Ultra-concentrated formulation of bioactive agents

Abstract

The present disclosure provides ultra-concentrated formulations of one or more bioactive agents in an administration solvent, which may include a bioactive agent present in a higher concentration than a solution of the bioactive agent in the administration solvent formulated for subcutaneous administration to a subject. The present disclosure also provides methods of preparing the ultra-concentrated formulations, and methods of using the ultra-concentrated formulations to treat or prevent a disease.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 217,462, filed July 1, 2021, which is incorporated by reference in its entirety. [Technical field]

[0002] The present disclosure relates to ultra-concentrated bioactive agent formulations and methods for their preparation.

[0003] Delivery of biologics to treat diseases or conditions is most commonly by intravenous (IV) administration. Biologics are usually considered therapeutic molecules with high molecular weight (e.g., 500 Da or greater) and / or similar to molecules normally found in the body (e.g., antibodies). Monoclonal antibodies (mABs) make up the majority of biologics currently approved for IV administration. There are many representative examples of mABs approved for IV infusion, including muromonab CD3 for treating organ transplant rejection, infliximab for treating rheumatoid arthritis, among other diseases, rituximab for treating non-Hodgkin's lymphoma, and alemtuzamab for treating B-cell chronic lymphocytic leukemia, among many other such mABs. Other biologics that require IV administration are also now known, including, but not limited to, aldesleukin, among others, for treating melanoma. From a practical standpoint, antibody therapies often require large therapeutic doses (e.g., 8 mg / kg for trastuzumab and 375 mg / m for rituximab). 2 ), the required injection volume to completely solubilize the antibody, which can only be administered via IV.

[0004] One limitation of IV infusion is the potential for rapid clearance of the therapeutic agent. Clearance of biologics from the circulation occurs either by renal filtration or nonspecific binding and absorption (e.g., endothelial pinocytosis). Short circulatory half-lives can result in weekly to daily dosing, necessitating frequent injections. This increases patient discomfort, the overall cost of the treatment regimen, and the risk of patient nonadherence.

[0005] To extend plasma half-life, a strategy known as PEGylation has been developed, which involves covalently attaching a hydrophilic polymer (polyethylene glycol; "PEG") to a therapeutic agent. This increases the hydrodynamic size of the therapeutic molecule, thereby decreasing the renal clearance rate and significantly increasing the persistence in the circulation by up to several hundred-fold. However, there have been limited successful FDA-approved PEGylated biologics for IV administration, such as pegloticase (a uric acid metabolizing enzyme) to treat chronic gout.

[0006] Another limitation of IV delivery is the potential toxic side effects in non-target organs due to intravenous administration of high levels of biologics. For example, aldesleukin therapy requires a significantly smaller dose (0.037 mg / kg per dose) than antibody therapy, but systemic infusion of aldesleukin is associated with severe, potentially fatal, side effects, necessitating inpatient administration. Another recombinant cytokine therapy that has proven extremely difficult to optimize in terms of balancing efficacy with minimal toxicity is the use of tumor necrosis factor alpha (TNF-α) as an immune-stimulating anti-cancer treatment. TNF-α is thought to act preferentially on tumor endothelium, inducing hyperpermeability that causes hemorrhagic necrosis of tumor tissue. However, exposure to high systemic levels of TNF-α can cause severe toxicities, such as hypotension and septic shock-like syndrome.

[0007] The problems of toxicity and rapid clearance following IV delivery of biologics are both related to the almost immediate systemic availability of the infused therapeutic. Although such rapid availability may be necessary to achieve therapeutic benefit, many biologics benefit from a slow pharmacokinetic distribution in the body (while maintaining systemic availability). Furthermore, IV infusion carries the risk of catheter-borne infection, and the emergence of resistant spores, especially in the hospital environment, can result in significant pain and discomfort, necessitating supervised inpatient administration.

[0008] Subcutaneous (SC) delivery of biologics is a very interesting method for more patient-friendly administration and to allow self-administration. Currently, many approved biologics are administered SC, and the success of this administration route is crucial for biologic therapies used to manage chronic medical conditions or symptoms, especially when combined with delivery into prefilled syringes (PFS), pens, or autoinjector devices that allow self-administration or home administration. In fact, many PEGylated biologics have been approved for SC injection recently, including COVID-19 vaccines from Pfizer and Moderna. Subcutaneous administration improves patient compliance and reduces the cost of treatment by avoiding hospitalization. Especially for biologics with short circulating half-lives that may require frequent administration, being able to self-administer therapeutics by SC injection, as opposed to the need for continuous hospitalized IV infusion, offers a tremendous benefit to the patient's quality of life.

[0009] Another situation in which the use of SC injections can improve IV delivery is in the case of biological drugs that have severe toxicity after systemic injection. For example, recombinant cytokine therapies such as α and β INF provide therapeutic benefit in many diseases such as various cancers, Hepatitis B and C, and multiple sclerosis, but IV delivery of such therapeutics can cause adverse events after rapid systemic diffusion of the therapeutic. SC administration has an advantage over IV infusion because it creates a relative accumulation effect at the injection site. The time required for the therapeutic to be cleared into the systemic circulation effectively extends the half-life of the drug in vivo and at the same time reduces the maximum drug concentration in various compartments. This reduces the required frequency of administration and reduces the frequency and severity of adverse events.

