352] In some embodiments, the present disclosure provides silk-like multi-block peptides that mimic repeating units of amino acids derived from natural spider silk proteins, such as Spidroin major 1 domain, Spidroin major 2 domain, or Spidroin minor 1 domain, and a profile of variation between repeating units without changing their three-dimensional conformation, wherein these silk-like multi-block peptides comprise repeating units of amino acids corresponding to one of the following sequences (I), (II), (III), and / or (IV).

[0353] [(XGG) w (XGA)(GXG) x (AGA) y (G) z AG] p (SEQ ID NO: 38) formula (I), wherein: X corresponds to tyrosine or to glutamine, w is an integer equal to 2 or 3, x is an integer from 1 to 3, y is an integer from 5 to 7, z is an integer equal to 1 or 2, and p is an integer, and has any weight average molecular weight described herein, and / or

[0354] [(GPG2YGPGQ2) a (X')2S(A) b ] p (SEQ ID NO: 39) Formula (II), wherein: X' corresponds to the amino acid sequence GPS or GPG, a is equal to 2 or 3, b is an integer from 7 to 10, p is an integer, and has any weight average molecular weight described herein, and / or

[0355] [(GR)(GA)l (A) m (GGX) n (GA) l (A) m ] p (SEQ ID NO: 40) Formula (III) and / or [(GGX") n (GA) m (A) l ] p (SEQ ID NO:41) Formula (IV), wherein: X" corresponds to tyrosine, glutamine or alanine, L is an integer from 1 to 6, m is an integer from 0 to 4, n is an integer from 1 to 4, and p is an integer.

[0356] In some embodiments, the recombinant spidroin protein or spidroin protein analog comprises amino acid repeating units of sequence (V):

[0357] [(Xaa Gly Gly) w (Xaa Gly Ala)(Gly Xaa Gly) x (Ala Gly Ala) y (Gly) z Ala Gly] p Formula (V), wherein Xaa is tyrosine or glutamine, w is an integer equal to 2 or 3, x is an integer from 1 to 3, y is an integer from 5 to 7, z is an integer equal to 1 or 2, and p is an integer.

[0358] In some embodiments, the recombinant spider silk protein of the present disclosure is selected from ADF-3 or a variant thereof, ADF-4 or a variant thereof, MaSpI or a variant thereof, MaSpII or a variant thereof, as described in US Patent No. 9,217,017.

[0359] In some embodiments, the present disclosure provides water-soluble recombinant spider silk proteins produced in mammalian cells. The solubility of spider silk proteins produced in mammalian cells can be attributed to the presence of COOH-terminal amino acids in these proteins, which make them more hydrophilic. These COOH-terminal amino acids are not present in spider silk proteins expressed in microbial hosts.

[0360] In some embodiments, the recombinant spider silk protein of the present disclosure comprises a water-soluble recombinant spider silk protein C16 modified with an amino or carboxyl terminal group selected from the group consisting of the amino acid sequences: GCGGGGGG (SEQ ID NO: 42), GKGGGGGG (SEQ ID NO: 43), GCGGSGGGGSGGGG (SEQ ID NO: 44), GKGGGGGGSGGGG (SEQ ID NO: 45), and GCGGGGGGSGGGG (SEQ ID NO: 46). 16 NR4, C 32 NR4, C16, C32, NR4C 16 NR4, NR4C 32 NR4, NR3C 16 NR3 or NR3C 32 NR3, so that the molecular weight of the protein is within the range described herein.

[0361] In some embodiments, the recombinant spider silk protein of the present disclosure includes as described in U.S. Patent No. 8,877,903. In some embodiments, the RSPF of the present disclosure includes a recombinant spider silk protein having a repeating peptide unit derived from a natural spider silk protein, such as Spidroin major 1 domain, Spidroin major 2 domain or Spidroin minor 1 domain, wherein the repeating peptide sequence is GSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 47) or SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 30), as described in U.S. Patent No. 8,367,803, which is incorporated herein by reference in its entirety.

[0362] In some embodiments, the present disclosure provides a recombinant spidroin protein consisting of repeating segments of GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY (SEQ ID NO: 32) and having a molecular weight as described herein.

[0363] As used herein, the term "recombinant silk" refers to recombinant spider silk and / or silk proteins or fragments thereof. In one embodiment, the spider silk protein is selected from the group consisting of swathing silk (Achniform silk), egg sac silk (Cylindriform silk), egg case silk (Tubuliform silk), non-viscous dragline silk (Ampullate silk), attaching thread silk (Piriform silk), sticky core silk fiber (Flagelliform silk), and sticky outer silk fiber (Polygonal silk). For example, recombinant spider silk proteins as described herein include proteins described in U.S. Patent Application 2016 / 0222174 and U.S. Patents 9,051,453, 9,617,315, 9,689,089, 8,173,772, and 8,642,734.

[0364] Some organisms produce a variety of silk fibers with unique sequences, structural elements, and mechanical properties. For example, orb-weaving spiders have six unique types of glands that produce different silk polypeptide sequences, which are polymerized into fibers adapted to their environment or lifecycle niche. These fibers are named after the glands from which they originate, and the polypeptides are labeled with glandular abbreviations (e.g., "Ma") and "Sp," short for spidroin (spidroin). In orb-weaving spiders, these types include major ampullate gland (MaSp, also known as dragline silk), minor ampullate gland (MiSp), flagellum gland (Flag), grape-shaped gland (AcSp), tubular gland (TuSp), and piriform gland (PySp). This combination of fiber types, structural domains, and variations in polypeptide sequences across organisms of different genera and species creates a wide range of potential properties that can be controlled through the commercial production of recombinant fibers. To date, the vast majority of work on recombinant silk has focused on major ampullate gland spidroin (MaSp).

[0365] Acetic (AcSp) filaments tend to have high toughness, which is the result of a combination of medium-to-high strength and medium-to-high ductility. AcSp filaments are characterized by large block ("monoblock repeat") size, which typically contains motifs of polyserine and GPX. Tubular (TuSp or Cylindrical) filaments tend to have large diameters, as well as moderate strength and high ductility. TuSp filaments are characterized by their polyserine and polythreonine content, and short segments of polyalanine. Large ampullate (MaSp) filaments tend to have high strength and moderate ductility. MaSp filaments can be one of two subtypes: MaSp1 and MaSp2. MaSp1 filaments are generally less ductile than MaSp2 filaments and are characterized by polyalanine, GX and GGX motifs. MaSp2 filaments are characterized by polyalanine, GGX and GPX motifs. Small ampullate (MiSp) filaments tend to have moderate strength and moderate ductility. MiSp filaments are characterized by GGX, GA, and poly A motifs and typically contain spacer units of approximately 100 amino acids. Flag filaments tend to have extremely high extensibility and moderate strength. Flag filaments are typically characterized by GPG, GGX, and a short spacer motif.

[0366] Silk polypeptides are uniquely composed of a repeat domain (REP) and non-repeat regions (e.g., C-terminal and N-terminal domains) on either side thereof. In one embodiment, the C-terminal and N-terminal domains are both 75-350 amino acids in length. The repeat domain exhibits a hierarchical structure. The repeat domain comprises a series of blocks (also referred to as repeat units). These blocks repeat in the silk repeat domain, sometimes perfectly, sometimes imperfectly (constituting quasi-repeat domains). The length and composition of the blocks vary between different silk types and between different species. Table 1 of U.S. Published Application 2016 / 0222174 (incorporated herein in its entirety) lists examples of block sequences from selected species and silk types, with further examples given in Rising, A. et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell Mol. Life Sci., 68:2, pg 169-184 (2011); and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pg.2603-2605 (2001). In some cases, blocks can be arranged in a regular pattern to form larger macro-repeats that occur multiple times (typically 2-8 times) in the repeat domain of the silk sequence. Repeat blocks within a repeat domain or macro-repeat and repeat macro-repeat within a repeat domain can be separated by spacer units.

[0367] The construction of certain spider silk block copolymer polypeptides from these block and / or large repeat domains according to certain embodiments of the present disclosure is described in U.S. Published Patent Application 2016 / 0222174.

[0368] The recombinant block copolymer polypeptide based on spider silk sequence made by gene expression in recombinant prokaryotic or eukaryotic system can be purified according to methods known in the art. In a preferred embodiment, commercially available expression / secretion system can be used to express the recombinant polypeptide thus, and thereafter secreted from host cell to easily purify from surrounding medium. If expression / secretion vector is not used, alternative method relates to purifying the recombinant block copolymer polypeptide from the cell lysate (cell residue after cell integrity destruction) of the prokaryotic or eukaryotic cell derived from the expressed polypeptide. The method for generating such cell lysate is well known to those skilled in the art. In some embodiments, the recombinant block copolymer polypeptide is separated from the cell culture supernatant.

[0369] The recombinant block copolymer polypeptide can be purified by affinity separation, for example, by immunointeraction with an antibody that specifically binds to the recombinant polypeptide, or by a nickel column for separating the recombinant polypeptide tagged with 6-8 histidine residues at its N-terminus or C-terminus, wherein the alternative tag can comprise a FLAG epitope or a hemagglutinin epitope. Alternative tags can comprise a FLAG epitope or a hemagglutinin epitope. Skilled practitioners often use such methods.

[0370] Solutions of such polypeptides (ie, recombinant silk proteins) can then be prepared and used as described herein.

[0371] In another embodiment, recombinant silk proteins can be prepared according to the methods described in US Patent No. 8,642,734, which is incorporated herein by reference in its entirety, and used as described herein.

[0372] In one embodiment, a recombinant spider silk protein is provided. The spider silk protein is generally composed of 170 to 760 amino acid residues, such as 170 to 600 amino acid residues, preferably 280 to 600 amino acid residues, such as 300 to 400 amino acid residues, and more preferably 340 to 380 amino acid residues. Small size is advantageous because longer spider silk proteins tend to form amorphous aggregates, which require the use of harsh solvents for dissolution and polymerization. The recombinant spider silk protein may comprise more than 760 residues, particularly in the case where the spider silk protein comprises more than two fragments derived from the N-terminal portion of the spider silk protein, the spider silk protein comprising an N-terminal fragment composed of at least one fragment (NT) derived from the corresponding portion of the spider silk protein, and a repeating fragment (REP) derived from the corresponding internal fragment of the spider silk protein. Optionally, the spider silk protein comprises a C-terminal fragment (CT) derived from the corresponding fragment of the spider silk protein. The spidroin protein typically comprises a single fragment (NT) derived from the N-terminal portion of the spidroin protein, but in a preferred embodiment, the N-terminal fragment comprises at least two, such as two, fragments (NT) derived from the N-terminal portion of the spidroin protein. Thus, the spidroin protein can be schematically represented by the formula NT m -REP or NT m The spidroin protein is represented by NT2-REP-CT, wherein m is 1 or higher, such as 2 or higher, preferably an integer in the range of 1-2, 1-4, 1-6, 2-4, or 2-6. Preferred spidroin proteins can be schematically represented by the formula NT2-REP or NT-REP, or NT2-REP-CT or NT-REP-CT. The protein fragments are typically covalently coupled via peptide bonds. In one embodiment, the spidroin protein consists of one or more NT fragments coupled to a REP fragment, which is optionally coupled to a CT fragment.

[0373] In one embodiment, the first step of the method for producing an isolated polymer of spider silk protein involves expressing a polynucleic acid molecule encoding the spider silk protein in a suitable host, such as Escherichia coli. The protein thus obtained is isolated using standard procedures. Optionally, lipopolysaccharide and other pyrogens are actively removed at this stage.

