HK40048921A - Composition for a molded body - Google Patents

Description

Composition for molded bodies

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application No. 62/717,622 filed on 8/10/2018, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates to a composition for a moulded body comprising a recombinant spider silk protein and a plasticizer. In addition, the present disclosure relates to a moulded body comprising a recombinant spider silk protein and a plasticizer and to a method for preparing said moulded body.

Background

As an alternative to petroleum-based products, biorenewable and biodegradable materials are receiving increasing attention. For this reason, considerable work has been done to develop methods for making materials and fibers from molecules derived from plants and animals. Fibers made from regenerated protein dates back to the 90's of the 19 th century and were made using various conventional wet spinning techniques.

Wet spinning uses a solvent and a coagulation bath to produce fibers. This is disadvantageous because the chemicals used as solvents and in the coagulation bath need to be extracted from the fiber after the spinning process and subjected to a closed loop process to provide a sustainable and reliable process. Although melt spinning offers an attractive option for wet spinning because it does not require a solvent and a coagulation bath, melt spinning also requires: (i) the polymer should produce a homogeneous melt composition that can be extruded to form commercial quality fibers, and (ii) the polymer should not degrade during the melting and extrusion steps.

Disclosure of Invention

According to some embodiments of the present invention, provided herein is a composition for a molded body, as well as a molded body comprising a recombinant spider silk protein and a plasticizer, wherein the composition may be substantially homogeneous after conversion to a molten or flowable state; and the recombinant spider silk protein is not substantially degraded or is degraded in an amount of less than 6.0 wt% after it is formed into a molded body.

Furthermore, the present disclosure provides a method for producing a molded body, comprising the steps of: applying pressure and/or shear force to a composition comprising recombinant spider silk protein and a plasticizer to form a substantially homogeneous melt composition; and molding the homogeneous melt composition to form the molded body. The substantially homogeneous melt composition should generally be in a flowable state and can be extruded, for example, to form fibers.

According to some embodiments, provided herein is a composition for a moulded body comprising a recombinant spider silk protein and a plasticizer, wherein the composition is capable of being induced into a flowable state, wherein the recombinant spider silk protein is not substantially degraded in the flowable state.

In some embodiments, the composition can be induced to a flowable state by the application of shear and pressure. In some embodiments, the composition can be induced to a flowable state by applying shear and pressure without the application of heat. In some embodiments, the composition is capable of being induced to a flowable state and squeezed multiple times, wherein the recombinant spider silk proteins within the composition remain substantially undegraded.

In some embodiments, the composition is thermoplastic.

In some embodiments, the composition can be induced to a flowable state by applying a shear force in the range of 1.5Nm to 13 Nm. In some embodiments, the composition can be induced to a flowable state by applying a shear force in the range of 2Nm to 6 Nm. In some embodiments, the composition can be induced to a flowable state by applying a pressure in the range of 1MPa to 300 MPa. In some embodiments, the composition can be induced to a flowable state by applying a pressure in the range of 5MPa to 75 MPa.

In some embodiments, the composition is capable of being induced to a flowable state at less than 120 ℃, less than 80 ℃, less than 40 ℃ or at room temperature. In some embodiments, the composition is substantially homogeneous.

In some embodiments, the recombinant spider silk protein comprises a repeat unit. In some embodiments, the recombinant spider silk protein comprises in the range of 2 to 20 repeating units having an amino acid length in the range of 60 to 100 amino acids. In some embodiments, the molecular weight of the recombinant spider silk protein ranges from 20 to 2000 kDa.

In some embodiments, the recombinant spider silk protein comprises a repeating unit that occurs at least twice, said repeating unit comprising: more than 150 amino acid residues and a molecular weight of at least 10 kDa; an alanine-rich region having 6 or more contiguous amino acids comprising an alanine content of at least 80%; and a glycine-rich region having 12 or more contiguous amino acids comprising a glycine content of at least 40% and an alanine content of less than 30%.

In some embodiments, the plasticizer is selected from a polyol, water, and/or urea. In some embodiments, the polyol comprises glycerol. In some embodiments, the plasticizer comprises water. In some embodiments, the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder, and wherein the weight ratio of plasticizer to recombinant silk polypeptide powder ranges from 0.05 to 1.50: 1. In some embodiments, the recombinant spider silk protein is present in the recombinant spider silk polypeptide powder, and the weight ratio of plasticizer to recombinant silk polypeptide powder ranges from 0.20 to 0.70: 1.

In some embodiments, the recombinant spider silk protein is present in the recombinant spider silk polypeptide powder, and the amount of recombinant spider silk polypeptide powder in the composition ranges from 1 to 90 wt% recombinant spider silk protein. In some embodiments, the recombinant spider silk protein is present in the recombinant spider silk polypeptide powder, and the amount of recombinant spider silk polypeptide powder in the composition ranges from 20 to 41 wt% recombinant spider silk protein. In some embodiments, the composition comprises glycerol as a plasticizer in the range of 1 to 60 weight percent. In some embodiments, the composition comprises in the range of 15 to 30 weight percent glycerin as a plasticizer. In some embodiments, the composition comprises water as a plasticizer in the range of 5 to 80 weight percent. In some embodiments, the composition comprises water as a plasticizer in the range of 19 to 27 weight percent.

In some embodiments, the recombinant spider silk protein degrades in a flowable state in an amount of less than 10.0 wt.%. In some embodiments, the recombinant spider silk protein degrades in a flowable state in an amount of less than 6.0 wt.%. In some embodiments, the recombinant spider silk protein degrades in a flowable state in an amount of less than 2.0 wt.%. In some embodiments, degradation of the recombinant spider silk protein is assessed by measuring the amount of full-length recombinant spider silk protein present in the composition before and after the flowable state is induced. In some embodiments, the amount of full length recombinant spider silk protein is measured using size exclusion chromatography.

According to some embodiments of the present invention, also provided herein is a moulded body comprising a composition for a moulded body, said composition comprising a recombinant spider silk protein and a plasticizer, wherein said composition is capable of being induced into a flowable state, wherein the recombinant spider silk protein is not substantially degraded in the flowable state.

In some embodiments, the molded body is a fiber. In some embodiments, the strength of the fiber is in the range of 100Pa to 1.2 GPa. In some embodiments, the fibers have a birefringence in the range of 5X 10-5 to about 0.04 as measured by polarized light microscopy.

Also provided herein, according to some embodiments of the present invention, is a method for preparing a molded body, comprising the steps of: applying pressure and shear force to a composition comprising recombinant spider silk protein and a plasticizer to convert the composition into a flowable state; and extruding the composition in the flowable state to form a molded body.

In some embodiments, extruding the composition to form a molded body comprises extruding the composition to form a fiber. In some embodiments, extruding the composition to form a fiber comprises extruding the composition through a spinneret. In some embodiments, extruding the composition to form a molded body comprises extruding the composition into a die.

In some embodiments, the method for preparing a molded body further comprises: (a) applying pressure and shear force to the molded body to convert the molded body into a composition in a flowable state, and (b) extruding the composition in the flowable state to form a second molded body. In some embodiments, the method further comprises repeating steps (a) and (b) of the second molded body at least once.

In some embodiments, the shear force is 1.5 to 13N × m. In some embodiments, the pressure is from 1MPa to 300 MPa. In some embodiments, the shear and pressure are applied to the composition using a capillary rheometer or a twin screw extruder. In some embodiments, the screw speed of the twin screw extruder ranges from 10 to 300RPM during the application of the pressure and shear forces.

In some embodiments, the apparatus for applying shear and pressure comprises a mixing chamber coupled to and proximal to the extrusion chamber. In some embodiments, the composition is heated in a mixing chamber. In some embodiments, the composition is heated in an extrusion chamber. In some embodiments, the composition is heated to a temperature of less than 120 ℃. In some embodiments, the composition is heated to a temperature of less than 80 ℃. In some embodiments, the composition is heated to a temperature of less than 40 ℃. In some embodiments, the extrusion chamber tapers proximal to the orifice through which the composition is extruded. In some embodiments, the extrusion chamber is temperature controlled. In some embodiments, the residence time of the composition in the mixing chamber ranges from 3 to 7 minutes.

In some embodiments, the water content of the molded body after extrusion is lost less than 15% compared to the composition prior to extrusion. In some embodiments, the water content of the molded body after extrusion is lost less than 10% compared to the composition prior to extrusion.

In some embodiments, the molded body is a fiber, and the fiber is manually drawn. In some embodiments, the molded body is a fiber, and the fiber is drawn through multiple steps.

In some embodiments, the recombinant spider silk protein is not substantially degraded in the moulded body. In some embodiments, the recombinant spider silk protein is degraded in the moulded body in an amount of less than 10 wt%. In some embodiments, the recombinant spider silk protein is degraded in the moulded body in an amount of less than 6 wt%. In some embodiments, the recombinant spider silk protein is degraded in the moulded body in an amount of less than 2 wt.%. In some embodiments, degradation of the recombinant spider silk protein is assessed by measuring the amount of full-length recombinant spider silk protein present in the composition before and after extrusion. In some embodiments, the amount of full length recombinant spider silk protein is measured using size exclusion chromatography.

In some embodiments, the molded body has a minimum birefringence as measured by polarized light microscopy.

Drawings

The foregoing and other objects, features, and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings.

Figure 1 shows size exclusion chromatography data for a P49W21G30 melt composition extruded under selected thermal and RPM conditions according to various embodiments of the present invention.

Figure 2 shows size exclusion chromatography data for a P65W20G15 melt composition extruded under selected thermal and RPM conditions according to various embodiments of the present invention.

Fig. 3 shows size exclusion chromatography data for a P71W19G10 melt composition extruded under selected thermal and RPM conditions according to various embodiments of the invention.

Fig. 4 shows the water loss scale during extrusion for a P49W21G30 melt composition extruded under selected thermal and RPM conditions according to various embodiments of the present invention, as measured by thermogravimetric analysis (TGA). The data show the% moisture in the starting pellets before extrusion and samples extruded under selected conditions after extrusion.

Fig. 5 shows the water loss scale during extrusion for a P65W20G15 melt composition extruded under selected thermal and RPM conditions according to various embodiments of the present invention, as measured by thermogravimetric analysis (TGA). The data show the% moisture in the starting pellets before extrusion and samples extruded under selected conditions after extrusion.

Fig. 6 shows the water loss scale during extrusion for a P71W19G10 melt composition extruded under selected thermal and RPM conditions according to various embodiments of the present invention, as measured by thermogravimetric analysis (TGA). The data show the% moisture in the starting powder before extrusion and in the samples extruded under selected conditions after extrusion.

Fig. 7 shows the beta sheet content of P49W21G30 samples extruded under selected thermal and RPM conditions, as measured by Fourier Transform Infrared Spectroscopy (FTIR). The samples were compared to reference controls of starting protein powder and starting pellets.

Fig. 8 shows the beta sheet content of P65W20G15 samples extruded under selected thermal and RPM conditions, as measured by fourier transform infrared spectroscopy (FTIR). The samples were compared to reference controls of starting protein powder and starting pellets.

Fig. 9 shows the beta sheet content of P71W19G10 samples extruded under selected thermal and RPM conditions, as measured by fourier transform infrared spectroscopy (FTIR). The samples were compared to reference controls of starting protein powder and starting pellets.

FIG. 10 shows an image of a selected extruded product produced at 10, 100, 200 or 300RPM at 20 ℃ using polarized light microscopy.

FIG. 11 shows images of selected extruded products produced at 95 ℃ at 10, 100, 200, or 300RPM, captured using polarized light microscopy.

Fig. 12 shows the glycerol loss scale during extrusion for P49W21G30 extrudates extruded under selected heat and RPM conditions according to various embodiments of the present invention, as measured by HPLC. The data show% glycerol loss in the starting powder or pellets prior to extrusion and in the samples after extrusion under selected conditions.

Fig. 13 shows the glycerol loss scale during extrusion for P65W20G15 extrudates extruded under selected heat and RPM conditions according to various embodiments of the present invention, as measured by HPLC. The data show% glycerol loss in the starting powder or pellets prior to extrusion and in the samples after extrusion under selected conditions.

