WO2014035798A1 - Silk lyogels for sustained release of protein therapeutics and methods of making and uses - Google Patents

Description

SILK LYOGELS FOR SUSTAINED RELEASE OF PROTEIN THERAPEUTICS AND METHODS

OF MANUFACTURING AND USES

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Serial No.

61/695,112, filed August 30, 2012, and French Application No. 1356305, filed June 28, 2013, each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to silk lyogels and their use in extended release drug administration. More specifically, the silk lyogels have hydrophobicity, ionic, or stability modifications and the drug administered is a protein therapeutic.

BACKGROUND

The availability of suitable sustained/local delivery systems for protein therapeutics continues to be limited. A thorough understanding of protein-matrix interactions, matrix manufacturing preparation and protein stability is critical for the development of such a system.

Sustained local delivery systems for therapeutic proteins are pharmaceutically appealing targets. Historically, protein therapeutics have been delivered systemically through periodic intravenous or intramuscular dosing. Maintaining appropriate plasma

concentrations, between the therapeutic effect threshold and toxicity level, is difficult using these approaches. Systemic delivery is also inefficient if the target is a specific organ or tissue, costly when dealing with expensive protein therapeutics, and creates an increased risk of toxicity.

Monoclonal antibodies are a subset of protein therapeutics that could benefit from sustained local delivery systems. Antibodies are excellent therapeutic targets due to their specificity, modular structure and their ability to leverage the patient's own immune system. A wide range of indications in oncology, immune mediated disorders and wound healing stand to benefit from the development of antibody based therapies. Long-term repetitive dosing is common for antibody therapies therefore drug efficacy and patient compliance would improve significantly from the availability of sustained local delivery options.

Monoclonal antibodies are also attractive model systems for study thanks to their highly conserved structure. While numerous systems and devices are available for sustained local delivery of small molecule therapeutics, none currently exist for monoclonal antibodies despite their broad therapeutic appeal.

Compared to small molecule therapeutics, protein therapeutics present unique obstacles for sustained local delivery. Significant challenges exist around the manufacturing of therapeutic proteins due to their limited inherent stability. Common chemical modifications such as oxidation or deamidation or subtle changes in secondary, tertiary or quaternary structure can lead to a loss of function. Protein chemical and physical stability is sensitive to stresses such as temperature, light, shear, air-water interface, and extremes in solution conditions. The intrinsic sensitivity of proteins highlights the need for novel, versatile and well characterized delivery systems whose preparation is sufficiently gentle to preserve structural, chemical and functional stability. Beyond the generation of desirable release profiles, a thorough characterization of protein-matrix interactions, protein retention, and protein stability is required for the development of viable delivery systems.

Hydrogel-based matrices and micro/nanoparticles produced from biodegradable polymers have been the predominant systems investigated for sustained local delivery of proteins. These delivery systems have been engineered from both synthetic and natural polymers. The material properties of synthetic materials such as poly(D,L-lactide-co- glycolide) (PLGA), and polyvinyl alcohol) (PVA) can be fine-tuned by introducing functional groups, altering polymerization conditions and changing the composition of monomers. While highly customizable, synthetic polymers are often incompatible with protein therapeutics because of harsh microclimates caused by matrix degradation products and dramatic hydrophobicity differences. Natural polymers such as gelatin, collagen, fibrin, alginate, chitosan, and hyaluronic acid are biocompatible and biodegradable but their use can be restricted by batch to batch variability and a smaller range of controllable physical properties.

There is a need in the art for an improved composition for sustained release of proteins including antibodies.

SUMMARY OF THE INVENTION

The present disclosure provides insight into the mechanisms governing silk-solvent and silk-antibody interactions. Such insights offer to further refine this new stabilization and delivery protocol for antibodies in silk matrices. The current work describes a series of mechanistic studies on antibody loaded silk lyogels. The relationship between silk density, hydration behavior and antibody recovery was confirmed and characterized. Release medium studies were used to characterize the nature of silk-antibody interactions. The presence of a surfactant was used to evaluate the role of hydrophobic interactions and hydration behavior, while variable salt and pH studies were used to probe the involvement of ionic interactions. Finally, a unified mechanism describing the factors that impact antibody release from silk lyogels is proposed.

The disclosure provides a silk lyogel comprising a hydrophobicity modification and/or an ionic modification. In certain embodiments, the silk lyogel facilitates sustained release of a therapeutic protein. In other embodiments, the hydrophobicity modification modulates lyogel swelling when the lyogel is added to an aqueous medium, thereby facilitating sustained release of the therapeutic protein. In other embodiments, the ionic modification decreases repulsive ionic interactions in the lyogel, thereby facilitating sustained release of the therapeutic protein. The stability modification increases stability of the therapeutic protein in the silk lyogel, thereby facilitating sustained release of the therapeutic protein.

In certain embodiments, the silk lyogel also includes the therapeutic protein. The therapeutic protein can be an antibody. The antibody can be an IgG antibody.

In other embodiments, the silk lyogel includes a hydrophobicity modification. The hydrophobicity modification can include compressing the silk lyogel to a high density. This high density can be between 100 and 1500 mg/cm³; 500 and 1400 mg/cm³; 800 and 1100

3 3

mg/cm ; or 1200 and 1500 mg/cm . The hydrophobicity modification can be the addition of a surfactant. The surfactant can be selected from a non-ionic surfactant, an anionic surfactant, a cationic surfactant and a zwitterionic surfactant.

In certain embodiments, the surfactant is a non-ionic surfactant. Non-ionic surfactant can be selected from polysorbates, poloxamers, polyols, and polyoxyethylene sorbitan monoethers. In certain embodiments, the surfactant is an anionic surfactant. The anionic surfactant can be selected from fatty acid soap, higher alkyl sulfate ester salt, alkyl ether sulfate ester salt, N-acyl sarcosinic acid, higher fatty acid amide sulfonate, phosphate ester salt, sulfosuccinate, alkylbenzene sulfonate, higher fatty acid ester sulfate ester salt, N-acyl glutamate, sulfonated oil, POE-alkyl ether carboxylic acid, POE-alkyl aryl ether carboxylate, a-olefine sulfonate, higher fatty acid ester sulfonate, secondary alcohol sulfate ester salt, higher fatty acid alkylolamide sulfate ester salt, sodium lauroyl monoethanolamide succinate, N-palmitoyl asparaginate ditriethanolamine and sodium casein. In certain embodiments, the surfactant is a cationic surfactant. The cationic surfactant can be selected from alkyltrimethyl ammonium salt, alkylpyridinium salt, distearyldimethyl ammonium chloride, dialkyldimethyl ammonium salt, poly (N,N'-dimethyl-3,5-methylenepiperidinium) chloride, alkyl quaternary ammonium salt, alkyldimethylbenzyl ammonium salt, alkylisoquinolinium salt,

dialkylmorphonium salt, POE-alkylamine, alkylamine salt, polyamine fatty acid derivative, amyl alcohol fatty acid derivative, benzalkonium chloride and benzethonium chloride. In certain embodiments, the surfactant is a zwitterionic surfactant. The zwitterionic surfactant is selected from the group consisting of alkyl amine oxides, alkyl betaines, alkyl amidopropyl betaines, alkyl sulpho betaines (sultaines), alkyl glycinates, alkyl carboxyglycinates, alkyl ampho acetates, alkyl amphopropionates, alkylamphoglycinates, alkyl amidopropyl hydroxysultaines, acyl taurates and acyl glutamates. Surfactants can also be selected from octyl maltoside, decyl maltoside, dodecyl β-D-maltoside, tridecyl maltoside, tetradecyl maltoside and sucrose dodecanoate.

In other embodiments, the silk lyogel comprises an ionic modification. The ionic modification can be the addition of a buffering agent. In certain embodiments, the buffering agent maintains the solution that is subject to lyophilization to form the lyogel at a pH of between 3 and 12. The buffering agent can be selected from HEPES, Tris, MES, sodium phosphate, potassium phosphate, sodium acetate, sodium citrate, potassium nitrate, histidine, and succinate.

The ionic modification can also be addition of a salt. In certain embodiments, the salt maintains the solution that is subject to lyophilization to form the lyogel at an osmolarity of between 1 mM and 5 M. The salt can be selected from NaCl, NaK, MgCb, and CaCb.

In other embodiments, the silk lyogel is produced from a silk solution of between 2 and 18% w/w. In other embodiments, the silk lyogel includes silk fibroin with 20-65% β- sheet content. In other embodiments, the silk lyogel includes silk fibroin with a monomer of sequence (Gly-Ser-Gly-Ala-Gly-Ala)„, wherein n is greater than or equal to 5 and less than or equal to 70. In other embodiments, the fibroin polymers comprise between 40 and 60% glycine content. In other embodiments, the fibroin polymers have an average molecular weight between 0.5 and 400 kDa.

In other embodiments, the silk lyogel further comprises a stability modification. The stability modification can include the addition of an anti-oxidation agent to the silk lyogel. The anti-oxidation agent can be selected from methionine, thiosulfate, N-Acetyl-L- methionine, N- Acetyl tryptophan.

In certain embodiments, the silk lyogel releases between 0.5 and 100% or 0.5 and 5% of the antibody when exposed to an aqueous environment for 160 days. In other

embodiments, the silk lyogel has a swelling ratio between 0.2 and 20 or 0.2 and 1 when exposed to an aqueous environment for 160 days.

The disclosure also provides a method of making a silk lyogel including the steps of sonicating silk fibroin; cooling the sonicated solution at room temperature; and lyophilizing the cooled solution, wherein the method further comprises making a modification selected from a hydrophobicity modification, an ionic modification, or a stability modification. In certain embodiments, the silk fibroin solution includes between 2 and 18% or 3 and 12 % silk fibroin w/w. In other embodiments, room temperature is between 18 and 26 °C.

In certain embodiments, the method also includes adding a protein to the sonicated silk fibroin. The protein can be an antibody. The antibody can be an IgG antibody.

In certain embodiments, the modification is a hydrophobicity modification. The hydrophobicity modification is compressing the lyophilized solution at between 900 and 1100 or 2,400 and 2,600 psi. In other embodiments, the silk lyogel has silk at a density between 100 and 1500, 500 and 1400, 800 and 1100 or 1200 and 1500 mg/cm³.