[0374] In the second step of the method for producing isolated spidroin protein polymers, a solution of the spidroin protein in a liquid medium is provided. The terms "soluble" and "in solution" mean that the protein does not appreciably aggregate at 60,000 × g and does not precipitate from the solvent. The liquid medium can be any suitable medium, such as an aqueous medium, preferably a physiological medium, typically a buffered aqueous medium such as a 10-50 mM Tris-HCl buffer or a phosphate buffer. The liquid medium has a pH of 6.4 or higher and / or an ionic composition that prevents the polymerization of the spidroin protein. In other words, the liquid medium has a pH of 6.4 or higher, an ionic composition that prevents the polymerization of the spidroin protein, or both.

[0375] A skilled artisan can readily prepare an ionic composition that prevents the polymerization of spider silk proteins using the methods disclosed herein. Preferred ionic compositions that prevent the polymerization of spider silk proteins have an ionic strength greater than 300 mM. Specific examples of ionic compositions that prevent the polymerization of spider silk proteins include greater than 300 mM NaCl, 100 mM phosphate, and combinations of these ions that have the desired preventive effect on spider silk protein polymerization, such as a combination of 10 mM phosphate and 300 mM NaCl.

[0376] The presence of the NT fragment improves the stability of the solution and prevents polymer formation under these conditions. This is advantageous when immediate polymerization may not be ideal, such as during protein purification, in large-scale production, or when other conditions need to be optimized. Preferably, the pH of the liquid medium is adjusted to 6.7 or higher, such as 7.0 or higher, or even 8.0 or higher, such as up to 10.5, to achieve high solubility of the spider silk protein. It is also advantageous to adjust the pH of the liquid medium to a range of 6.4-6.8, which provides sufficient solubility of the spider silk protein, but it is advantageous to subsequently adjust the pH to 6.3 or lower.

[0377] In the third step, the properties of the liquid medium are adjusted to a pH of 6.3 or lower and an ionic composition that allows polymerization. That is, if the liquid medium in which the spider silk protein is dissolved has a pH of 6.4 or higher, the pH is lowered to 6.3 or lower. The skilled person is familiar with various ways to achieve this, which generally involve the addition of a strong acid or a weak acid. If the liquid medium in which the spider silk protein is dissolved has an ionic composition that prevents polymerization, the ionic composition is changed to allow polymerization. The skilled person is familiar with various ways to achieve this, such as dilution, dialysis, or gel filtration. If necessary, this step involves lowering the pH of the liquid medium to 6.3 or lower and changing the ionic composition to allow polymerization. Preferably, the pH of the liquid medium is adjusted to 6.2 or lower, such as 6.0 or lower. In particular, from a practical point of view, it may be advantageous to limit the pH from 6.4 or 6.4-6.8 in the previous step to 6.3 or 6.0-6.3 in this step, such as 6.2. In a preferred embodiment, the pH of the liquid medium in this step is 3 or higher, such as 4.2 or higher. The resulting pH range, eg, 4.2-6.3, promotes rapid polymerization.

[0378] In the fourth step, spider silk proteins are polymerized in a liquid medium having a pH of 6.3 or less and an ionic composition that permits spider silk protein polymerization. Although the presence of the NT fragment improves the solubility of spider silk proteins at a pH of 6.4 or higher and / or an ionic composition that prevents spider silk protein polymerization, it accelerates polymer formation at a pH of 6.3 or lower when the ionic composition permits spider silk protein polymerization. The resulting polymers are preferably solid and macroscopic, and they are formed in a liquid medium having a pH of 6.3 or lower and an ionic composition that permits spider silk protein polymerization. In a preferred embodiment, the pH of the liquid medium in this step is 3 or higher, such as 4.2 or higher. The resulting pH range, for example, 4.2-6.3, promotes rapid polymerization, and the resulting polymers can be provided in the molecular weights described herein and prepared in solution form, which can be used for coating articles, if necessary.

[0379] A skilled artisan can readily prepare an ionic composition that allows spidroin polymerization using the methods disclosed herein. Preferred ionic compositions that allow spidroin polymerization have an ionic strength of less than 300 mM. Specific examples of ionic compositions that allow spidroin polymerization include 150 mM NaCl, 10 mM phosphate, 20 mM phosphate, and combinations of these ions that lack a preventive effect on spidroin polymerization, such as a combination of 10 mM phosphate or 20 mM phosphate and 150 mM NaCl. The ionic strength of the liquid medium is preferably adjusted to a range of 1-250 mM.

[0380] Without wishing to be bound by any particular theory, it is believed that the NT fragment has oppositely charged poles and changes in environmental pH affect the charge balance on the protein surface and subsequently polymerization, whereas salt inhibits the same event.

[0381] At neutral pH, the energetic cost of burying the excess negative charge of the acidic pole is expected to prevent polymerization. However, as the dimer approaches its isoelectric point at lower pH, attractive electrostatic forces eventually dominate, explaining the observed salt- and pH-dependent polymerization properties of NT and NT-containing minispidroins. It is proposed that, in some embodiments, pH-induced NT polymerization and the enhanced fiber assembly efficiency of NT-minispidroins are due to changes in surface electrostatic potential, and that clusters of acidic residues at one pole of NT change their charge balance, resulting in a polymerization transition at pH values ​​of 6.3 or lower.

[0382] In a fifth step, the resulting, preferably solid, spider silk protein polymer is separated from the liquid medium. Optionally, this step involves the active removal of lipopolysaccharides and other pyrogens from the spider silk protein polymer.

[0383] Without wishing to be bound by any particular theory, it has been observed that the formation of spider silk protein polymers proceeds via the formation of water-soluble spider silk protein dimers. The present disclosure therefore also provides a method for producing isolated spider silk protein dimers, wherein the first two method steps are as described above. The spider silk protein is present as a dimer in a liquid medium having a pH of 6.4 or higher and / or an ionic composition that prevents the polymerization of the spider silk protein. The third step involves isolating the dimer obtained in the second step and optionally removing lipopolysaccharides and other pyrogens. In a preferred embodiment, the spider silk protein polymers of the present disclosure consist of polymerized protein dimers. The present disclosure therefore provides novel uses of spider silk proteins, preferably those disclosed herein, for producing spider silk protein dimers.

[0384] According to another aspect, the present disclosure provides polymers of spider silk proteins as disclosed herein. In one embodiment, polymers of such proteins can be obtained by any of the methods used therefor according to the present disclosure. Therefore, the present disclosure provides various uses of recombinant spider silk proteins, preferably those disclosed herein, for producing spider silk protein polymers as recombinant silk-based coatings. According to one embodiment, the present disclosure provides novel uses of dimers of spider silk proteins, preferably those disclosed herein, for producing isolated spider silk protein polymers as recombinant silk-based coatings. Among these uses, it is preferred that the polymer is made in a liquid medium having a pH of 6.3 or lower and an ionic composition that allows the polymerization of the spider silk protein. In one embodiment, the pH of the liquid medium is 3 or higher, such as 4.2 or higher. The resulting pH range, for example 4.2-6.3, promotes rapid polymerization.

[0385] Using one or more of the methods of the present disclosure, the polymerization process can be controlled, and this enables optimization of parameters to obtain silk polymers with desired properties and shapes.

[0386] In one embodiment, the recombinant silk proteins described herein include those described in US Patent No. 8,642,734, which is incorporated herein by reference in its entirety.

[0387] In another embodiment, the recombinant silk proteins described herein can be prepared according to the methods described in US Patent No. 9,051,453, which is incorporated herein by reference in its entirety.

[0388] The amino acid sequence represented by SEQ ID NO: 52, also described in U.S. Patent No. 9,051,453, is equivalent to the amino acid sequence consisting of the C-terminal 50 amino acid residues of the amino acid sequence of ADF3 (NCBI Accession No.: AAC47010, GI: 1263287). The amino acid sequence represented by SEQ ID NO: 53, also described in U.S. Patent No. 9,051,453, is equivalent to the amino acid sequence represented by SEQ ID NO: 52, also described in U.S. Patent No. 9,051,453, from which 20 residues have been removed from the C-terminus. The amino acid sequence represented by SEQ ID NO: 54, also described in U.S. Patent No. 9,051,453, is equivalent to the amino acid sequence represented by SEQ ID NO: 52, from which 29 residues have been removed from the C-terminus.

[0389] An example of a polypeptide containing a unit of the amino acid sequence represented by Formula 1: REP1-REP2 (1) and having at the C-terminus an amino acid sequence represented by any one of SEQ ID NOs: 52 to 54 or an amino acid sequence having 90% or greater homology to the amino acid sequence represented by any one of SEQ ID NOs: 52 to 54 (also described in U.S. Patent No. 9,051,453) is a polypeptide having an amino acid sequence represented by SEQ ID NO: 65 (also described in U.S. Patent No. 9,051,453, which is incorporated herein by reference in its entirety). The polypeptide having the amino acid sequence represented by SEQ ID NO: 65, also described in U.S. Patent 9,051,453, was obtained by the following mutations: in the amino acid sequence of ADF3 (NCBI Accession No.: AAC47010, GI: 1263287), an amino acid sequence consisting of a start codon, a His 10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 66, also described in U.S. Patent 9,051,453) was added to its N-terminus, the first to 13 repeat regions were approximately doubled, and translation ended at amino acid residue 1154. In the polypeptide having the amino acid sequence represented by SEQ ID NO: 65, also described in U.S. Patent 9,051,453, the C-terminal sequence is equivalent to the amino acid sequence represented by SEQ ID NO: 54.

[0390] In addition, the polypeptide containing a unit of the amino acid sequence represented by Formula 1: REP1-REP2 (1) and having at the C-terminus an amino acid sequence represented by any one of SEQ ID NOs: 52 to 54 also described in U.S. Patent 9,051,453 or an amino acid sequence having 90% or greater homology to the amino acid sequence represented by any one of SEQ ID NOs: 52 to 54 also described in U.S. Patent 9,051,453 may be a protein having the amino acid sequence represented by SEQ ID NO: 65 also described in U.S. Patent 9,051,453, in which one or more amino acids have been substituted, deleted, inserted and / or added and having a repeating region consisting of a crystalline region and a non-crystalline region.

[0391] In addition, an example of a polypeptide containing two or more units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) is a recombinant protein derived from ADF4 having an amino acid sequence represented by SEQ ID NO: 67 (also described in U.S. Patent No. 9,051,453, which is incorporated herein by reference in its entirety). The amino acid sequence represented by SEQ ID NO: 67, also described in U.S. Patent No. 9,051,453, is an amino acid sequence obtained by adding an amino acid sequence consisting of a start codon, a His 10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 66, also described in U.S. Patent No. 9,051,453) to the N-terminus of a partial amino acid sequence of ADF4 (NCBI Accession No.: AAC47011, GI: 1263289) obtained from the NCBI database. In addition, the polypeptide containing two or more units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) may be a polypeptide having the amino acid sequence represented by SEQ ID NO: 67, which is also described in U.S. Patent No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted and / or added and has a repeat region consisting of a crystalline region and an amorphous region. In addition, an example of a polypeptide containing two or more units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) is a recombinant protein derived from MaSp2 having the amino acid sequence represented by SEQ ID NO: 68 (also described in U.S. Patent No. 9,051,453, which is incorporated herein by reference in its entirety). The amino acid sequence represented by SEQ ID NO: 68, also described in U.S. Patent No. 9,051,453, is an amino acid sequence obtained by adding an amino acid sequence consisting of a start codon, a His 10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 66, also described in U.S. Patent No. 9,051,453) to the N-terminus of a partial sequence of MaSp2 (NCBI accession number: AAT75313, GI: 50363147) obtained from the NCBI online database. In addition, a polypeptide containing two or more units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) may be a polypeptide having the amino acid sequence represented by SEQ ID NO: 68, also described in U.S. Patent No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted, and / or added and having a repeating region consisting of a crystalline region and an amorphous region.