Fig. 14 shows the glycerol loss scale during extrusion for P71W19G10 extrudates extruded under selected heat and RPM conditions according to various embodiments of the present invention, as measured by HPLC. The data show% glycerol loss in the starting powder or pellets prior to extrusion and in the samples after extrusion under selected conditions.

Detailed Description

The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. The terms "a" and "an" include plural references unless the context dictates otherwise. Generally, the nomenclature used in connection with and the techniques below are those well known and commonly used in the art: biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein.

Definition of

Unless otherwise indicated, the following terms are to be understood to have the following meanings:

the term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides that are at least 10 bases in length. The term includes DNA molecules (e.g., cDNA, or genomic DNA, or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of the DNA or RNA that contain non-natural nucleotide analogs, non-natural internucleoside linkages, or both. The nucleic acid may be in any topological conformation. For example, a nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplex, partially double-stranded, branched, hairpin, circular, or in a padlock (padlocked) conformation.

Unless otherwise indicated, and as an example of all sequences described herein in the general format "SEQ ID No.: the" nucleic acid comprising SEQ ID No. 1 "refers to a nucleic acid at least a portion of which has the following sequence: (i) the sequence SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO: 1. The choice between the two is determined by the context. For example, if a nucleic acid is used as a probe, the choice between the two is determined by the requirement that the probe be complementary to the desired target.

An "isolated" RNA, DNA, or mixed polymer is one that is substantially separated from other cellular components that naturally accompany a native polynucleotide in its native host cell, such as ribosomes, polymerases, and genomic sequences with which it is naturally associated.

An "isolated" organic molecule (e.g., silk protein) is one that is substantially separated from the cellular components (membrane lipids, chromosomes, proteins) of the host cell from which it is derived or the medium in which the host cell is cultured. The term does not require that the biomolecule be separated from all other chemicals, but certain isolated biomolecules may be purified to near homogeneity.

The term "recombinant" refers to a biological molecule (e.g., a gene or protein) that: (1) has been removed from its naturally occurring environment, (2) is not associated with all or part of a polynucleotide to which the gene is found in nature, (3) is operably linked to a polynucleotide to which it is not linked in nature, or (4) does not occur in nature. The term "recombinant" may be used with respect to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biosynthesized from heterologous systems, as well as proteins and/or mrnas encoded by such nucleic acids.

In this context, an endogenous nucleic acid sequence (or the encoded protein product of that sequence) in the genome of an organism is considered "recombinant" if the heterologous sequence is placed adjacent to the endogenous nucleic acid sequence such that expression of the endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally contiguous with an endogenous nucleic acid sequence, whether the heterologous sequence itself is endogenous (derived from the same host cell or progeny thereof) or exogenous (derived from a different host cell or progeny thereof). For example, a promoter sequence can replace (e.g., by homologous recombination) a native promoter of a gene in the genome of a host cell such that the gene has an altered expression pattern. The gene will now become "recombinant" in that it is separated from at least some of the sequences that naturally flank it.

A nucleic acid is also considered "recombinant" if it contains any modifications that do not naturally occur in the corresponding nucleic acid in the genome. For example, an endogenous coding sequence is considered "recombinant" if it contains an insertion, deletion, or point mutation that is artificially introduced (e.g., by human intervention). "recombinant nucleic acid" also includes nucleic acids that integrate into the host cell chromosome at a heterologous site and nucleic acid constructs that exist as episomes.

The term "peptide" as used herein refers to short polypeptides, e.g., short polypeptides that are typically less than about 50 amino acids in length and more typically less than about 30 amino acids in length. The term as used herein includes analogs and mimetics that mimic structure and thus biological function.

The term "polypeptide" encompasses both naturally occurring and non-naturally occurring proteins, as well as fragments, mutants, derivatives, and analogs thereof. The polypeptide may be monomeric or polymeric. In addition, a polypeptide may comprise a plurality of different domains, each domain having one or more different activities.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, due to its origin or derivative origin: (1) non-associated with the naturally associated component with which it is associated in its natural state, (2) present in a purity not found in nature, wherein purity can be judged with respect to the presence of other cellular material (e.g., free of other proteins from the same species), (3) expressed by cells from a different species, or (4) absent in nature (e.g., which is a fragment of a polypeptide found in nature, or which includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system different from the cell from which it is naturally derived will be "separated" from its naturally associated components. The polypeptide or protein may also be made substantially free of naturally associated components using protein purification techniques well known in the art. As defined herein, "isolated" does not necessarily require that the protein, polypeptide, peptide, or oligopeptide as described herein has been physically removed from its natural environment.

The term "polypeptide fragment" refers to a polypeptide having a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion, as compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding position in the naturally occurring sequence. Fragments are typically at least 5, 6, 7, 8, 9 or 10 amino acids in length, preferably at least 12, 14, 16 or 18 amino acids, more preferably at least 20 amino acids, more preferably at least 25, 30, 35, 40 or 45 amino acids, even more preferably at least 50 or 60 amino acids, and even more preferably at least 70 amino acids.

A protein is "homologous" to or "homologous" to a second protein if the nucleic acid sequence encoding the protein has a similar sequence to the nucleic acid sequence encoding the second protein. Alternatively, if a protein has a "similar" amino acid sequence to a second protein, then the two proteins share homology. (thus, the term "homologous protein" is defined to mean that two proteins have similar amino acid sequences.) as used herein, homology between two regions of an amino acid sequence (particularly with respect to predicted structural similarity) is interpreted to imply functional similarity.

When "homologous" is used in relation to a protein or peptide, it will be appreciated that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which an amino acid residue is substituted with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. The manner in which this adjustment is made is well known to those of ordinary skill in the art. See, e.g., Pearson,1994, Methods mol. biol.24:307-31 and 25:365-89 (incorporated herein by reference).

The twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (compiled by Golub and Gren, Sinauer Associates, Sunderland, Mass., 2 nd edition, 1991), which is incorporated herein by reference. Stereoisomers of twenty conventional amino acids, unnatural amino acids (e.g., alpha-disubstituted amino acids, N-alkyl amino acids) and other non-conventional amino acids (e.g., D-amino acids) may also be suitable components of the polypeptides of the invention. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamate, epsilon-N, N, N-trimethyllysine, epsilon-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine and other similar amino and imino acids (e.g., 4-hydroxyproline). In the polypeptide symbols used herein, the left-hand end corresponds to the amino-terminus and the right-hand end corresponds to the carboxy-terminus, according to standard usage and convention.

The following six groups each contain amino acids that are conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), alanine (a), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

Sequence homology, also sometimes referred to as percent sequence identity, of polypeptides is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center,910University Avenue, Madison, Wis.53705. Protein analysis software uses a measure of homology assigned to various substitutions, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG contains programs such as "Gap" and "Bestfit" that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides (e.g., homologous polypeptides from different species of organism) or between a wild-type protein and its mutein. See, e.g., GCG version 6.1.

One useful algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al, J.Mol.biol.215: 403-.

Preferred parameters for BLASTp are: desired values: 10 (default); a filter: seg (default); vacancy opening Cost (Cost to open a gap): 11 (default); vacancy extension Cost (Cost to open a gap): 1 (default); highest alignment: 100 (default); word length: 11 (default); description numbers (No. of descriptions): 100 (default); penalty matrix: BLOWSUM 62.

Preferred parameters for BLASTp are: desired values: 10 (default); a filter: seg (default); void opening cost: 11 (default); void extension cost: 1 (default); highest alignment: 100 (default); word length: 11 (default); description number: 100 (default); penalty matrix: BLOWSUM 62. The length of polypeptide sequences to which homology comparisons are made will typically be at least about 16 amino acid residues, typically at least about 20 residues, more typically at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching databases containing sequences from a large number of different organisms, it is preferred to compare amino acid sequences. Database searches using amino acid sequences can be measured by algorithms known in the art other than blastp. For example, polypeptide sequences can be compared using FASTA (the program in GCG version 6.1). FASTA provides alignments and percent sequence identity of the regions of optimal overlap between the query sequence and the search sequence. Pearson, Methods Enzymol.183:63-98(1990) (incorporated herein by reference). For example, percent sequence identity between amino acid sequences can be determined using FASTA as provided in GCG version 6.1 (incorporated herein by reference) with its default parameters (word length 2, PAM250 scoring matrix).

Throughout the specification and claims, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

As defined herein, the term "molded body" refers to a body made by using a shaped liquid or pliable raw material called a rigid frame of a mold, such as a molding process (including but not limited to extrusion molding, injection molding, compression molding, blow molding, lamination, matrix molding, rotational molding, spin casting, transfer molding, thermoforming, and the like).

As defined herein, the term "fiber" refers to an elongated molded body, typically the fiber should have the form of a filament.

As used herein, the term "melt spinning" refers to a process of forming fibers from a polymer, wherein the polymer is converted to a meltable or flowable state and then solidified by cooling after extrusion from a spinneret.

As used herein, the term "drawing" with respect to a fiber refers to applying a force to elongate the spun fiber along its longitudinal axis during or after extrusion of the fiber. The term "undrawn fiber" refers to a fiber that has been extruded but has not been subjected to any drawing. The term "draw ratio" is a term of art generally defined as the ratio between the take-up rate and the feed rate. At constant volume, it can be measured from the initial diameter (D) of the fiber_i) And finallyDiameter (D)_f) Ratio of (i.e., D)_i/D_f) To be determined.

As used herein, the term "glass transition" refers to the transformation of a substance or composition from a hard, rigid, or "glassy" state to a more flexible, "rubbery," or "sticky" state.

As used herein, the term "glass transition temperature" refers to the temperature at which a substance or composition undergoes a glass transition.

As used herein, the term "melt transition" refers to the transformation of a substance or composition from a rubbery state to a less ordered liquid phase or flowable state.

As used herein, the term "melting temperature" refers to the temperature range in which a substance undergoes a melt transition.

As used herein, the term "plasticizer" refers to any molecule that interacts with a polypeptide sequence to prevent the polypeptide sequence from forming tertiary structures and bonds and/or to increase the mobility of the polypeptide sequence.

As used herein, the term "flowable state" refers to a composition having substantially the same properties as a liquid (i.e., transitioning from a rubbery state to a more liquid state).

Although exemplary methods and materials are described below, methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention, and will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

SUMMARY

Provided herein is a composition for a moulded body comprising a recombinant spider silk protein and a plasticizer, wherein the composition is homogeneous or substantially homogeneous in the molten or flowable state; and the recombinant spider silk protein is not substantially degraded (e.g., degraded in an amount of less than 10% by weight or often less than 6% by weight) after it is formed into a molded body.

Recombinant silk proteins

The present disclosure describes embodiments of the invention, including fibers synthesized from synthetic protein copolymers (i.e., recombinant polypeptides). Suitable protein copolymers are discussed in U.S. patent publication No. 2016/0222174, published on 45/2016, 2018/0111970, published on 26/4/2018, and 2018/0057548, published on 3/1/2018, each of which is incorporated herein by reference in its entirety.

In some embodiments, the synthetic protein copolymer is made from a filamentous polypeptide sequence. In some embodiments, the filamentous polypeptide sequence is 1) a block copolymer polypeptide composition produced by mixing and matching repeating domains derived from the filamentous polypeptide sequence, and/or 2) recombinant expression of a block copolymer polypeptide having a sufficiently large size (about 40kDa) to form useful molded bodies by secretion from an industrial scalable microorganism. Large (about 40kDa to about 100kDa) block copolymer polypeptides (including sequences from almost all of the disclosed amino acid sequences of spider silk polypeptides) engineered by silk repeat domain fragments can be expressed in the modified microorganisms described herein. In some embodiments, the silk polypeptide sequences are matched and designed to produce highly expressed and secreted polypeptides capable of forming a molded body.

In some embodiments, the block copolymer is engineered from a combinatorial mixture of silk polypeptide domains spanning the silk polypeptide sequence space. In some embodiments, the block copolymer is made by expression and secretion in an expandable organism (e.g., yeast, fungi, and gram positive bacteria). In some embodiments, the block copolymer polypeptide comprises 0 or more N-terminal domains (NTD), 1 or more repeat domains (REP), and 0 or more C-terminal domains (CTD). In some aspects of embodiments, the block copolymer polypeptide is >100 amino acids of a single polypeptide chain. In some embodiments, the block copolymer polypeptide comprises a domain that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a block copolymer polypeptide sequence disclosed in international publication No. WO/2015/042164, "Methods and Compositions for Synthesizing Improved Silk Fibers," which is incorporated by reference in its entirety.

