Charged amino acids for rapid resuspension of materials of interest

Abstract

Embodiments of the present disclosure generally relate to compositions that are rapidly suspended in a liquid, to articles comprising the compositions, and to process for making the compositions and articles. Embodiments of the present disclosure also generally relate to uses of the compositions and articles. In an embodiment, a composition is provided. The composition includes a first amino acid having a side chain group that is negatively charged at a pH of 7. The composition further includes a second amino acid having a side chain group that is positively charged at a pH of 7. The composition further includes a material of interest, the material of interest different from the first amino acid and the second amino acid. The composition is characterized as being suspendible or resuspendible in an aqueous material in a period of less than 20 minutes.

Description

TITLE: Charged Amino Acids for Rapid Resuspension of Materials of InterestINVENTORS: Alan Stenquist; John OakeyCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 655,483, filed on June 3, 2024, which is incorporated herein by reference in its entirety.GOVERNMENT RIGHTS

[0002] This invention was made with government support under Grant Number CBET- 1254608 awarded by the National Science Foundation. The government has certain rights in the invention.FIELD

[0003] Embodiments of the present disclosure generally relate to compositions that are rapidly suspended in a liquid, to articles comprising the compositions, and to process for making the compositions and articles. Embodiments of the present disclosure also generally relate to uses of the compositions and articles.BACKGROUND

[0004] Improving protein stability during removal of water and drying, such as lyophilization, is a subject of great interest to the pharmaceutical industry. However, there is little work done on improving protein stability for suspension or resuspension. Upon suspension or resuspension, problems may arise from factors such as protein aggregation, denaturation, or improper buffer conditions. Another challenge is the difficulty in dissolving the solid protein pellet after drying or freeze drying the protein.

[0005] There is a need for new compositions that may be rapidly suspended in a liquid, for articles comprising the compositions, and to process for making such compositions and articles.SUMMARY

[0006] Embodiments of the present disclosure generally relate to compositions that are rapidly suspended in a liquid, to articles comprising the compositions, and to process for making the compositions and articles. Embodiments of the present disclosure also generally relate to uses of the compositions and articles described herein.

[0007] In an embodiment, a composition is provided. The composition includes a first amino acid having a side chain group that is negatively charged at a pH of 7. The compositionfurther includes a second amino acid having a side chain group that is positively charged at a pH of 7. The composition further includes a material of interest, the material of interest different from the first amino acid and the second amino acid. The composition is characterized as being suspendible or resuspendible in an aqueous material in a period of less than 20 minutes.

[0008] In another embodiment, a suspendible fibrinogen composition. The suspendible fibrinogen composition includes a first amino acid having a side chain group that is negatively charged at a pH of 7. The suspendible fibrinogen composition further includes a second amino acid having a side chain group that is positively charged at a pH of 7. The suspendible fibrinogen composition further includes fibrinogen. The suspendible fibrinogen composition is characterized as being suspendible or resuspendible in an aqueous material in a period of less than 20 minutes.

[0009] In another embodiment, a process is provided. The process includes introducing an aqueous material with a composition comprising a material of interest, a first amino acid, and a second amino acid to form a suspension or solution of the material of interest, the first amino acid, and the second amino acid in the aqueous material. The material of interest is different from the first amino acid and the second amino acid, the first amino acid has a side chain group that is negatively charged at a pH of 7, the second amino acid has a side chain group that is positively charged at a pH of 7, the suspension or solution is characterized as being free of aggregated material of interest or free of aggregated fibrinogen.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] So that the manner in which the above recited features of the present disclosure may be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments and are therefore not to be considered limiting of its scope, may admit to other equally effective embodiments.

[0011] FIGS. 1A-1D show dynamic light scattering (DLS) analysis data of fibrinogen in phosphate buffered saline (PBS), an AG buffer solution (arginine, glutamic acid, and PBS), or a DIAG solution (arginine, glutamic acid, and distilled water). FIG. 1A: A solution of 1.5% w / v fibrinogen in PBS was determined to have a poly dispersity coefficient of about 0.23. FIG. IB: A solution of 1.5% w / v fibrinogen in the AG buffer solution was determined to have alower poly dispersity coefficient of about 0.13. FIG. 1C: In higher concentrations (10% w / v fibrinogen) in the AG buffer solution, the poly dispersity coefficient increased to about 0.47, but was still low enough to be considered homogenous. FIG. ID: A solution of 1.5% w / v fibrinogen in the DIAG solution had a poly dispersity coefficient of about 0.98, indicating aggregation. Under the conditions investigated, fibrinogen did not dissolve to appreciable levels in DI water alone. In addition, 10% w / v fibrinogen in PBS was determined to be too polydisperse and aggregated for the DLS instrument to accurately read.

[0012] FIG. 2A-2D show DLS analysis data of bovine serum albumin (BSA) or gelatin in the AG buffer solution or PBS. FIG. 2 A: A solution of 10% w / v BSA in PBS was determined to have a poly dispersity coefficient of about 3.34. FIG. 2B: A solution of 10% w / v BSA in the AG buffer solution was determined to have a significantly lower poly dispersity coefficient of about 0.73. FIGS. 2C and 2D: 10% gelatin in PBS or the AG buffer solution were determined to have similar poly dispersity coefficients.

[0013] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.DETAILED DESCRIPTION

[0014] Embodiments of the present disclosure generally relate to compositions that are rapidly suspended in a liquid, to articles comprising the compositions, and to process for making the compositions and articles. Embodiments of the present disclosure also generally relate to uses of the compositions and articles. As used herein, a “composition” may include component(s) of the composition, reaction product(s) of two or more components of the composition, a remainder balance of remaining starting component(s), or combinations thereof. As described herein the compositions are suspendible, resuspendible, or reconstitutible. Suspendible, resuspendible, or reconstitutible generally refers to the process of dissolving a pellet or dried protein powder into solution. The pellet or dried protein powder may be in a dry state as described herein. Suspendible, resuspendible, or reconstitutible may be referred to interchangeably unless specified to the contrary or the context clearly indicates otherwise.

[0015] The suspendible composition generally includes a material of interest and amino acids. Such suspendible compositions may be quickly suspended in the field prior to use. After suspending the suspendible composition in a liquid, the material of interest remains dissolvedin the liquid. Such suspendible compositions may have a high content of the material of interest. For example, after suspending the suspendible composition in the liquid, an amount of the material that remains dissolved in the aqueous material may include a physiological amount or a supraphy si ologi cal amount of the material of interest.

[0016] Moreover, the suspendible compositions may be shelf-stable such that the material of interest of the suspendible composition is stabilized or preserved by a component present in the suspendible composition. With conventional technologies, however, the degradation or decomposition of the material of interest under non-ideal conditions (for example, without refrigeration or the cold chain) represents a loss of, for example, food, agricultural products, and pharmaceuticals, among other materials and presents an enormous economic and logistical burden. In contrast, suspendible compositions of the present disclosure are shelf-stable under non-ideal conditions.

[0017] A purpose of this present disclosure is the use of two oppositely charged amino acids dissolved in solution with a material(s) of interest (for example, protein(s)). With respect to proteins, the charged amino acids may alter the hydration shell of the protein, may suppress intermolecular interactions between protein monomers, and may limit aggregation. Once dried (for example, lyophilized), the charged amino acids may allow for the rapid and near instantaneous dissolution of the lyophilized protein. An example reference protein utilized herein is fibrinogen, though other proteins are contemplated. The inventors found that rapid reconstitution of fibrinogen to a concentration of about 100 mg / mL may occur within seconds after the solvent, such as water, is introduced.

[0018] Solutions containing a high concentration of a material of interest may be made in an amino acid solution. The amino acid solution may include amino acids at any suitable concentration. For example, an amino acid solution may include about 50 mM of a positively charged amino acid (for example, arginine) and about 50 mM of a negatively charged amino acid (for example, glutamic acid). With respect to proteins, and while not wishing to be bound by theory, it is believed that the charged amino acids may interact with the protein of interest via hydrogen bonding and / or other interaction and may alter the dynamics of the water solvation shell around the protein of interest, thereby greatly increasing the free energy required for intermolecular interactions between proteins. For highly concentrated protein solutions, this may create a more monodisperse solution with less aggregation. If the solution is lyophilized, the charged amino acids allows for the rapid reconstitution of the protein using an aqueoussolvent (for example, distilled water) with minimal mixing and little-to-no aggregation of the protein.

[0019] Improving protein stability during removal of water and lyophilization is a subject of great interest to the pharmaceutical industry, however there is little work done on improving protein stability for resuspension. Towards these efforts, the inventors have found that drying (e.g., lyophilization) of a material of interest (for example, a protein) in the presence of an amino acid solution allows for the resulting lyophilized composition to be rapidly resuspended.