[0392] Examples of polypeptides derived from whip silk protein include polypeptides containing 10 or more units of the amino acid sequence represented by Formula 2: REP3(2), preferably polypeptides containing 20 or more units thereof, and more preferably polypeptides containing 30 or more units thereof. In the case of producing recombinant proteins using microorganisms such as Escherichia coli as hosts, the molecular weight of the polypeptide derived from whip silk protein is preferably 500 kDa or less, more preferably 300 kDa or less, and further preferably 200 kDa or less, in view of productivity.

[0393] In formula (2), REP 3 refers to an amino acid sequence consisting of Gly-Pro-Gly-Gly-X (SEQ ID NO: 69), wherein X refers to an amino acid selected from the group consisting of Ala, Ser, Tyr and Val.

[0394] A key characteristic of spider silk is that whip silk lacks crystalline regions but instead contains repeating regions composed of amorphous regions. Since main dragline silk and other silks have repeating regions composed of crystalline and amorphous regions, they are expected to possess high stress and stretchability. Meanwhile, whip silk, while not as stress-resistant as main dragline silk, exhibits high stretchability. This is believed to be because most whip silk consists of amorphous regions.

[0395] An example of a polypeptide containing 10 or more units of the amino acid sequence represented by Formula 2: REP3(2) is a recombinant protein derived from whipworm silk protein having the amino acid sequence represented by SEQ ID NO: 70 (also described in U.S. Patent 9,051,453, which is incorporated herein by reference in its entirety). The amino acid sequence represented by SEQ ID NO: 70, also described in U.S. Patent No. 9,051,453, is an amino acid sequence obtained by combining a partial sequence of a whip gland silk protein from the genus Nephila obtained from the NCBI database (NCBI Accession No.: AAF36090, GI: 7106224), particularly its amino acid sequence from residues 1220 to 1659 at the N-terminus (corresponding to the repeat region and motif) (referred to as a PR1 sequence), with a partial sequence of a whip gland silk protein from the genus Nephila obtained from the NCBI database (NCBI Accession No.: AAC38847, GI: 2833649), particularly its C-terminal amino acid sequence from residues 816 to 907 at the C-terminus, and thereafter adding an amino acid sequence consisting of a start codon, a His 10 tag, and an HRV3C protease recognition site (SEQ ID NO: 66, also described in U.S. Patent No. 9,051,453) to the N-terminus of the combined sequence. In addition, the polypeptide containing 10 or more units of the amino acid sequence represented by Formula 2: REP3(2) may be a polypeptide having the amino acid sequence represented by SEQ ID NO: 70, which is also described in U.S. Patent 9,051,453, in which one or more amino acids have been substituted, deleted, inserted and / or added and has a repeat region consisting of an amorphous region.

[0396] The polypeptide can be produced using a host transformed with an expression vector containing a gene encoding the polypeptide. The method for producing the gene is not particularly limited, and it can be prepared by amplifying a gene encoding a natural spider silk protein from a spider-derived cell by polymerase chain reaction (PCR) or the like and cloning it, or it can be chemically synthesized. The method for chemically synthesizing the gene is also not particularly limited, and it can be synthesized as follows, for example, by ligating oligonucleotides automatically synthesized using AKTA oligopilot plus 10 / 100 (GE Healthcare Japan) based on information on the amino acid sequence of the natural spider silk protein obtained from the NCBI online database or the like by PCR. At this time, in order to facilitate the purification and observation of the protein, a gene encoding a protein having an amino acid sequence to which an amino acid sequence consisting of a start codon and a His 10 tag has been added to its N-terminus can be synthesized.

[0397] The example of expression vector comprises plasmid, phage, virus etc. that can express protein based on DNA sequence.Plasmid type expression vector is not particularly limited, as long as it allows to express target gene in host cell and can amplify itself.For example, when using Escherichia coli Rosetta (DE3) as host, pET22b (+) plasmid vector, pCold plasmid vector etc. can be used.Among these, considering the productivity of protein, pET22b (+) plasmid vector is preferably used.The example of host comprises animal cell, plant cell, microorganism etc.

[0398] The polypeptide used in the present disclosure is preferably a polypeptide derived from ADF3, one of the two major dragline silk proteins of the spider Araneus cross. This polypeptide has the advantages of basically having high strength-elongation and toughness and being easy to synthesize.

[0399] Thus, recombinant silk proteins (e.g., recombinant spider silk-based proteins) used in accordance with the embodiments, articles, and / or methods described herein can include one or more of the proteins described above or in U.S. Patents 8,173,772, 8,278,416, 8,618,255, 8,642,734, 8,691,581, 8,729,235, 9,115,204, 9,157,070, 9,309,299, 9,644,012, 9,708,376, 9,051,453, 9,617,315, 9,968,682, 9,689,089, 9,732,125, 9,856,308, 9,926,348, 10,065,997, 10,316,069, and 10,329,332;and U.S. Patent Publications 2009 / 0226969, 2011 / 0281273, 2012 / 0041177, 2013 / 0065278, 2013 / 0115698, 2013 / 0316376, 2014 / 0058066, 2014 / 0079674, 2014 / 0245923, 2015 / 0087046, 2015 / 0119554, 2015 / 0141618, 2015 / 0291673, 2015 / 0291674, 2015 / 02395 87, 2015 / 0344542, 2015 / 0361144, 2015 / 0374833, 2015 / 0376247, 2016 / 0024464, 2017 / 0066804, 2017 / 0066805, 2015 / 0293076, 2016 / 0222174, 2017 / 0283474, 2017 / 0088675, 2019 / 0135880, 2015 / 0329587, 2019 / 0040109, 2019 / 0135881, 2 019 / 0177363, 2019 / 0225646, 2019 / 0233481, 2019 / 0031842, 2018 / 0355120, 2019 / 0186050, 2019 / 0002644, 2020 / 0031887, 2018 / 0273590, 20191 / 094403, 2019 / 0031843, 2018 / 0251501, 2017 / 0066805, 2018 / 0127553, 2019 / 0329526, 2020 / 0031886, 2018 / 0080147, 2019 / 0352349, 2020 / 0043085, 2019 / 0144819, 2019 / 0228449, 2019 / 0340666, 2020 / 0000091, 2019 / 0194710, 2019 / 0151505, 2018 / 0265555, 2019 / 0352330, 2019 / 0248847, and 2019 / 0378191 (which are incorporated herein by reference in their entireties).

[0400] fibroin-like protein fragments

[0401] The recombinant silk protein in the present disclosure comprises a synthetic protein based on the repeating unit of natural silk protein. In addition to the synthetic repeating silk protein sequence, these can additionally comprise one or more natural non-repeating silk protein sequences. "Silk fibroin-like protein fragment" as used herein refers to a protein fragment having a molecular weight and polydispersity as defined herein and a certain degree of homology with a protein selected from natural silk protein, silk fibroin heavy chain, silk fibroin light chain or any protein comprising one or more GAGAGS (SEQ ID NO: 2) six amino acid repeating units. In some embodiments, the degree of homology is selected from about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, or less than 75%.

[0402] As described herein, proteins such as natural silk proteins, fibroin heavy chains, fibroin light chains, or any proteins comprising one or more GAGAGS (SEQ ID NO: 2) six amino acid repeating units comprise from about 9% to about 45% glycine, or about 9% glycine, or about 10% glycine, about 43% glycine, about 44% glycine, about 45% glycine, or about 46% glycine. As described herein, proteins such as natural silk proteins, fibroin heavy chains, fibroin light chains, or any proteins comprising one or more GAGAGS (SEQ ID NO: 2) six amino acid repeating units comprise from about 13% to about 30% alanine, or about 13% alanine, or about 28% alanine, or about 29% alanine, or about 30% alanine, or about 31% alanine. As described herein, proteins, such as natural silk proteins, fibroin heavy chains, fibroin light chains, or any protein comprising one or more GAGAGS (SEQ ID NO: 2) six amino acid repeating units, contain 9% to about 12% serine, or about 9% serine, or about 10% serine, or about 11% serine, or about 12% serine.

[0403] In some embodiments, the fibroin-like protein described herein comprises about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about About 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, or about 55% glycine. In some embodiments, the fibroin-like protein described herein comprises about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38% or about 39% alanine. In some embodiments, the fibroin-like proteins described herein comprise about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, or about 22% serine. In some embodiments, the fibroin-like proteins described herein may independently comprise any amino acid known to be contained in natural fibroin. In some embodiments, the fibroin-like proteins described herein may independently not comprise any amino acid known to be contained in natural fibroin. In some embodiments, an average of 2 / 6 amino acids, 3 / 6 amino acids, or 4 / 6 amino acids in the fibroin-like proteins described herein are glycine. In some embodiments, an average of 1 / 6 amino acids, 2 / 6 amino acids, or 3 / 6 amino acids in the fibroin-like proteins described herein are alanine. In some embodiments, an average of 0 / 6 amino acids, 1 / 6 amino acids, or 2 / 6 amino acids in the fibroin-like proteins described herein are serine.

[0404] Sericin or sericin fragments

[0405] The main component of raw silk is fibroin fiber, which is coated with the adhesive substance sericin. Sericin is a gelatinous silk protein that covers the surface of the silk thread and is composed of chemically reactive, large steric amino acids (such as serine, threonine and aspartic acid, as well as glycine and alanine). In the multiple processes of producing silk from raw silk, sericin is important in controlling the dissolution of silk and producing high-quality silk. In addition, it plays an extremely important role as an adhesive functional protein. When silk fibers are used as clothing materials, most of the sericin covering the silk thread is removed and discarded, so sericin is a valuable unused resource.

[0406] In some embodiments, the silk protein fragments described herein include sericin or sericin fragments. Methods for preparing sericin or sericin fragments and their applications in various fields are known and described herein, and are also described in, for example, U.S. Patents 7,115,388, 7,157,273, and 9,187,538, all of which are incorporated herein by reference in their entirety.

[0407] In some embodiments, sericin, such as that removed from raw silk cocoons in a degumming step, can be collected and used in the methods described herein.Sericin can also be reconstituted from a powder and used in the compositions and methods of the present disclosure.

[0408] Other properties of SPF

[0409] The compositions of the present disclosure are "biocompatible" or exhibit "biocompatibility", meaning that the compositions are compatible with living tissues or living systems because they are non-toxic, harmless, or have no physiological reactivity and do not cause immune rejection or inflammatory responses. Such biocompatibility can be demonstrated by participants applying the compositions of the present disclosure topically on their skin for an extended period of time. In one embodiment, the extended period of time is about 3 days. In one embodiment, the extended period of time is about 7 days. In one embodiment, the extended period of time is about 14 days. In one embodiment, the extended period of time is about 21 days. In one embodiment, the extended period of time is about 30 days. In one embodiment, the extended period of time is selected from the group consisting of: about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely. For example, in some embodiments, the coating described herein is a biocompatible coating.

[0410] In some embodiments, the compositions described herein (which may be biocompatible compositions) (e.g., biocompatible coatings comprising silk) can be evaluated and conform to the international standard ISO 10993-1 entitled "Biological evaluation of medical devices—Part 1: Evaluation and testing within a risk management process." In some embodiments, the compositions described herein (which may be biocompatible compositions) can be evaluated for one or more of cytotoxicity, sensitization, hemocompatibility, pyrogenicity, implantation, genotoxicity, carcinogenicity, reproductive and developmental toxicity, and degradation according to ISO 106993-1.