Several types of natural spider silk have been identified. It is believed that the mechanical properties of each natural spinning type are closely related to the molecular composition of the filaments. See, e.g., Garb, j.e., et al, angling spray site with spray terminal domains, BMC evol. biol.,10:243 (2010); bittencourt, d. et al, Protein families, natural history and biotechnology industries of spreader silk, genet. mol.res.,11:3 (2012); rising, A. et al, Spider batch proteins, recovery enhancements in recovery products, structure-function relationships and biological applications, cell. mol. Life Sci.,68:2, pp.169 and 184 (2011); and Humenik, M. et al, spreader talk: understating the structure-function relationship of a natural fiber, prog.mol.biol.Transl.Sci.103, pages 131-85 (2011). For example:

the wires of the botryoid glands (AcSp) tend to have a high tenacity, which is the result of a combination of a suitably high strength and a suitably high ductility. The AcSp filaments are characterized by a large block ("overall repeat") size, which often incorporates motifs for polyserines and GPX. Tubular gland (TuSp or cylindrical) filaments tend to have large diameters, with moderate strength and high ductility. TuSp filaments are characterized by their polyserine and polyserine content, as well as short strands of polyalanine. Major ampullate gland (MaSp) filaments tend to have high strength and moderate ductility. MaSp filaments can be one of two subtypes: MaSp1 and MaSp 2. The MaSp1 filaments are generally less ductile than the MaSp2 filaments and are characterized by polypropionic, GX and GGX motifs. The MaSp2 filament is characterized by polypropionic, GGX and GPX motifs. Small ampullate gland (MiSp) filaments tend to have moderate strength and moderate ductility. The MiSp filaments are characterized by GGX, GA, and poly A motifs, and often contain a spacer element of about 100 amino acids. Flagellar (Flag) filaments tend to have very high ductility and moderate strength. Flag filaments are generally characterized by GPG, GGX and short spacer motifs.

The properties of each silk type may vary from species to species and different lifestyles (e.g., sedentary web shooter versus free hunter spider) or evolutionarily older spiders may produce silk with properties different from those described above (for descriptions of spider diversity and classification, see Hormiga, g. and Griswold, c.e., Systematics, phylogeny, and evolution of orb-weighted spiders, annu. rev. entol.59, 487-512(2014), and black, t.a. et al, constracting web evolution and spider conversion in molecular plant, proc. ad. sci. u.s.a. 106, 5213.31: 5234, 5234). However, synthetic block copolymer polypeptides having sequence similarity and/or amino acid composition similarity to the repeating domains of native silk proteins can be used to produce consistent mouldings on a commercial scale with properties that replicate the corresponding mouldings made from native silk polypeptides.

In some embodiments, a list of putative silk sequences can be compiled by searching GenBank for related terms, such as "spidroin", "fibroin", "MaSp", and those sequences can be pooled with additional sequences obtained by independent sequencing efforts. The sequence is then translated into amino acids, the repeated entries are filtered and manually resolved into domains (NTD, REP, CTD). In some embodiments, the candidate amino acid sequence is reverse translated into a DNA sequence optimized for expression in pichia (Komagataella) yeast. The DNA sequences were each cloned into an expression vector and transformed into Pichia pastoris (Komagataella). In some embodiments, the various silk domains that show successful expression and secretion are subsequently assembled in a combinatorial fashion to construct a silk molecule capable of forming a molded body.

A silk polypeptide characteristically consists of a repeat domain (REP) flanked by non-repeat regions (e.g., a C-terminal domain and an N-terminal domain). In one embodiment, the C-terminal domain and the N-terminal domain are between 75 and 350 amino acids in length. The repeat domains exhibit a hierarchical architecture, as shown in FIG. 1. The repeating domain comprises a series of blocks (also referred to as repeating units). The blocks are repeating, sometimes perfectly repeating, sometimes imperfectly repeating (constituting a quasi-repeating domain) throughout the silk repeating domain. The length and composition of the blocks vary between different filament types and among different species. Table 1A lists examples of block sequences from selected species and silk types, with other examples given in the following references: rissing, A. et al, spacer-size proteins, recovery enhancements in a recovery process, structure-function relationships and biological applications, Cell mol. Life Science, 68:2, pp.169-184 (2011), and gateway, J. et al, expression sensitivity, registration, and registration of spacer-size fiber sequences, Science,291:5513, pp.2603-2605 (2001). In some cases, the blocks may be arranged in a regular pattern, forming large macroscopic repeats (macro-repeats) that occur multiple times (typically 2 to 8 times) in the repeating structural domain of the silk sequence. Repeating blocks within repeating domains or macro-repeating bodies, and repeating macro-repeating bodies within repeating domains, may be separated by spacer elements. In some embodiments, the block sequence comprises a glycine-rich region followed by a polyA region. In some embodiments, a short (about 1 to 10) amino acid motif occurs multiple times within a block. For the purposes of the present invention, blocks from different native silk polypeptides may be selected without reference to the circular arrangement (i.e., otherwise similar identifying blocks between silk polypeptides may not be aligned due to the circular arrangement). Thus, for example, for the purposes of the present invention, a "block" SGAGG (SEQ ID NO:494) is identical to a GSGAG (SEQ ID NO:495) and to a GGSGA (SEQ ID NO: 496); all of which are arranged in a ring with each other. The particular arrangement selected for a given silk sequence may be determined by, among other things, convenience (usually starting with G). Silk sequences obtained from NCBI databases can be divided into block and non-repeat regions.

Table 1A: samples of Block sequences

According to certain embodiments of the present invention, fiber-forming block copolymer polypeptides from block and/or macro-repeat domains are described in international publication No. WO/2015/042164 (incorporated by reference). Native silk sequences obtained from protein databases (e.g., GenBank) or by de novo sequencing were resolved according to domain (N-terminal domain, repeat domain, and C-terminal domain). The N-terminal domain and C-terminal domain sequences selected for the purpose of synthesis and assembly into fibers or molded bodies include the natural amino acid sequence information and other modifications described herein. The repeat domain is broken down into a repeat sequence containing representative blocks, typically 1 to 8, depending on the type of silk, which capture critical amino acid information while reducing the size of the DNA encoding the amino acids to a readily synthesized fragment. In some embodiments, a suitably formed block copolymer polypeptide comprises at least one repeat domain comprising at least 1 repeat sequence, and optionally flanked by an N-terminal domain and/or a C-terminal domain.

In some embodiments, the repeat domain comprises at least one repeat sequence. In some embodiments, the repeat sequence is 150 to 300 amino acid residues. In some embodiments, the repeating sequence comprises a plurality of blocks. In some embodiments, the repeat sequence comprises a plurality of macroscopic repeats. In some embodiments, the block or macrorepeat is segmented into a plurality of repeating sequences.

In some embodiments, the repeat sequence begins with glycine and cannot end with phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine (C), histidine (H), asparagine (N), methionine (M), or aspartic acid (D) to meet DNA assembly requirements. In some embodiments, some of the repeated sequences may be altered from the native sequence. In some embodiments, the repeat sequence may be altered, for example, by adding a serine to the C-terminus of the polypeptide (to avoid terminating at F, Y, W, C, H, N, M or D). In some embodiments, the repeat sequence may be modified by filling in the incomplete block with homologous sequences from another block. In some embodiments, the repeat sequence may be modified by rearranging the order of the blocks or macroscopic repeats.

In some embodiments, non-repetitive N-terminal and C-terminal domains can be selected for synthesis. In some embodiments, the N-terminal domain may be obtained by removal of, for example, a leader signal sequence as identified by SignalP (Peterson, T.N. et al, SignalP 4.0: discrete signal peptides from transmembrane regions, nat. methods,8:10, pages 785 and 786 (2011).

In some embodiments, the N-terminal domain, repeat sequence, or C-terminal domain sequence may be from an infundibular spider (agilenopsis aperta), alitypus gulosus, costaphylocentrotus magnus (aphanopelma seemani), brachylodon brevis AS217, brachylodon brevis AS220, aranthus spicata (Araneus diadematus), catnip, aragonia ventricosa (Araneus ventricosus), euglenopsis grandiflora (aranus ventricosus), euglenopsis clavata (argonaea amoena), argentum argentea (argiophylla argentata), rhabdorachus striatus (argiophenope bruuennis), trichoderma triphyllum, atheoides, brazianum maculatus, arachnidus maculatus, garphus maculatus, garnetus digera (Avicularia), gynura lutea, gynura pacifica (gynura), gynura pacifica, gynura pacifica, gynura, nephila filipes, Nephilengys cruentata, Palawegian bistriata (Parawixia bistriata), Green lynx spider (Peucetia virridans), original carnivorous spider, Indian Hualilin spider (Poecilothia regalis), Long paw Green shepherd moth spider or holomorphic spider.

In some embodiments, the silk polypeptide nucleotide coding sequence may be operably linked to an alpha mating factor nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence may be operably linked to another endogenous or heterologous secretion signal coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operably linked to a 3X FLAG nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence is operably linked to other affinity tags, such as 6 to 8 His residues.

In some embodiments, the recombinant spider silk polypeptide is based on a recombinant spider silk protein fragment sequence derived from, for example, MaSp2 from the species chrysoideus venomorphus. In some embodiments, the synthetic fiber contains a protein molecule comprising two to twenty repeat units, wherein the molecular weight of each repeat unit is greater than about 20 kDa. Within each repeat unit of the copolymer there are more than about 60 amino acid residues organized into a number of "quasi-repeat units", typically ranging from 60 to 100 amino acids. In some embodiments, the repeat unit of a polypeptide described in the present disclosure has at least 95% sequence identity to a MaSp2 dragline silk protein sequence.

The repeating units forming the protein block copolymer having good mechanical properties can be synthesized using a portion of the silk polypeptide. These polypeptide repeat units contain alanine-rich and glycine-rich regions and are 150 amino acids or longer in length. Some exemplary sequences that can be used as repeat sequences in the protein block copolymers of the present disclosure are provided in commonly owned PCT publication WO 2015/042164, which is incorporated by reference in its entirety and demonstrated to be expressed using the pichia pastoris expression system.

In some embodiments, the spider silk protein comprises: a repeating unit that occurs at least twice, the repeating unit comprising: more than 150 amino acid residues and a molecular weight of at least 10 kDa; an alanine-rich region having 6 or more contiguous amino acids comprising an alanine content of at least 80%; a glycine-rich region having 12 or more contiguous amino acids comprising a glycine content of at least 40% and an alanine content of less than 30%; and wherein the fiber comprises at least one property selected from the group consisting of: an elastic modulus of greater than 550cN/tex, a ductility of at least 10% and an ultimate tensile strength of at least 15 cN/tex.

In some embodiments, wherein the recombinant spider silk protein comprises repeat units, wherein each repeat unit has at least 95% sequence identity to a sequence comprising 2 to 20 quasi-repeat units; each quasi-repeat unit comprises { GGY- [ GPG-X [)₁]_n1-GPS-(A)_n2-wherein for each quasi-repeat unit; x₁Independently selected from the group consisting of SGGQQ, GAGQQ, GQGOPY, AGQQ, and SQ; and n1 is 4 to 8 and n2 is 6 to 10. The repeat unit is comprised of a plurality of quasi-repeat units.

In some embodiments, 3 "long" quasi repeating units are followed by 3 "short" quasi repeating units. As mentioned above, short quasi-repeat units are those in which n1 ═ 4 or 5. Long quasi-repeat units are defined as those in which n1 is 6, 7 or 8. In some embodiments, all short quasi-repeats have the same X at the same position within each quasi-repeat unit of the repeat unit₁And (c) a motif. In some embodiments, no more than 3 of the 6 quasi repeat units have the same X₁And (c) a motif.