[0020] Currently, there is a $2 billion dollar a year industry built around fibrinogen-based tissue sealants, with almost all use in clinical hospital settings. The inventors have shown that fibrinogen may be stabilized and rapidly resuspended for the creation of a field deployable tissue-sealant that may be, for example, reconstituted a few minutes prior to use as opposed the 30-minute reconstitution protocol that conventional clinical tissue sealants require. While the inventors utilize fibrinogen as a model protein, this technology has applications with many lyophilized proteins, lyophilized drugs, lyophilized food products, any suitable dried protein, among other materials of interest.

[0021] Embodiments of the present disclosure generally relate to compositions that are rapidly suspended or resuspended in a liquid. The suspendible composition may include a material of interest and charged amino acids.

[0022] Materials of interest may include a biological material, a biologically-derived material, a synthetic material, or combinations thereof. A biological material may be derived from a living organism, in its natural state or its modified state, and / or synthesized. The biological material, also referred to as a biological material of interest may include any suitable biological material such as at least one or more of the following: a peptide, a polypeptide, a protein, an enzyme, an antibody, a globular protein, a hormone, an antibiotic, a nucleic acid, a nucleotide, a lipid, a polylipid, a fat, a monosaccharide, a polysaccharide, a carbohydrate, a cell, a tissue, an organ, a natural product, a food product, an agricultural product, a therapeutic agent, a diagnostic agent, an agent used for research purposes, a pharmaceutical such as a protein-based pharmaceutical, a derivative thereof, or combinations thereof, among others.

[0023] Biologically-derived materials may include materials that are, for example, derived from or produced from a biological material, as well as biological materials that have been modified. Such biologically-derived materials may include, for example, a biologic, a vaccine, a food product, an agricultural product, a therapeutic agent, a diagnostic agent, an agent usedfor research purposes, a pharmaceutical such as a protein-based pharmaceutical, a derivative thereof, or combinations thereof, among others. The inventor contemplates that certain examples of biological materials and biologically-derived materials may fall within or outside both categories; however, such biological materials and biologically-derived materials may be included in the compositions described herein.

[0024] Other illustrative, but non-limiting, examples of biological materials and / or biologically-derived materials may include a nucleic acid-based biologic, a nucleic acid-based therapeutic, a nucleic acid-based vaccine such as an mRNA vaccine; protein-based biologies, protein-based therapeutics, and protein-based diagnostics (for example, vaccines, antibodies, enzymes, et cetera). Other biological materials and / or biologically-derived materials are contemplated.

[0025] Synthetic compounds may include synthetic (for example, chemically synthesized) counterparts of biological materials, biologically-derived materials, or combinations thereof. Synthetic compounds may also include pharmaceuticals and drugs, such as antibiotics.

[0026] The material of interest may include a protein, a recombinant protein, a pharmaceutical, a food product, an agricultural product, or combinations thereof. The protein or the recombinant protein may be any suitable protein that may be dissolved in an aqueous suspension. For example, the material of interest may include fibrinogen, BSA, gelatin, or combinations thereof.

[0027] One or more materials of interest may be present in the suspendible composition.

[0028] Charged amino acids refer to amino acids having ionizable side chain groups that are charged at certain pH levels, for example, charged at a physiologically relevant pH, a pH of about 7, and / or a pH relative to a pKa of their amino acid side chain group. Such charged amino acids may include negatively charged amino acids (for example, glutamic acid, aspartic acid, tyrosine, and / or cysteine), positively charged amino acids (for example, arginine, lysine, and / or histidine), or combinations thereof. For example, the side chain group of aspartic acid, glutamic acid, tyrosine, and histidine is negatively charged at pH levels above the pKa of their amino acid side chain group. In contrast, the side chain group of lysine, arginine, and histidine, is positively charged at pH levels below the pKa of their amino acid side chain group.

[0029] The charged amino acids may include a first amino acid having a side chain group that is negatively charged at a pH of 7, for example, aspartic acid or glutamic acid, and a secondamino acid having a side chain group that is positively charged at a pH of 7, such as arginine or glycine.

[0030] In some embodiments, which may be combined with other embodiments, a weight ratio of the material of interest to the total amino acid in the composition may be in a range that is from about 0.1 : 1 to about 10: 1, such as from about 0.5: 1 to about 9: 1, such as from about 1 : 1 to about 5: 1, though other weight ratios are contemplated.

[0031] A weight ratio of the first amino acid to the second amino acid in the suspendible composition may be in a range from about 1 : 100 to about 100: 1, such as from about 1 :50 to about 50: 1, such as from about 1 :25 to about 25: 1, such as from about 1 : 10 to about 10: 1, such as from about 10:90 to about 90: 10, such as from about 20:80 to about 80:20, such as from about 30:70 to about 70:30, such as from about 40:60 to about 60:40, such as from about 45:55 to about 55:45, such as from about 46:54 to about 54:46, such as from about 47:53 to about 53 :47, such as from about 48:52 to about 52:48, such as from about 49:51 to about 51 :49, such as about 50:50 (first amino acid:second amino acid).

[0032] Besides the material of interest and the amino acids, suspendible compositions described herein may further include water. The water may come from the material of interest. Suspendible compositions described herein may have an amount of water that is 0 wt% or more, about 15 wt% or less, or combinations thereof, such as in a range from greater than 0 wt% to about 15 wt% or less, from about 2 wt% to about 12 wt%, from about 5 wt% to about 15 wt%, from about 6 wt% to about 14 wt%, such as from about 7 wt% to about 12 wt%, such as from about 8 wt% to about 10 wt% based on a total wt% of the suspendible composition. The total wt% of the suspendible composition is 100 wt%.

[0033] Suspendible composition described herein may further include any suitable optional additive. Such optional additives may include those additives that aid in preventing or at least mitigating decomposition, degradation, and / or polymerization of the material of interest. Additionally, or alternatively, optional additives may include excipients or carriers useful in pharmaceutical compositions, such a salt, an antioxidant, a preservative, other suitable excipients, other suitable carriers, or combinations thereof.

[0034] After the composition is made (e.g., the material of interest, the amino acids, the water, and any optional additive), the suspendible or resuspendible composition may be subjected to any suitable method for reducing the water content such as lyophilization, air drying, dehydration, desiccation, vacuum desiccation, vacuum drying, spray drying, freezedrying, spray-freeze drying, foam drying or combinations thereof. Accordingly, and in some embodiments, the suspendible or resuspendible composition may be a lyophilized composition, an air-dried composition, a dehydrated composition, a desiccated composition, a vacuum desiccated composition, a vacuum dried composition, a spray-dried composition, a freeze-dried composition, spray-freeze dried composition, a foam-dried composition, or combinations thereof.

[0035] The suspendible or resuspendible composition may be in the form of a solid powder, or a reduced water content composition to be suspended.

[0036] In use, the suspendible or resuspendible composition is brought into contact with any suitable aqueous material. Aqueous materials for suspending suspendible compositions described herein may include water, distilled water, a buffer solution such as phosphate buffered saline (PBS), and / or a (4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid) (HEPES) containing aqueous solution, or combinations thereof. A co-solvent such as an alcohol, for example, ethanol, may form at least a portion of the aqueous solution.

[0037] The suspendible composition may be characterized as being suspendible or resuspendible in an aqueous material (for example, water, distilled water, PBS, HEPES, other buffer, or combinations thereof) in a period of less than 1 hour, such as less than 45 minutes, such as less than 30 minutes, such as less than 20 minutes, such as less than 15 minutes, such as less than 10 minutes, such as less than 5 minutes, such as less than 2 minutes, such as less than 1 minute, such as less than 30 seconds, such as less than 10 seconds. The suspendible composition may be suspendible or resuspendible without sonication, vortexing, or other mixing procedures, though sonication, vortexing, or other mixing procedures may be utilized to aid in suspending or resuspending the suspendible composition. In contrast, conventional technologies require suspension or re-suspension periods of at least 20 minutes or more along with vortexing, sonication, or other mixing procedures.

[0038] Embodiments of the present disclosure enable suspension or resuspension of materials of interest to higher concentrations than with conventional technologies.

[0039] The suspendible composition may be characterized as stabilizing the material of interest of the suspendible composition at a temperature of ambient (room) temperature or higher. Ambient temperature refers to a temperature of about 20°C. As such, embodiments described herein can stabilize or preserve the material of interest, or at least limit degradation of the material of interest.

[0040] As used herein, “stabilizing” a material of interest refers to maintaining the structure and / or the function of the material of interest under either aqueous conditions or dried conditions, or after being frozen and / or dried and then thawed and / or rehydrated. In some embodiments, the material of interest in the suspendible composition may be stable at a temperature from about -80°C to about 100°C once the at least one material of interest is introduced or contacted with the amino acids. At least about 10% to about 100% (for example, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any range of values therein) of the structure and function of the stabilized material of interest is maintained. Thus, in some embodiments, about 10% to about 90%, about 10 to about 85% about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 30% to about 90%, about 30 to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 40% to about 90%, about 40 to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 90%, about 50 to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 60%, and the like, of the structure and function of the material of interest is maintained.