In other embodiments, the hydrophobicity modification is addition of a surfactant prior to lyophilization. The surfactant can be selected from a non-ionic surfactant, an anionic surfactant, a cationic surfactant and a zwitterionic surfactant. The non-ionic surfactant can be selected from polysorbates, poloxamers, polyols, and polyoxyethylene sorbitan monoethers. The anionic surfactant can be selected from fatty acid soap, higher alkyl sulfate ester salt, alkyl ether sulfate ester salt, N-acyl sarcosinic acid, higher fatty acid amide sulfonate, phosphate ester salt, sulfo succinate, alkylbenzene sulfonate, higher fatty acid ester sulfate ester salt, N-acyl glutamate, sulfonated oil, POE-alkyl ether carboxylic acid, POE-alkyl aryl ether carboxylate, a-olefine sulfonate, higher fatty acid ester sulfonate, secondary alcohol sulfate ester salt, higher fatty acid alkylolamide sulfate ester salt, sodium lauroyl

monoethanolamide succinate, N-palmitoyl asparaginate ditriethanolamine and sodium casein. The cationic surfactant can be selected from alkyltrimethyl ammonium salt, alkylpyridinium salt, distearyldimethyl ammonium chloride, dialkyldimethyl ammonium salt, poly (Ν,Ν'- dimethyl-3,5-methylenepiperidinium) chloride, alkyl quaternary ammonium salt, alkyldimethylbenzyl ammonium salt, alkylisoquinolinium salt, dialkylmorphonium salt, POE-alkylamine, alkylamine salt, polyamine fatty acid derivative, amyl alcohol fatty acid derivative, benzalkonium chloride and benzethonium chloride. The zwitterionic surfactant can be selected from alkyl amine oxides, alkyl betaines, alkyl amidopropyl betaines, alkyl sulphobetaines (sultaines), alkyl glycinates, alkyl carboxyglycinates, alkyl amphoacetates, alkyl amphopropionates, alkylamphoglycinates, alkyl amidopropyl hydro xysultaines, acyl taurates and acyl glutamates. Surfactants can also be selected from octyl maltoside, decyl maltoside, dodecyl β-D-maltoside, tridecyl maltoside, tetradecyl maltoside and sucrose dodecanoate.

In other embodiments, the modification is an ionic modification. The ionic modification can be addition of a buffering agent prior to lyophilization. In certain embodiments, the buffering agent maintains the solution that is subject to lyophilization to form the lyogel at a pH of between 2 and 12. The buffering agent can be selected from HEPES, Tris, MES, sodium phosphate, potassium phosphate, sodium acetate, sodium citrate, potassium nitrate, histidine, and succinate.

In another embodiment, the ionic modification is addition of a salt prior to lyophilization. In certain embodiments, the salt maintains the solution that is subject to lyophilization to form the lyogel at an osmolarity of between 1 mM and 5 M. The salt can be selected from NaCl, NaK, MgCl₂, and CaCl₂.

In other embodiments, the silk lyogel is present at between 2 and 18% w/w. In other embodiments, the silk lyogel releases between 0.5 and 100%; 0.5 and 5% or 10 and 20% of the antibody when exposed to an aqueous environment for 160 days. In other embodiments, the swelling ratio of the silk lyogel is between 0.2 and 20 or 0.2 and 1 when exposed to an aqueous environment for 160 days.

In other embodiments, the method includes making a stability modification to the lyogel. The stability modification can include the addition of an anti-oxidant to the lyogel. The anti-oxidant is selected from methionine, thiosulfate, N-Acetyl-L-methionine, N-Acetyl tryptophan.

The disclosure also provides a method of determining the rate of release of an antibody from a silk lyogel comprising exposing the high density silk lyogel to aqueous solution, wherein the aqueous solution comprises a surfactant, for a set period of time; and detecting the amount of antibody in the aqueous solution at the end of the set period of time. The surfactant can be present at a concentration of 0.001-1.0% (w/v). The disclosure also provides a method of determining the rate of release of an antibody from a silk lyogel including the steps of exposing the silk lyogel to an aqueous solution, wherein the pH of the aqueous solution is between 2 and 12, for a set period of time; and detecting the amount of antibody in the aqueous solution at the end of the set period of time.

The disclosure also provides a method of determining the rate of release of an antibody from a silk lyogel including the steps of exposing the silk lyogel to an aqueous solution, wherein the aqueous solution comprises a salt at a concentration between 0 and 3 M, for a set period of time; and detecting the amount of antibody in the aqueous solution at the end of the set period of time.

The disclosure also provides a sustained release dosage form including a silk lyogel as described above and a pharmaceutically acceptable carrier or excipient. The sustained release dosage form can also include a protein. The protein can be an antibody. The antibody can be an IgG antibody. The sustained release can last for 1 -160 days.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a line graph showing antibody release related to matrix density altered by varying silk solution concentration from 3.1% (w/w) to 6.7% (w/w) representing silk densities from 52 mg cm^"3 to 80 mg cm^"3. Release data was truncated at approximately 85% of cumulative release observed at plateau. Lines were added as a visual aid.

Figure IB is a line graph showing antibody release related to matrix density altered by pressing a 3.2% (w/w) silk lyogel representing silk densities from 45 mg cm^"3 to 1 ,321 mg cm^"3. Release data was truncated at approximately 85% of cumulative release observed at plateau. Lines were added as a visual aid.

Figure 2A is a line graph showing silk lyogel hydration behavior modeled by swelling ratio as a function of silk matrix density.

Figure 2B is a line graph showing antibody recovery as a function of swelling ratio for variable density silk lyogels fit to a four parameter sigmoidal model. Residuals represent the difference between experimental and predicted values.

Figure 3A is a line graph showing antibody release at increasing levels of polysorbate

80. Release data was truncated at approximately 85% of cumulative release observed at plateau. Lines were added as a visual aid. Release data was truncated at approximately 85% of cumulative release observed at plateau. Lines were added as a visual aid. Figure 3B is a line graph showing antibody release at increasing sodium chloride concentration. Release data was truncated at approximately 85% of cumulative release observed at plateau. Lines were added as a visual aid.

Figure 3C is a line graph showing antibody release at variable pH. Release data was truncated at approximately 85% of cumulative release observed at plateau. Lines were added as a visual aid.

Figure 4A is a line graph showing thermal characterization of variable density silk lyogels by mDSC in a non reversing heat flow thermogram.

Figure 4B is a line graph showing thermal characterization of variable density silk lyogels by mDSC in a non reversing heat flow thermogram.

Figure 5A is a bar graph showing secondary structure composition of silk lyogels determined by FTIR as a function of variable silk density.

Figure 5B is a bar graph showing secondary structure composition of silk lyogels determined by FTIR after exposure to various release medium conditions.

Figure 6A is a line graph showing antibody recovery as a function of swelling ratio with variable polysorbate 80 compared to the model describing the same relationship for variable silk density.

Figure 6B is a line graph showing antibody recovery as a function of swelling ratio with variable sodium chloride compared to the model describing the same relationship for variable silk density.

Figure 6C is a line graph showing antibody recovery as a function of swelling ratio with variable pH compared to the model describing the same relationship for variable silk density.

Figure 7 is a schematic representation of silk-antibody interactions governing release and recovery. In the normal condition (physiological PBS) hydrophobic attraction combined with an opposing ionic repulsion control antibody release. In the high silk density state hydration is decreased minimizing the disruption of hydrophobic interactions decreasing recovery. Addition of polysorbate 80 increases hydration and shields hydrophobic interactions improving recovery. High salt and low pH decrease recovery by eliminating the repulsive forces through ionic shielding and charge neutralization, respectively.

Figure 8 are line graphs depicting modification of lyogel antibody release profile following modification with a surfactant (Polysorbate 80). Polysorbate 80 was used in the release medium (see Figure 8A) or directly incorporated into the lyogel matrix (see Figure 8B).

Figure 9 depicts the results of antibody lyogel modification with an anti-oxidant modifier (methionione). Chromatogram overlays of the oxidized marker peptide at 47.5 minutes (Figure 9A) and oxidized methionine marker peptide levels (Figure 9B) were measured as a function of varying methionine levels incorporated in silk lyogels.

DETAILED DESCRIPTION

Silk fibroin is a naturally occurring protein polymer which can be processed into a wide range of useful biomaterial formats including sponges, films, micro/nanoparticles, coatings and hydrogels with a high degree of control. The use of silk fibroin as a versatile biomaterial, specifically its biocompatibility, all aqueous manufacturing process, controllable degradation rates, impressive mechanical properties, and favorable immunological properties are well documented. Specifically, silk based materials have been successfully used for sustained small molecule delivery, and enzyme, antibiotic and vaccine stabilization.

Lyogels leverage the use of sonication in the absence of antibody to induce a time- delayed gelation of silk fibroin through physical cross-links. The addition of antibody post sonication but prior to gelation results in an antibody loaded hydrogel. The ability to avoid antibody exposure to the heat and shear stresses caused by sonication generated a manufacturing process which maintained antibody biological function. The antibody loaded silk hydrogels are subsequently lyophilized, resulting in sustained release from the lyogel upon rehydration. Lyophilization is also an excellent approach for stabilization of proteins for long-term storage.

This disclosure provides methods and compositions based upon mechanisms controlling antibody release and recovery from silk lyogels. One driving force controlling antibody release and recovery from silk lyogels is the hydration behavior of the silk lyogel matrix. In certain embodiments, this hydration behavior is affected by silk matrix density or by the addition of a hydrophobicity modifier. Limited solvent penetration and availability for the disruption of silk-antibody hydrophobic interactions decrease antibody recovery. Further, ionic repulsions play a role in antibody recovery and release.

The disclosure provides silk lyogels and methods of using these lyogels as sustained delivery systems for therapeutic proteins such as monoclonal antibodies. The gentle manufacturing process and lyophilized final dosage form create a device compatible with fragile protein therapeutics. The mechanistic understanding of silk-antibody interactions governing release and recovery described herein defines the silk lyogels used as an optimized sustained delivery system for a given protein therapeutic.

This disclosure describes the protein-protein interactions which govern antibody release from silk lyogels. Silk hydration behavior, controlled in part by silk density, is one control point for antibody release. Antibody release is also impacted by changes to several lyogel parameters such as pH, ionic strength, levels of surfactant and presence of antioxidants.

Antibody release and recovery decreased significantly upon lyophilization and subsequent hydration, making modification of hydration properties an attractive target for controlling release from silk lyogels. Using swelling ratio as an indicator of hydration properties, a strong relationship between silk density and hydration was observed (Figure 2A). Both approaches for altering silk matrix density, namely increased silk solution concentration or pressing, resulted in significant changes to the hydration properties.

Differences in hydration behavior resulting from silk matrix density changes are independent of matrix structure as characterized by DSC (Figure 4) and FTIR (Figure 5). One hypothesis suggests that the primary mechanism of this relationship is the hydrophobicity of silk β- sheets that leads to the exclusion of water. A high density network of hydrophobic, water excluding β-sheets decreases the propensity for replacement water removed during lyophilization. Altering silk hydration and solubilization properties by various materials processing techniques such as water, solvent or vapor annealing has been previously demonstrated.