In additional embodiments, the repeat unit consists of quasi-repeat units that use the same X in rows within the repeat unit₁Not more than twice. In additional embodiments, the repeat unit consists of a quasi-repeat unit, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 quasi-repeat units use the same X in a single quasi-repeat unit of the repeat unit₁Not more than 2 times.

In some embodiments, the recombinant spider silk polypeptide comprises the polypeptide sequence SEQ ID NO:1 (i.e., 18B). In some embodiments, the repeat unit is a polypeptide comprising SEQ ID NO 2. These sequences are provided in table 1B:

TABLE 1B-recombinant proteins and exemplary polypeptide sequences of the repeat units

In some embodiments, the structure of the fiber formed by the recombinant spider silk polypeptide forms a beta-sheet structure, a beta-turn structure, or an alpha helix structure. In some embodiments, the secondary, tertiary, and quaternary protein structures of the formed fibers are described as having nanocrystalline β -sheet regions, amorphous β -turn regions, amorphous α -helical regions, randomly spatially distributed nanocrystalline regions embedded in an amorphous matrix, or randomly oriented nanocrystalline regions embedded in an amorphous matrix. Although not wishing to be bound by theory, the structural properties of proteins within spider silks are theoretically related to the fiber mechanical properties. Crystalline regions in the fiber have been correlated with the tensile strength of the fiber, while amorphous regions have been correlated with the extensibility of the fiber. Major ampullate gland (MA) filaments tend to have higher strength and lower elongation than flagellar filaments, and also MA filaments have higher crystalline region volume fractions than flagellar filaments. Furthermore, theoretical models based on the molecular dynamics of the crystalline and amorphous regions of spider silk proteins support the determination that the crystalline regions have been correlated with fiber tensile strength, while the amorphous regions have been correlated with fiber extensibility. In addition, theoretical modeling supports the importance of secondary, tertiary, and quaternary structures for the mechanical properties of the RPF. For example, the assembly of random, parallel, and serial spatial distributions and the strength of the interaction forces between the entanglement chains within the amorphous region and between the amorphous and nanocrystalline regions all affect the theoretical mechanical properties of the resulting fiber.

In some embodiments, the molecular weight of the silk protein may range from 20kDa to 2000kDa, or greater than 20kDa, or greater than 10kDa, or greater than 5kDa, or 5 to 400kDa, or 5 to 300kDa, or 5 to 200kDa, or 5 to 100kDa, or 5 to 50kDa, or 5 to 500kDa, or 5 to 1000kDa, or 5 to 2000kDa, or 10 to 400kDa, or 10 to 300kDa, or 10 to 200kDa, or 10 to 100kDa, or 10 to 50kDa, or 10 to 500kDa, or 10 to 1000kDa, or 10 to 2000kDa, or 20 to 400kDa, or 20 to 300kDa, or 20 to 200kDa, or 40 to 300kDa, or 40 to 500kDa, or 20 to 100, or 20 to 50, or 20 to 500kDa, or 20 to 1000kDa, or 20 to 2000 kDa.

Characterization of purity and degradation of recombinant spider silk polypeptide powder

Different recombinant spider silk polypeptides have different physiochemical properties such as melting temperature and glass transition temperature, based on the strength and stability of the secondary and tertiary structures formed by the protein. The silk polypeptide forms a beta sheet structure in monomeric form. In the presence of other monomers, the silk polypeptide forms a three-dimensional lattice of beta sheet structures. The beta sheet structure is separated from and interspersed with amorphous regions of the polypeptide sequence.

The beta sheet structure is extremely stable at high temperatures, with the melting temperature of the beta sheet being about 257 ℃ as measured by rapid scanning calorimetry. See, Cebe et al, coating the Heat-Fast Scanning means Silk Beta Sheet Crystals, Nature Scientific Reports3:1130 (2013). Since the beta sheet structure is believed to remain intact above the glass transition temperature of the silk polypeptide, the structural transformation seen at the glass transition temperature of recombinant silk polypeptides is assumed to be due to the increased mobility of the amorphous regions between the beta sheets.

Plasticizers lower the glass transition temperature and melting temperature of silk proteins by increasing the mobility of the amorphous regions and potentially disrupting beta sheet formation. Suitable plasticizers for this purpose include, but are not limited to, water and polyols (polyols) such as glycerol, triglycerol, hexaglycerol, and decaglycerol. Other suitable plasticizers include, but are not limited to: dimethyl isosorbide; bisamides of dimethylaminopropylamine and adipic acid; 2,2, 2-trifluoroethanol; amides of dimethylaminopropylamine and caprylic/capric acid; DEA acetamide; and any combination thereof. Other suitable Plasticizers are discussed in Ullsten et al, Chapter 5: Plasticizers for Protein Based Materials Viscoelectroplastics and Viscoslastic Materials (2016) (accessible by reference)https://www.intechopen.com/books/viscoelastic-and- viscoplastic-materials/plasticizers-for-protein-based-materialsObtained) and Vierra et al, Natural-based plastics and Polymer files: A review, European Polymer Journal 47(3):254-63(2011), which are incorporated herein by reference in their entirety.

Since the hydrophilic part of the silk polypeptide can bind to the ambient water present in the air as humidity, water will be present almost always and the bound ambient water can make the silk polypeptide plastic. In some embodiments, a suitable plasticizer may be glycerin, alone or in combination with water or other plasticizers. Other suitable plasticizers are discussed above.

In addition, in the case where the recombinant spider silk polypeptide is produced by fermentation and recovered therefrom as recombinant spider silk polypeptide powder, impurities may be present in the recombinant spider silk polypeptide powder that act as plasticizers or otherwise inhibit the formation of tertiary structures. For example, residual lipids and sugars can act as plasticizers and thus influence the glass transition temperature of proteins by interfering with the formation of tertiary structures.

Various well-established methods can be used to assess the purity and relative composition of the recombinant spider silk polypeptide powder or composition. Size exclusion chromatography separates each molecule based on the relative size of the molecule and can be used to analyze the relative amounts of full length polymers and monomer-formed recombinant spider silk polypeptides as well as the amounts of high, low and medium molecular weight impurities in the recombinant spider silk polypeptide powder. Similarly, flash high performance liquid chromatography can be used to measure various compounds present in solution, such as recombinant spider silk polypeptides in monomeric form. Ion exchange liquid chromatography can be used to assess the concentration of various trace molecules in solution, including impurities such as lipids and sugars. Other methods of chromatography and quantification of various molecules, such as mass spectrometry, are well established in the art.

According to embodiments, the recombinant spider silk polypeptide may have a purity calculated based on the amount by weight of the recombinant spider silk polypeptide in monomeric form relative to the other components of the recombinant spider silk polypeptide powder. In various instances, the purity can range from 50 to 90 wt% depending on the type of recombinant spider silk polypeptide and the technique used to recover, isolate and post-process the recombinant spider silk polypeptide powder.

Size exclusion chromatography and reverse phase high performance liquid chromatography can be used to measure full length recombinant spider silk polypeptides, making it a useful technique for determining whether a processing step degrades a recombinant spider silk polypeptide by comparing the amount of the full length spider silk polypeptide in the composition before and after processing. In various embodiments of the invention, the amount of full length recombinant spider silk polypeptide present in the composition before and after treatment may undergo minimal degradation. The amount of degradation may range from 0.001 wt% to 10 wt%, or from 0.01 wt% to 6 wt%, for example less than 10 wt%, or 8 wt%, or 6 wt%, or less than 5 wt%, less than 3 wt%, or less than 1 wt%.

Melt rheology, secondary and tertiary structure

Rheology is commonly used in fiber spinning to analyze the physicochemical properties of materials (such as polymers) spun into fibers. Different rheological properties can affect the ability to spin the material into fibers as well as the mechanical properties of the spun fibers. Rheology can also be used to indirectly study secondary and tertiary structures formed by recombinant spider silk polypeptides and/or plasticizers under different pressures, temperatures and conditions. According to embodiments, a shear rheometer and/or an extensional rheometer may be used to analyze different rheological properties by oscillatory rheology and extensional rheology.

In some embodiments, the glass transition and/or melt transition of the composition comprising the recombinant spider silk polypeptide powder and the plasticizer is characterized using a capillary rheometer. These compositions prior to conversion to a molten or flowable state are referred to herein as "recombinant spider silk compositions". Furthermore, when the recombinant spider silk compositions are in a molten or flowable state, these compositions are referred to herein as "recombinant spider silk melt compositions".

In some embodiments, the melt transition and/or glass transition of a recombinant spider silk composition can be characterized using a capillary rheometer by extruding the recombinant spider silk composition under different pressure ranges and "ramps" created by increasing the shear rate. According to embodiments and examples, the ramp may start at about 300m/s to 1500 m/s. According to embodiments, the pressure may vary from 1MPa to 125MPa, often from 6MPa to 50 MPa.

In some embodiments, the glass transition and/or melt transition temperature of the recombinant spider silk polypeptide and/or the fiber containing the recombinant spider silk polypeptide is determined using differential scanning calorimetry. In particular embodiments, the glass transition and/or melt transition temperatures are measured using modulated differential scanning calorimetry.

Depending on the embodiment and type of recombinant spider silk polypeptide, the glass transition and/or melt transition temperature may have various value ranges. However, the measured glass transition and/or melting transition temperature is much lower than the temperatures typically observed for recombinant spider silk polypeptides in solid form, which may indicate the presence of impurities or other plasticizers.

Additionally, Fourier Transform Infrared (FTIR) spectroscopy data may be combined with rheological data to provide direct characterization of tertiary structure in the reconstituted silk powder and/or compositions containing the reconstituted silk powder. FTIR can be used to quantify secondary structure in a silk polypeptide and/or a composition comprising a silk polypeptide, as discussed below in the section entitled "Fourier Transform Infrared (FTIR) spectroscopy".

According to said embodiment FTIR may be used to quantify beta sheet structures present in the recombinant spider silk polypeptide powder and/or the composition containing said recombinant spider silk polypeptide powder. Additionally, in some embodiments, FTIR may be used to quantify impurities such as sugars and lipids present in the recombinant spider silk polypeptide powder. However, the various chaotropic agents and solubilizers used in the different protein preprocessing methods can reduce the number of tertiary structures in the recombinant spider silk polypeptide powder or the composition containing the recombinant spider silk polypeptide powder. Thus, there may be no correspondence between the amount of beta sheet structure in the recombinant spider silk polypeptide powder before and after moulding or spinning the recombinant spider silk polypeptide powder into fibres. Similarly, there is little correspondence between the glass transition temperatures of the powders before and after they are molded or spun into fibers.

In some embodiments, rheological data characterizing a recombinant spider silk polypeptide can be combined with FTIR to analyze secondary and tertiary structures formed in the polypeptide. In particular embodiments, rheological data may be captured in conjunction with FTIR spectroscopy. An exemplary method of combining rheology and FTIR is described in Boulet-Audet et al, Silk protein aggregation kinetics reconstructed by Rheo-IR, Acta biomaterials 10: 776-784 (2014), which is incorporated herein by reference in its entirety.

Fourier Transform Infrared (FTIR) spectroscopy can be used to assess the tertiary structure of proteins present in the polypeptide powder and/or fibers. In particular, FTIR spectroscopy can be used to determine the amount of β -sheet present in fibers that are subjected to different spinning and post-processing conditions. Thus, FTIR spectroscopy can be used to determine the relative amount of beta sheet structure based on different techniques. Alternatively, the FTIR spectra may be compared to natural insect silk.

According to embodiments, FTIR spectra at different wavenumbers may be used to assess different tertiary structures present in the fiber. In various embodiments, the wavenumbers corresponding to the amide I and amide II bands can be used to assess various protein structures, such as turns, β sheets, α helices, and side chains. The wavenumbers corresponding to the structure are well known in the art.