[0041] When dried (for example, as a solid composition) or when in solution (for example, as a liquid composition), the material of interest of the suspendible composition may be stabilized over a range of temperatures from about -80°C to about 100°C, such as over a range of temperatures from about -80°C to about 50°C, such as from about 20°C to about 45°C, though other temperatures are contemplated.

[0042] Suspendible compositions described herein may be stored under ideal conditions or non-ideal conditions. For example, when the material of interest is intended to be stored at an ideal temperature of 4°C according to conventional methods, suspendible compositions described herein comprising the material of interest and the amino acids may be stored at thisideal temperature or a non-ideal temperature (for example, greater than about 4°C or less than about 4°C).

[0043] Suspendible compositions described herein may be characterized as stabilizing the material of interest in a dry state. “Dry state” when referring to a suspendible composition described herein refers to a suspendible composition that has 15 wt% or less water based on the total wt% of the suspendible composition. A suspendible composition in a dry state may have an amount of water that is from 0 wt% or more, about 15 wt% or less, or combinations thereof, such as in a range from greater than 0 wt% to about 15 wt%, from about 2 wt% to about 12 wt%, from about 5 wt% to about 15 wt%, from about 6 wt% to about 14 wt%, such as from about 7 wt% to about 12 wt%, such as from about 8 wt% to about 10 wt% based on a total wt% of the suspendible composition. The total wt% of the suspendible composition is 100 wt%.

[0044] Suspendible compositions described herein may be characterized as being shelfstable.

[0045] Suspendible compositions described herein may be characterized as having a high content of material of interest. The content of the material of interest in the suspendible composition may be an amount relative to physiological amount or concentration. That is, an amount or concentration of the material of interest in the suspendible composition, when the suspendible composition is suspended or resuspended in the aqueous material, may be a physiological amount or a supraphysiological amount. The composition, upon suspension or re-suspension in an aqueous material, may include about 1% w / v or more, such as about 2% w / v material of interest or more, such as about 3% w / v or more, such as about 4% w / v or more, such as about 5% w / v or more, such as about 6% w / v or more, such as about 7% w / v or more, such as about 8% w / v or more, such as about 9% w / v or more, such as about 10% w / v or more. The suspendible composition, upon suspension in an aqueous material, may be characterized as being free of aggregates.

[0046] With respect to fibrinogen, physiological amounts of fibrinogen are between 0.2% w / v and 0.4% w / v. The suspendible composition, when suspended or resuspended in an aqueous material, may comprise an amount of fibrinogen that is greater than 0.4% weight / volume (w / v), such as about 1% w / v or more, such as about 2% w / v fibrinogen or more, such as about 3% w / v or more, such as about 4% w / v or more, such as about 5% w / v or more, such as about 6% w / v or more, such as about 7% w / v or more, such as about 8% w / v or more,such as about 9% w / v or more, such as about 10% w / v or more, about 15% w / v or more, or about 20% w / v or more and about 30% w / v or less, such as about 25% w / v or less, such as about 20% w / v or less, such as about 15% w / v or less, such as about 10% w / v or less, such as about 5% w / v or less, or combinations thereof. Any suitable higher limit may be combined with any suitable lower limit of the aforementioned % w / v values. For example, the suspendible composition, when suspended or resuspended in an aqueous material, may comprise an amount of fibrinogen that is in a range from about 5 wt% to about 25 wt%, such as from about 7 wt% to about 20 wt%, such as from about 9 wt% to about 18 wt%, such as from about 10 wt% to about 15 wt%.

[0047] In an embodiment is provided a suspendible fibrinogen composition that includes fibrinogen and amino acids. The suspendible fibrinogen composition may be a shelf-stable composition. Suitable amino acids with the suspendible fibrinogen composition are described herein. As described above, the suspendible composition may be dried or at least partially dried such that an amount of water in the suspendible composition may be about 15 wt% or less based on the total wt% of the suspendible fibrinogen composition.

[0048] The composition may be rapidly and homogeneously reconstituted at concentrations exceeding current market products. In a specific, non-limiting example, an AG buffer solution enables fibrinogen to be resuspended using readily available aqueous materials (such as distilled water) avoiding any detrimental effects that additional salts may cause.

[0049] Compositions described herein may further include thrombin. Thrombin is the enzyme that polymerizes fibrinogen. Conventional technologies require thrombin to be separated from fibrinogen as well as refrigeration / cold-chain. In contrast, embodiments described herein enable compositions that include both fibrinogen and thrombin.

[0050] The suspendible or resuspendible fibrinogen composition may be utilized as a rapidly reconstituted anti-thrombogenic tissue sealant appropriate for field use at the point of need. The global fibrin glue market has almost no use as a field-deployable anti-hemorrhagic intervention indicating the utility of shelf-stable, field deployable sealant compositions described herein for use in clinical settings and outside of clinical settings. Controlling bleeding during battlefield trauma and emergency trauma is lifesaving. Additionally, avoiding the coldchain allows for fibrin sealants to be used in sub-optimal surgical settings in the developing world. The super-solvation effects of AG buffer that allow for extreme fibrinogen concentrations could find additional uses in inhibiting protein precipitation throughout thepharmaceutical industry, food processing, and regenerative medicine. The avoidance of the cold chain for storage and transportation further enhances the commercial appeal of this technology. By eliminating the need for refrigeration, these sealants become more accessible and practical for use in field surgeries and remote locations, significantly expanding their market reach.

[0051] Embodiments described herein provide for suspendible compositions that include a protein (such as fibrinogen) that may be made without the use of a droplet generator.

[0052] In another embodiment is provided an article that includes a composition described herein. The article may be in the form of a substrate impregnated or coated with a composition described herein. The article may include a medical dressing, a wound dressing, a hemostatic tissue sealant, or a closure for a bleeding wound. The substrate of the article may include any suitable substrate such as a bandage, an adhesive bandage, gauze, cloth, tampon, membrane, or sponge. The substrate may be any suitable substrate for application to a wound or any suitable substrate to stem bleeding. The article may be 3D-printed. The 3D-printed article is easy to use, is simple to apply to a wound site, and concentrations of components in the composition may be modulated.

[0053] Embodiments of the present disclosure also generally relate to processes for making the suspendible compositions and articles described herein. Processes for making the suspendible composition may include introducing or contacting the material of interest with the amino acids to form the suspendible composition comprising the material of interest and the amino acids. The process may further include adding optional additives to the material of interest and the amino acids.

[0054] The introducing may be performed under any suitable conditions such as conditions suitable for maintaining the stability of the material of interest. The conditions may include mixing the material of interest and the amino acids by any suitable mixing process.

[0055] In at least one embodiment of the process for making the suspendible composition, the material of interest may be in the form of a material of interest solution or suspension comprising the material of interest, and the amino acids may be in the form of an amino acid solution or suspension comprising the amino acids. The material of interest solution or suspension and the amino acid solution or suspension may be introduced with one another followed by an optional mixing process. The optional mixing process may be performed by any suitable method such as by vortexing, sonication, or other mixing method.

[0056] The amino acid solution or suspension may be formed by dissolving or suspending the material of interest in any suitable solvent such as an aqueous solvent. The aqueous solution or suspension comprising the amino acids may include water, distilled water, a buffer, a cosolvent such as ethanol, or combinations thereof. The aqueous solution or suspension comprising the two amino acids may include salts, such as alkali metal salts (for example, salts of Na, K, or combinations thereof) and alkaline earth metal salts (for example, salts of Mg, Ca, or combinations thereof). Corresponding anions for the alkali and alkali earth metal salts may include, but are not limited to phosphate ion, chlorine ion. The aqueous solution or suspension comprising the one more amino acids may be a buffer solution that includes phosphate buffered saline (PBS) and / or 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES). The pH of the aqueous solution or suspension comprising the amino acids may range from about 6 to about 10, from about 6.5 to about 9.5, from about 7 to about 9, from about 7 to about 8, from about 6.8 to about 8.2, from about 7 to about 8.5, or from about 7.2 to about 7.8. The pH of the aqueous solution or suspension comprising the one more amino acids may be adjusted by use of suitable acids or bases such as hydrochloric acid and sodium hydroxide.

[0057] An amount of the amino acids in the amino acid solution or suspension may include from about 0.1 millimolar (mM) to about 200 mM, such as from about 0.5 mM to about 150 mM, such as from about 1 mM to about 100 mM, such as from about 5 mM to about 95 mM, such as from about 10 mM to about 90 mM, such as from about 15 mM to about 85 mM, such as from about 20 mM to about 80 mM, such as from about 25 mM to about 75 mM, such as from about 30 mM to about 70 mM, such as from about 35 mM to about 65 mM, such as from about 45 mM to about 55 mM, such as about 50 mM. When more than one amino acid is utilized, equimolar amounts of the amino acids may be used or differing amounts of the amino acids can be used.

[0058] In at least one embodiment, the amino acid solution or suspension comprising the two amino acids is an aqueous buffer solution that includes PBS, about 50 mM arginine, about 50 mM glutamic acid, and optionally has a pH that is from about 6 to about 9.