[0411] The compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by having participants topically apply the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period of time is about 3 days. In one embodiment, the extended period of time is about 7 days. In one embodiment, the extended period of time is about 14 days. In one embodiment, the extended period of time is about 21 days. In one embodiment, the extended period of time is about 30 days. In one embodiment, the extended period of time is selected from the group consisting of: about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0412] In one embodiment, the stability of the composition of the present disclosure is about 1 day. In one embodiment, the stability of the composition of the present disclosure is about 2 days. In one embodiment, the stability of the composition of the present disclosure is about 3 days. In one embodiment, the stability of the composition of the present disclosure is about 4 days. In one embodiment, the stability of the composition of the present disclosure is about 5 days. In one embodiment, the stability of the composition of the present disclosure is about 6 days. In one embodiment, the stability of the composition of the present disclosure is about 7 days. In one embodiment, the stability of the composition of the present disclosure is about 8 days. In one embodiment, the stability of the composition of the present disclosure is about 9 days. In one embodiment, the stability of the composition of the present disclosure is about 10 days.

[0413] In one embodiment, the stability of the composition of the present disclosure is about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 days.

[0414] In one embodiment, the stability of the composition of the present disclosure is from 10 days to 6 months. In one embodiment, the stability of the composition of the present disclosure is from 6 months to 12 months. In one embodiment, the stability of the composition of the present disclosure is from 12 months to 18 months. In one embodiment, the stability of the composition of the present disclosure is from 18 months to 24 months. In one embodiment, the stability of the composition of the present disclosure is from 24 months to 30 months. In one embodiment, the stability of the composition of the present disclosure is from 30 months to 36 months. In one embodiment, the stability of the composition of the present disclosure is from 36 months to 48 months. In one embodiment, the stability of the composition of the present disclosure is from 48 months to 60 months.

[0415] In one embodiment, the SPF composition of the present disclosure is insoluble in aqueous solution due to the crystallinity of the protein. In one embodiment, the SPF composition of the present disclosure is soluble in aqueous solution. In one embodiment, the SPF of the composition of the present disclosure comprises approximately 2 / 3 crystalline and approximately 1 / 3 amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises approximately half crystalline and approximately half amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 99% crystalline and 1% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 95% crystalline and 5% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 90% crystalline and 10% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 85% crystalline and 15% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 80% crystalline and 20% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 75% crystalline and 25% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 70% crystalline and 30% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 65% crystalline and 35% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 60% crystalline and 40% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 50% crystalline and 50% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 40% crystalline and 60% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 35% crystalline and 65% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 30% crystalline and 70% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 25% crystalline and 75% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 20% crystalline and 80% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 15% crystalline and 85% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 10% crystalline portion and 90% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 5% crystalline portion and 90% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 1% crystalline portion and 99% amorphous region.

[0416] As used herein, the term "substantially free of inorganic residues" means that the composition exhibits 0.1% (w / w) or less residues. In one embodiment, substantially free of inorganic residues refers to a composition that exhibits 0.05% (w / w) or less residues. In one embodiment, substantially free of inorganic residues refers to a composition that exhibits 0.01% (w / w) or less residues. In one embodiment, the amount of inorganic residues is from 0 ppm ("not detectable" or "ND") to 1000 ppm. In one embodiment, the amount of inorganic residues is from ND to about 500 ppm. In one embodiment, the amount of inorganic residues is from ND to about 400 ppm. In one embodiment, the amount of inorganic residues is from ND to about 300 ppm. In one embodiment, the amount of inorganic residues is from ND to about 200 ppm. In one embodiment, the amount of inorganic residues is from ND to about 100 ppm. In one embodiment, the amount of inorganic residues is from 10 ppm to 1000 ppm.

[0417] As used herein, the term "substantially free of organic residues" means that the composition exhibits 0.1% (w / w) or less residues, and in one embodiment, substantially free of organic residues means that the composition exhibits 0.05% (w / w) or less residues. In one embodiment, substantially free of organic residues means that the composition exhibits 0.01% (w / w) or less residues. In one embodiment, the amount of organic residues is from 0 ppm ("not detectable" or "ND") to 1000 ppm. In one embodiment, the amount of organic residues is from ND to about 500 ppm. In one embodiment, the amount of organic residues is from ND to about 400 ppm. In one embodiment, the amount of organic residues is from ND to about 300 ppm. In one embodiment, the amount of organic residues is from ND to about 200 ppm. In one embodiment, the amount of organic residues is from ND to about 100 ppm. In one embodiment, the amount of organic residues is from 10 ppm to 1000 ppm.

[0418] The compositions of the present disclosure exhibit "biocompatibility", meaning that the compositions are compatible with living tissues or life systems due to being non-toxic, harmless or having no physiological reactivity and not causing immune rejection. Such biocompatibility can be demonstrated by participants applying the compositions of the present disclosure topically on their skin for a prolonged period of time. In one embodiment, the prolonged period of time is about 3 days. In one embodiment, the prolonged period of time is about 7 days, in one embodiment, the prolonged period of time is about 14 days, in one embodiment, the prolonged period of time is about 21 days. In one embodiment, the prolonged period of time is about 30 days. In one embodiment, the prolonged period of time is selected from the group consisting of: about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months and indefinite.

[0419] The compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by having participants topically apply the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period of time is about 3 days. In one embodiment, the extended period of time is about 7 days. In one embodiment, the extended period of time is about 14 days. In one embodiment, the extended period of time is about 21 days. In one embodiment, the extended period of time is about 30 days. In one embodiment, the extended period of time is selected from the group consisting of: about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0420] The following are non-limiting examples of suitable ranges for various parameters in and for the preparation of the silk solutions of the present disclosure. The silk solutions of the present disclosure may include one or more, but not necessarily all, of these parameters and may be prepared using various combinations of ranges for such parameters.

[0421] In one embodiment, the SPF Percent in solution is less than 30.0% by weight. In one embodiment, the SPF Percent in solution is less than 25.0% by weight. In one embodiment, the SPF Percent in solution is less than 20.0% by weight. In one embodiment, the SPF Percent in solution is less than 19.0% by weight. In one embodiment, the SPF Percent in solution is less than 18.0% by weight. In one embodiment, the SPF Percent in solution is less than 17.0% by weight. In one embodiment, the SPF Percent in solution is less than 16.0% by weight. In one embodiment, the SPF Percent in solution is less than 15.0% by weight. In one embodiment, the SPF Percent in solution is less than 14.0% by weight. In one embodiment, the SPF Percent in solution is less than 13.0% by weight. In one embodiment, the SPF Percent in solution is less than 12.0% by weight. In one embodiment, the SPF Percent in solution is less than 11.0% by weight. In one embodiment, the SPF Percent in solution is less than 10.0% by weight. In one embodiment, the SPF Percent in solution is less than 9.0% by weight. In one embodiment, the SPF Percent in solution is less than 8.0% by weight. In one embodiment, the SPF Percent in solution is less than 7.0% by weight. In one embodiment, the SPF percentage in the solution is less than 6.0% by weight. In one embodiment, the SPF percentage in the solution is less than 5.0% by weight. In one embodiment, the SPF percentage in the solution is less than 4.0% by weight. In one embodiment, the SPF percentage in the solution is less than 3.0% by weight. In one embodiment, the SPF percentage in the solution is less than 2.0% by weight. In one embodiment, the SPF percentage in the solution is less than 1.0% by weight. In one embodiment, the SPF percentage in the solution is less than 0.9% by weight. In one embodiment, the SPF percentage in the solution is less than 0.8% by weight. In one embodiment, the SPF percentage in the solution is less than 0.7% by weight. In one embodiment, the SPF percentage in the solution is less than 0.6% by weight. In one embodiment, the SPF percentage in the solution is less than 0.5% by weight. In one embodiment, the SPF percentage in the solution is less than 0.4% by weight. In one embodiment, the SPF percentage in the solution is less than 0.3% by weight. In one embodiment, the SPF percentage in the solution is less than 0.2% by weight. In one embodiment, the SPF percentage in the solution is less than 0.1% by weight.

[0422] In one embodiment, the SPF percentage in the solution is greater than 0.1% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.2% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.3% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.4% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.5% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.6% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.7% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.8% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.9% by weight. In one embodiment, the SPF percentage in the solution is greater than 1.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 2.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 3.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 4.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 5.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 6.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 7.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 8.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 9.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 10.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 11.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 12.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 13.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 14.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 15.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 16.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 17.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 18.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 19.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 20.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 25.0% by weight.

[0423] In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 30.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 25.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 20.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 15.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 10.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 9.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 8.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 7.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 6.5 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 6.0 weight %. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 5.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 5.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 4.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 4.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 3.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 3.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 2.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 2.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 2.4% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 5.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 4.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 4.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 3.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 3.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 2.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 4.0% by weight.In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 3.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 3.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 2.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 2.4% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 2.0% by weight.

[0424] In one embodiment, the SPF percentage in the solution ranges from about 20.0% to about 30.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 2% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 6.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 6.0% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 6.0% to about 8.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 6.0% to about 9.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 10.0% to about 20.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 11.0% to about 19.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 12.0% to about 18.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 13.0% to about 17.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 14.0% to about 16.0% by weight. In one embodiment, the SPF percentage in the solution is about 1.0% by weight. In one embodiment, the SPF percentage in the solution is about 0.5% by weight. In one embodiment, the SPF percentage in the solution is about 1.5% by weight. In one embodiment, the SPF percentage in the solution is about 2.0% by weight. In one embodiment, the SPF percentage in the solution is about 2.4% by weight. In one embodiment, the SPF percentage in the solution is 3.0% by weight. In one embodiment, the SPF percentage in the solution is 3.5% by weight. In one embodiment, the SPF percentage in the solution is about 4.0% by weight. In one embodiment, the SPF percentage in the solution is about 4.5% by weight. In one embodiment, the SPF percentage in the solution is about 5.0% by weight. In one embodiment, the SPF percentage in the solution is about 5.5% by weight. In one embodiment, the SPF percentage in the solution is about 6.0% by weight. In one embodiment, the SPF percentage in the solution is about 6.5% by weight. In one embodiment, the SPF percentage in the solution is about 7.0% by weight. In one embodiment, the SPF percentage in the solution is about 7.5% by weight. In one embodiment, the SPF percentage in the solution is about 8.0% by weight. In one embodiment, the SPF percentage in the solution is about 8.5% by weight.In one embodiment, the SPF percentage in the solution is about 9.0% by weight. In one embodiment, the SPF percentage in the solution is about 9.5% by weight. In one embodiment, the SPF percentage in the solution is about 10.0% by weight.

[0425] In one embodiment, the percentage of sericin in the solution is between undetectable and 25.0% by weight. In one embodiment, the percentage of sericin in the solution is between undetectable and 5.0% by weight. In one embodiment, the percentage of sericin in the solution is 1.0% by weight. In one embodiment, the percentage of sericin in the solution is 2.0% by weight. In one embodiment, the percentage of sericin in the solution is 3.0% by weight. In one embodiment, the percentage of sericin in the solution is 4.0% by weight. In one embodiment, the percentage of sericin in the solution is 5.0% by weight. In one embodiment, the percentage of sericin in the solution is 10.0% by weight. In one embodiment, the percentage of sericin in the solution is 25.0% by weight.