In most embodiments, FTIR spectra at wavenumbers corresponding to β sheets will be used to assess the amount of β sheet structure in the polypeptide powder and/or fibers. In a specific embodiment, at 982-^-1(CH₂Swinging vibration (A)_n)、1695-1690cm^-1(amide I), 1620-1625cm^-1(amide I), 1440-substituted 1445cm^-1(asymmetric CH)₃Curved) and/or 1508cm^-1FTIR spectra under (amide II) were used to determine the amount of β sheets present. According to embodiments, different wavenumbers and ranges may be measured to determine the amount of beta sheet present. In some embodiments, the use is made at 982-^-1FTIR spectra below in order to eliminate interference from the corresponding peaks. Exemplary methods for obtaining spectra at the wavenumbers described are discussed in detail in Boudet-Audet et al, Identification and classification of solids using isolated spectroscopy, journalnal of Experimental Biology,218:3138-3149(2015), which is incorporated herein by reference in its entirety.

Similarly, various methods of characterizing impurities in recombinant silk powders can be combined with rheological and/or FTIR data to analyze the relationship between the presence of impurities and the formation of secondary and/or tertiary structures.

Recombinant spider silk melt compositions

The present invention is directed to making various recombinant spider silk compositions that are capable of being converted into a molten or flowable state (i.e., capable of being converted into a recombinant spider silk melt composition) according to the methods described herein. In various embodiments, the concentrations of recombinant spider silk polypeptide powder and plasticizer in the composition may be varied based on the properties of the recombinant spider silk polypeptide powder (e.g., the purity of the recombinant spider silk polypeptide powder), the type of plasticizer used, and the desired fiber properties. In some embodiments, the concentration may be adjusted based on rheological data, such as data from a capillary rheometer.

In some embodiments, the melt flow index is used to determine whether a recombinant spider silk melt composition is capable of being drawn into fibers. In particular, the melt flow index can be used to measure the 'melt strength' of the recombinant spider silk melt composition or the ability to draw the recombinant spider silk melt composition as it is extruded. In various embodiments, the concentration of recombinant spider silk polypeptide and plasticizer may be varied based on the desired melt strength.

In some embodiments, various agents may be added to the recombinant spider silk composition to alter the rheological properties of the recombinant spider silk composition, such as elongation viscosity, shear viscosity, and linear viscoelasticity. Suitable agents for altering elongational viscosity include polyethylene glycol (PEG), Tween (polysorbate), sodium lauryl sulfate, polyethylene, or any combination thereof. Other suitable agents are well known in the art.

In some embodiments, a second polymer may be added to produce a polymer blend or a two-component fiber with the recombinant spider silk composition. In these cases, it may be useful to include a second polymer whose melting temperature makes it suitable to melt in tandem with the recombinant spider silk composition itself without degrading the amorphous regions of the recombinant spider silk polypeptide. In various embodiments, the melting temperature (Tm) of the polymer suitable for blending with a recombinant spider silk polypeptide should be below 200 ℃, 180 ℃, 160 ℃, 140 ℃, 120 ℃ or 100 ℃. Often, the melting temperature of the recombinant spider silk polypeptide should be above 20 ℃ or 25 ℃ or 50 ℃. A non-limiting list of exemplary polymers and melting temperatures is included in the table below.

TABLE 1C-Polymer

According to an embodiment, suitable concentrations by weight of the recombinant spider silk polypeptide powder in the recombinant spider silk composition are in the range: 1 to 90 wt%, 3 to 80 wt%, 5 to 70 wt%, 10 to 60 wt%, 15 to 50 wt%, 18 to 45 wt%, or 20 to 41 wt%.

In case glycerol is used as plasticizer, suitable concentrations of glycerol by weight in the recombinant spider silk composition are in the range: 1 to 60 wt%, 10 to 50 wt%, 10 to 40 wt%, 15 to 40 wt%, 10 to 30 wt%, or 15 to 30 wt%.

In case water is used as plasticizer, suitable concentrations of water by weight in the recombinant spider silk composition are in the range: 5 to 80 wt%, 15 to 70 wt%, 20 to 60 wt%, 25 to 50 wt%, 19 to 43 wt%, or 19 to 27 wt%. When water is used in combination with another plasticizer, it may be present in a range of 5 to 50 weight percent, 15 to 43 weight percent, or 19 to 27 weight percent.

In some embodiments, the water may evaporate during the extruder and/or cooling process depending on the treatment and/or die size used. In some embodiments, the post-molding water loss amount can range from 1 to 50 weight percent, 3 to 40 weight percent, 5 to 30 weight percent, 7 to 20 weight percent, 8 to 18 weight percent, or 10 to 15 weight percent, based on the total water amount. Typically the loss will be less than 15%, in some cases less than 10%, for example 1 to 10% by weight. The evaporation may be deliberate or due to the applied treatment. The degree of evaporation can be readily controlled, for example, by selection of operating temperature, flow rate, and applied pressure, as will be understood in the art.

In some embodiments, suitable plasticizers may include polyols (e.g., glycerol), water, lactic acid, methyl hydroperoxide, ascorbic acid, 1, 4-dihydroxybenzene (1, 4-benzenediol) benzene-1, 4-diol, phosphoric acid, ethylene glycol, propylene glycol, triethanolamine, acid acetate, propane-1, 3-diol, or any combination thereof.

In various embodiments, the amount of plasticizer may vary depending on the purity and relative composition of the recombinant spider silk polypeptide powder. For example, higher purity powders may have fewer impurities, such as low molecular weight compounds that may act as plasticizers, and thus require the addition of a higher weight percentage of plasticizer.

In particular embodiments, the various ratios (weight ratios) of plasticizer (e.g., a combination of glycerol and water) to recombinant spider silk polypeptide powder may range from 0.5 or 0.75 to 350 wt% plasticizer: recombinant spider silk polypeptide powder, 1 or 5 to 300 wt% plasticizer: recombinant spider silk polypeptide powder, 10 to 300 wt% plasticizer: recombinant spider silk polypeptide powder, 30 to 250 wt% plasticizer: recombinant spider silk polypeptide powder, 50 to 220 wt% plasticizer: recombinant spider silk protein, 70 to 200 wt% plasticizer: recombinant spider silk polypeptide powder, or 90 to 180 wt% plasticizer: recombinant spider silk polypeptide powder. As used herein, reference to 0.5 to 350 wt% plasticizer to recombinant spider silk polypeptide powder corresponds to a ratio of 0.5:1 to 350: 1.

Without intending to be limited by theory, in various embodiments of the invention, inducing transformation of a recombinant spider silk composition into a flowable state (e.g., inducing a recombinant spider silk melt composition) may be used as a pre-processing step in any formulation where it is advantageous to include the recombinant spider silk polypeptide in monomeric form. More specifically, the inducing recombinant spider silk melt composition may be used in applications where it is desired to prevent aggregation of monomeric recombinant spider silk polypeptides into their crystalline polymeric form or where it is desired to control the conversion of recombinant spider silk polypeptides into their crystalline polymeric form at a later stage of processing. In a particular embodiment, the recombinant spider silk melt composition can be used to prevent aggregation of the recombinant spider silk polypeptide prior to blending the recombinant spider silk polypeptide with the second polymer. In another specific embodiment, the recombinant spider silk melt composition can be used to produce a base for a cosmetic or skin care product in which the recombinant spider silk polypeptide is present in monomeric form in the base. In this embodiment, having the recombinant spider silk polypeptide in monomeric form in the base allows for controlled aggregation of the monomers into their crystalline polymeric form upon contact with the skin or by various other chemical reactions.

Inducing melt or flowable state

According to some embodiments of the invention, the recombinant spider silk composition is converted into a molten or flowable state by the application of shear and/or pressure (typically both). Suitable devices for generating a combination of shear and pressure include, but are not limited to: single screw extruders, twin screw extruders, melt flow extruders and capillary rheometers.

In some embodiments, a twin screw extruder is used to provide the necessary pressure and shear to convert the recombinant spider silk composition into a molten or flowable composition. In some embodiments, the twin screw extruder is configured to provide the following ranges of shear force: 1.5 to 13, 2 to 10, 2 to 8, or 2 to 6 newton meters (Nm). In some embodiments, the shear provided by the twin screw extruder is dependent on the rpm of the twin screw extruder. In various embodiments and configurations, the twin screw extruder may have a range of Revolutions Per Minute (RPM) from 10RPM to 300 RPM. In various embodiments, the twin screw extruder is configured to provide a pressure in the range of 1MPa to 300MPa and a shear force.

In an optional embodiment, the twin screw extruder is configured to apply heat to the recombinant spider silk composition before and/or after converting the recombinant spider silk composition into a recombinant spider silk melt composition. In some embodiments, the barrel of the twin screw extruder (i.e., the barrel in which the twin screws mix the composition) is subjected to heat. In other embodiments, the portion of the twin screw extruder adjacent to the spinneret (i.e., the spinneret orifice through which the reconstituted spider silk melt composition is extruded) is subjected to heating. Alternatively, without the application of heat, the melt/flowable state is entirely induced through the heat generated in the twin screw extruder by the shear forces applied to the recombinant spider silk composition. For example, in some embodiments, the amount of heat applied to achieve a melt/flowable state will be similar to equal to ambient room temperature (e.g., about greater than 20 ℃).

In various embodiments, the temperature at which the recombinant spider silk melt composition is heated will be minimized in order to minimize or completely prevent degradation of the recombinant spider silk polypeptide. In specific embodiments, the recombinant spider silk melt is heated to a temperature of less than 120 ℃, less than 100 ℃, less than 80 ℃, less than 60 ℃, less than 40 ℃ or less than 20 ℃. The temperature of the melt during processing is often in the range of 10 ℃ to 120 ℃,10 ℃ to 100 ℃, 15 ℃ to 80 ℃, 15 ℃ to 60 ℃, 18 ℃ to 40 ℃ or 20 ± 2 ℃.

In other embodiments, other devices may be used to provide the pressure and shear necessary to convert the recombinant spider silk composition into a molten or flowable state. As discussed above, capillary rheometers may also be used to provide the shear and pressure necessary to convert the recombinant spider silk composition into a flowable or molten state.

In some embodiments, the recombinant spider silk composition is optionally heated after the recombinant spider silk composition is in a molten or flowable state and/or prior to extrusion of the molten or flowable recombinant spider silk melt composition. When heating is required, possibly because the recombinant spider silk composition has a high glass transition temperature, the means for providing shear and pressure to convert the recombinant spider silk composition into a molten or flowable state may be coupled directly or indirectly to a heated extrusion device. In particular embodiments, the twin screw extruder is coupled (directly or indirectly) to a heated extrusion device. Depending on the embodiment and configuration of the heated extrusion apparatus, the heated extrusion apparatus may be maintained at a temperature in the following range: 20 to 120 ℃, 80 to 110 ℃, 85 to 100 ℃, 85 to 95 ℃ and/or 90 to 95 ℃.

The extruded recombinant spider silk melt composition is referred to herein as a "recombinant spider silk extrudate". The diameter of the spinneret from which the extrudate is extruded may vary depending on the application of the recombinant spider silk extrudate. For example, in embodiments where the reconstituted spider silk extrudate is extruded into a die to form a molded article, the diameter of the spinneret may be greater than 200mm, greater than 150mm, greater than 100mm, greater than 50mm, for example in the range 100mm to 500mm, 150mm to 400mm, or 200mm to 300 mm. As discussed below, in some embodiments, the recombinant spider silk extrudate can be processed into pellets, which can be reprocessed by subjecting the pellets again to shear and pressure sufficient to convert the spider silk extrudate into a recombinant spider silk melt composition. In embodiments where the recombinant spider silk extrudate is processed into pellets, the diameter of the spinneret may be greater than 2mm, greater than 1.5mm or greater than 1mm, for example the diameter may range from 1mm to 5mm, 1.5mm to 4mm or 2mm to 3 mm.

In embodiments where the reconstituted spider silk extrudate is made into fibers, the spinneret may have spinneret holes of less than 500 μm (e.g., in the range of 10 μm to 500 μm). The recombinant spider silk protein melt composition can be extruded through spinnerets having different orifice sizes, depending on the desired initial denier of the extruded fiber. In particular embodiments, the range of the spinneret holes can be 25 μm to 500 μm, 50 μm to 250 μm, or 75 μm to 125 μm. In some embodiments, the desired orifice size should be based on the final draw ratio of the fiber. For example, extruded fibers of higher initial denier may experience higher draw ratios.