Lyogel hydration propensity is hypothesized to impact antibody release in two ways. The limited ability of the release medium to successfully penetrate the three dimensional matrix will directly impact antibody release. Also, a water- limited environment is not favorable for the reversal of silk-antibody interactions. Hydrophobic silk-antibody interactions replace silk-water and antibody-water interactions disrupted during the lyophilization process. The sigmoidal relationship between lyogel swelling and antibody recovery (Figure 2B) empirically describes the primary mechanism governing antibody release from silk lyogels - hydration resistance. A minimum threshold hydration level is required for sufficient solvent penetration into the matrix to initiate antibody release. This threshold occurred at a swelling ratio of 3, corresponding to a silk density of approximately 185 mg cm^" . Antibody recovery varied less with density above a swelling ratio of 11, a silk density of approximately 60 mg cm^"3. The decreased variance indicates a slow approach to saturating levels of solvent penetration for maximum diffusional release and displacement of silk-antibody interactions.

To determine if hydration resistance is the sole parameter controlling antibody release and recovery a series of release media modification studies were performed. Polysorbate 80 (Figure 3 A) incrementally increased antibody recovery while increasing levels of sodium chloride (Figure 3B) and decreasing pH (Figure 3C) both decreased antibody recovery. Swelling ratio versus recovery results for each of the release media modifiers were overlaid with the sigmoidal model describing the hydration/recovery relationship (Figure 6). A linear regression was performed for the swelling/recovery data from each release media modifier. The analysis was intended primarily for visualization purposes. The empirical

swelling/recovery slope with varying polysorbate 80 levels was similar to the model over the same swelling ratio range. The similarity in slope implies polysorbate 80, as well as other surfactants, alters antibody recovery by modifying lyogel hydration properties in a manner consistent with silk matrix density changes. In contrast, the empirical swelling/recovery slopes for sodium chloride and pH were significantly higher than the model over the same swelling ratio range. This observation implies that swelling behavior had little effect on antibody release indicating a secondary mechanism to hydration resistance.

This suggests a multi-modal mechanism controlling antibody release and recovery from silk lyogels (Figure 7). Hydrophobic attraction is one driving force which traps the antibody preventing release. The ability to disrupt these interactions is governed by hydration resistance which in turn is controlled by silk matrix density. Repulsive ionic interactions, which mitigate the hydrophobic attraction, are the other mechanism. The isoelectric points for silk fibroin and the antibody are 4.5 and 5.0, respectively. At a physiological pH of 7.4 both molecules are negatively charged, resulting in a repulsive force. This counterbalance of hydrophobic attraction and ionic repulsion is diagrammed in the "normal" state of Figure 7.

In the high silk density state hydration is decreased, water available for interaction is decreased, silk-antibody interactions are not displaced and an increase in antibody retention is observed. In the presence of surfactant, hydrophobic regions are shielded. Hypothetically, the primary effect is an increase in hydration as the amphiphilic polysorbate molecules bind to the most hydrophobic domains of the silk matrix facilitating the uptake of water.

Surfactant is known to prevent protein-protein interactions and could therefore directly interfere with silk-antibody interactions facilitating release and recovery. Neither variable silk density nor varying levels of surfactant had a detectable impact on silk matrix structure.

Antibody recovery behavior in the presence of salt and low pH both support the hypothesis of secondary ionic interactions. At high levels of salt, ionic repulsion is shielded, effectively increasing antibody retention through the hydrophobic attraction. At low pH, as both molecules approach their isoelectric point, their net negative charge neutralizes. In the absence of surface charge, the ionic repulsion is again eliminated, increasing the hydrophobic attraction. No changes in silk matrix structure were observed after storage in high salt or low pH solutions.

Combined, this mechanistic understanding indicates that antibody release can be controlled by altering either hydration or charge properties of the silk matrix. A variety of strategies, either individually or in combination, are employed, as described herein, to manipulate these properties. In certain embodiments, the lyogel matrix is modified indirectly by changing processing parameters such as silk pH, antibody pH, freezing rates,

lyophilization cycle, and residual moisture or through the incorporation of excipients such as salts, surfactants, hydrophilic molecules or other stabilizers to prevent silk-antibody interaction. Direct matrix modification can also be accomplished through chemical modification or the introduction of a copolymer. Finally, the therapeutic molecule could be selected or engineered with different hydrophobicity or surface charge to aid in optimization of the sustained local delivery system.

The silk lyogel can have other modifications made to improve its interaction with proteins contained and later released from the lyogel. One example of these modifications is a protein stability modifier. In certain embodiments, this modifier is an antibody stability modifier. Specifically, this antibody stability modifier can be an anti-oxidant. As shown in Figure 9, the addition of an anti-oxidant to a silk lyogel prevented antibody oxidation of methionine within the lyogel that prevented antibody release.

In certain embodiments, the silk lyogel is modified via a hydrophobicity modification, an ionic modification a stability modification or any combination of the three. A

hydrophobicity modification increases or decreases the ability of a silk lyogel to exclude water. For example, compressing a silk lyogel, or increasing the concentration of silk in the lyogel can modify the hydrophobicity of the lyogel, thus making the lyogel more resistant to hydration and allowing a protein associated with the lyogel to elute more slowly from the lyogel when it is placed in aqueous solution. Another hydrophobicity modification is the addition of a surfactant to the hydrogel. The surfactant interacts with hydrophobic moieties in the silk lyogel, blocking them from repelling water from the lyogel. Thus, incorporation of surfactants to lyogel increases the rate of protein elution from the lyogel when they are placed in aqueous solution.

An ionic modification decreases the repulsive force between negatively charged antibody and silk, allowing the antibody to elute more slowly from the silk lyogel. Thus, increasing the amount of salt present in a silk lyogel decreases the repulsion between a protein, e.g. an antibody, and thus decreases its rate of elution. Similarly, decreasing the pH of the solution that is lyophilized to form a silk lyogel has the same effect.

A stability modification increases the stability of the proteins within the lyogel. For example, a stability modification can be the addition of an anti-oxidant which prevents oxidizing interactions between the protein and the lyogel. This tends to increase release of the protein from the lyogel.

Preparation of Silk

The silk used herein can be prepared using any method known in the art. For example, an all-aqueous process for producing silk biomaterials, e.g., electrospun silk fibers, films, foams and mats can be used as disclosed in International Publication No. WO

2004/000915, which is incorporated by reference herein in its entirety. This process includes adding a biocompatible polymer to an aqueous solution of a silk protein. The solution is then processed to form a silk bio material.

The silk protein suitable for use in the present invention can be fibroin or related proteins {i.e., silks from spiders). Preferably, fibroin or related proteins are obtained from a solution containing a dissolved silkworm silk or spider silk. The silkworm silk is obtained, for example, from Bombyx mori. Spider silk may be obtained from Nephila clavipes. In the alternative, the silk protein suitable for use in the present invention can be obtained from a solution containing a genetically engineered silk, such as from bacteria, yeast, mammalian cells, transgenic animals or transgenic plants. See, for example, WO 97/08315 and US Patent 5,245,012, incorporated herein by reference in their entireties.

The silk protein solution can be prepared by any conventional method known to one skilled in the art. For example, B. mori cocoons can be boiled for about 30-60 minutes in an aqueous solution. Preferably, the aqueous solution is about 0.02 M Na₂C0₃. The cocoons can be rinsed, for example, with water to extract the sericin proteins. The extracted silk can then be dissolved in an aqueous salt solution. Salts useful for this purpose include lithium bromide, lithium thiocyanate, calcium nitrate or other chemical capable of solubilizing silk. A strong acid such as formic or hydrochloric may also be used. Preferably, the extracted silk is dissolved in about 9-12 M LiBr solution. The salt is consequently removed using, for example, dialysis. In other embodiments, purification of the silk fibroin by removal of sericin protein was achieved by boiling approximately 4 cm² silk cocoon pieces in a 0.02 M sodium carbonate solution for 60 m. The silk fibroin was rinsed with ambient UPW three times and air dried at ambient temperature for a minimum of 12 h. After drying, the fibroin was solubilized at 20% (w/v) in a 9 M aqueous lithium bromide solution at 60°C for 60 m. The resulting solution was dialyzed against UPW for 48 h using a 3500 MWCO Slide-A-Lyzer cassette (Thermo Fisher Scientific Inc., Rockford, IL). The silk concentration was determined by comparing the mass of 0.2 mL of aqueous silk and dried silk after storage at 60°C for a minimum of 12 h. The stock silk solution was diluted with UPW to prepare lower concentration silk solutions and dialyzed against 20% (w/v) polyethylene glycol (10,000 g mol^"1) at room temperature to produce high concentration silk solutions. A silk to PEG ratio of 1 :33 was necessary for proper dialysis to higher concentration silk solutions. Silk fibroin solutions were stored at 5°C.

The silk fibroin can be made up of monomers comprising the amino acid sequence of (Gly-Ser-Gly-Ala-Gly-Ala)„, wherein n is between 5 and 70. In other embodiments, n is between 10 and 60, 15 and 55, 20 and 50, 25 and 45, 30 and 40, 10 and 50, 10 and 40, 10 and 30, 10 and 20, 20 and 70, 30 and 70, 40 and 70, 50 and 70 or 60 and 70. In other

embodiments, n is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 and 70. In other embodiments, silk fibroin is made up of between 40 and 60% glycine. In other embodiments, the silk fibroin is made up of between 40 and 55, 45 and 60 or 45 and 55% glycine. In yet other embodiments, the silk fibroin is about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60% glycine. The silk fibroin can have a molecular weight between 0.5 and 400 kDa. In other embodiments, the molecular weight of the silk fibroin is between 0.5 and 400, 50 and 350, 100 and 300, 150 and 300 and 200 and 250 kDa. In other embodiments, the molecular weight of the silk fibroin is about 0.5, 50, 100, 150, 200, 250, 300, 350 or 400 kDa.

Preparation ofLyogels

Lyogels can be prepared according to any method known in the art. According to certain embodiments, lyogels are formed through lyophilization of a sonicated silk fibroin solution. The silk fibroin solution can be between 2 and 18% silk. In other embodiments, the silk fibroin solution is between 3 and 15, 5 and 10, 6 and 8%, 3-4 and 2.8-3.5% silk. In other embodiments, the silk fibroin solution is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17 or 18% silk. In certain embodiments, first the silk fibroin is sonicated, then it is cooled and then lyophilized. Lyophilization can be performed according to any method known in the art.

The therapeutic agent is generally added to the silk fibroin solution prior to lyophilization. It can be added before or after sonication. However, adding the therapeutic after lyophilization prevents the therapeutic from undergoing heating and shear stress during sonication.

Hydrophobicity modifications and/or ionic modifications can be made before or after lyophilization. When the hydrophobicity modification is compressing the lyogel to increase density, this is generally done after lyophilization.