[0426] In some embodiments, the silk fibroin fragments of the present disclosure are storage stable (they do not slowly or spontaneously gel when stored in aqueous solution and there is no aggregation of fragments over time, and therefore no increase in molecular weight) for 10 days to 3 years, depending on the storage conditions, SPF percentage, and the number and conditions of shipments. In addition, the pH can be modified to extend the storage life and / or support the shipping conditions by preventing premature folding and aggregation of the silk. In one embodiment, the stability of the LiBr-silk fragment solution is 0 to 1 year. In one embodiment, the stability of the LiBr-silk fragment solution is 0 to 2 years. In one embodiment, the stability of the LiBr-silk fragment solution is 0 to 3 years. In one embodiment, the stability of the LiBr-silk fragment solution is 0 to 4 years. In one embodiment, the stability of the LiBr-silk fragment solution is 0 to 5 years. In one embodiment, the stability of the LiBr-silk fragment solution is 1 to 2 years. In one embodiment, the stability of the LiBr-silk fragment solution is 1 to 3 years. In one embodiment, the stability of the LiBr-silk fragment solution is 1 to 4 years. In one embodiment, the stability of the LiBr-silk fragment solution is 1 to 5 years. In one embodiment, the stability of the LiBr-silk fragment solution is 2 to 3 years. In one embodiment, the stability of the LiBr-silk fragment solution is 2 to 4 years. In one embodiment, the stability of the LiBr-silk fragment solution is 2 to 5 years. In one embodiment, the stability of the LiBr-silk fragment solution is 3 to 4 years. In one embodiment, the stability of the LiBr-silk fragment solution is 3 to 5 years. In one embodiment, the stability of the LiBr-silk fragment solution is 4 to 5 years.

[0427] In one embodiment, the stability of the composition of the present disclosure is from 10 days to 6 months. In one embodiment, the stability of the composition of the present disclosure is from 6 months to 12 months. In one embodiment, the stability of the composition of the present disclosure is from 12 months to 18 months. In one embodiment, the stability of the composition of the present disclosure is from 18 months to 24 months. In one embodiment, the stability of the composition of the present disclosure is from 24 months to 30 months. In one embodiment, the stability of the composition of the present disclosure is from 30 months to 36 months. In one embodiment, the stability of the composition of the present disclosure is from 36 months to 48 months. In one embodiment, the stability of the composition of the present disclosure is from 48 months to 60 months.

[0428] In one embodiment, the composition of the present disclosure having an SPF has an undetectable level of LiBr residuals. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is 10 ppm to 1000 ppm. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is 10 ppm to 300 ppm. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is less than 25 ppm. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is less than 50 ppm. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is less than 75 ppm. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is less than 100 ppm. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is less than 200 ppm. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is less than 300 ppm. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is less than 400 ppm. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is less than 500 ppm. In one embodiment, the amount of LiBr residuals in the composition of the present disclosure is less than 600 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 700 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 800 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 900 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is less than 1000 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 500 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 450 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 400 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 350 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 300 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 250 ppm. In one embodiment, the amount of LiBr residues in the composition of the present disclosure is undetectable to 200 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from undetectable to 150 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from undetectable to 100 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from 100 ppm to 200 ppm.In one embodiment, the amount of LiBr residual in the composition of the present disclosure is 200 ppm to 300 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is 300 ppm to 400 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is 400 ppm to 500 ppm.

[0429] In one embodiment, the composition of the present disclosure having an SPF has an undetectable level of Na2CO3 residuals. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 100 ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 200 ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 300 ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 400 ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 500 ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 600 ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 700 ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 800 ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 900 ppm. In one embodiment, the amount of Na2CO3 residuals in the composition of the present disclosure is less than 1000 ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 500ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 450ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 400ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 350ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 300ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 250ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 200ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 150ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is less than detectable to 100ppm. In one embodiment, the amount of Na2CO3 residues in the composition of the present disclosure is 100ppm to 200ppm. In one embodiment, the amount of Na2CO3 residual in the composition of the present disclosure is 200 ppm to 300 ppm. In one embodiment, the amount of Na2CO3 residual in the composition of the present disclosure is 300 ppm to 400 ppm. In one embodiment, the amount of Na2CO3 residual in the composition of the present disclosure is 400 ppm to 500 ppm.

[0430] A unique feature of the SPF compositions of the present disclosure is their shelf stability (they do not slowly or spontaneously gel when stored in aqueous solution and do not aggregate over time, thus increasing molecular weight) of 10 days to 3 years, depending on the storage conditions, the percentage of filaments, and the number and conditions of shipments. In addition, the pH can be varied to extend the shelf life and / or support the shipping conditions by preventing premature folding and aggregation of the filaments. In one embodiment, the SPF solution compositions of the present disclosure have a shelf stability of up to 2 weeks at room temperature (RT). In one embodiment, the SPF solution compositions of the present disclosure have a shelf stability of up to 4 weeks at RT. In one embodiment, the SPF solution compositions of the present disclosure have a shelf stability of up to 6 weeks at RT. In one embodiment, the SPF solution compositions of the present disclosure have a shelf stability of up to 8 weeks at RT. In one embodiment, the SPF solution compositions of the present disclosure have a shelf stability of up to 10 weeks at RT. In one embodiment, the SPF solution compositions of the present disclosure have a shelf stability of up to 12 weeks at RT. In one embodiment, the SPF solution compositions of the present disclosure have a shelf stability of from about 4 weeks to about 52 weeks at RT.

[0431] Table R below shows the results of storage stability testing of embodiments of the SPF compositions of the present disclosure.

[0432]

[0433] In some embodiments, the water solubility of silk films derived from fibroin fragments as described herein can be altered by solvent annealing (water annealing or methanol annealing), chemical cross-linking, enzymatic cross-linking, and heat treatment.

[0434] In some embodiments, the annealing process may involve inducing beta fold formation in a solution of fibroin fragments used as a coating material. Techniques for annealing (e.g., increasing crystallinity) or otherwise promoting "molecular stacking" of fibroin-based fragments have been described. In some embodiments, the amorphous silk film is annealed in the presence of a solvent selected from water or an organic solvent to introduce beta folds. In some embodiments, the amorphous silk film is annealed in the presence of water to introduce beta folds (water annealing). In some embodiments, the amorphous silk fragment film is annealed in the presence of methanol to introduce beta folds. In some embodiments, annealing (e.g., beta fold formation) is initiated by adding an organic solvent. Suitable organic solvents include, but are not limited to, methanol, ethanol, acetone, isopropanol, or a combination thereof.

[0435] In some embodiments, annealing is performed by so-called "water annealing" or "water vapor annealing", in which water vapor is used as an intermediate plasticizer or catalyst to promote the stacking of beta sheets. In some embodiments, the water annealing method can be performed under vacuum. Suitable such methods have been described in Jin HJ et al. (2005), Water-stable Silk Films with Reduced Beta-Sheet Content, Advanced Functional Materials, 15: 1241-1247; Xiao H et al. (2011), Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing, Biomacromolecules, 12 (5): 1686-1696.

[0436] An important feature of the water annealing method is to drive the formation of crystalline β-sheets in the peptide chains of the fibroin fragments so that the fibroin can self-assemble into a continuous film. In some embodiments, the crystallinity of the fibroin fragment film is controlled by controlling the temperature of the water vapor and the duration of annealing. In some embodiments, annealing is performed at a temperature of about 65°C to about 110°C. In some embodiments, the temperature of the water is maintained at about 80°C, and annealing is performed at a temperature selected from about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, about 100°C, about 105°C, and about 110°C.

[0437] In some embodiments, in some embodiments, the annealing process continues for a time period selected from the group consisting of: about 1 minute to about 40 minutes, about 1 minute to about 50 minutes, about 1 minute to about 60 minutes, about 1 minute to about 70 minutes, about 1 minute to about 80 minutes, about 1 minute to about 90 minutes, about 1 minute to about 100 minutes, about 1 minute to about 110 minutes, about 1 minute to about 120 minutes, about 1 minute to about 130 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 110 minutes, about 5 minutes to about 120 minutes , about 5 minutes to about 130 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 70 minutes, about 10 minutes to about 80 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 100 minutes, about 10 minutes to about 110 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 130 minutes, about 15 minutes to about 40 minutes, about 15 minutes to about 50 minutes, about 15 minutes to about 60 minutes, about 15 minutes to about 70 minutes, about 15 minutes to about 80 minutes, about 15 minutes to about 90 minutes, about 15 minutes to about 100 minutes, about 15 minutes to about 110 minutes, about 15 minutes to about 120 minutes, from about 15 minutes to about 130 minutes, from about 20 minutes to about 40 minutes, from about 20 minutes to about 50 minutes, from about 20 minutes to about 60 minutes, from about 20 minutes to about 70 minutes, from about 20 minutes to about 80 minutes, from about 20 minutes to about 90 minutes, from about 20 minutes to about 100 minutes, from about 20 minutes to about 110 minutes, from about 20 minutes to about 120 minutes, from about 20 minutes to about 130 minutes, from about 25 minutes to about 40 minutes, from about 25 minutes to about 50 minutes, from about 25 minutes to about 60 minutes, from about 25 minutes to about 70 minutes, from about 25 minutes to about 80 minutes, from about 25 minutes to about 90 minutes, from about 25 minutes to about 100 minutes, from about 25 minutes to about 110 minutes, from about 25 minutes to about 120 minutes, from about 25 minutes to about 130 minutes, from about 30 minutes to about 40 minutes, from about 30 minutes to about 50 minutes, from about 30 minutes to about 60 minutes, from about 30 minutes to about 70 minutes, from about 30 minutes to about 80 minutes, from about 30 minutes to about 90 minutes, from about 30 minutes to about 100 minutes, from about 30 minutes to about 110 minutes, from about 30 minutes to about 120 minutes, from about 30 minutes to about 130 minutes, from about 35 minutes to about 40 minutes, from about 35 minutes to about 50 minutes, from about 35 minutes to about 60 minutes, from about 35 minutes to about 70 minutes, from about 35 minutes to about 80 minutes, from about 35 minutes to about 90 minutes, from about 35 minutes to about 100 minutes, from about 35 minutes to about 110 minutes, from about 35 minutes to about 120 minutes,In some embodiments, the annealing process lasts for a period of time of about 1 minute to about 60 minutes. In some embodiments, the annealing process lasts for a period of time of about 45 minutes to about 60 minutes. A longer water annealing post-treatment corresponds to an increased crystallinity of the fibroin fragments.

[0438] In some embodiments, the annealed silk fibroin fragment membrane is prepared by immersing the wet silk fibroin fragment membrane in 100% methanol at room temperature for 60 minutes. Methanol annealing changes the composition of the silk fibroin fragment membrane from a mainly amorphous random coil to a crystalline antiparallel β-sheet structure.

[0439] In some embodiments, SPF as described herein can be used to prepare SPF microparticles by precipitation with methanol. Alternative flash drying, fluidized bed drying, spray drying, or vacuum drying can be applied to remove water from the silk solution. The SPF powder can then be stored and handled without refrigeration or other special handling procedures. In some embodiments, the SPF powder comprises low molecular weight silk fibroin fragments. In some embodiments, the SPF powder comprises medium molecular weight silk fibroin fragments. In some embodiments, the SPF powder comprises a mixture of low molecular weight silk fibroin fragments and medium molecular weight silk fibroin fragments.