In most embodiments of the invention, the recombinant spider silk melt composition and the recombinant spider silk extrudate will be substantially homogeneous, meaning that the material does not have any inclusions or precipitates as examined by light microscopy. In some embodiments, birefringence can be measured using an optical microscope, which can be used as an alternative to aligning recombinant spider silks into a three-dimensional lattice. Birefringence is the refractive index that depends on the polarization of light and the optical properties of the propagating material. In particular, a high degree of axial order as measured by birefringence may be associated with a high tensile strength. In some embodiments, the recombinant spider silk melt extrudate will have minimal birefringence.

According to the present invention, a uniform flowable state can be induced by applying only shear and pressure, but optionally heat can be applied. Without the application of heat or, optionally, heat, it has been found that the combination of shear and pressure alone provides a composition that is not degraded during processing of the recombinant spider silk polypeptide in the recombinant spider silk melt composition and the recombinant spider silk extrudate. This is desirable and advantageous, as retaining the full length recombinant spider silk polypeptide in the extrudate composition results in optimized material properties, such as crystallinity, resulting in a higher quality product. In embodiments of the invention, the reconstituted spider silk melt extrudate achieved from the application of shear and pressure (and optionally heat) has minimal or negligible degradation.

The amount of degradation of recombinant spider silk polypeptides can be measured using various techniques. As discussed above, the amount of degradation of the recombinant spider silk polypeptide can be measured using size exclusion chromatography to measure the amount of full length recombinant spider silk polypeptide present. In various embodiments, the composition degrades in an amount less than 6.0 wt% after the composition is formed into a molded body. In another embodiment, after molding, the composition degrades in an amount of less than 4.0 wt.%, less than 3.0 wt.%, less than 2.0 wt.%, or less than 1.0 wt.% (such that the amount of degradation can range from 0.001 wt.% to 10 wt.%, 8 wt.%, 6 wt.%, 4 wt.%, 3 wt.%, 2 wt.%, or 1 wt.%, or from 0.01 wt.% to 6 wt.%, 4 wt.%, 3 wt.%, 2 wt.%, or 1 wt.%). In another embodiment, the recombinant spider silk protein in the extrudate and/or melt composition is not substantially degraded.

Drawn fiber

Where the extrudate is used in fiber formation, the precursor fibers may be drawn to increase the orientation of the fibers and promote a three-dimensional crystalline structure. Applying force during stretching facilitates alignment of the molecules on the fiber axis. When forced through the spinneret orifice, the polymer molecules (such as polypeptides) are partially aligned. The fibers may be drawn by hand or by machine. Hand stretching often provides well-aligned fibers with low birefringence and minimal reduction in fiber diameter.

In the present invention, alignment can be optimized by passing the precursor fibers over a uniform hot surface while drawing the fibers. As used herein, the term "hot surface" refers to a surface that provides substantially uniform heat and a substantially uniform surface. The use of a hot surface as a heat source eliminates the variability seen with ambient heat sources, resulting in greater uniformity of results and thus scalability of the process for commercial mass production of fibers. In some embodiments, the hot surface will be a metal rod or other metal surface. In other embodiments, the hot surface may be made of ceramic or other materials. According to embodiments, the hot surface may be curved or otherwise configured to facilitate movement of the fibers over the hot surface.

In embodiments of the invention, the undrawn extruded fiber may be moved on a hot surface while it is being drawn. According to embodiments, the temperature of the hot surface may range from 160 to 210 ℃, 180 to 210 ℃, 190 to 210 ℃, 195 to 205 ℃, or 200 to 205 ℃.

According to embodiments, the undrawn extruded fiber may be subjected to different draw ratios while being drawn on its hot surface. According to embodiments, the stretch ratio may range from 2 to 7. In some embodiments, the maximum stable draw ratio may depend on the temperature of the hot surface.

In some embodiments, the temperature of the hot surface is calculated as a function of the glass transition temperature of the undrawn extruded fiber. For example, the temperature of the hot surface may be calculated as being above 5 ℃,10 ℃, 15 ℃, 20 ℃ or 25 ℃ higher than the glass transition temperature of the recombinant silk protein powder and/or the undrawn extruded fiber. In other words, in the range of 0 or 0.1 ℃ to 25 ℃ higher than the glass transition temperature of the gravity histone powder, often in the range of 0 to 10 ℃, 15 ℃, 20 ℃ higher.

According to embodiments and the rate at which the fibers are passed over a uniform hot surface (referred to herein as the "reel rate"), the length of the hot surface may be varied (i.e., the size in cm of the hot surface over which the fibers are drawn), thus varying the duration of time the undrawn extruded fibers are subjected to heat and deformation. In most embodiments, the width of the hot bar is not less than 1 cm. However, in various embodiments, the width of the hot surface can range from 1 to 50cm, 1 to 2cm, 1 to 3cm, 1 to 5cm, 5 to 38cm, 38 to 50 cm. According to embodiments, the spool speed may range from 1 to 60 minutes per minute.

The total residence time on the hot surface may vary depending on the spool speed and the length of the hot surface. In most embodiments, the total residence time can range from 0.2 seconds to 3 seconds.

Further, the undrawn fibers may experience different forces that provide different draw ratios. In most embodiments, the tension will be provided by a godet roll. In some embodiments, the godets will be positioned such that the fibers passing over the hot surface are at an angle relative to the hot surface. For example, in the case where the hot surface is curved, the godet rolls may be positioned such that the fibers passing over the hot surface are at an angle of 10 to 40 degrees relative to the hot surface.

In various embodiments, the rate of deformation of the undrawn fiber (i.e., the amount of deformation the fiber undergoes due to heat and drawing) may vary based on the above factors. The rate of deformation can be calculated based on the rate at which the undrawn fiber is fed to the hot surface and the rate at which the fiber is collected from the hot surface. For example, the fibers may be fed to the hot surface at a rate of 1 meter/minute and collected from the hot surface at a rate of 5 meters/minute. In a specific embodiment, the rate of deformation is calculated using the equation in which the rate at which the fiber is fed to the hot surface is expressed as v₁The rate of fiber collection from the hot surface is v₂And the length over which the deformation occurs is L₀：

Equation 1：

According to embodiments, the stretching on the hot surface may be performed in one step or in multiple (i.e., two, three, or four) steps. At each step, parameters such as strain rate, deformation rate, reel speed, temperature of the hot surface, and length of the hot surface may be varied or otherwise varied. Stretching over multiple steps can affect the overall strain rate of the fiber, which can enhance the formation of crystalline β -sheet structures, often improving fiber strength.

Post-processed fiber

Various post-processing methods may be employed to improve the molecular alignment of the fibers. Depending on the amount of plasticizer and/or recombinant spider silk present in the fiber, the fiber may be heat treated (e.g., using water vapor or thermal annealing). In other cases, the fibers may be treated with various solvents to anneal the fibers and improve the crystallinity of the protein (e.g., 18B protein) in the fibers. In some cases, the fibers may be annealed using an alcohol (such as methanol). In particular embodiments, alcohol vapor may be used to anneal the fibers.

In some cases, treating the fiber or textile with one or more conditioning, lubricating, surfactant, emulsifying, anti-adhesive, or annealing agents prior to treating the fiber with water will alter the hand or drape of the textile after treatment with water. In particular embodiments, cyclopentasiloxane or PDMS is used as a conditioning agent. In particular embodiments, annealing the fibers or textiles formed from the fibers with an alcohol improves the hand and drape of the water-treated fibers or textiles.

Remelting and re-extruding extrudates

In some embodiments of the invention, the method for preparing a recombinant spider silk extrudate may further comprise reprocessing the molded body comprising the recombinant spider silk extrudate (e.g., pellets, fibers or other molded articles formed from the recombinant spider silk extrudate). In these embodiments, the recombinant spider silk extrudate is subjected to shear forces and pressure sufficient to convert the recombinant spider silk extrudate into a molten or flowable state.

Without intending to be limited by theory, subjecting the recombinant spider silk polypeptide to shear forces and pressure in the presence of a plasticizer such as glycerol causes the recombinant spider silk polypeptide to become an "open form recombinant spider silk polypeptide" in which the recombinant spider silk polypeptide unfolds and forms an interaction with glycerol. This "open form recombinant spider silk polypeptide" forms fewer intermolecular and intramolecular beta-sheet interactions due to interactions with glycerol. In particular, the open form of the recombinant spider silk polypeptide is prevented from forming intermolecular interactions to form an irreversible three-dimensional lattice.

Because the recombinant spider silk polypeptide has minimal degradation, if any, during the melting and extrusion process, the recombinant spider silk extrudate can be converted back into the recombinant spider silk melt composition and re-extruded any number of times. In this sense, the composition is "thermoplastic" in that it can be heated, cooled, and hardened many times without significant degradation of the protein or composition. In various embodiments, the recombinant spider silk extrudate may be remelted and re-extruded at least 20 times, at least 10 times, or at least 5 times. In these embodiments, the degradation seen in multiple remelting and re-extrusion steps can be as low as 10%. The option of re-extruding without degradation allows for the production of a substantially uniform composition and also allows for the re-planning or redesign of products formed from the composition. For example, a molded product of poor quality can be re-extruded and re-molded. End-of-life product recovery is also possible.

Examples

Example 1: purity of recombinant 18B polypeptide powder

Recombinant spider silk, i.e., an 18B polypeptide sequence comprising a FLAG tag (SEQ ID NO:1), was produced by various batches of large scale fermentations, recovered and dried to a powder ("18B powder"). The amount by weight of 18B polypeptide monomer in the powder was measured using reverse phase high performance liquid chromatography ("RP-HPLC"). The sample was dissolved using 5M guanidine thiocyanate (GdSCN) reagent and injected into an Agilent Poroshell 300SB c32.1x75mm 5 μ M column to separate the components based on hydrophobicity. The detection mode was UV absorbance of peptide bonds at 215nm (360nm reference). The sample concentration of the 18B-FLAG monomer was determined by comparison to an 18B-FLAG powder standard, the 18B-FLAG monomer concentration having been previously measured using size exclusion chromatography (SEC-HPLC)

The sample powder was found to include 57.964 mass% of 18B monomer.

Example 2: production of reconstituted filament powder extrudates

The reconstituted silk powder of example 1 was mixed using a household spice mill. Water and glycerol were added to the recombinant silk powder ("18B powder") at a ratio to produce recombinant spider silk compositions with different ratios of protein powder to plasticizer, as listed in table 2 below.

Lots of 10 to 100 grams of the recombinant spider silk composition (i.e., "formulation") listed in table 2 below were compounded using an Xceptional Instruments Twin Screw Extruder (TSE) (item No. TT-ZE5-MSMS-3HT) which was used for all TSE experiments. The stainless steel (S316) extruder barrel had 3 heating zones each about 5cm in length. The screw used was a pair of standard stainless steel (S316) co-rotating screws having a length of 180mm and a diameter of 9mm and (L/D ratio of 20: 1). The pitch of the screw was 9 mm.

For the P25W05G70, P49W21G30 and P65W20G15 formulations listed below, the recombinant spider silk composition was first extruded into pellets, which were reprocessed in the following experiments by re-extruding the pellets. To make pellets, a recombinant spider silk composition comprising an 18B/water/glycerol mixture was introduced into the TSE using a metal funnel and pushed into continuous contact with the twin screw using a tamping device for several minutes while the TSE was run at 300RPM at a temperature of about 90 ℃ to 95 ℃ across all three barrel zones, including the initial barrel zone, the middle barrel zone and the final barrel zone. The material in the molten state (i.e., as a reconstituted spider silk melt composition) was extruded through a 0.5mm die whose orifices were at a 180 ° angle to the screw axis to form a reconstituted spider silk extrudate.

0.5mm recombinant spider silk extrudate emerges from the die in the form of continuous elastic "noodles" of about >10 meters in length. Pellets were generated by placing a corresponding extrudate composition in a kitchen flavor mill in an amount of 5 to 10g and subjecting it to 5 second pulses for a total of 6 pulses (30 seconds total). The pellets were inspected to ensure that their length did not exceed 5mm, with the average length of the pellets being about 2.5 mm.

For the P71W19G10 formulation listed below, an 18B/water/glycerol recombinant spider silk mixture (i.e., not first extruded as pellets) was previously mixed under the conditions described in example 2 to form a recombinant spider silk extrudate.