Compression can be accomplished by applying pressure on the silk lyogel. In certain embodiments, the lyogel is pressed between two flat objects applying pressure to the lyogel. In certain embodiments, the pressure ranges from 200-4000 psi. In other embodiments, the pressure ranges between 200-4000, 500-3500 or 1000-2500 psi. In other embodiments, the pressure ranges between 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600- 1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-2900, 2900-3000, 3000-3100, 3100- 3200, 3200-3300, 3300-3400, 3400-3500, 3500-3600, 3600-3700, 3700-3800, 3800-3900 or 3900-4000 psi. The pressure can be applied for 1-1000 seconds. In other embodiments, the pressure is applied for 1-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850- 900, 900-950 or 950-1000 seconds. The pressure can be applied for 1-20, 5-15 or 7-12 seconds. The pressure can be applied for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 seconds.

When the hydrophobicity modification is addition of a surfactant, this modification is generally performed prior to lyophilization. It can occur prior to or after sonication. In certain embodiments, the surfactant is a non-ionic surfactant, an anionic surfactant, a cationic surfactant or a zwitterionic surfactant. Non-ionic surfactants include polysorbates, poloxamers and polyoxyethylene sorbitan monoethers. Polysorbates include polysorbate 20, 40, 60 or 80. Poloxamers include all versions of Pluronics®. Anionic surfactants include fatty acid soap, higher alkyl sulfate ester salt, alkyl ether sulfate ester salt, N-acyl sarcosinic acid, higher fatty acid amide sulfonate, phosphate ester salt, sulfosuccinate, alkylbenzene sulfonate, higher fatty acid ester sulfate ester salt, N-acyl glutamate, sulfonated oil, POE- alkyl ether carboxylic acid, POE-alkyl aryl ether carboxylate, a-olefine sulfonate, higher fatty acid ester sulfonate, secondary alcohol sulfate ester salt, higher fatty acid alkylolamide sulfate ester salt, sodium lauroyl monoethanolamide succinate, N-palmitoyl asparaginate ditriethanolamine and sodium casein. Fatty acid soaps include those having the formula R— C(0)OM, wherein R is to C22 alkyl and M is a cation. Cationic surfactants include alkyltrimethyl ammonium salt, alkylpyridinium salt, distearyldimethyl ammonium chloride, dialkyldimethyl ammonium salt, poly (N,N'-dimethyl-3,5-methylenepiperidinium) chloride, alkyl quaternary ammonium salt, alkyldimethylbenzyl ammonium salt, alkylisoquinolinium salt, dialkylmorphonium salt, POE-alkylamine, alkylamine salt, polyamine fatty acid derivative, amyl alcohol fatty acid derivative, benzalkonium chloride and benzethonium chloride. Zwitterionic surfactants include alkyl amine oxides, alkyl betaines, alkyl amidopropyl betaines, alkyl sulpho betaines (sultaines), alkyl glycinates, alkyl

carboxyglycinates, alkyl amphoacetates, alkyl amphopropionates, alkylamphoglycinates, alkyl amidopropyl hydroxysultaines, acyl taurates and acyl glutamates. Surfactants can also be selected from octyl maltoside, decyl maltoside, dodecyl β-D-maltoside, tridecyl maltoside, tetradecyl maltoside and sucrose dodecanoate.

Ionic modifications are generally made prior to lyophilization. Ionic modifications include changing the pH of the silk solution to be lyophilized. This pH adjustment can be performed by any method known in the art. In one embodiment, a buffer is added to adjust pH. Buffer can be added prior to lyophilization and before or after sonication. Buffers include HEPES, Tris, MES, sodium phosphate, potassium phosphate, sodium acetate, sodium citrate, histidine, succinate, and potassium nitrate. pH can be adjusted to between 3 and 12. In other embodiments, the pH is adjusted to between 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 1 1 , or 11 and 12. In other embodiments, the pH is adjusted to between 4 and 8, 5 and 8, 6 and 8, 4 and 7, or 5 and 7. In other embodiments, the pH is adjusted to about 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

Ionic modifications also include adding salt of the silk solution to be lyophilized to increase the osmolarity of the silk solution to be lyophilized. Adding a salt can be done prior to lyophilization and before or after sonication. Appropriate salts include NaCl and KC1.

Salt can be added to adjust the osmolarity of the silk solution to be lyophilized to be between 1 mM and 5 M. In other embodiments, the osmolarity is adjusted to be between 1 mM and 5M, 150 mM and 3 M, 300 mM and 2 M, 500 mM and 1 M, 700 mM and 900 mM or 600 mM to 800 mM. In other embodiments, the osmolarity is adjusted to be about 1, 25, 50, 75, 100, 125, 150, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750 and 5000 mM.

Stability modifications are generally made prior to lyophilization. It can occur prior to or after sonication. Stability modifications include the addition of anti-oxidants. Antioxidants include methionine, thiosulfate, N-Acetyl-L-methionine and N-Acetyl tryptophan.

The silk lyogel has a significant amount of β-sheet content. This β-sheet content is one reason why silk lyogels are so water resistant. In certain embodiments, the silk lyogel has between 20 and 65% β-sheet content. In other embodiments, the silk lyogel has between 30 and 50, 35 and 45, 35 and 60, 40 and 60, 50 and 65 or 45 and 60% β-sheet content. In other embodiments, the silk lyogel has 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65% β-sheet content.

Sustained release

Sustained release permits dosages to be administered over time, with sustained release kinetics. In some instances, delivery of the therapeutic agent is continuous to the site where treatment is needed, for example, over several weeks. Sustained release over time, for example, over several days or weeks, or longer, permits continuous delivery of the therapeutic agent to obtain optimal treatment. The controlled delivery vehicle is advantageous because it protects the therapeutic agent from degradation in vivo in body fluids and tissue, for example, by proteases.

Sustained release from the silk lyogels described herein may be designed to occur over time, for example, for greater than about 12 or 24 hours. The time of release may be selected, for example, to occur over a time period from about 12 hours to 24 hours; from about 12 hours to 42 hours; or, e. g., from about 12 to 72 hours. In another embodiment, release may occur for example on the order of about 2 to 120 days, for example, from about 3 to 90 days. In another embodiment, the release may occur for between 60 and 120; 80 and 120; 100 and 120; 60 and 100; 60 and 80; 80 and 100; and 100 and 120 days. In one embodiment, the therapeutic agent is delivered locally over a time period of about 7-21 days, or about 3 to 10 days. In other instances, the therapeutic agent is administered over 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20 or more weeks in a controlled dosage. The sustained release time may be selected based on the condition treated. For example, longer times may be more effective for wound healing, whereas shorter delivery times may be more useful for some cardiovascular applications.

Sustained release of the therapeutic agent from the fibroin article in vivo may occur, for example, in the amount of about 1 ng to 1 mg/day, for example, about 50 ng to 500 ng/day, or, in one embodiment, about 100 ng/day. Delivery systems comprising therapeutic agent and a carrier may be formulated that include, for example, 10 ng to 1 mg therapeutic agent, or in another embodiment, about 1 μg to 500 μg, or, for example, about 10 μg to 100 μg, depending on the therapeutic application.

Sustained release can involve the release of between 0.5 and 100% of a therapeutic agent from a fibroin article over 160 days. In other embodiments, the amount of release of a therapeutic agent from a fibroin article is between 0.5 and 5, 10 and 20, 0.2 and 20, 10 and 100, 20 and 100, 30 and 100, 40 and 100, 50 and 100, 60 and 100, 70 and 100, 80 and 100 or 90 and 100% over 160 days. In other embodiments, the amount of release of a therapeutic agent from a fibroin article is about 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% after 160 days.

The silk lyogel may be administered by a variety of routes known in the art including topical, oral, parenteral (including intravenous, intraperitoneal, intramuscular and subcutaneous injection as well as intranasal or inhalation administration) and implantation. The delivery may be systemic, regional, or local. Additionally, the delivery may be intrathecal, e. g., for CNS delivery. For example, administration of the pharmaceutical formulation for the treatment of wounds may be by topical application, systemic administration by enteral or parenteral routes, or local or regional injection or implantation. The silk-based vehicle may be formulated into appropriate forms for different routes of administration as described in the art, for example, in "Remington: The Science and Practice of Pharmacy", Mack Publishing Company, Pennsylvania, 1995, the disclosure of which is incorporated herein by reference.

The silk lyogel may include excipients available in the art, such as diluents, solvents, buffers, solubilizers, suspending agents, viscosity controlling agents, binders, lubricants, surfactants, preservatives and stabilizers. The formulations may include bulking agents, chelating agents, and antioxidants. Where parenteral formulations are used, the formulation may additionally or alternately include sugars, amino acids, or electrolytes.

Excipients include polyols, for example of a molecular weight less than about 70,000 kD, such as sucrose, trehalose, mannitol, and polyethylene glycol. See for example, U. S. Patent No. 5,589,167, the disclosure of which is incorporated herein. Exemplary surfactants include nonionic surfactants, such as polysorbates, such as polysorbate 20 or 80, etc. , and the poloxamers, such as poloxamer 184 or 188, ethylene/polypropylene block polymers, etc. Buffers include Tris, citrate, succinate, acetate, or histidine buffers. Preservatives include phenol, benzyl alcohol, metacresol, methyl paraben, propyl paraben, benzalconium chloride, and benzethonium chloride. Other additives include carboxymethylcellulose, dextran, and gelatin. Stabilizing agents include heparin, pentosan polysulfate and other heparinoids, and divalent cations such as magnesium and zinc.

The pharmaceutical formulation of the present invention may be sterilized using conventional sterilization process such as radiation based sterilization (i.e. gamma-ray), chemical based sterilization (ethylene oxide),, autoclaving, or other appropriate procedures. Preferably the sterilization process will be with ethylene oxide at a temperature between 52 - 55° C for a time of 8 or less hours. After sterilization the formulation may be packaged in an appropriate sterilize moisture resistant package for shipment.

Therapeutic agents

The variety of different therapeutic agents that can be used in conjunction with the formulations of the present invention is vast and includes small molecules, proteins, peptides and nucleic acids. In general, therapeutic agents which may be administered via the invention include, without limitation: anti-infectives such as antibiotics and antiviral agents;

chemotherapeutic agents (i.e. anticancer agents); anti-rejection agents; analgesics and analgesic combinations; anti- inflammatory agents; hormones such as steroids; growth factors (bone morphogenic proteins (i.e. BMP's 1-7), bone morphogenic-like proteins (i.e. GFD-5, GFD-7 and GFD-8), epidermal growth factor (EGF), fibroblast growth factor (i.e. FGF 1-9), platelet derived growth factor (PDGF), insulin like growth factor (IGF-I and IGF-II), transforming growth factors (i.e. TGF- .beta. -Ill), vascular endothelial growth factor (VEGF)); anti-angiogenic proteins such as endostatin, and other naturally derived or genetically engineered proteins, polysaccharides, glycoproteins, or lipoproteins. Growth factors are described in The Cellular and Molecular Basis of Bone Formation and Repair by Vicki Rosen and R. Scott Thies, published by R. G. Landes Company, hereby incorporated herein by reference. Additionally, the silk based devices of the present invention can be used to deliver any type of molecular compound, such as, pharmacological materials, vitamins, sedatives, steroids, hypnotics, antibiotics, chemotherapeutic agents, prostaglandins, and

radiopharmaceuticals. The delivery system of the present invention is suitable for delivery of the above materials and others including but not limited to proteins, peptides, nucleotides, carbohydrates, simple sugars, cells, genes, anti-thrombotics, anti-metabolics, growth factor inhibitor, growth promoters, anticoagulants, antimitotics, fibrinolytics, anti-inflammatory steroids, and antibodies. Antibodies include monoclonal and polyclonal antibodies.