[0059] The material of interest solution or suspension may be formed by dissolving or suspending the material of interest in any suitable solvent. Suitable solvents may include those components of the aqueous solution or suspension useful to form the amino acid solution or suspension. An amount of the material of interest in the material of interest solution or suspension may include greater than 0.1% w / v, less than 50% w / v, or combinations thereof,such as greater than 0.4% w / v, such as about 1% w / v or more, such as about 2% w / v or more, such as about 5% w / v or more, about 10% w / v or more, about 15% w / v or more, or about 20% w / v or more and about 30% w / v or less, such as about 25% w / v or less, such as about 20% w / v or less, such as about 15% w / v or less, such as about 10% w / v or less, such as about 5% w / v or less, or combinations thereof. Any suitable higher limit may be combined with any suitable lower limit of the aforementioned % w / v values.

[0060] After the introducing and the optional mixing, the resulting product mixture (for example, a solution or suspension) comprises any suitable amount of the material of interest and any suitable amount of the amino acids.

[0061] An amount of the material of interest in the resulting product mixture comprising the material of interest and the amino acids may include those % w / v amounts of the material of interest described herein for the material of interest solution or suspension. For example, with conventional processes, it may be possible to resuspend a lyophilized protein solution to 3% w / v. With the two charged amino acids described herein, it is possible to reduce the volume of fluid needed for resuspension, yielding a 20% solution.

[0062] An amount of the amino acids in the resulting product mixture comprising the material of interest and the amino acids may include those millimolar amounts of the amino acids described herein for the amino acid solution or suspension.

[0063] Processes for forming the suspendible composition may further include introducing an additive to the resulting mixture comprising the material of interest and the amino acids. Suitable additives are described herein.

[0064] Processes for forming the suspendible composition may further include removing at least a portion of the liquid content (for example, water and / or co-solvent) from the product mixture comprising the material of interest, the amino acids, and the optional additive. Removing at least a portion of the liquid content from the product mixture may include drying, or at least partially drying, the suspendible composition to a dry state. Any suitable method of drying or partially drying may be utilized, such as, for example, lyophilizing, air drying, evaporating, dehydrating, desiccating, vacuum desiccating, vacuum drying, spray drying, freeze drying, spray-freeze drying, foam drying, or combinations thereof, among other suitable methods.

[0065] Following the drying or at least partially drying, the suspendible composition is formed and may be in a dry state such that the water content of the suspendible compositionmay be about 15 wt% or less based on a total wt% of the suspendible composition. The suspendible composition may be self-stable. The suspendible composition is characterized as stabilizing the material of interest of the suspendible composition in a dry state and / or characterized as stabilizing the material of interest of the suspendible composition at a temperature of ambient temperature or higher, though lower temperatures are contemplated.

[0066] As described above, the suspendible composition is characterized as being suspendible or resuspendible in an aqueous material as described above. Aqueous materials for suspending or resuspending the suspendible composition may include water, distilled water, or combinations thereof. Additionally, or alternatively, aqueous materials for suspending or resuspending the suspendible composition may include an aqueous solution useful for forming the amino acid solution or suspension.

[0067] To make articles that include a suspendible composition described herein, the suspendible composition may be applied to the substrate of the article. Applying the suspendible composition to the substrate may be performed by impregnating or coating the substrate with the suspendible composition by any suitable method.

[0068] To apply the suspendible composition to the substrate during processes for making the article, the suspendible composition may be in a solid form (such as particles and / or powders) and / or a liquid form (such as the combined solution of suspension resulting from the introduction of the material of interest with the amino acids). When the suspendible composition is in solid form, the solid form of the suspendible composition may be dissolved or suspended in a suitable solvent and then applied to the substrate.

[0069] Following the applying the suspendible composition to the substrate, the substrate coated or impregnated with the suspendible composition may be dried by any suitable method such as by lyophilizing, air drying, evaporating, dehydrating, desiccating, vacuum desiccating, vacuum drying, spray drying, freeze drying, spray-freeze drying, foam drying, or combinations thereof, among other suitable methods.

[0070] Embodiments of the present disclosure also generally relate to methods of using the suspendible compositions described herein. In an embodiment is provided a process that includes introducing an aqueous material with a suspendible composition described herein to form a suspension or solution that includes the material of interest, the amino acids, and an optional additive. The suspension or solution may be characterized as being free of aggregated material of interest even at supraphysiological amounts of the material of interest. Thesuspendible compositions dissolve rapidly, such as in a period of less than 1 hour, such as less than 45 minutes, such as less than 30 minutes, such as less than 20 minutes, such as less than 15 minutes, such as less than 10 minutes, such as less than 5 minutes, such as less than 2 minutes, such as less than 1 minute, such as less than 30 seconds, such as less than 10 seconds. The suspendible composition may be suspendible or resuspendible without sonication, vortexing, or other mixing procedures, though sonication, vortexing, or other mixing procedures may be utilized to aid in suspending or resuspending the composition. In contrast, conventional technologies require suspension or re-suspension periods of at least 20 minutes or more along with vortexing, sonication, or other mixing procedures

[0071] Aqueous materials for suspending or resuspending suspendible compositions described herein may include those materials described herein. Additionally, or alternatively, aqueous materials for suspending or resuspending suspendible compositions described herein may include those aqueous solutions useful for forming the amino acid solution or suspension.

[0072] Methods of use may include methods of treating a patient, such as treating various medical conditions or diseases in a patient utilizing suspendible compositions described herein or articles described herein. The term “patient”, “subject”, or “individual” are used interchangeably herein and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans.

[0073] Treatment” and “treating” includes the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a pathological condition, disease, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a pathological condition, disease, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated pathological condition, disease, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the pathological condition, disease, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated pathological condition, disease, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated pathological condition, disease, or disorder. It is understood that treatment, while intended to cure, ameliorate, stabilize, or prevent a pathological condition, disease, or disorder, need not actually result in the cure, ameliorization, stabilization or prevention. The effects of treatment can bemeasured or assessed as described herein and as known in the art as is suitable for the pathological condition, disease, or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitative terms. Thus, for example, characteristics or features of a pathological condition, disease, or disorder and / or symptoms of a pathological condition, disease, or disorder can be reduced to any suitable effect or to any suitable amount.

[0074] In an embodiment is provided a method of treating a wound in a patient. The method includes administering to the patient a suspendible composition described herein or an article described herein. As described above, the suspendible composition is suspendible or resuspendible in an aqueous material. Aqueous materials for suspending or resuspending the suspendible composition may include water, distilled water, or combinations thereof. Additionally, or alternatively, aqueous materials for suspending or resuspending the suspendible composition may include an aqueous solution useful for forming the amino acid solution or suspension.

[0075] Accordingly, the method of treating a wound in the patient may include introducing the aqueous material to the suspendible composition or the article prior to the administering to the patient the suspendible composition or the article.

[0076] In another embodiment is provided a method to control bleeding in a patient. The method includes administering to the patient a suspendible composition described herein or an article described herein. The method to control bleeding in a patient may further include introducing the aqueous material to the suspendible composition or the article prior to the administering to the patient the suspendible composition or the article.

[0077] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure, and are not intended to limit the scope of embodiments of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used but some experimental errors and deviations should be accounted for.Examples

[0078] Materials of interest for the examples included fibrinogen, gelatin, and BSA. Other materials of interest may be used. In various examples, the amino acid solution may be a buffer that includes arginine and glutamic acid. Other amino acid solutions containing different amino acids may be used.Example 1 : Effects of Buffer on the Solubility of Several Model Proteins1.1. Introduction

[0079] Conventional methods of generating fibrin particles by a fibrin droplet generator may require high concentrations of fibrinogen to achieve recoverable particles due to the dilution caused by the additional stream of buffer between the fibrinogen and thrombin. However, creating high concentration fibrinogen solutions is challenging. Particles mechanically robust enough to withstand the centrifugation involved in the recovery process require over 50 mg / mL or 5% w / v of fibrinogen. However, precipitates routinely occur when creating working solutions in PBS that obstruct the microchannels of the fibrin droplet generator.

[0080] The inventors found that charged amino acids may solubilize several proteins with poor solubility. The inventors also found that charged amino acids may limit fibrinogen aggregation allowing for the high concentration solutions our droplet generator required. Charged amino acids avoid issues that could be caused by excess salt in biological systems. Experimental and computational indicate that charged amino acids alter the hydration shell of a protein by creating an alternating layer of positively and negatively charged amino acids through hydrogen bonding. This layer of amino acids suppresses intermolecular interactions between proteins by increasing the free energy required for interaction. This increase in free energy limits how highly charged regions on proteins can interact. Embodiments described herein provide for suspendible compositions that include a protein, such as fibrinogen, and the compositions are made without the use of a droplet generator.1.2. Materials and Methods

[0081] Arginine, glutamic acid, fibrinogen, and gelatin were obtained from Alfa Aesar. PBS was obtained from Genesee.1.2.1. Solution Preparation

[0082] AG buffer solution: An AG buffer solution was prepared by dissolving 50 mM arginine (A) and 50 mM glutamic acid (G) in PBS. The AG buffer solution may be interchangeably referred to as “A / G” or “A / G buffer solution. The AG buffer solution represents a mixed amino acid solution. Other mixed amino acid solutions may be utilized.