[0440] As used herein, the term "substantially free of sericin" or "substantially sericin-free" refers to silk fibers from which a majority of the sericin has been removed. In one embodiment, substantially free of sericin refers to silk fibers having from about 0.01% to about 10.0% sericin by weight. In one embodiment, substantially free of sericin refers to silk fibers having from about 0.01% to about 9.0% sericin by weight. In one embodiment, substantially free of sericin refers to silk fibers having from about 0.01% to about 8.0% sericin by weight. In one embodiment, substantially free of sericin refers to silk fibers having from about 0.01% to about 7.0% sericin by weight. In one embodiment, substantially free of sericin refers to silk fibers having from about 0.01% to about 6.0% sericin by weight. In one embodiment, substantially free of sericin refers to silk fibers having from about 0.01% to about 5.0% sericin by weight. In one embodiment, silk fibroin that is substantially free of sericin refers to silk fibroin having from about 0% to about 4.0% sericin by weight. In one embodiment, silk fibroin that is substantially free of sericin refers to silk fibroin having from about 0.05% to about 4.0% sericin by weight. In one embodiment, silk fibroin that is substantially free of sericin refers to silk fibroin having from about 0.1% to about 4.0% sericin by weight. In one embodiment, silk fibroin that is substantially free of sericin refers to silk fibroin having from about 0.5% to about 4.0% sericin by weight. In one embodiment, silk fibroin that is substantially free of sericin refers to silk fibroin having from about 1.0% to about 4.0% sericin by weight. In one embodiment, silk fibroin that is substantially free of sericin refers to silk fibroin having from about 1.5% to about 4.0% sericin by weight. In one embodiment, silk fibroin that is substantially free of sericin refers to silk fibroin having from about 2.0% to about 4.0% sericin by weight. In one embodiment, the silk fibroin that is substantially free of sericin refers to a silk fibroin having from about 2.5% to about 4.0% sericin by weight. In one embodiment, the silk fibroin that is substantially free of sericin refers to a silk fibroin having a sericin content of from about 0.01% to about 0.1% by weight. In one embodiment, the silk fibroin that is substantially free of sericin refers to a silk fibroin having a sericin content of less than about 0.1% by weight. In one embodiment, the silk fibroin that is substantially free of sericin refers to a silk fibroin having a sericin content of less than about 0.05% by weight.In one embodiment, a degumming loss of about 26.0 wt% to about 31.0 wt% is obtained when the silk source is added to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes.

[0441] The following are non-limiting examples of suitable ranges for various parameters in and for the preparation of the silk solutions of the present disclosure. The silk solutions of the present disclosure may include one or more, but not necessarily all, of these parameters and may be prepared using various combinations of ranges for such parameters.

[0442] In one embodiment, the SPF Percent in solution is less than 30.0% by weight. In one embodiment, the SPF Percent in solution is less than 25.0% by weight. In one embodiment, the SPF Percent in solution is less than 20.0% by weight. In one embodiment, the SPF Percent in solution is less than 19.0% by weight. In one embodiment, the SPF Percent in solution is less than 18.0% by weight. In one embodiment, the SPF Percent in solution is less than 17.0% by weight. In one embodiment, the SPF Percent in solution is less than 16.0% by weight. In one embodiment, the SPF Percent in solution is less than 15.0% by weight. In one embodiment, the SPF Percent in solution is less than 14.0% by weight. In one embodiment, the SPF Percent in solution is less than 13.0% by weight. In one embodiment, the SPF Percent in solution is less than 12.0% by weight. In one embodiment, the SPF Percent in solution is less than 11.0% by weight. In one embodiment, the SPF Percent in solution is less than 10.0% by weight. In one embodiment, the SPF Percent in solution is less than 9.0% by weight. In one embodiment, the SPF Percent in solution is less than 8.0% by weight. In one embodiment, the SPF Percent in solution is less than 7.0% by weight. In one embodiment, the SPF percentage in the solution is less than 6.0% by weight. In one embodiment, the SPF percentage in the solution is less than 5.0% by weight. In one embodiment, the SPF percentage in the solution is less than 4.0% by weight. In one embodiment, the SPF percentage in the solution is less than 3.0% by weight. In one embodiment, the SPF percentage in the solution is less than 2.0% by weight. In one embodiment, the SPF percentage in the solution is less than 1.0% by weight. In one embodiment, the SPF percentage in the solution is less than 0.9% by weight. In one embodiment, the SPF percentage in the solution is less than 0.8% by weight. In one embodiment, the SPF percentage in the solution is less than 0.7% by weight. In one embodiment, the SPF percentage in the solution is less than 0.6% by weight. In one embodiment, the SPF percentage in the solution is less than 0.5% by weight. In one embodiment, the SPF percentage in the solution is less than 0.4% by weight. In one embodiment, the SPF percentage in the solution is less than 0.3% by weight. In one embodiment, the SPF percentage in the solution is less than 0.2% by weight. In one embodiment, the SPF percentage in the solution is less than 0.1% by weight.

[0443] In one embodiment, the SPF percentage in the solution is greater than 0.1% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.2% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.3% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.4% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.5% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.6% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.7% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.8% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.9% by weight. In one embodiment, the SPF percentage in the solution is greater than 1.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 2.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 3.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 4.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 5.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 6.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 7.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 8.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 9.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 10.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 11.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 12.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 13.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 14.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 15.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 16.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 17.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 18.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 19.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 20.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 25.0% by weight.

[0444] In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 30.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 25.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 20.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 15.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 10.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 9.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 8.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 7.0 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 6.5 weight %. In one embodiment, the SPF percentage range in the solution is from about 0.1 weight % to about 6.0 weight %. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 5.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 5.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 4.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 4.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 3.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 3.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 2.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 2.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 2.4% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 5.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 4.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 4.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 3.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 3.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.5% to about 2.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 4.0% by weight.In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 3.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 3.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 2.5% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 2.4% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 2.0% by weight.

[0445] In one embodiment, the SPF percentage in the solution ranges from about 20.0% to about 30.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 1.0% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 2% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 0.1% to about 6.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 6.0% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 6.0% to about 8.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 6.0% to about 9.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 10.0% to about 20.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 11.0% to about 19.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 12.0% to about 18.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 13.0% to about 17.0% by weight. In one embodiment, the SPF percentage in the solution ranges from about 14.0% to about 16.0% by weight. In one embodiment, the SPF percentage in the solution is about 1.0% by weight. In one embodiment, the SPF percentage in the solution is about 1.5% by weight. In one embodiment, the SPF percentage in the solution is about 2.0% by weight. In one embodiment, the SPF percentage in the solution is about 2.4% by weight. In one embodiment, the SPF percentage in the solution is 3.0% by weight. In one embodiment, the SPF percentage in the solution is 3.5% by weight. In one embodiment, the SPF percentage in the solution is about 4.0% by weight. In one embodiment, the SPF percentage in the solution is about 4.5% by weight. In one embodiment, the SPF percentage in the solution is about 5.0% by weight. In one embodiment, the SPF percentage in the solution is about 5.5% by weight. In one embodiment, the SPF percentage in the solution is about 6.0% by weight. In one embodiment, the SPF percentage in the solution is about 6.5 wt %. In one embodiment, the SPF percentage in the solution is about 7.0 wt %. In one embodiment, the SPF percentage in the solution is about 7.5 wt %. In one embodiment, the SPF percentage in the solution is about 8.0 wt %. In one embodiment, the SPF percentage in the solution is about 8.5 wt %. In one embodiment, the SPF percentage in the solution is about 9.0 wt %.In one embodiment, the SPF percentage in the solution is about 9.5% by weight. In one embodiment, the SPF percentage in the solution is about 10.0% by weight.

[0446] In one embodiment, the percentage of sericin in the solution is between undetectable and 25.0% by weight. In one embodiment, the percentage of sericin in the solution is between undetectable and 5.0% by weight. In one embodiment, the percentage of sericin in the solution is 1.0% by weight. In one embodiment, the percentage of sericin in the solution is 2.0% by weight. In one embodiment, the percentage of sericin in the solution is 3.0% by weight. In one embodiment, the percentage of sericin in the solution is 4.0% by weight. In one embodiment, the percentage of sericin in the solution is 5.0% by weight. In one embodiment, the percentage of sericin in the solution is 10.0% by weight. In one embodiment, the percentage of sericin in the solution is 25.0% by weight.

[0447] In some embodiments, the fibroin-based protein fragments of the present disclosure are storage stable (they do not slowly or spontaneously gel when stored in aqueous solution and there is no aggregation of fragments over time, so there is no increase in molecular weight) for 10 days to 3 years, depending on the storage conditions, SPF percentage, and the number and conditions of shipments. In addition, the pH can be modified to extend the storage life and / or support the shipping conditions by preventing premature folding and aggregation of the silk. In one embodiment, the stability of the LiBr-silk fragment solution is 0 to 1 year. In one embodiment, the stability of the LiBr-silk fragment solution is 0 to 2 years. In one embodiment, the stability of the LiBr-silk fragment solution is 0 to 3 years. In one embodiment, the stability of the LiBr-silk fragment solution is 0 to 4 years. In one embodiment, the stability of the LiBr-silk fragment solution is 0 to 5 years. In one embodiment, the stability of the LiBr-silk fragment solution is 1 to 2 years. In one embodiment, the stability of the LiBr-silk fragment solution is 1 to 3 years. In one embodiment, the stability of the LiBr-silk fragment solution is 1 to 4 years. In one embodiment, the stability of the LiBr-silk fragment solution is 1 to 5 years. In one embodiment, the stability of the LiBr-silk fragment solution is 2 to 3 years. In one embodiment, the stability of the LiBr-silk fragment solution is 2 to 4 years. In one embodiment, the stability of the LiBr-silk fragment solution is 2 to 5 years. In one embodiment, the stability of the LiBr-silk fragment solution is 3 to 4 years. In one embodiment, the stability of the LiBr-silk fragment solution is 3 to 5 years. In one embodiment, the stability of the LiBr-silk fragment solution is 4 to 5 years.

[0448] In one embodiment, the stability of the composition of the present disclosure is from 10 days to 6 months. In one embodiment, the stability of the composition of the present disclosure is from 6 months to 12 months. In one embodiment, the stability of the composition of the present disclosure is from 12 months to 18 months. In one embodiment, the stability of the composition of the present disclosure is from 18 months to 24 months. In one embodiment, the stability of the composition of the present disclosure is from 24 months to 30 months. In one embodiment, the stability of the composition of the present disclosure is from 30 months to 36 months. In one embodiment, the stability of the composition of the present disclosure is from 36 months to 48 months. In one embodiment, the stability of the composition of the present disclosure is from 48 months to 60 months.

[0449] In one embodiment, the selected properties of the SPF coated article that may be enhanced compared to an uncoated article may include one or more of the following: dimensional stability to washing, dimensional stability to dry cleaning, appearance after washing, appearance after dry cleaning, color fastness to washing, color fastness to dry cleaning, color fastness to non-chlorine bleaching, seam torque / spiral (for woven fabrics), color fastness to bleeding, color fastness to rubbing, color fastness to water, color fastness to light, color fastness to perspiration, color fastness to chlorinated swimming pool water, color fastness to sea water, tensile strength, seam slippage, tear strength, seam breaking strength, abrasion resistance, pilling resistance, stretch recovery, burst strength, mold rotation resistance during storage, Color fastness to color transfer (labels), color fastness to ozone, lint retention, bend and tilt resistance, color fastness to saliva, anti-snagging, wrinkle resistance (e.g., garment appearance, fabric crease retention, fabric appearance smoothness), water repellency, water resistance, stain resistance (e.g., water repellency, oil repellency, water / alcohol repellency), vertical wicking, water absorption, drying rate, stain release, breathability, wicking, antimicrobial properties, UV protection, torque resistance, odor resistance, biocompatibility, wetting time, absorption rate, spreading speed, cumulative unidirectional transport, flame retardant properties, coloring properties, fabric softening properties, pH adjustment properties, anti-felting properties, and overall moisture management capabilities.