Antibodies can be IgA, IgD, IgE, IgG or IgM isotypes. A silk lyogel described herein can contain one or more different types of antibody. The concentration of antibody in a silk lyogel can range from 1 ng/mL to 320 mg/mL. The antibodies can specifically bind any therapeutically relevant target. Antibodies also include the following: abagovomab (95), afelimomab (80), altumomab (80), anatumomab mafenatox (86), arcitumomab (74), bectumomab (81), besilesomab (92), biciromab (66), capromab (80), detumomab (80), dorlimomab aritox (66), edobacomab (80), edrecolomab (74), elsilimomab (89), enlimomab (80), enlimomab pegol (77), epitumomab (82), epitumomab cituxetan (89), faralimomab (81), gavilimomab (84), ibritumomab tiuxetan (86), igovomab (86), imciromab (66), inolimomab (80), lemalesomab (86), maslimomab (66), minretumomab (80), mitumomab (82), nacolomab tafenatox (80), nerelimomab (81), odulimomab (81), oregovomab (86), satumomab (81), sulesomab (86), taplitumomab paptox (84), technetium (99mTc) fanolesomab (86), technetium (99mTc) nofetumomab merpentan (81), technetium (99mTc) pintumomab (86), telimomab aritox (66), tositumomab (80), vepalimomab (80), zolimomab aritox (80), adalimumab (85), adecatumumab (90), atorolimumab (80), belimumab (89), bertilimumab (88), denosumab (94), efungumab (95), exbivirumab (91), golimumab (91), ipilimumab (94), iratumumab (94), lerdelimumab (86), lexatumumab (95), libivirumab (91), mapatumumab (93), metelimumab (88), morolimumab (79), nebacumab (66), ofatumumab (93), panitumumab (91), pritumumab (89), raxibacumab (92), regavirumab (80), sevirumab (66), stamulumab (95), ticilimumab (95), tuvirumab (66), votumumab (80), zalutumumab (93), zanolimumab (92), ziralimumab (84), abciximab (80), basiliximab (81), bavituximab (95), cetuximab (82), clenoliximab (77), ecromeximab (87), galiximab (89), infliximab (77), keliximab (81), lumiliximab (90), pagibaximab (93), priliximab (80), rituximab (77), teneliximab (87), vapaliximab (87), volociximab (93), alemtuzumab (83), apolizumab (87), aselizumab (88), bapineuzumab (93), bevacizumab (86), bivatuzumab (86), cantuzumab mertansine (89), cedelizumab (81), certolizumab pegol (90), daclizumab (78), eculizumab (87), efalizumab (85), epratuzumab (82), erlizumab (84), felvizumab (77), fontolizumab (87), gemtuzumab (83), inotuzumab ozogamicin (92), labetuzumab (85), lintuzumab (86), matuzumab (88), mepolizumab (81), motavizumab (95), natalizumab (79), nimotuzumab (94), ocrelizumab (95), omalizumab (84), palivizumab (79), pascolizumab (87), pertuzumab (89), pexelizumab (86), ranibizumab (90), reslizumab (85), rovelizumab (81), ruplizumab (83), sibrotuzumab (86), siplizumab (87), sontuzumab (94), tadocizumab (94), talizumab (89), tefibazumab (92), tocilizumab (90), toralizumab (87), trastuzumab (78), tucotuzumab celmoleukin (95), urtoxazumab (90), visilizumab (84), yttrium 90Y tacatuzumab tetraxetan (93). The number following each antibody is the number corresponding to the list on the International Nonproprietary Name (INN) list of names produced by the World Health Organization. The mentioned lists are incorporated by reference herein in their entireties.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. In addition, the materials, methods and examples are illustrative only and not intended to be limiting. In case of conflict, the present specification, including definitions, controls.

EXAMPLES

Methods

Materials

Bombyx mori silkworm cocoons were purchased from Tajima Shoji Co., LTD (Sumiyashicho, Naka-Ku, Yokohama, Japan) and used to produce silk fibroin solutions and. All studies were conducted using purified murine Immunoglobulin G type 1 (IgGl) monoclonal antibody provided by Genzyme Corporation (Framingham, MA). All chemicals used in the production of silk and the preparation of solutions were reagent grade and purchased from either Sigma- Aldrich (St. Louis, MO) or Mallinckrodt Baker, Inc.

(Phillipsburg, NJ). Lyophilization of the antibody was performed in clear type I borosilicate glass serum vials obtained from Wheaton Industries, Inc. (Millville, NJ). All aqueous solutions were prepared using ultrapure water (UPW) with <5 ppb total organic carbon (TOC) and an 18.2 ΜΩ resistivity produced by a Millipore Milli-Q Advantage A10 purification system (Billerica, MA). Lyophilized antibody powders

Lyophilized antibody powders were prepared using the lyophilization method described previously. Antibody vials were prepared by placing 2.5 mL at 5 mg mL^"1 in 0.02 M histidine, 0.5% (w/v) sucrose, pH 6.0 into a 5 mL serum vial with a vented silicone stopper. Lyophilization was performed using a LyoStar II tray freeze dryer (FTS Systems, Stone Ridge, NY). The initial freezing step was performed at -45 °C for 8 h. Primary drying was executed at -20°C, 100 mTorr for 40 h. Secondary drying was executed at 35°C, 100 mTorr for 11 h. At the end of the lyophilization cycle, the silicone stoppers were depressed under a vacuum of 600,000 mTorr and the vials were sealed using aluminum tear off caps. The lyophilized antibody samples were stored at 5°C ± 3°C immediately following lyophilization.

Preparation of concentrated silk fibroin solution

Concentrated silk fibroin solutions were produced using the aqueous process described previously. (Kim et al., Structure and Properties of Silk Hydrogels.

Biomacromolecules 2004, 5, (3), 786-792, incorporated by reference herein in its entirety). Briefly, purification of the silk fibroin by removal of sericin protein was achieved by boiling approximately 4 cm silk cocoon pieces in a 0.02 M sodium carbonate solution for 60 m. The silk fibroin was rinsed with ambient UPW three times and air dried at ambient temperature for a minimum of 12 h. After drying, the fibroin was solubilized at 20% (w/v) in a 9 M aqueous lithium bromide solution at 60°C for 60 m. The resulting solution was dialyzed against UPW for 48 h using a 3500 MWCO Slide-A-Lyzer cassette (Thermo Fisher Scientific Inc., Rockford, IL). The silk concentration was determined by comparing the mass of 0.2 mL of aqueous silk and dried silk after storage at 60°C for a minimum of 12 h. The stock silk solution was diluted with UPW to prepare lower concentration silk solutions and dialyzed against 20% (w/v) polyethylene glycol (10,000 g mol^"1) at room temperature to produce high concentration silk solutions. A silk to PEG ratio of 1 :33 was necessary for proper dialysis to higher concentration silk solutions. Silk fibroin solutions were stored at 5°C. Preparation of silk hydrogels and lyogels

The silk hydrogels and lyogels were prepared using the methods described previously. Sonication was performed on 8 mL of silk fibroin solution in a 15 mL conical tube using a Branson 450D sonifier equipped with a 3.175 mm diameter tapered microtip (Branson Ultrasonics Co., Danbury, CT). Sonication power was adjusted for each silk concentration from 20% to 65% amplitude to achieve a sol-gel transition within 2 h. The sonicated solutions were cooled in a room temperature water bath for 1 m after each sonication step, 30 s and 10 s, sequentially. A single hydrogel pellet was prepared by using a positive displacement pipette to place 0.2 mL of sonicated fibroin solution into a 96 well plate. The plate was allowed to sit at room temperature uncovered until successful sol-gel transition, in which the solution turned opaque and water droplets appeared on the surface. The lyogels were prepared by lyophilizing the hydrogels in the 96 well plates. To maximize the heat transfer from the lyophilizer shelf, aluminum blocks were placed under the plastic plate. Lyophilization was performed using the parameters described above.

For hydrogels and lyogels containing antibody, lyophilized antibody was added to the sonicated solutions to a target concentration of 5 mg mL^"1. The solution was gently inverted to promote homogeneity and dissolution of lyophilized antibody into the solution. After sufficient mixing, the solution was transferred to 96 well plates and allowed to gel as previously described.

Lyogels were pressed into discs using a 12-ton EZ press and 6 mm and 13 mm die sets (Crystal Laboratories International, Garfield, NJ). The lyogels were pressed with the 6 mm die at 1 ,000 psi for 10 s. Some samples were pressed a second time with the 13 mm die at 2,500 psi for 10 s. Antibody release studies

Antibody release studies were performed in 0.5 mL of release buffer stored in a 1.2 mL polystyrene screw top vial at 37°C. Pressed lyogels were stored in 1.5 mL conical micro centrifuge tubes. Each lyogel was placed in release medium and transferred to a new vial with fresh release buffer at various time points. Prior to each transfer, excess buffer was removed from each hydrogel or lyogel by contact with the inside surface of the vial.

The standard PBS release medium consisted of 0.02 M sodium phosphate, 0.15 M sodium chloride, pH 7.4, and 0.01% (w/v) sodium azide. Variable polysorbate 80 release buffers were made by spiking stock solutions to the standard release media to final concentration of 0.001 , 0.01, 0.1 and 1.0% (w/v). Variable pH release media was prepared using a multi component buffer system consisting of 0.01 M succinate, 0.01M histidine, 0.01M phosphate, 0.15M sodium chloride and 0.01% (w/v) sodium azide titrated to pH values of 4, 5, 6, 7 and 8. Solutions containing 0.02 M sodium phosphate, 0.01 % (w/v) sodium azide with varying levels of sodium chloride, 0, 0.15, 0.5, 1 and 3M were titrated to pH 7.0 for use in the variable salt release studies.

Quantitation of antibody release

Affinity chromatography was used to determine the amount of antibody released at each time point using a Protein G cartridge (Applied Biosystems, Carlsbad, CA) and a 1200 series HPLC (Agilent Technologies, Santa Clara, CA). The binding and elution mobile phases were 0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.3 and 0.012 M hydrochloric acid, 0.15 M sodium chloride, pH 2.0, respectively. A flow rate of 2 mL min^"1 was used. Antibody standards were employed to generate a standard curve for each assay instance to determine the antibody concentrations at each release time point. Protein G recovery from the various release buffers was confirmed using spiked samples with known antibody concentrations. With the exception of the sample containing no sodium chloride, all recoveries were within 10% of the target value. Raw data from the samples containing no sodium chloride was adjusted to account for the 80% recovery consistently observed.