[0083] DIAG solution: A DIAG solution was prepared by dissolving 50 mM arginine (A) and 50 mM glutamic acid (G) in distilled (DI) water.

[0084] Fibrinogen solution: Fibrinogen solutions were prepared by adding commercially obtained fibrinogen to AG buffer solution, DIAG solution or DI water.

[0085] Bovine serum albumin (BSA) solution: BSA solutions were prepared by dissolving BSA in PBS or AG buffer solution.

[0086] Gelatin solution: A gelatin solution was prepared by dissolving gelatin in PBS or AG buffer solution.1.2.2. Dynamic Light Scattering (DLS) of Protein Solutions

[0087] Supraphy si ologi cal fibrinogen solutions were created using AG buffer solution, DIAG solution, PBS, or DI water, and added to a 1 mL spectrophotometry cuvette. A Brookhaven Instruments NanoBrook 173 Plus instrument running Brookhaven Instruments Particle Solutions software was used to measure solution polydispersity. All solutions were measured using default software settings at 25°C.1.2.3. Lyophilization and Resuspension

[0088] 10 mL of 2.5 mg / mL fibrinogen solution in AG buffer solution, DIAG solution, orPBS were prepared, frozen at -80°C for 30 minutes and lyophilized under vacuum overnight in 15 mL conical tubes. After lyophilization, a large fibrinogen mass is present in the bottom of the 15 mL conical tubes. To prevent confounding effects of additional salts, the lyophilized fibrinogen masses were resuspended with DI water. 10 mL of DI water is added to the lyophilized mass and images were taken 5 minutes later. No shaking, vortexing, or other mixing procedures were performed before imaging.1.3. Non-limiting Results

[0089] Typically, fibrinogen is difficult to keep soluble at concentrations over about 2% w / v in conventional buffers. Such low solubility limits the ability to modulate properties of the fibrinogen, and later, the fibrin network by changing the mass fraction in solution.

[0090] In contrast, the AG buffer solution — comprising 50 mM arginine and 50 mM glutamic acid in PBS — allows for the rapid suspension or resuspension of supraphy si ologi cal fibrinogen solutions, such as those greater than about 1% weight / volume (w / v). In some examples, the inventors demonstrate that the AG buffer solution allows for the rapid suspension or resuspension of supraphy si ologi cal fibrinogen solutions of up to at least 250 mg / mL (at least 25% w / v).

[0091] With minimal mixing, the inventors observed formation of fibrinogen solutions of up to 100 mg / mL with room temperature reagents. Vortexing or sonication allowed for solutions of at least 250 mg / mL. Fibrinogen does not dissolve in distilled water, but readilydissolves in the DIAG solution that includes 50 mM arginine and 50 mM glutamic acid in distilled water.

[0092] Overall, the results indicated that various solutions that include amino acids can be utilized to solubilize fibrinogen at much higher concentrations than those known in the art. In some examples, solutions of about 25% w / v fibrinogen or more can be created in minutes, even with using cold reagents, and with little or no precipitation. Such solutions enable supraphysiological levels of fibrinogen to be in solution and a high dynamic range in fibrin content enables altering of the mechanical properties of the particle by adjusting the mass fraction of fibrin.

[0093] DLS data confirmed that fibrinogen solutions made in the AG buffer solution are superior in their poly dispersity coefficient. Poly dispersity is a figure calculated by the DLS software and the larger the poly dispersity coefficient, the larger the overall coefficient the more variation of particle size is present in the solution. A poly dispersity coefficient >0.5 indicate a homogenous solution. As shown in FIGS. 1A and IB, when fibrinogen solutions were mixed / dissolved with PBS alone, the fibrinogen showed a higher poly dispersity coefficient (about 0.23) than a fibrinogen solution that was mixed / dissolved with the AG buffer solution (polydispersity coefficient of about 0.13).

[0094] As shown in FIG. 1C, supraphysiological fibrinogen (for example, 100 mg) solutions made with the AG buffer solution showed a higher poly dispersity coefficient (about 0.47) than solutions made with lower concentrations of fibrinogen, but the poly dispersity coefficient of 0.47 was low enough to be considered homogenous.

[0095] Solutions of -100 mg / mL of fibrinogen made in PBS alone could not be read by the DLS instrument due to the high degree of precipitation and flocculation present (data not shown), preventing direct comparison of poly dispersity between PBS and the AG buffer solution at supraphysiological concentrations. It was determined that 50 mM arginine and 50 mM glutamic acid added to DI water (DIAG solution) will dissolve fibrinogen solutions, but have high poly dispersity (FIG. ID).

[0096] While the AG buffer solution enabled the rapid dissolution of supraphysiological fibrinogen solutions at room temperature, the inventors investigated whether the solvation effect that the AG buffer solution could be expanded upon. Here, the inventors investigated whether the efficient solubilization of commercial fibrinogen powders could be extended to fibrinogen lyophilized in the AG buffer solution.

[0097] Lyophilization is a process that removes water from a material through freezing and sublimation under vacuum. The reduced water content allows for stable, long-term storage of many otherwise temperature sensitive materials of interest. Lyophilization of 25 mg / mL fibrinogen masses overnight yielded large, solid fibrinogen masses. These large masses had considerably less surface area compared to the commercially sourced powders previously used. Attempting to dissolve fibrinogen (25 mg / mL) in DI water yielded a precipitated mass that predictably did not re-suspend in DI water after lyophilization. Fibrinogen (25 mg / mL) dissolved in the DIAG solution, and the lyophilized mass at least partially resuspended in DI water. Fibrinogen (25 mg / mL) that was dissolved in PBS, lyophilized, and resuspended in DI water was found to dissolve, but left large flocculants floating in solution. A fibrinogen mass (25 mg / mL) that was prepared by first dissolving in the AG buffer solution readily goes into solution with no visible flocculation. The inventors also found that a 50 mg / mL fibrinogen solution did not precipitate in a 10% ethanol / DI water solution.

[0098] With these positive results from creating high concentration monodisperse fibrinogen solutions, the inventors investigated whether similar results could be obtained with other model proteins. BSA, a globular protein derived from bovine blood, is commonly used as a blocking protein due to its solubility and low cost. When dissolved in PBS, BSA showed a poly dispersity coefficient of about 3.34, indicating an extremely polydisperse solution (FIG. 2A). When BSA solutions were made in AG buffer, they showed a poly dispersity coefficient of about 0.73 (FIG. 2B). Although the poly dispersity of BSA dissolved in the AG buffer solution was still high enough to indicate large aggregates, the nearly 5-fold reduction in poly dispersity coefficient is noteworthy.

[0099] Gelatin, denatured collagen derived from byproduct connective tissue, is another commonly used protein for bulk laboratory use. Gelatin dissolved in PBS showed a poly dispersity coefficient of about 4.09 (FIG. 2C). Gelatin dissolved in the AG buffer solution showed a poly dispersity coefficient of about 4.22 (FIG. 2D). These high poly dispersity coefficients of gelatin may be due to the highly heterogeneous nature of gelatin mitigating the effects of the AG buffer solution to alter the poly dispersity coefficient.1.4. Non-limiting Discussion

[0100] The inventors found that, when applied to protein solubility, the charged amino acids arginine and glutamic acid greatly increase the solubility of fibrinogen allowing for facile creation of supraphy si ologi cal solutions. The mechanical properties of fibrin are directlyrelated to the mass fraction of the fibrinogen solution and commercial fibrin sealants use 40- 100 mg / mL concentrations of fibrinogen. The rapid and homogenous resuspension of fibrinogen with the AG buffer solution could benefit tissue sealants without the use or concern for additional salts.

[0101] The long-term stability provided by lyophilization combined with the rapid resuspension provided by the AG buffer solution enables fibrinogen-based sealants that do not require a cold chain, as current fibrinogen sealants require. The most widespread method of biologic stabilization is cold storage using what is known as the “cold-chain”, a system of refrigerators and freezers used during the production, transportation, and storage of a biologic to help maintain its viability. Under ideal circumstances, cold stabilization can be effective, however, in remote or developing parts of the world, purchasing, and maintaining the necessary infrastructure such as freezers, electrical systems, and backup generators needed for the coldchain to work seamlessly can be close to impossible. The cold-chain is currently essential for the stabilization of most biologies. This is because above certain temperatures (for example, about 8°C for most vaccines), molecular dynamics and rearrangements are accelerated leading to hastened breakdown of these materials. At too cold of temperatures (for example, about 2°C for most vaccines), water begins to freeze, leading to the formation of ice crystals that can irreparably damage sensitive biologies. Overall, the cold-chain, while effective, represents a serious economic and logistical hurdle for deploying materials of interest (such as biologies) in remote, underdeveloped, or austere settings where access to cold-chain infrastructure ranging from refrigerators and freezers to stable electricity is limited.