TABLE 2 recombinant spider silk preparation compositions by weight

Preparation	18B powder weight%	Weight% of water	Weight% of glycerin
				P25W05G70	25％	5％	70％
P49W21G30	49％	21％	30％
				P65W20G15	65％	20％	15％
P71W19G10	71％	19％	10％

Example 3: production of reconstituted filament extrudates with minimal degradation

To assess degradation under a variety of different conditions, the recombinant spider silk formulations listed in example 2 were subjected to various temperatures and to various amounts of pressure and shear during extrusion. Specifically, the rpm of the twin screw extruded pellets was varied to provide variable amounts of torque and shear. Included below are various temperature and RPM combinations for converting the recombinant spider silk formulation to a melt state and extruding different samples.

Extruded pellets of the P49W21G30 and P65W20G15 formulations listed in table 1 were again subjected to extrusion using Xceptional Instruments TSE at various RPMs and temperatures. Other parameters used to operate the Xceptional Instruments TSE are the same as those described above with respect to example 2.

P71W19G10 formulations were also extruded at various RPM and temperatures using an Xceptional Instruments TSE as described in example 2. Other parameters used to operate the Xceptional Instruments TSE are the same as those described above with respect to example 2.

Data characterizing relative amounts of high molecular weight, low molecular weight, and medium molecular weight impurities, monomer 18B, and aggregate 18B were collected using Size Exclusion Chromatography (SEC) as follows: the 18B powder was dissolved in 5M guanidine thiocyanate and injected into a Yarra SEC-3000SEC-HPLC column to separate the components based on molecular weight. The refractive index is used as the detection mode. Quantification of 18B aggregates, 18B monomers, low molecular weight (1-8kDa) impurities, medium molecular weight impurities (8-50kDa) and high molecular weight impurities (110-150 kDa). The relevant compositions are reported as mass% and area%. Using BSA as a common protein standard, it was assumed that > 90% of all proteins demonstrated dn/dc values (response factors for refractive index) within about 7% of each other. Poly (ethylene oxide) was used as retention time standard and BSA calibrator was used as check standard to ensure consistent performance of the method.

Tables 3 to 5 below list various SEC analyses of extrudates produced at various RPM and temperatures. The fifth column included the difference in 18B monomer (area%) reported in the starting pellets and extrudates (P49W21G30 and P65W20G15) and the difference in 18B monomer (area%) reported in the starting powder and extrudates (P71W19G 10). Fig. 1 to 3 are described in detail below and include diagrams corresponding to tables 3 to 5, respectively. As can be seen from the above, degradation is minimal at all temperatures and RPMs tested, indicating flexibility in processing conditions and general robustness to processing using extrusion methods.

TABLE 3 SEC analysis for P49W21G30

TABLE 4 SEC analysis for P65W20G15

TABLE 5 SEC analysis for P71W19G10

Figure 1 shows the SEC data for the P49W21G30 sample listed in table 3 above under extrusion conditions of 20 ℃, 40 ℃, 60 ℃, 80 ℃, 95 ℃, or 120 ℃, where the operating parameters of 10, 100, 200, or 300RPM were used to obtain extrudates at each temperature. The 18B monomer (black bars), medium molecular weight impurities (grey bars) and low molecular weight impurities (cross-hatched bars) are shown as area%.

Figure 2 shows the SEC data for the P65W20G15 samples listed in table 4 above under extrusion conditions of 20 ℃, 40 ℃, 60 ℃, 95 ℃, or 140 ℃, where the operating parameters of 10, 100, 200, or 300RPM were used to obtain extrudates at each temperature. The 18B monomer (black bars), medium molecular weight impurities (grey bars) and low molecular weight impurities (cross-hatched bars) are shown as area%.

Figure 3 shows SEC data for the P71W19G10 sample listed in table 5 above under extrusion conditions of 90 ℃ or 120 ℃, where extrudates at each temperature were obtained using operating parameters of 10, 100, 200 or 300 RPM. The 18B monomer (black bars), medium molecular weight impurities (grey bars) and low molecular weight impurities (cross-hatched bars) are shown as area%.

Example 4: thermogravimetric analysis-P49W 21G30

To analyze the amount of water lost during extrusion, the water content of the recombinant spider silk composition before extrusion and the recombinant spider silk extrudate after extrusion was analyzed by TGA (thermogravimetric analysis) using a TA brand TGA Q500 instrument. For the P49W21G30 and P65W20G15 samples, the water content of the pellets used in the extrusion experiments described in example 3 was used as a reference sample for measuring water loss. For the P71W19G10 sample, the water content of the recombinant spider silk composition used in the extrusion experiment described in example 3 was used as a reference sample for measuring water loss.

For each sample, 10mg +/-1mg of powder or pellet containing the formulation listed above was analyzed. To measure water content, the samples were run "in air" compared to "in nitrogen". The sample was continuously introduced into the TGA furnace using an equipped autosampler. The temperature was programmed to increase from room temperature at a rate of 20 ℃/min using the TA brand software program suite until it reached 110 ℃. The sample was then held at that temperature for 45 minutes. The sample was then removed from the oven and the oven was flushed with air for 15 minutes before the next run was started.

Tables 6 to 8 below list various measured values for the reference sample (i.e., starting pellet or powder) and the extruded sample. Fig. 4 to 6 include graphs of data respectively included in tables 6 to 8. From this data, it can be seen that the water loss during extrusion is low and well within acceptable limits for the extrusion process, typically in the range of 2% to 18%.

Water loss in tables 6-P49W 21G30

Water loss in tables 7-P65W 20G15

Water loss in tables 8-P71W 19G10

Figure 4 shows TGA data for the samples listed in table 6 above generated under extrusion conditions of 20 ℃, 40 ℃, 95 ℃, and 120 ℃, where 10, 100, 200, and 300RPM operating parameters were used to obtain extrudates at each temperature. Fig. 4 also shows TGA data for a reference sample of starting pellets used to generate the sample. The data shows% water content across all treated samples, with water loss ranging from about 1% to 13% when compared to the starting pellets.

Figure 5 shows TGA data for the samples listed in table 7 above generated under extrusion conditions of 20 ℃, 40 ℃, 60 ℃, and 140 ℃, where extrudates at various temperatures were obtained using 10, 100, 200, and 300RPM operating parameters. Fig. 5 also shows TGA data for a reference sample of starting pellets used to generate the sample. The data shows% water content across all treated samples, with water loss ranging from about 1% to 8% when compared to the starting pellets.

Figure 6 shows TGA data for the samples listed in table 8 above generated under extrusion conditions of 90 ℃ and 120 ℃, where 10, 100, 200, and 300RPM operating parameters were used to obtain extrudates at each temperature. Figure 5 also shows TGA data for a reference sample of starting powder used to generate the sample. The data shows% water content across all treated samples, with water loss ranging from about 1.5% to 4% when compared to the starting powder.

Example 5: beta sheet content analysis using Fourier transform Infrared Spectroscopy

To assess the formation of secondary and tertiary structures in the extrudates, the beta sheet content was measured by FTIR (fourier transform infrared spectroscopy). FTIR was performed on the extrudates using a Bruker Alpha spectrometer equipped with a diamond attenuated total reflection accessory followed by a wire grid polarizer with a major selection of S (perpendicular) polarized light. Recombinant polypeptide powder and precursor fiber were included as controls. For quantitative molecular alignment, at 4000 to 600cm^-14cm of^-1Three spectra were collected at resolution in 32 scans for each orientation (0 and 90 ° with respect to the polarizing electric field).

The calculation corresponded to 982-949cm based on the following procedure^-1Average of the peaks of (a). By subtracting 1900 and 1800cm without bands^-1Average between to cancel out the absorbance value. Then by removing 1350 and 1315cm strips corresponding to isotropic (non-oriented) side chain vibration^-1Average value in between to normalize the spectrum. Beta sheet content measurements were taken as 982 and 949cm^-1Taking the average of the integrated absorbance values in between.

The beta sheet (i.e., "sample beta sheet") content of the recombinant spider silk extrudate was compared to: i) beta sheet content in the starting recombinant spider silk polypeptide powder (i.e., "reference prehydrated powder") used to generate the recombinant spider silk composition, and ii) beta sheet content in the starting pellets (P49W21G30 and P65W20G15) (i.e., "reference pellets"). Tables 9 to 11 below list the reference samples and the measured values of the extrudates produced under the conditions listed in the tables below. Fig. 7 to 9 include graphs of the data shown in tables 9 to 11. As can be seen, there was no significant change in the beta sheet content from the starting recombinant silk polypeptide powder to the material of the recombinant spider silk extrudate, indicating that the method is capable of polarizing and migrating the amorphous protein domain without disrupting the beta sheet, as would be the case if solvent processing were used.

Beta sheet formation in Table 9-P49W 21G30

Beta sheet formation in Table 10-P65W20G15

Beta sheet formation in Table 11-P71W 19G10

Fig. 7 shows FTIR data for the samples listed in table 9 above generated under extrusion conditions of 20 ℃, 40 ℃, 60 ℃, 80 ℃, 95 ℃, or 120 ℃, where the extrudate was obtained for each temperature using operating parameters of 10, 100, 200, or 300 RPM. The data was obtained from 949-.

Fig. 8 shows FTIR data for the samples listed in table 10 above generated under extrusion conditions of 20 ℃, 40 ℃, 60 ℃, 95 ℃, or 140 ℃, where the extrudates at each temperature were obtained using operating parameters of 10, 100, 200, or 300 RPM. The data was obtained from the 949-982 band and showed no clear trend compared to the starting pellets.

Fig. 9 shows FTIR data for the samples listed in table 11 above generated under extrusion conditions of 90 ℃ or 120 ℃, where extrudates at various temperatures were obtained using operating parameters of 10, 100, 200, or 300 RPM. The data was obtained from 949-.

Example 6: polarized light microscope

The smoothness and uniformity of the various extrudates was examined using polarized light microscopy (PL). Light and Polarized Light (PL) images were obtained using a Leica DM750P polarized light microscope, using a 4X PL objective. The microscope was coupled to a complementary PC-based image analysis Leica Application Suite, LAS V4.9. TSE extrudates of about 20 to 30mm length are carefully placed along the long axis of a standard microscope slide and horizontally (east-west; i.e., 0) over the microscope aperture. The sample is initially brought to focus at its edge and then focused overall on the sample. The sample was initially viewed under white light, controlled by illuminating control buttons, and an image captured using the appropriate scale included. In all cases, the automatic brightness feature of the LAS V4.9 software is turned off.

The analyzer/boeholder lens module is then engaged by flipping its lower arm to the right ("a" position/inside analyzer), while ensuring that its upper arm is flipped to the left ("O" position/outside boeholder lens). This arrangement allows analysis in a "cross-polarization mode", which is an optically aligned state where the allowed vibration direction of light passing through the polarizer and analyzer is oriented at 90 °.

To control the light intensity value background fluctuation, all samples were initially viewed and the brightness of the background was reduced using the illumination control button until it just reached complete darkness. Each eyepiece is then covered with an eyepiece light blocking attachment during an image capture sequence to prevent ambient light from passing through. Images were captured at 0 ° and 45 ° orientations using the LAS V4.9 software package. A 45 ° image was obtained by rotating the glass side to a 45 ° angle using a circular rotating table with which this microscope was equipped.

Fig. 10 and 11 are images of exemplary samples captured using polarized light microscopy. The images show that smooth fibers with low melt fracture can be obtained using the required process. Thus, the conditions are clearly suited for melt flow and extrusion. In addition, under many conditions, qualitative birefringence was observed, as was axial alignment.

FIG. 10 shows pictures generated from samples P49W21G30-1, P49W21G30-2, P49W21G30-3, and P49W21G30-4, all generated at 20 ℃ with different RPMSs. Under these conditions, the extrudate is smooth with low melt fracture. Polarized light microscopy showed the preferred axial alignment according to the conditions (check for 45 ° difference), with 100RPM producing the maximum axial alignment.