Swelling properties

The swelling properties of each lyogel were tracked at each time point. Prior to transferring the lyogel to fresh release medium, excess buffer was removed from the lyogel by contact with the inside surface to the polystyrene vial and the weight of the rehydrated lyogel was determined (W_r). The swelling ratio was calculated using the following equation:

W -

Swelling ratio =— ^:—

Where WA is the mass of the dried lyogel. The reported swelling ratio is the first

reading after a plateau in fluid uptake was observed, typically after 1-2 days.

Fourier transform infrared spectroscopy (FTIR) Secondary structure of the silk matrix was analyzed by Fourier transform infrared spectroscopy (FTIR) on an MB series spectrometer (ABB Bomem, Quebec, Canada) equipped with a MIRacle attenuated total reflection (ATR) diamond crystal (Pike

Technologies, Madison, WI). Data acquisition and analysis were performed using PROTA (Bio Tools, Inc., Jupiter, FL) together with RazorTools 6.0 (Spectrum Square Associates, Inc., Ithaca, NY). Each spectrum was collected as a 400 scan interferogram with a 4 cm^"1 resolution in single-beam mode from 400 to 4000 cm^"1. Silk lyogels were pressed onto the crystal using the attached high-pressure clamp equipped with a flat tip. Protein absorbance spectra in the amide I and amide II regions were obtained by water and water vapor subtraction procedures established previously.

Fourier self-deconvolution (FSD) of the amide I region (1590-1710) was performed using a Lorentzian peak shape with a half-bandwidth of 25 cm^"1 and a K factor of 2.0 to quantify secondary structure composition. Second derivative analysis with five point Savitsky-Golay smoothing of the non-FSD spectrum was performed to identify contributing bands. The baselines of the FSD spectra were manually corrected and a maximum entropy Gaussian curve fitting algorithm was executed using peak positions identified in the second derivative ± 2 cm^"1, a maximum peak width of 20 cm^"1, and variable peak height. Structural assignment to peak positions was made using wave number ranges described by Lu, Q., et al, Water-insoluble silk films with silk I structure. Acta biomaterialia 2010, 6, (4), 1380-1387, incorporated herein by reference in its entirety. Structural composition was determined by relative areas of the fit cures.

Modulated differential scanning calorimetry (mDSC)

Thermal properties of silk lyogels were investigated using temperature modulated differential scanning calorimetry on a Q2000 DSC equipped with the RCS90 refrigeration system (TA Instruments, New Castle, DE). The instrument was operated with a nitrogen purge at 50 mL min^"1. Calibration for temperature was performed using indium and for heat capacity using sapphire. Silk lyogel samples, 5-10 mg, were loaded into Tzero aluminum pans (TA Instruments, New Castle, DE) and sealed. The samples were heated at 2°C min^"1 from -40°C to 350°C with a modulation period of 60 s and temperature amplitude of 0.318°C.

Peptide map Antibody post translational modifications, such as oxidation, were analyzed by LC- MS peptide map using a reduced, alkylated, single enzyme (Trypsin) digestion protocol. Briefly, 100 μg of each sample was diluted with reduction and alkylation buffer (6 M

Guanidine-HCl, 0.25 M Tris-Cl pH 8.5). Reduction of disulfides was accomplished by the addition of 1M DTT and the incubation for 30 minutes at 60°C (dark). Free cysteines were alkylated with 2 M Iodoacetic acid for one hour at room temperature (dark). Biospin columns were used to desalt and buffer exchange the sample into 50 mM Tris, pH 7.1.

Digestion with trypsin (1 :25) occurred for 2 hours at 37°C, and was quenched with 2% TFA. Digests were frozen and thawed at room temperature before analysis.

Chromatographic separation was performed with an Acquity UPLC® (Waters,

Milford, MA) equipped with a BEH300 C18 column (2.1 x 150 mm, 1.7 μιη) (Waters, Milford, MA) using a water/acetonitrile. Digests containing 25 μg of antibody were injected and the column was equilibrated to 45°C.

Mass spectrometry analysis was performed using an LCT Premiere™ ESI TOF instrument (Waters, Milford, MA) driven by MassLynx 4.1 (Waters, Milford, MA). The collected mass range was 200 to 2000. Desolvation gas flow was 600.0. The capillary, sample cone and aperture 1 voltage was 3500.0, 35.0 and 2.5 V respectively. Quantitation was performed using QuanLynx^1M (Waters, Milford, MA). Statistical analysis

Each data point is the average of five individual lyogels in a specified release medium and is reported ± standard deviation (SD), unless noted otherwise.

Example 1. Antibody release from variable density silk lyogels.

The impact of silk density on IgGl monoclonal antibody release was evaluated.

Initially, 1.0 mg of lyophilized antibody was loaded at a target concentration of 5 mg mL^"1 into sonicated silk solutions ranging in concentration from 3.1 % (w/w) to 6.7% (w/w). In this configuration, the ratio of silk to antibody increased with increasing silk density. To simulate physiological conditions, PBS at pH 7.4 was used as a release medium. The release medium was exchanged at pre-determined intervals. Antibody released was measured by protein G- ID affinity chromatography. For all release studies, data presented in the figures was truncated for simplified visualization when cumulative release approached approximately 85% of the final cumulative release observed, which is reported in the tables. Both release rate and cumulative release amount decreased incrementally with increasing silk concentration (Figure 1A). In the 3.1 % (w/w) silk lyogel samples, release was nearly complete at day eight with 79.6% of the loaded antibody recovered. In contrast, at day eight only 17.9% of the loaded antibody was released from 6.7% (w/w) silk lyogels.

Antibody release from 6.7% (w/w) lyogels continued through 160 days reaching a final cumulative release of 36% (Table 1). Lyogel hydration, as measured by the swelling ratio, decreased incrementally from 16.0 to 6.7 with increasing silk concentration (Table 1). The reported swelling ratio is the first mass reading after a plateau in fluid uptake was observed, typically after 1 -2 days.

Silk density was further altered by compacting 3.2% (w/w) antibody loaded silk lyogels using a hydraulic press at either 1,500 or 2,500 psi. This procedure increased silk density 44.9 mg cm^"3 to 1,045.3 mg cm^"3 and 1 ,320.5 mg cm^"3 for the low and high pressure condition respectively (Table 1 ). Table 1. Numerical summary of swelling and antibody recovery from variable silk density lyogels

Sample Silk density (mg cm^" ) Swelling ratio Antibody recovery (%)

3.1% silk 51.6 (+2.6) 16.0 (+0.6) 81.3 (+7.7)

4.3% silk 56.4 (+2.7) 13.8 (+1.6) 74.2 (+2.3)

5.5% silk 63.3 (+1.8) 9.1 (+0.9) 52.2 (+3.5)

6.7% silk 80.4 (+5.1) 6.7 (+0.3) 36.0 (+2.0)

Not pressed 44.9 (+3.7) 14.5 (+1.0) 69.8 (+7.2)

1500 psi 1045.3 (+36.8) 0.6 (+0.01) 15.6 (+1.6)

2500 psi 1320.5 (+40.5) 0.5 (+0.08) 3.0 (+0.6)

In this configuration the silk to antibody ratio remained constant, while the silk density increased. Antibody release (Figure IB) and swelling ratio (Table 1) both decreased significantly in the pressed lyogel samples. Antibody release was suppressed to only 3% in the high pressure sample compared to 69.8% in the control (Table 1). The swelling ratio decreased to 0.5 for the 2,500 psi sample compared to 14.5 in the control. Intermediate values for swelling ratio and antibody recovery were observed in the 1,500 psi sample.

Silk density versus swelling data from 19 lyogel preparations was compared (Figure 2A) to further characterize this relationship. Increasing silk density significantly decreased the swelling ratio of silk lyogels. Silk densities below 200 mg cm^"3 were obtained by varying silk concentration from 3.1 % (w/w) to 12.4% (w/w). The variable silk concentration lyogels produced swelling ratios from 17.3 to 3.3. Silk densities of approximately 1,000 mg cm^" were obtained by pressing. The high density pressed matrices further reduced swelling ratios to 0.8 to 0.4.

The relationship between hydration and antibody recovery was characterized by comparing release to hydration from historical silk lyogel studies (Figure 2A). The release/hydration data was well described empirically by the four parameter sigmoidal curve described below. a

y ⁼ o ^"I - p

1 + e ^{ b >

The values of a, b, yo and xo are 70.4, 1.7, 3.0 and 6.9, respectively. Maximum and minimum antibody recovery shows asymptotic behavior as a function of swelling ratio. Antibody recovery did not change significantly at swelling ratios below 3 and above 11. The most dynamic antibody recovery response is observed between swelling ratios of 3 and 11 with recoveries of approximately 10% and 67%, respectively. Based on the relationship described in Figure 2A, the swelling ratio range above correlates to silk densities of approximately 60 to 185 mg cm^" .

Example 2. Antibody release into variable release medium.

To characterize the nature of interactions governing antibody release from lyogels, a series of experiments was performed evaluating the impact of various modifiers on antibody release. Three solution variables were investigated: varying surfactant levels, varying ionic strength and varying solution pH. Antibody release studies were performed as described in the methods sections with the exception of the altered release media. These experiments relied on changing release medium rather than incorporating each modifier directly into the lyogel matrix. Incorporating each of these modifiers directly into the matrix would result in a high probability of altering the matrix itself. By only changing the release medium, a consistent starting point for each level of modifier was ensured. As they demonstrate an intermediate release and recovery profile, silk lyogels at 6.8% (w/w) were used for these experiments.

Silk lyogels at 6.8% (w/w) were immersed into PBS at pH 7.4 with varying levels of the non-ionic surfactant polysorbate 80. Polysorbate 80 levels in the range of 0.001 % to

1.0% were evaluated. Increasing levels of polysorbate 80 incrementally increased the amount of antibody released from the lyogels (Figure 3A). Antibody recovery improved from 39.5% in the absence of polysorbate 80 to 68.8% in the presence of 1.0% polysorbate in the release medium (Table 2).