[0102] Overall, the data described herein indicates that embodiments described herein may be utilized to overcome the major obstacle of the cold chain. The data also indicates that embodiments described herein may be used to make suspendible compositions that include a material of interest being free of aggregates even at supraphy si ologi cal amounts of the material of interest.

[0103] The AG buffer solution likely has utility beyond creating supraphy si ologi cal fibrinogen solutions. For example, the data shows the utility of the AG buffer solution in increasing the monodispersity of proteins even at extreme concentrations. Limiting interprotein interactions through use of the AG buffer solution shows utility by the facile creation of supraphy si ologi cal fibrin solutions and in the formulation of a rapid-resuspension mass. It is likely that AG buffer’s solubilization and aggregation limiting effects have myriad uses. Forexample, the inventors are investigating the effects of an AG buffer solution in lowering the melting temperature of gelatin solutions, overcoming the frustrating effect of room temperature gelation.Example 2: Shelf-Stable Fibrinogen-Based Tissue Sealants2.1. Introduction

[0104] Protein solubility is an area of tremendous concern to various industries, for example, the pharmaceutical, biotechnology, food processing, and agricultural industries as well as to researchers who study proteins. Protein solubility is characterized by problems such as protein aggregation and precipitation under non-ideal conditions. Conventional buffers used to stabilize proteins under non-ideal conditions show limited protein solubility, limiting the total amount of dissolved protein that can be made available. As protein concentration increases, proteins seek the lowest free energy state and aggregate or precipitate out of solution. Aggregation is often irreversible and can render commercially valuable proteins useless.2.2. Non-limiting Technology Description

[0105] To address these and other issues, the inventors developed a novel buffer solution that may include millimolar concentrations of arginine and glutamic acid in PBS (an AG buffer solution). While not wishing to be bound by any theory, it is believed that the positive and negative charges on arginine and glutamic acid side chains, respectively, may alter the hydration shell of a protein by interacting with peptide residues of opposing charges. This interaction may greatly increase the free energy required for inter-protein interactions, thereby preventing highly charged regions on proteins from interacting and thus limiting aggregation. Moreover, arginine and glutamic acid have a similar effect to increasing the salt concentration of the solution, assisting in protein solubility without the deleterious cytocompatibility concerns of high salt. That is, arginine and glutamic acid may mimic the effects of high salt concentrations in solutions, aiding in protein solubility without the harmful effects of high salt formulations conventionally used.

[0106] Results described herein indicated that an AG buffer solution rapidly dissolved large quantities of proteins, such as the glycoprotein fibrinogen. The use of the AG buffer solution enabled the near-instantaneous creation of aggregate free solutions at concentrations of hundreds of milligrams per milliliter. Such concentrations represented an order of magnitude greater than current state of the art. Such concentrations also significantly exceeded the physiological concentration of fibrinogen in blood.

[0107] A goal of the investigation was to develop a buffer and a composition that stabilizes fibrinogen and that may be used instead of conventional cryoprotectants and stabilizers. The buffer and the composition that stabilizes fibrinogen may be used in field-deployable fibrinogen tissue sealants for field trauma that do not require a cold chain, with increased mechanical properties compared to current sealants, and rapid, homogenous resuspension immediately prior to use.

[0108] Fibrinogen is the primary protein responsible for the formation of blood clots and therefore maintaining hemostasis. Fibrinogen is the primary component of tissue sealants or Fibrin Glues, and is used in surgeries worldwide to control unwanted or excessive bleeding. 500 million years of evolution has granted fibrinogen unique and unmatched properties to control bleeding and fibrin tissue sealants are responsible for greatly enhancing the safety of surgeries since their introduction. Despite the utility and efficacy of tissue sealants to control bleeding, their use is generally limited to hospitals as resuspension of tissue sealants is a timeconsuming process with a limited pot-life once resuspended. For example, conventional compositions require 20 minutes or more of re-suspension time which may not be available during a surgery and is too long for traumatic field use.

[0109] These limitations such as short pot-life, inability to have a high fibrinogen concentration product, and time-consuming resuspension may arise from its poor solubility in biocompatible buffers. As a result, there is a need for a shelf-stable fibrinogen-containing composition that has high amounts of fibrinogen and that may be rapidly suspended or dissolved. Such shelf-stable rapid-resuspension fibrinogen-containing compositions may be used as fibrinogen-based tissue sealants. The shelf-stable rapid-resuspension fibrinogen- containing compositions may be formed by lyophilizing a solution that includes fibrinogen, amino acids, and an aqueous buffer. For resuspension, an aqueous solvent may be introduced with the composition. The resuspension may be free of vortexing or sonicating, though vortexing and sonicating are contemplated. The resuspension of the composition enables supraphy si ologi cal concentrations of the fibrinogen in, for example, an AG buffer solution.

[0110] Results described herein demonstrated the creation of lyophilized fibrinogen compositions, such as fibrinogen-based sealants, that resuspended to supraphy si ologi cal concentrations within seconds at room temperature without aggregation or unwanted gelling. This significant improvement in re-suspension time (conventional technologies require more than 20 minutes along with vortexing or sonicating) enabled more efficient use and easierdeployment of the lyophilized fibrinogen compositions described herein as tissue sealants. Sonicated fibrin is the conventional route to provide blood clotting. However, sonication produces fibrinogen particles (and resulting fibrin particles) of various dimensions and sizes, resulting in poor mechanical properties and limiting their use.[OHl] Moreover, the lyophilized fibrinogen compositions described herein are useful in the field and do not require the cold-chain or refrigeration. Further, the lyophilized fibrinogen compositions described herein are stable over a variety of fibrinogen concentrations with none of the protein aggregation and gelling observed with conventional technologies.

[0112] Overall, the development of shelf-stable, rapidly-suspendible compositions (and articles thereof) described herein addresses a significant unmet need in emergency and battlefield trauma care, where controlling bleeding quickly and effectively can be life-saving. The avoidance of the cold chain for storage and transportation further enhances the commercial appeal of this technology. By eliminating the need for refrigeration and the cold-chain, sealant compositions described herein become more accessible and practical for use in field surgeries and remote locations, significantly expanding their reach. Embodiments described herein may be used in a variety of applications such as wound care in combat zones where speed and product stability in diverse environments and in healthcare and emergency response sectors.

[0113] This novel technology described herein significantly impacts the efficiency and effectiveness of wound treatment in field medicine by providing immediate access to high- concentration sealants without the delays and logistical challenges associated with reconstitution and refrigeration. This will ultimately save lives by enabling faster control of bleeding and stabilization of wounds than currently possible.

[0114] In addition, buffer solutions comprising amino acids as described herein may be utilized for stabilizing other materials of interest during lyophilization, such as pharmaceuticals, food products, and agricultural products, among other materials of interest. For example, livestock feed stability, food emulsification, and recombinant protein solubility are problems seen in food processing, agriculture, and pharmaceutical industries, respectively. Embodiments Listing

[0115] The present disclosure provides, among others, the following embodiments, each of which may be considered as optionally including any alternate embodiments:

[0116] Clause Al. A composition, comprising: a first amino acid having a side chain group that is negatively charged at a pH of 7;a second amino acid having a side chain group that is positively charged at a pH of7; a material of interest, the material of interest different from the first amino acid and the second amino acid; and the composition characterized as being suspendible or resuspendible in an aqueous material in a period of less than 20 minutes.

[0117] Clause A2. The composition according to Clause 1, wherein the composition is a lyophilized composition, an air-dried composition, a dehydrated composition, a desiccated composition, a vacuum desiccated composition, a vacuum dried composition, a spray-dried composition, a freeze-dried composition, spray-freeze dried composition, a foam-dried composition, or combinations thereof.

[0118] Clause A3. The composition according to any one of the preceding Clauses, wherein the material of interest comprises a protein, a recombinant protein, or combinations thereof.

[0119] Clause A4. The composition according to any one of the preceding Clauses, wherein the material of interest comprises a protein that is dissolvable in an aqueous suspension.

[0120] Clause A5. The composition according to any one of the preceding Clauses, wherein the material of interest comprises fibrinogen, BSA, gelatin, or combinations thereof.

[0121] Clause A6. The composition according to any one of the preceding Clauses, wherein: when the composition is suspended or re-suspended in the aqueous material, the material of interest remains dissolved in the aqueous material; an amount of the material of interest that remains dissolved in the aqueous material comprises a supraphy si ologi cal amount of the material of interest; or a combination thereof.

[0122] Clause A7. The composition according to any one of the preceding Clauses, wherein the material of interest comprises fibrinogen.

[0123] Clause A8. The composition according to any one of the preceding Clauses, wherein: the first amino acid comprises glutamic acid, aspartic acid, or combinations thereof; the second amino acid comprises arginine, lysine, or combinations thereof; or a combination thereof.

[0124] Clause A9. The composition according to any one of the preceding Clauses, wherein: a weight ratio of the first amino acid to the second amino acid is in a range from about 45:55 to about 55:45.