FIG. 11 shows pictures produced from samples P49W21G30-17, P49W21G30-18, P49W21G30-19, and P49W21G30-20, all produced at 95 ℃ with different RPMSs. The extrudate showed moderate melt fracture/surface defects. Polarized light microscopy showed an increase in axial alignment from 10-100 RPM. The samples show similar differences from each other from 100-300RPM when examined at 0 and 45 °.

Example 7: metabolite analysis of glycerol content

To determine glycerol loss from recombinant spider silk compositions during extrusion, glycerol content was analyzed using a Benson polymeric150x7.8 mm H +7110-0HPLC column equipped with a Phenomenex Security Guard car H + protective column, using a mobile phase of 0.004M sulfuric acid. A glycerol calibrator was initially run for quantification. To measure the amount of glycerol in the 18B based samples, the glycerol present in the composition was measured before (i.e., as pellets or powder) and after extrusion. For each sample, 25mg of the powder or pellet was dissolved in 1ml of 0.004M sulfuric acid and sonicated for 1 h. The samples were then vortexed and placed in HPLC vials for subsequent runs of each condition/treatment.

Tables 12 to 14 below list various measurements of extrudates produced under the conditions listed below. Fig. 12-14 include plots of the same samples. As can be seen from the figure, the glycerol content in the composition is stable across the range of conditions tested, as evidenced by minimal loss during testing.

TABLE 12 Glycerol loss in the extrudates-P49W 21G30

TABLE 13 Glycerol loss in the extrudates-P65W 20G15

TABLE 14 Glycerol loss in the extrudates-P71W 19G10

Results within error of the test instrument

Fig. 12 shows metabolite data for the samples listed in table 12 above generated under extrusion conditions of 20 ℃, 40 ℃, 60 ℃, 80 ℃, 95 ℃ and 120 ℃, where the extrudates at each temperature were obtained using operating parameters of 10, 100, 200 and 300 RPM. Glycerol loss was negligible in all treatments.

Fig. 13 shows metabolite data for the samples listed in table 13 above generated under extrusion conditions of 20 ℃, 40 ℃, 60 ℃, 95 ℃ and 140 ℃, where the extrudates at each temperature were obtained using operating parameters of 10, 100, 200 and 300 RPM. Glycerol loss was negligible in all treatments.

Fig. 14 shows metabolite data for the samples listed in table 14 above generated under extrusion conditions of 90 ℃ and 120 ℃, where extrudates at each temperature were obtained using operating parameters of 10, 100, 200, or 300 RPM. Glycerol loss was negligible in all treatments.

Example 8: metabolite analysis of glycerol content

The P49W21G30 and P25W05G70 filament powder compositions were mixed and twin screw extruded as described in example 2. The extrudate was cut into pellets and subjected to Melt Flow Indexing (MFI). MFI was carried out on a Goettfert melt indexer, model MI-40, No. 10005563. The barrel diameter was 9.5320mm, the die length was 8.015mm, and the orifice diameter was 2.09 mm. Two minutes of preheating is utilized. The flow rate of the thermoplastic was measured by an extrusion plastometer according to ASTM D1238 standard test method. The test was carried out at 95 ℃ under a load of 2.16kg or 21.6 kg.

Table 15 shows the melt flow index values obtained from the respective material compositions. Tested at 2.1 and 21.1Kg respectively, n is 3 for P49W21G30 and 6 for P25W05G 70. '+/-' indicates the standard deviation between n samples. The data indicate that protein/glycerol/water based pellets exhibit MFI values (20g/10min) in a similar range as for example polypropylene. Higher flow rates are obtained at lower protein compositions.

TABLE 15 melt flow index

	2.1Kg	21.1Kg
			P49W21G30	-	7.10+/-2.58
P25W05G70	14.18+/-3.07	-

Claims (67)

1. A composition for moulded bodies comprising a recombinant spider silk protein and a plasticizer, wherein the composition is capable of being induced into a flowable state, wherein the recombinant spider silk protein is not substantially degraded in the flowable state.

2. The composition of claim 1, wherein said composition can be induced into said flowable state by the application of shear and pressure.

3. The composition of claim 2, wherein the composition can be induced into the flowable state by applying shear and pressure without the application of heat.

4. The composition of claim 3, wherein the composition is capable of being induced into the flowable state and extruded multiple times, wherein the recombinant spider silk protein remains substantially undegraded within the composition.

5. The composition of claim 1, wherein the composition is thermoplastic.

6. The composition of claim 2, wherein the composition can be induced into the flowable state by applying a shear force in the range of 1.5Nm to 13 Nm.

7. The composition of claim 2, wherein the composition is capable of being induced into the flowable state by application of a shear force in the range of 2Nm to 6 Nm.

8. The composition of claim 2, wherein the composition can be induced into the flowable state by applying a pressure ranging from 1MPa to 300 MPa.

9. The composition of claim 2, wherein the composition is inducible into the flowable state by application of a pressure in the range of 5MPa to 75 MPa.

10. The composition of any one of claims 6 to 9, wherein the composition is capable of being induced to the flowable state at less than 120 ℃, less than 80 ℃, less than 40 ℃ or at room temperature.

11. The composition of any one of claims 1 to 10, wherein the composition has a melt flow index of at least 0.5, at least 1, at least 2, or at least 5, as tested according to ASTM D1238 at 95 ℃ with a load of 2.16 kg.

12. The composition of any one of claims 1 to 10, wherein the composition has a melt flow index of at least 0.5, at least 1, at least 2, or at least 5, as tested according to ASTM D1238 at 95 ℃ with a load of 21.6 kg.

13. The composition of claim 1, wherein the composition is substantially homogeneous.

14. The composition of claim 1, wherein the recombinant spider silk protein comprises a repeating unit.

15. The composition of claim 1, wherein the recombinant spider silk protein comprises in the range of 2 to 20 repeating units having an amino acid residue length in the range of 60 to 100 amino acids.

16. The composition of claim 1, wherein the molecular weight of the recombinant spider silk protein ranges from 20 to 2000 kDa.

17. The composition of claim 1, wherein the recombinant spider silk protein comprises a repeating unit that occurs at least twice, said repeating unit comprising:

more than 150 amino acid residues and a molecular weight of at least 10 kDa;

an alanine-rich region having 6 or more contiguous amino acids comprising an alanine content of at least 80%; and

a glycine-rich region having 12 or more contiguous amino acids comprising a glycine content of at least 40% and an alanine content of less than 30%.

18. The composition of claim 1, wherein the plasticizer is selected from polyols, water and/or urea.

19. The composition of claim 16, wherein the polyol comprises glycerol.

20. The composition of claim 1, wherein the plasticizer comprises water.

21. The composition of claim 1, wherein the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder, and wherein the weight ratio of plasticizer to recombinant silk polypeptide powder ranges from 0.05:1 to 4: 1.

22. The composition of claim 1, wherein the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder and the weight ratio of plasticizer to recombinant silk polypeptide powder ranges from 0.20:1 to 0.70: 1.

23. The composition of claim 1, wherein the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder, and the amount of recombinant spider silk polypeptide powder in the composition ranges from 1 to 90 wt% of recombinant spider silk protein.

24. The composition of claim 1, wherein the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder, and the amount of recombinant spider silk polypeptide powder in the composition ranges from 20 to 41 wt% of recombinant spider silk protein.

25. The composition of claim 1, comprising glycerol as a plasticizer in the range of 1 to 90 wt.%.

26. The composition of claim 1, comprising glycerol as a plasticizer in the range of 15 to 30 wt.%.

27. The composition of claim 1, comprising water as a plasticizer in the range of 5 to 80 wt.%.

28. The composition of claim 1, comprising water as a plasticizer in the range of 19 to 27 weight percent.

29. The composition of claim 1, wherein the recombinant spider silk protein degrades in the flowable state in an amount of less than 10.0 wt%.

30. The composition of claim 1, wherein the recombinant spider silk protein degrades in the flowable state in an amount of less than 6.0 wt%.

31. The composition of claim 1, wherein the recombinant spider silk protein degrades in the flowable state in an amount of less than 2.0 wt%.

32. The composition of any one of claims 29 to 31, wherein the degradation of the recombinant spider silk protein is assessed by measuring the amount of full length recombinant spider silk protein present in the composition before and after induction of the flowable state.

33. The composition of claim 32, wherein the amount of full length recombinant spider silk protein is measured using size exclusion chromatography.

34. A molded body comprising the composition of any one of claims 1 to 33.

35. A moulded body as claimed in claim 34, in which the moulded body is a fibre.

36. The molded body of claim 35, wherein the fibers have a strength in the range of 100Pa to 1.2 GPa.

37. The molded body of claim 35, wherein said fibers have a birefringence of 5 x 10 as measured by polarized light microscopy^-5To about 0.04.

38. A method for producing a molded body, comprising the steps of:

(a) applying pressure and shear force to a composition comprising recombinant spider silk protein and a plasticizer to convert the composition into a flowable state; and

(b) extruding the composition in the flowable state to form a molded body.

39. The method of claim 38, wherein extruding the composition to form a molded body comprises extruding the composition to form a fiber.

40. The method of claim 39, wherein extruding the composition to form a fiber comprises extruding the composition through a spinneret.

41. The method of claim 38, wherein extruding the composition to form a molded body comprises extruding the composition into a mold.

42. The method of claim 38, further comprising:

(a) applying pressure and shear force to the molded body to convert the molded body into a composition in a flowable state; and

(b) extruding the composition in the flowable state to form a second molded body.

43. The method of claim 42, further comprising repeating steps (a) and (b) of the second molded body at least once.

44. The method of any one of claims 38 to 43, wherein the shear force is from 1.5 to 13 Nm.

45. The method of any one of claims 38 to 43, wherein the pressure is from 1MPa to 300 MPa.

46. The method of any one of claims 38 to 45, wherein the shear and pressure are applied to the composition using a capillary rheometer or a twin screw extruder.

47. The method of claim 46, wherein the screw speed of the twin screw extruder during the application of the pressure and shear force ranges from 10 to 300 RPM.

48. The method of any one of claims 38-45, wherein the instrument for applying the shear force and pressure comprises a mixing chamber coupled to and proximal to an extrusion chamber.

49. The method of claim 48, wherein the composition is heated in the mixing chamber.

50. The method of claim 48, wherein the composition is heated in the extrusion chamber.

51. The method of claim 49 or claim 50, wherein the composition is heated to a temperature of less than 120 ℃.

52. The method of claim 49 or claim 50, wherein the composition is heated to a temperature of less than 80 ℃.

53. The method of claim 49 or claim 50, wherein the composition is heated to a temperature of less than 40 ℃.

54. The method of any one of claims 38-53, wherein the water content of the molded body after extrusion is lost less than 15% compared to the composition prior to extrusion.

55. The method of any one of claims 38-53, wherein the water content of the molded body after extrusion is lost less than 10% as compared to the composition prior to extrusion.

56. The method of claim 48, wherein the residence time of the composition in the mixing chamber ranges from 3 to 7 minutes.

57. The method of claim 48, wherein the extrusion chamber is tapered proximal to the orifice where the composition is extruded.

58. The method of claim 48, wherein the extrusion chamber is temperature controlled.

59. The method of any one of claims 48 to 58, wherein the moulded body is a fibre and the fibre is manually drawn.

60. The method of any one of claims 48 to 59, wherein the molded body is a fiber and the fiber is drawn in multiple steps.

61. The method of any one of claims 48 to 60, wherein the recombinant spider silk protein is not substantially degraded in the moulded body.

62. The method of claim 61, wherein the recombinant spider silk protein is degraded in the moulded body in an amount of less than 10% by weight.

63. The method of claim 61, wherein the recombinant spider silk protein is degraded in the moulded body in an amount of less than 6 wt%.

64. The method of claim 61, wherein the recombinant spider silk protein is degraded in the moulded body in an amount of less than 2% by weight.

65. The method of any one of claims 61-64, wherein the degradation of the recombinant spider silk protein is assessed by measuring the amount of full length recombinant spider silk protein present in the composition before and after extrusion.

66. The method of claim 65, wherein the amount of full length recombinant spider silk protein is measured using size exclusion chromatography.

67. The method of any one of claims 38-66, wherein the molded body has a minimum birefringence as measured by polarized light microscopy.