Table 2. Numerical summary of swelling and antibody recovery from variable release medium silk lyogels

Sample Silk density (mg cm^"3) Swelling ratio Antibody recovery (%)

0 PS80 85.3 (+4.8) 6.4 (+0.4) 39.5 (+8.4)

0.001% PS80 81.0 (+1.6) 6.8 (+0.4) 48.8 (+5.9)

0.01% PS80 88.0 (+4.2) 7.8 (+0.5) 58.1 (+8.3)

0.1% PS80 83.2 (+2.6) 8.3 (+0.7) 63.4 (+6.3)

1.0% PS80 85.1 (+3.2) 7.7 (+0.4) 68.8 (+6.7)

0 mM NaCl 79.4 (+4.1) 7.1 (+0.1) 40.8 (+6.3)

150 mM NaCl 75.8 (+2.7) 7.0 (+0.7) 41.2 (+1.8)

500 mM NaCl 76.7 (+3.7) 6.5 (+0.2) 36.6 (+4.9)

l .O M NaCl 74.5 (+1.4) 6.6 (+0.4) 30.5 (+1.6)

3.0 M NaCl 77.1 (+3.2) 5.5 (+0.4) 6.7 (+1.3)

pH 4.0 76.2 (+1.8) 6.5 (+0.5) 4.6 (+0.5)

pH 5.0 81.6 (+6.1) 6.3 (+0.5) 12.3 (+1.5)

pH 6.0 85.6 (+6.9) 6.5 (+0.4) 30.6 (+2.4)

pH 7.0 83.3 (+0.6) 7.1 (+0.4) 38.2 (+1.3)

pH 8.0 85.6 (+5.8) 6.9 (+0.2) 39.9 (+1.8)

The swelling ratio increased from 6.4 to 8.3 with increasing polysorbate concentration (Table 2).

Solution ionic strength was altered by increasing levels of sodium chloride in a 20 mM sodium phosphate solution at pH 7.4. Sodium chloride levels of 150 mM, 300 mM, 1.5 M and 3.0 M were evaluated. Increasing levels of sodium chloride incrementally decreased antibody release from the lyogels (Figure 3B). Antibody recovery decreased from 40.8% in the absence of sodium chloride to 6.7% at 3 M sodium chloride. The swelling ratio decreased incrementally from 7.1 to 5.5 (Table 2).

Solution pH was varied from 4.0 to 8.0 in 1.0 unit increments. The varying pH was achieved using a multi-component buffering system (10 mM succinate, 10 mM histidine, 10 mM phosphate) to ensure comparable solution composition across the pH range. Decreasing the pH from 8.0 to 4.0 resulted in a significant and incremental reduction in antibody release (Figure 3C). Antibody recovery was highest at pH 8.0 (39.9%) and lowest at pH 4.0 (4.6%) (Table 2). The swelling ratio remained constant across the pH range (Table 2). Example 3. Structural characterization of variable density silk lyogels

Temperature modulated differential scanning calorimetry (mDSC) was performed to characterize the bound water, glass transition and degradation of varying density silk lyogels. Lyogels ranging in density from 48.6 mg cm^"3 (3.1% (w/w)) to 950 mg cm^"3 (6.7% (w/w) pressed) were evaluated from -50°C to 350°C. Two transitions were observed in the non- reversing heat flow thermograms (Figure 4A). The first endothermic transition at approximately 75°C is associated with the evaporation of bound non-freezing water. The amount of residual water, as measured by the transition enthalpy, did not change as a function of increasing silk density. While the enthalpy of the pressed sample water transition was similar to the not pressed samples, the peak width increased considerably. The change in peak shape can be attributed to the increased packing density of the pressed matrix, making escape of the trapped water more difficult. The second, larger endothermic transition at approximately 275°C represents the thermal degradation of the silk matrix. The degradation of the matrix occurred at similar temperatures regardless of silk density. A single endothermic transition at approximately 195°C representing the glass transition of pure silk was observed in the reversing heat flow thermogram (Figure 4B). No differences in the glass transition temperature were observed across the range of silk densities.

To understand if silk structure plays a role in antibody release, FTIR characterization was performed. The infrared (IR) region of 1700-1500 cm^"1 corresponds to peptide backbone absorption for amide I (1700-1600 cm^"1) and amide II (1600-1500 cm^"1) bands. Absorbance spectra in this region are used for evaluating the secondary structure composition of various proteins, including silk fibroin.⁴⁰' ⁴¹ Calculation of secondary structure composition through deconvolution of the amide I band was performed. Silk lyogels of varying density (Figure 5A) and lyogels exposed to each modifier (Figure 5B) were evaluated. In all samples silk lyogels demonstrated primarily β-sheet structure, accounting for approximately 60% of the structural composition. Neither increasing silk density (Figure 5A) nor exposure to solution modifiers (Figure 5B) significantly altered the structural composition. Example 4. Silk matrix modifications for improved antibody release

In this example, release buffer modifications were employed to study the nature of silk-antibody interactions. In particular, one of the release medium modifiers shown to improve antibody recovery was the surfactant Polysorbate 80. The amphiphilic nature of this surfactant was proposed to improve silk lyogel hydration properties, leading to higher antibody recovery. As human plasma cannot be made to contain 1% polysorbate 80, this knowledge had to be applied to modify the matrix itself to produce a similar effect. The most direct application of this information was to incorporate varying levels of Polysorbate 80 directly into the sonicated silk solution, prior to addition of the antibody (see Figure 8).

The increase of surfactant levels in the sonicated silk solution was found to decrease silk gelation propensity. Sonication time and amplitude were optimized to account for this effect. Similar changes in release profiles were observed when polysorbate 80 was used in the release medium (see Figure 8A) or directly incorporated into the lyogel matrix (see

Figure 8B). In both cases, increasing levels of polysorbate 80 improved antibody recovery while maintaining the sustained release profile over approximately 80 days. These data support the notion that the knowledge gained through release medium modification studies can be used to guide modifications directly to the lyogel matrix.

Example 5. Silk matrix modifications for improved antibody stability

In addition to controlling release profiles, the silk lyogel matrix may be modified to prevent antibody degradation. In particular, antibody methionine oxidation was identified as a source of instability resulting from antibody entrapment in the silk lyogel matrix. Addition of an anti-oxidant or scavenger excipient such as L-methionine to protein liquid formulations was explored as a strategy for preventing methionine oxidation. For example, the possibility of incorporating excess methionine directly into the silk lyogel matrix to act as an antioxidant was investigated. Varying levels of methionine were added to sonicated silk solution prior to addition of the antibody lyophilized powder. The antibody samples were released and subsequently analyzed for methionine oxidation (see Figure 9). A significant increase in oxidized marker peak area was observed for the lyogel encapsulated antibody sample in the absence of methionine (see Figure 9A). The addition of methionine to the lyogel matrix incrementally decreased the amount of antibody methionine oxidation observed. In the absence of methionine, antibody methionine oxidation increased to 28% (see Figure 9B). Antibody methionine oxidation decreased to 9.3%, 7.3%, and 5.6% for 3 mM, 10 mM and 30 mM added methionine respectively. Antibody methionine oxidation in the control sample was 6.8%. Addition of excess methionine to the lyogel matrix was able to inhibit antibody oxidation caused by interaction with the silk lyogel matrix.

In conclusion, an anti-oxidation strategy has been shown effective for modifying the silk lyogel matrix to improve antibody recovery and inhibit antibody degradation. In this approach, modifying excipients are incorporated into the sonicated silk solution prior to addition of lyophilized antibody powered and the preparation of lyogels. The strategy of adding modifying excipients can be adapted to alter different lyogel properties, such as pH or surface charge, which have been identified as critical for controlling antibody release and stability.

The compositions and methods of the present disclosure are not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims

CLAIMS What is claimed is

1. A silk lyogel comprising a hydrophobicity modification, an ionic modification and/or a stability modification which facilitates sustained release of a therapeutic protein, wherein the hydrophobicity modification modulates lyogel swelling when the lyogel is added to an aqueous medium, wherein the ionic modification decreases repulsive ionic interactions in the lyogel, and wherein the stability modification increases stability of the therapeutic protein in the silk lyogel, thereby facilitating sustained release of the therapeutic protein.

2. The silk lyogel of claim 1 , further comprising the therapeutic protein.

3. The silk lyogel of claim 2, wherein the therapeutic protein is an antibody.

4. The silk lyogel of claim 3, wherein the antibody is an IgG antibody.

5. The silk lyogel of claim 1 , wherein the silk lyogel comprises a hydrophobicity modification.

6. The silk lyogel of claim 5, which is a lyogel, wherein the hydrophobicity modification is compressing the silk lyogel to a high density.

7. The silk lyogel of claim 6, comprising silk at a density of between 100 and 1500 mg/cm³.

8. The silk lyogel of claim 6, wherein the silk is at a density between 500 and 1400 mg/cm³.

9. The silk lyogel of claim 6, wherein the silk is at a density between 800 and 1100 mg/cm³.

10. The silk lyogel of claim 6, wherein the silk is at a density between 1200 and 1500 mg/cm³.

11. The silk lyogel of claim 5, wherein the hydrophobicity modification is addition of a surfactant.

12. The surfactant-modified silk lyogel of claim 11, wherein the surfactant is selected from the group consisting of a non-ionic surfactant, an anionic surfactant, a cationic surfactant and a zwitterionic surfactant.

13. The surfactant-modified silk lyogel of claim 12, wherein the surfactant is a non-ionic surfactant.

14. The surfactant-modified silk lyogel of claim 13, wherein the non- ionic surfactant is selected from the group consisting of polysorbates, poloxamers, polyols, and

polyoxyethylene sorbitan monoethers.

15. The surfactant-modified silk lyogel of claim 12, wherein the surfactant is an anionic surfactant.

16. The surfactant-modified silk lyogel of claim 15, wherein the anionic surfactant is selected from the group consisting of fatty acid soap, higher alkyl sulfate ester salt, alkyl ether sulfate ester salt, N-acyl sarcosinic acid, higher fatty acid amide sulfonate, phosphate ester salt, sulfosuccinate, alkylbenzene sulfonate, higher fatty acid ester sulfate ester salt, N- acyl glutamate, sulfonated oil, POE-alkyl ether carboxylic acid, POE-alkyl aryl ether carboxylate, a-olefine sulfonate, higher fatty acid ester sulfonate, secondary alcohol sulfate ester salt, higher fatty acid alkylolamide sulfate ester salt, sodium lauroyl monoethanolamide succinate, N-palmitoyl asparaginate ditriethanolamine and sodium casein.

17. The surfactant-modified silk lyogel of claim 12, wherein the surfactant is a cationic surfactant.

18. The surfactant-modified silk lyogel of claim 17, wherein the cationic surfactant is selected from the group consisting of alkyltrimethyl ammonium salt, alkylpyridinium salt, distearyldimethyl ammonium chloride, dialkyldimethyl ammonium salt, poly (N,N'-dimethyl- 3,5-methylenepiperidinium) chloride, alkyl quaternary ammonium salt, alkyldimethylbenzyl ammonium salt, alkylisoquinolinium salt, dialkylmorphonium salt, POE-alkylamine, alkylamine salt, polyamine fatty acid derivative, amyl alcohol fatty acid derivative, benzalkonium chloride and benzethonium chloride.