[0450] In any of the preceding embodiments, at least one property of the article is improved, wherein the improved property is dimensional stability to washing, and wherein the amount of improvement in the property relative to the uncoated article is selected from the group consisting of: at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, and at least 500%.

[0451] In any of the preceding embodiments, at least one property of the article is improved, wherein the improved property is wash resistance size retention, and wherein the amount of improvement in the property relative to the uncoated article is selected from the group consisting of: at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at le...

Claims

1. A plurality of substantially solid silk fibroin particles comprising silk fibroin fragments, said particles being characterized by at least one of bulk density, surface area, pore size, pore volume, aspect ratio, and / or Hausner ratio.

2. The plurality of substantially solid silk fibroin particles of claim 1, wherein the substantially solid silk fibroin particles are annealed.

3. The plurality of substantially solid silk fibroin particles of claim 1 or 2, wherein the substantially solid silk fibroin particles are ground.

4. The plurality of substantially solid fibroin particles of any one of claims 1 to 3, wherein the bulk density of the substantially solid fibroin particles is less than 0.03 g / ml, less than 0.04 g / ml, less than 0.05 g / ml, less than 0.06 g / ml, less than 0.07 g / ml, less than 0.08 g / ml, less than 0.09 g / ml, less than 0.10 g / ml, less than 0.11 g / ml, less than 0.12 g / ml, less than 0.13 g / ml, less than 0.14 g / ml, less than 0.15 g / ml, less than 0.16 g / ml, less than 0.17 g / ml, less than 0.18 g / ml, less than 0.19 g / ml, less than 10 ... Less than 0.17g / ml, less than 0.18g / ml, less than 0.19g / ml, less than 0.20g / ml, less than 0.21g / ml, less than 0.22g / ml, less than 0.23g / ml, less than 0.24g / ml, or less than 0.25g / ml, less than 0.26g / ml, less than 0.27g / ml, less than 0.28g / ml, less than 0.29g / ml, less than 0.30g / ml, less than 0.31g / ml, less than 0.32g / ml, less than 0.33g / ml, less than 0.34g / ml, or less than 0.35g / ml.

5. The plurality of substantially solid fibroin particles of any one of claims 1 to 3, wherein the substantially solid fibroin particles have an average bulk density of about 0.03 g / ml, about 0.04 g / ml, about 0.05 g / ml, about 0.03 g / ml, about 0.04 g / ml, about 0.05 g / ml, about 0.06 g / ml, about 0.07 g / ml, about 0.08 g / ml, about 0.09 g / ml, about 0.10 g / ml, about 0.11 g / ml, about 0.12 g / ml, about 0.13 g / ml, about 0.14 g / ml, about 0.15g / ml, about 0.16g / ml, about 0.17g / ml, about 0.18g / ml, about 0.19g / ml, about 0.20g / ml, about 0.21g / ml, about 0.22g / ml, about 0.23g / ml, about 0.24g / ml, about 0.25g / ml, about 0.26g / ml, about 0.27g / ml, about 0.28g / ml, about 0.29g / ml, about 0.30g / ml, about 0.31g / ml, about 0.32g / ml, about 0.33g / ml, about 0.34g / ml or about 0.35g / ml.

6. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 5, wherein the substantially solid silk fibroin particles have a Hausner Ratio of 1.00 to 1.11, 1.12 to 1.18, 1.19 to 1.25, 1.26 to 1.34, or 1.35 to 1.

45.

7. The plurality of substantially solid fibroin particles of any one of claims 1 to 6, wherein the substantially solid fibroin particles have an average diameter of about 3 mm to about 10 mm.

8. A plurality of substantially solid silk fibroin particles as described in any one of claims 1 to 6, wherein the average diameter of the substantially solid silk fibroin particles is about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm or about 10 mm.

9. The plurality of substantially solid fibroin particles of any one of claims 1 to 8, wherein the substantially solid fibroin particles have an aspect ratio of 1 to about 1.

45.

10. A plurality of substantially solid silk fibroin particles as described in any one of claims 1 to 8, wherein the substantially solid silk fibroin particles have an aspect ratio of 1, about 1.10, about 1.15, about 1.20, about 1.25, about 1.30, about 1.35, about 1.40 or about 1.

45.

11. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 8, wherein the substantially solid silk fibroin particles are substantially spherical.

12. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 11, wherein the substantially solid silk fibroin particles are mesoporous.

13. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 11, wherein the substantially solid silk fibroin particles have a BET (Brunauer-Emmett-Teller) surface area of ​​about 2.50 m 2 / g to about 6.50m 2 / g.

14. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 11, wherein the substantially solid silk fibroin particles have a BET (Brunauer-Emmett-Teller) surface area of ​​about 2.50 m 2 / g to about 3.00m 2 / g, about 3.00m 2 / g to about 3.50m 2 / g, about 3.50m 2 / g to about 4.00m 2 / g, about 4.00m 2 / g to about 4.50m 2 / g, about 4.50m 2 / g to about 5.00m 2 / g, about 5.00m 2 / g to about 5.50m 2 / g, about 5.50m 2 / g to about 6.00m 2 / g, or about 6.00m 2 / g to about 6.50m 2 / g.

15. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 14, wherein the substantially solid silk fibroin particles have an average pore size of about to about 16. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 14, wherein the substantially solid silk fibroin particles have an average pore size of about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about 1 ,about to about about to about or about to about 17. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 16, wherein the substantially solid silk fibroin particles comprise a plurality of radially oriented microchannels.

18. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise an emulsifier, a surfactant, a buffer, an amino acid, or a sugar.

19. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise polysaccharides, polysorbates, glycosides, PBS, arginine, trehalose, glucose, or sucrose.

20. A plurality of substantially solid silk fibroin particles as described in any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise a surfactant selected from the group consisting of sucrose esters, cetearyl glucoside, capryl / capryl glucoside, sucrose laurate, sucrose palmitate, sucrose stearate, sucrose cocoate, sorbitan monostearate, and combinations thereof.

21. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise an additional protein or peptide, a C12-C24 fatty alcohol, a glycolipid, or a lipid.

22. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise a protein, a peptide, a sugar surfactant, a biosurfactant, a lipid, or a combination.

23. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise sugar fatty acid esters, sugar fatty acid monoesters, sugar fatty diesters, sugar fatty triesters, or sugar fats and polyesters.

24. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise sucrose fatty acid esters, sorbitan fatty acid esters or sorbitol fatty acid esters, alkyl glucosides, alkyl polyglucosides, or combinations thereof.

25. A plurality of substantially solid silk fibroin particles as described in any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise KCl, NaCl, MgCl2, CaCl2, PBS, Tris, polysorbate 20, polysorbate 80, decanoyl glucoside, sucrose, histidine, glycine, or arginine.

26. A silk fibroin nanoclay composite or film comprising silk fibroin fragments and clay, wherein the % (w / w) of clay in the composite is from about 1% to about 99%.

27. The nanoclay composite or film of claim 26, wherein the clay is bentonite.

28. The nanoclay composite or film of claim 26 or 27, wherein the concentration of clay in the composite or film is from about 20% (w / w) to about 33% (w / w), from about 33% (w / w) to about 50% (w / w), or from about 50% (w / w) to about 67% (w / w).

29. The nanoclay composite or film of any one of claims 26 to 28, wherein the water vapor permeability (WVP) of the composite is inversely proportional to the concentration of clay in the composite.

30. The nanoclay composite or film of any one of claims 26 to 28, wherein the composite has a water vapor permeability (WVP, g / m 2 *Pa*24h) is about 0.20 to about 0.30, about 0.30 to about 0.35, about 0.35 to about 0.40, about 0.40 to about 0.45, about 0.45 to about 0.50, about 0.50 to about 0.55, about 0.55 to about 0.60, about 0.60 to about 0.65, about 0.65 to about 0.70, about 0.70 to about 0.75, about 0.75 to about 0.80, about 0.80 to about 0.

85.

31. A stable silk fibroin solution, comprising silk fibroin fragments and a stabilizer, wherein: i) the solution has a z-average value that is lower than a substantially similar silk fibroin solution comprising silk fibroin fragments but not comprising the stabilizer; and / or ii) the solution has a z-average plateau value that is lower than a substantially similar silk fibroin solution comprising silk fibroin fragments but not comprising the stabilizer.

32. The stable silk fibroin solution of claim 31, wherein the z-average value is measured after the silk fibroin fragments and the stabilizer are co-formulated for a period of time, wherein the period of time ranges from 1 hour to 250 hours, from 1 hour to 350 hours, from 1 hour to 450 hours, from 1 hour to 550 hours, from 1 hour to 650 hours, or from 1 hour to 1000 hours.

33. A stable silk fibroin solution as described in claim 31, wherein the z-average value is measured after the silk fibroin fragments and the stabilizer are co-formulated for a time period, wherein the time period ranges from 1 minute to 10 minutes, 1 minute to 20 minutes, 1 minute to 30 minutes, 1 minute to 40 minutes, 1 minute to 50 minutes, 1 minute to 60 minutes, 1 minute to 70 minutes, or 1 minute to 80 minutes.

34. The stable silk fibroin solution of any one of claims 31 to 33, wherein the stabilizer is an emulsifier, a surfactant, a buffer, an amino acid or a sugar.

35. The stable silk fibroin solution of any one of claims 31 to 33, wherein the stabilizer is a polysaccharide, polysorbate, glycoside, PBS, arginine, trehalose, glucose or sucrose.

36. The stable silk fibroin solution of any one of claims 31 to 33, wherein the stabilizer is a surfactant selected from sucrose esters, cetearyl glucoside, capryl / capryl glucoside, sucrose laurate, sucrose palmitate, sucrose stearate, sucrose cocoate, sorbitan monostearate, and combinations thereof.

37. The stable silk fibroin solution of any one of claims 31 to 33, wherein the stabilizer is another protein or peptide, a C12-C24 fatty alcohol, a glycolipid, or a lipid.

38. The stable silk fibroin solution of any one of claims 31 to 33, wherein the stabilizer is a protein, a peptide, a sugar surfactant, a biosurfactant, a lipid, or a combination.

39. The stable silk fibroin solution of any one of claims 31 to 33, wherein the stabilizer is a sugar fatty acid ester, a sugar fatty acid monoester, a sugar fatty diester, a sugar fatty triester, or a sugar fat and polyester.

40. The stable silk fibroin solution of any one of claims 31 to 33, wherein the stabilizer is sucrose fatty acid ester, sorbitan fatty acid ester or sorbitol fatty acid ester, alkyl glucoside, alkyl polyglucoside or a combination thereof.

41. The stable silk fibroin solution of any one of claims 31 to 33, wherein the stabilizer is KCl, NaCl, MgCl2, CaCl2, PBS, Tris, polysorbate 20, polysorbate 80, decanoyl glucoside, sucrose, histidine, glycine or arginine.

42. The stable silk fibroin solution of any one of claims 31 to 41, wherein the solution is sprayable.

43. A liquid suspension in air comprising a plurality of droplets comprising the stabilized silk fibroin solution of claim 42, wherein the droplets are sufficiently stable for a period of time required to reach a surface after spraying.

44. A plurality of droplets or droplets comprising the stable silk fibroin solution of any one of claims 31 to 41, wherein the droplets or droplets are sufficiently stable after formation for the period of time required to reach a surface.

45. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 25, the silk fibroin nanoclay composite or film of any one of claims 26 to 30, the stable silk fibroin solution of any one of claims 31 to 42, the liquid suspension in air of claim 43, or the plurality of droplets or droplets of claim 44, wherein the silk fibroin fragments have a molecular weight selected from the group consisting of about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 14 kDa to about 30 kDa, about 15 kDa to about 20 kDa, about 17 ... [0014] In some embodiments, the present invention comprises a weight average molecular weight of about 100 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, about 55 kDa to about 60 kDa, about 60 kDa to about 100 kDa, about 80 kDa to about 144 kDa, about 144 kDa to about 250 kDa, or about 250 kDa to about 350 kDa, and a polydispersity of 1 to about 5.

46. ​​A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in claim 45, wherein the polydispersity is 1 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, or about 4.5 to about 5.

0.

47. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in claim 45, further comprising from about 0.001% (w / w) to about 10% (w / w) sericin relative to the silk fibroin fragments.

48. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 45 to 47, wherein the silk fibroin fragments do not spontaneously or gradually gel and do not significantly change in color or turbidity in aqueous solution for at least 10 days before being formulated into the substantially solid silk fibroin particles, the silk fibroin nanoclay composites or films, or the stable silk fibroin solutions.

49. A plurality of substantially solid silk fibroin particles as described in any one of claims 1 to 25, a silk fibroin nanoclay composite or film as described in any one of claims 26 to 30, a stable silk fibroin solution as described in any one of claims 31 to 42, a liquid suspension in air as described in claim 43, or a plurality of droplets or droplets as described in claim 44, wherein the silk fibroin fragments comprise a plurality of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P and W, wherein at least one of the amino acids is modified, substituted or replaced.

50. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air or a plurality of droplets or droplets as described in claim 49, wherein the silk fibroin is a silk fibroin heavy chain, a silk fibroin light chain or a silk fibroin hexamer.

51. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 49 or 50, wherein the silk fibroin fragments comprise about 2 to about 100 amino acids.

52. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in claims 49 to 51, wherein the silk fibroin fragments comprise 1 to 5 modifications, substitutions and / or replacements.

53. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 49 to 52, wherein the modification, substitution and / or replacement is selected from the modification, substitution and / or replacement of asparagine to aspartic acid, the modification, substitution and / or replacement of glutamine to glutamic acid and the modification, substitution and / or replacement of methionine to oxymethionine.

54. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 49 to 53, wherein the silk fibroin is a silk fibroin heavy chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the silk fibroin heavy chain.

55. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in claim 54, wherein the modification, substitution and / or replacement is at Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 and / or N5262.

56. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 49 to 53, wherein the silk fibroin is a silk fibroin light chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 262 of the silk fibroin light chain.

57. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in claim 56, wherein the modification, substitution and / or replacement is at N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 and / or Q255.

58. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in any one of claims 49 to 53, wherein the silk fibroin is a silk fibroin hexamer (p25), and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 220 of the silk fibroin hexamer (p25).

59. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in claim 58, wherein the modification, substitution and / or replacement is at Q62, N93, M120, N149, N172, N174 and / or N202.

60. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 49 to 59, wherein in the silk fibroin fragment portion of the substantially solid silk fibroin particles, the silk fibroin nanoclay composites or films, or the stable silk fibroin solution composition, each modification, substitution and / or replacement independently ranges from about 1% to about 99%.

61. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 61, wherein the modification %, substitution % and / or replacement % is defined as (the number of peptides or protein fragments containing a modification, substitution and / or replacement at a specific position divided by the total number of peptides or protein fragments including the specific position, whether or not containing a modification, substitution and / or replacement) x 100.

62. A plurality of substantially solid silk fibroin particles as described in any one of claims 1 to 25, a silk fibroin nanoclay composite or film as described in any one of claims 26 to 30, a stable silk fibroin solution as described in any one of claims 31 to 42, a liquid suspension in air as described in claim 43, or a plurality of droplets or droplets as described in claim 44, wherein the silk fibroin fragments are contained in one or more fractions, each fraction independently comprising a plurality of silk fibroin heavy chain fragments, a plurality of silk fibroin light chain fragments and / or a plurality of silk fibroin hexamer (p25) fragments.

63. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 62, wherein the silk fibroin fragments have a weight average molecular weight (MW) selected from about 1 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, or about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 250 kDa. w ) and a polydispersity of 1 to about 1.

7.

64. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or plurality of droplets or droplets of claim 62, wherein the silk fibroin fragments have a weight average molecular weight (MW) selected from the group consisting of about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, or about 160 kDa to about 180 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.

2.

65. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 62, wherein the silk fibroin fragments in the fraction have a weight average molecular weight (MW) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.

2.

66. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets of claim 62, wherein the silk fibroin fragments in the fraction have a weight average molecular weight (MW) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 120 kDa. w ) and a polydispersity of 1 to about 1.

1.

67. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 62, wherein the silk fibroin fragments in the fraction have a weight average molecular weight (MW) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.

2.

68. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets of claim 62, wherein the silk fibroin fragments in the fraction have a weight average molecular weight (MW) selected from the group consisting of about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. w ) and a polydispersity of 1 to about 1.

1.

69. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or liquid droplets of claim 62, wherein the silk fibroin fragments in the fraction have a weight average molecular weight (MW) selected from about 20 kDa to about 40 kDa, or about 40 kDa to about 60 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.

2.

70. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 62, wherein the one or more fractions are selected from AS77, AS78, AS79, AS80 and AS81.

71. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 62, wherein the one or more fractions are selected from AS82, AS83, AS84, AS85, AS86, AS87, AS88 and AS89.

72. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 62, wherein the one or more fractions are selected from AS90, AS91, AS92, AS93 and AS94.

73. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 62, wherein the one or more fractions are selected from AS95, AS96, AS97, AS98, AS99 and AS100.

74. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or plurality of droplets or droplets of claim 62, wherein the silk fibroin fragments have a weight average molecular weight (MW) selected from the group consisting of about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 220 kDa. w ) and a polydispersity of 1 to about 1.

7.

75. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets of claim 62, wherein the silk fibroin fragments in the fraction have a weight average molecular weight (MW) selected from the group consisting of about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. w ) and a polydispersity of 1 to about 1.2 or 1 to about 1.

3.

76. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets of claim 62, wherein the silk fibroin fragments in the fraction have a weight average molecular weight (MW) selected from the group consisting of about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. w ) and a polydispersity of 1 to about 1.1 or 1 to about 1.

2.

77. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 62, wherein the silk fibroin fragments in the fraction have a weight average molecular weight (MW) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. w ) and a polydispersity of 1 to about 1.2 or 1 to about 1.

3.

78. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 62, wherein the one or more fractions are selected from AS101, AS102, AS103, AS104 and AS105.

79. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 62, wherein the one or more fractions are selected from AS106, AS107, AS108, AS109, AS110 and AS111.

80. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 62 to 79, wherein the silk fibroin fragments comprise one or more amino acid modifications, substitutions or replacements of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P and W.

81. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 62 to 80, wherein the silk fibroin fragments comprise about 2 to about 100 amino acids.

82. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 80 or 81, wherein the silk fibroin fragment comprises 1 to 5 modifications, substitutions and / or replacements.

83. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 80 to 82, wherein the silk fibroin is a silk fibroin heavy chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the silk fibroin heavy chain.

84. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 80 to 82, wherein the silk fibroin is a silk fibroin light chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 262 of the silk fibroin light chain.

85. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in any one of claims 80 to 82, wherein the silk fibroin is a silk fibroin hexamer (p25) chain, and wherein the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 220 of the silk fibroin hexamer (p25) chain.

86. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 80 to 85, wherein the modification, substitution and / or replacement is selected from the modification, substitution and / or replacement of asparagine to aspartic acid, the modification, substitution and / or replacement of glutamine to glutamic acid and the modification, substitution and / or replacement of methionine to oxymethionine.

87. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 80 to 86, wherein the modification, substitution and / or replacement is at a silk fibroin heavy chain position selected from Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216 and / or N5262.

88. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 80 to 86, wherein the modification, substitution and / or replacement is at a silk fibroin light chain position selected from N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248 and / or Q255.

89. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 80 to 86, wherein the modification, substitution and / or replacement is at a silk fibroin hexamer (p25) position selected from Q62, N93, M120, N149, N172, N174 and / or N202.

90. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stable silk fibroin solutions, liquid suspensions in air, or a plurality of droplets or droplets as described in any one of claims 80 to 89, wherein each modification, substitution and / or replacement in the composition is independently in the range of about 1% to about 99%.

91. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in claim 90, wherein the modification %, the substitution % and / or the replacement % is defined as (the number of peptides or protein fragments containing a modification, substitution and / or replacement at a specific position divided by the total number of peptides or protein fragments including the specific position, regardless of whether they contain a modification, substitution and / or replacement) x 100.

92. A plurality of substantially solid silk fibroin particles, a silk fibroin nanoclay composite or film, a stable silk fibroin solution, a liquid suspension in air, or a plurality of droplets or droplets as described in any one of claims 45 to 91, wherein the molecular weight is determined by MALS.

93. A pouch or laundry pod comprising a plurality of substantially solid silk fibroin particles as described in any one of claims 1 to 92.

94. The laundry pod of claim 93, wherein the plurality of substantially solid fibroin particles are compressed in a multi-particle disc.

95. A laundry pod as claimed in claim 93 or 94 comprising a dissolvable shell comprising polyvinyl alcohol (PVA) or a PVA derivative.

96. The pouch of claim 93, further comprising a shell comprising one or more of nylon, polyglycolide (PGA), polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), polycaprolactone (PCL), poly(butylene succinate) (PBS), polybutylene succinate adipate, poly(p-dioxanone) (PPDO), poly(butylene adipate-co-terephthalate) (PBAT), copolyesters of terephthalic acid and lactic acid, copolyesters of terephthalic acid and glycolic acid, copolyesters of terephthalic acid and succinic acid, poly(hydroxybutyrate), poly(hydroxyvalerate), polyhydroxyhexanoate, poly(hydroxyalkanoate) (PHA), polymethylene adipate / terephthalate.

97. A method for preparing a plurality of substantially solid silk fibroin particles as claimed in any one of claims 1 to 92, the method comprising dropping a solution comprising a plurality of said silk fibroin fragments into liquid nitrogen.

98. The method of claim 97, further comprising a freeze-drying step.

99. The method of claim 97 or 98, wherein the concentration of the fibroin fragments in the solution is from about 3% (w / w) to about 50% (w / w).

100. The method of any one of claims 97 to 99, wherein the fibroin fragments comprise one or more of the molecular weight, polydispersity and / or modifications, substitutions and / or replacements at specific amino acid positions as defined in any one of claims 45 to 91.

101. The method of any one of claims 97 to 100, wherein the solution is stable as defined in any one of claims 31 to 41.

102. A method of reconstructing a solution of silk fibroin fragments, the method comprising dissolving a plurality of substantially solid silk fibroin particles as described in any one of claims 1 to 92 in a solvent, wherein the particles have a reconstitution yield greater than 90%.

103. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 92, wherein the particles have a reconstitution yield greater than 90% in deionized water.

104. The method of claim 102, or the plurality of substantially solid silk fibroin particles of claim 103, wherein at least 90% of the reconstitution rate is maintained after a stability test comprising simulated aging of the plurality of substantially solid silk fibroin particles of any one of claims 1 to 92, the aging comprising storage at a temperature of about 40°C to about 60°C for a time period ranging from about 400 days to about 650 days.

105. The method of claim 104, wherein the simulated aging ranges from about 4 years to about 15 years.

Images