[0125] Clause A10. An article comprising: a substrate impregnated or coated with the composition according to any one of the preceding Clauses.

[0126] Clause Al l. A suspendible fibrinogen composition, comprising: a first amino acid having a side chain group that is negatively charged at a pH of 7; a second amino acid having a side chain group that is positively charged at a pH of 7; fibrinogen; and the suspendible fibrinogen composition characterized as being suspendible or resuspendible in an aqueous material in a period of less than 20 minutes.

[0127] Clause A12. The suspendible fibrinogen composition according to Clause Al l, wherein: when the suspendible fibrinogen composition is suspended or re-suspended in the aqueous material, the fibrinogen remains dissolved in the aqueous material; the suspendible fibrinogen composition is characterized as being suspendible or resuspendible in an aqueous material in a period of less than 5 minutes; or combinations thereof.

[0128] Clause Al 3. The suspendible fibrinogen composition according to any one of Clauses Al 1-A12, wherein an amount of the fibrinogen that remains dissolved in the aqueous material comprises a supraphy si ologi cal amount of the fibrinogen.

[0129] Clause A14. The suspendible fibrinogen composition according to any one of Clauses A11-A13, wherein the composition, upon suspension or re-suspension in an aqueous material, comprises about 5% w / v fibrinogen or more.

[0130] Clause Al 5. The suspendible fibrinogen composition according to any one of Clauses A11-A14, wherein the suspendible fibrinogen composition, upon suspension or resuspension in an aqueous material, comprises about 5% w / v to about 25% w / v fibrinogen.

[0131] Clause Al 6. The suspendible fibrinogen composition according to any one of Clauses A11-A15, wherein the suspendible fibrinogen composition is a lyophilized composition, an air-dried composition, a dehydrated composition, a desiccated composition, a vacuum desiccated composition, a vacuum dried composition, a spray-dried composition, a freeze-dried composition, spray-freeze dried composition, a foam-dried composition, or combinations thereof.

[0132] Clause Al 7. The suspendible fibrinogen composition according to any one of Clauses A11-A16, wherein: the first amino acid comprises glutamic acid, aspartic acid, or a combination thereof; the second amino acid comprises arginine, lysine, or a combination thereof; and a weight ratio of the first amino acid to the second amino acid is in a range from about 45:55 to about 55:45.

[0133] Clause Al 8. An article, comprising: a substrate impregnated or coated with the suspendible fibrinogen composition according to any one of Clauses Al 1-A17.

[0134] Clause A19. The article according to Clause A18, wherein the substrate comprises a bandage, an adhesive bandage, gauze, cloth, tampon, membrane, or sponge.

[0135] Clause A20. A process, comprising: introducing an aqueous material with a composition comprising a material of interest, a first amino acid, and a second amino acid to form a suspension or solution of the material of interest, the first amino acid, and the second amino acid in the aqueous material, the material of interest different from the first amino acid and the second amino acid, the first amino acid and the second amino acid are different, the first amino acid having a side chain group that is negatively charged at a pH of 7, the second amino acid having a side chain group that is positively charged at a pH of 7, the suspension or solution characterized as being free of aggregated material of interest or free of aggregated fibrinogen.

[0136] Clause A21. A method of treating a wound in a patient, comprising: administering to a patient the composition of any one of Clauses Al -Al 7 or the article of any one of Clauses A18-A19.

[0137] Clause A22. A method to control bleeding in a patient, comprising: administering to a patient the composition according to any one of Clauses Al -Al 7 or the article of any one of Clauses A18-A19.

[0138] Clause A23. The method according to any one of Clauses A21-A22, further comprising: introducing an aqueous material to the composition of any one of Clauses Al -Al 7 or the article of any one of Clauses A18-A19 prior to administering to the patient the composition or the article.

[0139] Clause A24. A process, comprising: introducing an aqueous material with the composition according to any one of Clauses Al -Al 7 to form a suspension or solution of the material of interest and the amino acids in the aqueous material, the suspension or solution characterized as being free of aggregated material of interest or free of aggregated fibrinogen.

[0140] Clause Bl. A composition, comprising: a material of interest; and charged amino acids, the composition characterized as being suspendible or resuspendible in an aqueous material in a period of less than five minutes.

[0141] Clause B2. The composition of Clause Bl, wherein: the material of interest comprises a protein, a recombinant protein, a pharmaceutical, a food product, an agricultural product, or combinations thereof; the composition is characterized as being suspendible or resuspendible in an aqueous material in a period of less than 2 minutes; or combinations thereof.

[0142] Clause B3. The composition of Clauses B1-B2, wherein, when the composition is suspended or re-suspended in the aqueous material, the material of interest remains dissolved in the aqueous material.

[0143] Clause B4. The composition of Clauses B1-B3, wherein an amount of the material of interest that remains dissolved in the aqueous material comprises a physiological amount or a supraphy si ologi cal amount of the material of interest.

[0144] Clause B5. The composition of any one of Clauses Bl -B4, wherein the composition is further characterized as stabilizing the material of interest of the composition at a temperature of ambient (room) temperature or higher.

[0145] Clause 6. The composition of any one of Clauses B1-B5, wherein the composition is further characterized as stabilizing the material of interest in a dry state.

[0146] Clause B7. The composition of any one of Clauses Bl -B6, wherein the composition comprises 15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%.

[0147] Clause B8. The composition of any one of Clauses B1-B7, wherein the material of interest comprises fibrinogen.

[0148] Clause B9. The composition of any one of Clauses 1-8, wherein the charged amino acids comprises: a first amino acid having a side chain group that is negatively charged at a pH of 7; and a second amino acid having a side chain group that is positively charged at a pH of 7.

[0149] Clause BIO. The composition of any one of Clauses B1-B9, wherein the charged amino acids comprises: glutamic acid, aspartic acid, or combinations thereof; arginine, lysine, or combinations thereof; or combinations thereof.

[0150] Clause B 11. The composition of any one of Clauses B 1 -B 10, wherein a weight ratio of the material of interest to the negatively charged amino acid in the composition is from about 0.1 :1 to about 10: 1.

[0151] Clause B 12. The composition of any one of Clauses Bl-Bl 1, wherein a weight ratio of the material of interest to the positively charged amino acid in the composition is from about 0.1 :1 to about 10: 1.

[0152] Clause B13. The composition of any one of Clauses B1-B12, wherein: the charged amino acids comprises a first amino acid having a side chain group that is negatively charged at a pH of 7; the charged amino acids comprises a second amino acid having a side chain group that is positively charged at a pH of 7; and a weight ratio of the first amino acid to the second amino acid is from about 0.1 : 1 to about 10: 1.

[0153] Clause B14. A suspendible fibrinogen composition, comprising: fibrinogen; and charged amino acids, the composition characterized as being suspendible or resuspendible in an aqueous material in a period of less than five minutes.

[0154] Clause Bl 5. A rapidly reconstituted anti-thrombogenic tissue sealant appropriate for field use at the point of need, comprising a composition comprising: fibrinogen; and amino acids.

[0155] Clause B16. The composition of any one of Clauses B14-B15, wherein: when the composition is suspended or re-suspended in the aqueous material, the fibrinogen remains dissolved in the aqueous material; the composition is characterized as being suspendible or resuspendible in an aqueous material in a period of less than 2 minutes; or combinations thereof.

[0156] Clause B17. The composition of Clauses B14-B16, wherein an amount of the fibrinogen that remains dissolved in the aqueous material comprises a physiological amount or a supraphy si ologi cal amount of the fibrinogen.

[0157] Clause Bl 8. The composition of any one of Clauses B14-B17, wherein the composition, upon suspension or re-suspension in an aqueous material, comprises: about 2% w / v fibrinogen or more; or about 10% w / v fibrinogen or more.

[0158] Clause B19. The composition of any one of Clauses B14-B18 wherein the composition, upon suspension or re-suspension in an aqueous material, comprises about 5% w / v to about 25% w / v fibrinogen.

[0159] Clause B20. The composition of any one of Clauses B14-B19, wherein the suspendible fibrinogen composition is a lyophilized fibrinogen composition.

[0160] Clause B21. The composition of any one of Clauses B14-B20, wherein the composition is further characterized as stabilizing the fibrinogen of the composition at a temperature of ambient (room) temperature or higher.

[0161] Clause B22. The composition of any one of Clauses B14-B21, wherein the composition is further characterized as stabilizing the fibrinogen of the composition in a dry state.

[0162] Clause B23. The composition of any one of Clauses B14-B22, wherein the composition comprises 15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%.

[0163] Clause B24. The composition of any one of Clauses Bl 4-B23, wherein the charged amino acids comprises: a first amino acid having a side chain group that is negatively charged at a pH of 7; and a second amino acid having a side chain group that is positively charged at a pH of 7.