19. The surfactant-modified silk lyogel of claim 12, wherein the surfactant is a zwitterionic surfactant.

20. The surfactant-modified silk lyogel of claim 19, wherein the zwitterionic surfactant is selected from the group consisting of alkyl amine oxides, alkyl betaines, alkyl amidopropyl betaines, alkyl sulpho betaines (sultaines), alkyl glycinates, alkyl carboxyglycinates, alkyl ampho acetates, alkyl amphopropionates, alkylamphoglycinates, alkyl amidopropyl hydroxysultaines, acyl taurates and acyl glutamates.

21. The silk lyogel of claim 1 , wherein the silk lyogel comprises an ionic modification.

22. The silk lyogel of claim 21 , wherein the ionic modification is addition of a buffering agent.

23. The buffered silk lyogel of claim 22, wherein the buffering agent maintains the solution that is subject to lyophilization to form the lyogel at a pH of between 3 and 12.

24. The buffered silk lyogel of claim 23, wherein the buffering agent is selected from the group consisting of HEPES, Tris, MES, sodium phosphate, potassium phosphate, sodium acetate, sodium citrate, potassium nitrate, histidine, and succinate.

25. The buffered silk lyogel of claim 21, wherein the ionic modification is addition of a salt.

26. The buffered silk lyogel of claim 25, wherein the salt maintains the solution that is subject to lyophilization to form the lyogel at an osmolarity of between 1 mM and 5 M.

27. The buffered silk lyogel of claim 25, wherein the salt is selected from the group consisting of NaCl, NaK, MgCl₂, and CaCl₂.

28. The silk lyogel of any one of the preceding claims, wherein the silk lyogel is produced from a silk solution of between 2 and 18% w/w.

29. The silk lyogel of any one of the preceding claims, wherein the silk lyogel comprises silk fibroin with 20-65% β-sheet content.

30. The silk lyogel of any one of the preceding claims, wherein the silk lyogel comprises silk fibroin with a monomer of sequence (Gly-Ser-Gly-Ala-Gly-Ala)„, wherein n is greater than or equal to 5 and less than or equal to 70.

31. The silk lyogel of any one of the preceding claims, wherein the fibroin polymers comprise between 40 and 60% glycine content.

32. The buffered silk lyogel of claim 31, wherein the fibroin polymers have an average molecular weight between 0.5 and 400 kDa.

33. The silk lyogel of any one of the preceding claims, comprising a stability

modification.

34. The silk lyogel of claim 33, wherein the stability modification comprises the addition of an anti-oxidation agent to the silk lyogel.

35. The silk lyogel of claim 34, wherein the anti-oxidation agent is selected from the group consisting of methionine, thiosulfate, N-Acetyl-L-methionine, N-Acetyl tryptophan.

36. A silk lyogel comprising an antibody, wherein the silk lyogel releases between 0.5 and 100% of the antibody when exposed to an aqueous environment for 160 days.

37. The silk lyogel of claim 36, wherein the silk lyogel releases between 0.5 and 5% of the antibody when exposed to an aqueous environment for 160 days.

38. The silk lyogel of claim 37, wherein the swelling ratio is between 0.2 and 20 when exposed to an aqueous environment for 160 days.

39. The silk lyogel of claim 37, wherein the swelling ratio is between 0.2 and 1 when exposed to an aqueous environment for at least 160 days.

40. A method of making a silk lyogel comprising

a) sonicating silk fibroin;

b) cooling the sonicated solution at room temperature; and

c) lyophilizing the cooled solution,

wherein the method further comprises making a modification selected from a hydrophobicity modification, an ionic modification, or a stability modification.

41. The method of claim 40, wherein the silk fibroin solution comprises between 2 and 18% silk fibroin w/w.

42. The method of claim 40, wherein the silk fibroin solution comprises between 3 and 12 % silk fibroin w/w.

43. The method of claim 40, wherein room temperature is between 18 and 26 °C.

44. The method of claim 40, further comprising adding a protein to the sonicated silk fibroin.

45. The method of claim 44, wherein the protein is an antibody.

46. The method of claim 45, wherein the antibody is an IgG antibody.

47. The method of claim 40, wherein the modification is a hydrophobicity modification.

48. The method of claim 48, wherein the hydrophobicity modification is compressing the lyophilized solution at between 900 and 1100 psi.

49. The method of claim 48, wherein the hydrophobicity modification is compressing the lyophilized solution at between 2,400 and 2,600 psi.

50. The method of claim 40, wherein the silk lyogel has silk at a density between 100 and 1500 mg/cm³.

51. The method of claim 40, wherein the silk lyogel has silk at a density between 500 and 1400 mg/cm³.

52. The method of claim 40, wherein the silk lyogel has silk at a density between 800 and 1100 mg/cm³.

53. The method of claim 40, wherein the silk lyogel has silk at a density between 1200 and 1500 mg/cm .

54. The method of claim 48, wherein the hydrophobicity modification is addition of a surfactant prior to lyophilization.

55. The method of claim 54, wherein the surfactant is selected from the group consisting of a non-ionic surfactant, an anionic surfactant, a cationic surfactant and a zwitterionic surfactant.

56. The method of claim 55, wherein the surfactant is a non- ionic surfactant.

57. The method of claim 56, wherein the non-ionic surfactant is selected from the group consisting of polysorbates, poloxamers, polyols, and polyoxyethylene sorbitan monoethers.

58. The method of claim 55, wherein the surfactant is an anionic surfactant.

59. The method of claim 58, wherein the anionic surfactant is selected from the group consisting of fatty acid soap, higher alkyl sulfate ester salt, alkyl ether sulfate ester salt, N- acyl sarcosinic acid, higher fatty acid amide sulfonate, phosphate ester salt, sulfosuccinate, alkylbenzene sulfonate, higher fatty acid ester sulfate ester salt, N-acyl glutamate, sulfonated oil, POE-alkyl ether carboxylic acid, POE-alkyl aryl ether carboxylate, a-olefine sulfonate, higher fatty acid ester sulfonate, secondary alcohol sulfate ester salt, higher fatty acid alkylolamide sulfate ester salt, sodium lauroyl monoethanolamide succinate, N-palmitoyl asparaginate ditriethanolamine and sodium casein.

60. The method of claim 55, wherein the surfactant is a cationic surfactant.

61. The method of claim 60, wherein the cationic surfactant is selected from the group consisting of alkyltrimethyl ammonium salt, alkylpyridinium salt, distearyldimethyl ammonium chloride, dialkyldimethyl ammonium salt, poly (N,N'-dimethyl-3,5- methylenepiperidinium) chloride, alkyl quaternary ammonium salt, alkyldimethylbenzyl ammonium salt, alkylisoquinolinium salt, dialkylmorphonium salt, POE-alkylamine, alkylamine salt, polyamine fatty acid derivative, amyl alcohol fatty acid derivative, benzalkonium chloride and benzethonium chloride.

62. The method of claim 55, wherein the surfactant is a zwitterionic surfactant.

63. The silk lyogel of claim 62, wherein the zwitterionic surfactant is selected from the group consisting of alkyl amine oxides, alkyl betaines, alkyl amidopropyl betaines, alkyl sulphobetaines (sultaines), alkyl glycinates, alkyl carboxyglycinates, alkyl amphoacetates, alkyl amphopropionates, alkylamphoglycinates, alkyl amidopropyl hydro xysultaines, acyl taurates and acyl glutamates.

64. The method of claim 40, wherein the modification is an ionic modification.

65. The method of claim 64, wherein the ionic modification is addition of a buffering agent prior to lyophilization.

66. The method of claim 65, wherein the buffering agent maintains the solution that is subject to lyophilization to form the lyogel at a pH of between 2 and 12.

67. The method of claim 66, wherein the buffering agent is selected from the group consisting of HEPES, Tris, MES, sodium phosphate, potassium phosphate, sodium acetate, sodium citrate, potassium nitrate, histidine, succinate.

68. The method of claim 64, wherein the ionic modification is addition of a salt prior to lyophilization.

69. The method of claim 68, wherein the salt maintains the solution that is subject to lyophilization to form the lyogel at an osmolarity of between 1 mM and 5 M.

70. The method of claim 69, wherein the salt is selected from the group consisting of NaCl, NaK, MgCl₂, and CaCl₂.

71. The method of any one of claims 40-70, wherein the silk lyogel is present at between 2 and 18% w/w.

72. The method of any one of claims 40-70, wherein the silk lyogel releases between 0.5 and 100% of the antibody when exposed to an aqueous environment for 160 days.

73. The method of any one of claims 40-70, wherein the silk lyogel releases between 0.5 and 5% of the antibody when exposed to an aqueous environment for 160 days.

74. The method of any one of claims 40-70, wherein the lyogel releases between 10 and 20% of the antibody when exposed to an aqueous environment for 160 days.

75. The method of any one of claims 40-70, wherein the swelling ratio of the silk lyogel is between 0.2 and 20 when exposed to an aqueous environment for 160 days.

76. The method of any one of claims 40-70, wherein the swelling ratio of the silk lyogel is between 0.2 and 1 when exposed to an aqueous environment for 160 days.

77. The method of any one of claims 40-70, wherein the method further comprises making a stability modification to the lyogel.

78. The method of claim 77, wherein the stability modification comprises the addition of an anti-oxidant to the lyogel.

79. The method of claim 78, wherein the anti-oxidant is selected from the group consisting of methionine, thiosulfate, N-Acetyl-L-methionine, N- Acetyl tryptophan.

80. A method of determining the rate of release of an antibody from a silk lyogel comprising

a) exposing the high density silk lyogel to aqueous solution, wherein the aqueous solution comprises a surfactant, for a set period of time; and

b) detecting the amount of antibody in the aqueous solution at the end of the set period of time.

81. The method of claim 80, wherein the surfactant is present at a concentration of 0.001- 1.0% (w/v).

82. A method of determining the rate of release of an antibody from a silk lyogel comprising

a) exposing the silk lyogel to an aqueous solution, wherein the pH of the aqueous solution is between 2 and 12, for a set period of time; and

b) detecting the amount of antibody in the aqueous solution at the end of the set period of time.

83. A method of determining the rate of release of an antibody from a silk lyogel comprising

a) exposing the silk lyogel to an aqueous solution, wherein the aqueous solution comprises a salt at a concentration between 0 and 3 M, for a set period of time; and

b) detecting the amount of antibody in the aqueous solution at the end of the set period of time.

84. A sustained release dosage form comprising a silk lyogel according to claim 1 and a pharmaceutically acceptable carrier or excipient.

85. The sustained release dosage form of claim 84, further comprising a protein.

86. The sustained release dosage form of claim 85, wherein the protein is an antibody.

87. The sustained release dosage form of claim 86, wherein the antibody is an IgG antibody.

88. The sustained release dosage form of claim 87, wherein the sustained release lasts for 1-160 days.