[0164] Clause B25. The composition of any one of Clauses Bl 4-B24, wherein the charged amino acids comprises: glutamic acid, aspartic acid, or combinations thereof; arginine, lysine, or combinations thereof; or combinations thereof

[0165] Clause B26. The composition of any one of Clauses B14-B25, wherein a weight ratio of the material of interest to the negatively charged amino acid in the composition is from about 0.1 : 1 to about 10: 1.

[0166] Clause B27. The composition of any one of Clauses B14-B26, wherein a weight ratio of the material of interest to the positively charged amino acid in the composition is from about 0.1 : 1 to about 10: 1.

[0167] Clause B28. The composition of any one of Clauses B14-B27, wherein: the charged amino acids comprises a first amino acid having a side chain group that is negatively charged at a pH of 7; the charged amino acids comprises a second amino acid having a side chain group that is positively charged at a pH of 7; and a weight ratio of the first amino acid to the second amino acid is from about 0.1 : 1 to about 10: 1.

[0168] Clause B29. The composition of any one of Clauses B14-B28, further comprising thrombin, a fibrinogen polymerization inhibitor, a thrombin inhibitor, or combinations thereof.

[0169] Clause B30. The composition of Clause B29, wherein the thrombin inhibitor comprises bivalirudin.

[0170] Clause B31. An article, comprising: a substrate impregnated or coated with the composition of any one of Clauses Bl -B30.

[0171] Clause B32. The article of Clause B31, wherein the article comprises a medical dressing, a wound dressing, a hemostatic tissue sealant, or a closure for a bleeding wound.

[0172] Clause B33. The article of any one of Clauses B31-B32, wherein the substrate comprises a bandage, an adhesive bandage, gauze, cloth, tampon, membrane, or sponge.

[0173] Clause B34. A method of treating a wound in a patient, comprising: administering to a patient the composition of any one of Clauses B1-B30 or the article of any one of Clauses B31-B33.

[0174] Clause B35. A method to control bleeding in a patient, comprising: administering to a patient the composition of any one of Clauses 1-30 or the article of any one of Clauses B31-B33.

[0175] Clause B36. The method of any one of Clauses B34-B35, further comprising: introducing an aqueous material to the composition of any one of Clauses 1-30 or the article of any one of Clauses B31-B33 prior to administering to the patient the composition or the article.

[0176] Clause 37. A process, comprising: introducing an aqueous material with the composition of any one of Clauses B1-B30 to form a suspension or solution of the material of interest and the amino acids in the aqueous material, the suspension or solution characterized as being free of aggregated material of interest or free of aggregated fibrinogen.

[0177] All documents described herein are incorporated by reference herein, including any priority documents and / or testing procedures to the extent they are not inconsistent with this text. As is apparent from the foregoing general description and the specific embodiments, while forms of the embodiments have been illustrated and described, various modifications may bemade without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the present disclosure be limited thereby. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise whenever a composition, an element, a group of elements, or a method is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition, method, or group of elements with transitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “Is” preceding the recitation of the composition, element, elements, or method, and vice versa, such as the terms “comprising,” “consisting essentially of,” “consisting of’ also include the product of the combinations of elements listed after the term.

[0178] In the foregoing, reference is made to embodiments of the disclosure. However, it should be understood that the disclosure is not limited to specific described embodiments. Instead, any combination of the following features and elements, whether related to different embodiments or not, is contemplated to implement and practice the disclosure. Furthermore, although embodiments of the disclosure may achieve advantages over other possible solutions and / or over the prior art, whether or not a particular advantage is achieved by a given embodiment is not limiting of the disclosure. Thus, the foregoing aspects, features, embodiments, and advantages are merely illustrative and are not considered elements or limitations of the appended claims except where explicitly recited in a claim(s). Likewise, reference to “the disclosure” shall not be construed as a generalization of any inventive subject matter disclosed herein and shall not be considered to be an element or limitation of the appended claims except where explicitly recited in a claim(s).

[0179] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error and variations that would be expected by a person having ordinary skill in the art. For the sake of brevity, only certain ranges are explicitly disclosed herein. However, ranges from any lower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the subranges 1 to 4, 1.5 to 4.5, 1 to 2, among other subranges. As anotherexample, the recitation of the numerical ranges 1 to 5, such as 2 to 4, includes the subranges 1 to 4 and 2 to 5, among other subranges. Additionally, within a range includes every point or individual value between its end points even though not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the numbers 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, among other numbers. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.

[0180] As used herein, the indefinite article “a” or “an” shall mean “at least one” unless specified to the contrary or the context clearly indicates otherwise. For example, embodiments comprising “an amino acid” include embodiments comprising one, two, or more amino acids, unless specified to the contrary or the context clearly indicates only one amino acid is included.

[0181] While the foregoing is directed to embodiments of the present disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

ClaimsWhat is claimed is:

1. A composition, comprising: a first amino acid having a side chain group that is negatively charged at a pH of 7; a second amino acid having a side chain group that is positively charged at a pH of 7; a material of interest, the material of interest different from the first amino acid and the second amino acid; and the composition characterized as being suspendible or resuspendible in an aqueous material in a period of less than 20 minutes.

2. The composition according to claim 1, wherein the composition is a lyophilized composition, an air-dried composition, a dehydrated composition, a desiccated composition, a vacuum desiccated composition, a vacuum dried composition, a spray-dried composition, a freeze-dried composition, spray-freeze dried composition, a foam-dried composition, or combinations thereof.

3. The composition according to claim 1, wherein the material of interest comprises a protein, a recombinant protein, or combinations thereof.

4. The composition according to claim 1, wherein the material of interest comprises a protein that is dissolvable in an aqueous suspension.

5. The composition according to claim 1, wherein the material of interest comprises fibrinogen, BSA, gelatin, or combinations thereof.

6. The composition according to claim 1, wherein: when the composition is suspended or re-suspended in the aqueous material, the material of interest remains dissolved in the aqueous material; an amount of the material of interest that remains dissolved in the aqueous material comprises a supraphysiological amount of the material of interest; or a combination thereof.

7. The composition according to claim 1, wherein the material of interest comprises fibrinogen.

8. The composition according to claim 1, wherein: the first amino acid comprises glutamic acid, aspartic acid, or combinations thereof; the second amino acid comprises arginine, lysine, or combinations thereof; or a combination thereof.

9. The composition according to claim 1, wherein: a weight ratio of the first amino acid to the second amino acid is in a range from about 45:55 to 55:45.

10. An article comprising: a substrate impregnated or coated with the composition according to claim 1.

11. A suspendible fibrinogen composition, comprising: a first amino acid having a side chain group that is negatively charged at a pH of 7; a second amino acid having a side chain group that is positively charged at a pH of 7; fibrinogen; and the suspendible fibrinogen composition characterized as being suspendible or resuspendible in an aqueous material in a period of less than 20 minutes.

12. The suspendible fibrinogen composition according to claim 11, wherein: when the suspendible fibrinogen composition is suspended or re-suspended in the aqueous material, the fibrinogen remains dissolved in the aqueous material; the suspendible fibrinogen composition is characterized as being suspendible or resuspendible in an aqueous material in a period of less than 5 minutes; or combinations thereof.

13. The suspendible fibrinogen composition according to claim 11, wherein an amount of the fibrinogen that remains dissolved in the aqueous material comprises a supraphy si ologi cal amount of the fibrinogen.

14. The suspendible fibrinogen composition according to claim 11, wherein the composition, upon suspension or re-suspension in an aqueous material, comprises about 5% w / v fibrinogen or more.

15. The suspendible fibrinogen composition according to claim 11 , wherein the suspendible fibrinogen composition, upon suspension or re-suspension in an aqueous material, comprises about 5% w / v to about 25% w / v fibrinogen.

16. The suspendible fibrinogen composition according to claim 11, wherein the suspendible fibrinogen composition is a lyophilized composition, an air-dried composition, a dehydrated composition, a desiccated composition, a vacuum desiccated composition, a vacuum dried composition, a spray-dried composition, a freeze-dried composition, spray-freeze dried composition, a foam-dried composition, or combinations thereof.

17. The suspendible fibrinogen composition according to claim 11, wherein: the first amino acid comprises glutamic acid, aspartic acid, or a combination thereof; the second amino acid comprises arginine, lysine, or a combination thereof; and a weight ratio of the first amino acid to the second amino acid is in a range from about 45:55 to 55:45.

18. An article, comprising: a substrate impregnated or coated with the suspendible fibrinogen composition according to claim 11.

19. The article according to claim 18, wherein the substrate comprises a bandage, an adhesive bandage, gauze, cloth, tampon, membrane, or sponge.

20. A process, comprising: introducing an aqueous material with a composition comprising a material of interest, a first amino acid, and a second amino acid to form a suspension or solution of the material of interest, the first amino acid, and the second amino acid in the aqueous material,the material of interest different from the first amino acid and the second amino acid, the first amino acid having a side chain group that is negatively charged at a pH of 7, the second amino acid having a side chain group that is positively charged at a pH of 7, the suspension or solution characterized as being free of aggregated material of interest or free of aggregated fibrinogen.

Images