[0010] Although SC injections can be used to achieve a flatter pharmacokinetic profile (lower peak plasma concentrations but longer duration at effective levels), a limitation of the SC route is the injection volume that can be administered painlessly, which is typically up to about 2-2.5 ml of liquid in humans. Some biologic regimens, such as therapeutic antibodies in oncology applications, require a 5 ml dose for the most concentrated form of the drug that can be prepared in a stable injectable form, making traditional SC injections problematic. To overcome this volumetric limitation, one strategy currently in clinical trials is to use recombinant human hyaluronidase to locally digest hyaluronic acid, a key polysaccharide component of the extracellular matrix (ECM) of connective tissues.

[0011] When hyaluronidase is co-injected with a biologic, it creates nanoscale porosity in the local ECM, allowing rapid drainage of fluids from the injection site and allowing the injection of large volumes of solution without pain. This change is only temporary, as the matrix repairs itself within approximately 24 hours. To improve patient compliance in long-term (>1 year) maintenance therapy, as indicated in situations such as the treatment of early stage breast cancer, several oncology-approved mABs (trastuzumab, rituximab) currently administered as IV infusions are being tested with this modified administration strategy.

[0012] Another motivation for local injection of biologics is to minimize systemic exposure and subsequent toxicity. An example of this is intravitreal (directly into the eye) injection of vascular endothelial growth factor (VEGF) antagonists to treat age-related macular degeneration (AMD). Pegaputinib, an RNA aptamer targeting VEGF, and the anti-VEGF mAbs bevacizumab and ranibizumab have all demonstrated the ability to slow the progression of AMD. However, systemic injection of VEGF antagonists may disrupt the normal function of VEGF in healthy vasculature throughout the body, potentially leading to increased risk of thromboembolism, hypertension, and impaired wound healing. Early clinical studies have shown that intravitreal injection of VEGF antagonists has an improved safety profile and reduced frequency of systemic adverse events compared to IV infusion.

[0013] Another important motivation for local injection of biological therapeutics is to maximize the local concentration of the therapeutic agent and improve the efficacy of treatment for a specific tissue or organ. For example, the bispecific antibody catumaxomab (anti-CD3 / anti-epithelial cell adhesion molecule EPCAM) is injected intraperitoneally to treat malignant ascites resulting from epithelial, gastric, or ovarian cancer. Preclinical pharmacokinetic studies confirmed that intraperitoneal (IP) administration of catumaxomab generates high local concentrations of antibody in the ascites while greatly limiting systemic exposure (lowest limit of detection in plasma is <5%), an important finding considering the potential toxicity of systemic anti-CD3 stimulation.

[0014] To take advantage of the aforementioned advantages of SC administration, highly concentrated solutions are necessary to keep injection volumes small. Highly concentrated protein formulations (HCPF) are generally, albeit imprecisely, applied to protein formulations in the 50-150 mg / mL range, although the physical properties of HCPF can also be applied to non-protein biologics. HCPF properties include, for example, increased viscosity, high opalescence, liquid-liquid phase separation, gel formation, or increased tendency to form protein particles. The main goals of highly concentrated formulations are protein stability and good injectability, the latter of which requires low to moderate viscosity. Chemical degradation mechanisms include deamidation, oxidation, and formation of isoaspartic acid. Insufficient colloidal stability leads to irreversible aggregation, precipitation, and phase separation.

[0015] A limited number of commercially available high-concentration biological products, with protein concentrations between 150 and 200 mg / mL, are supplied as lyophilized (freeze-dried) products. However, the development of high-concentration lyophilized protein formulations poses additional challenges, including lengthy reconstitution times and stability issues. Some properties observed at high protein concentrations pose special challenges for the development of lyophilized drug products.

[0016] Colloidal instability increases at higher protein concentrations. Liquid-liquid phase separation can be enhanced during the freezing step of lyophilization. Phase separation of excipients during lyophilization can also impair protein stability. Phase separation of excipients may be one of the reasons why the concept of "glassy immobilization" often used to describe protein stabilization in lyophilized solids does not always hold; i.e., the protein simply does not "see" the glass. Excipients that function as effective protein stabilizers not only form a chemically inert glass, but also "form a single phase with the protein. This requires a "just right" interaction with the protein surface to prevent separation but not denature the protein" (Pikal, M. J. Freeze-drying of proteins. Stable formulation and delivery of peptides and proteins; Cleland, J. L.; Langer, R., Eds., ACS Symposium Series, American Chemical Society: Washington, DC, 1994; pp 120-133). Excipients that remain hydrogen bonded to the protein during drying cannot be phase separated from the protein.

[0017] Protein behavior during freezing is another important aspect of freeze-drying. At high concentrations (approximately 50 mg / ml), freezing can lead to increased opalescence, accompanied by the formation of visible particles and a decrease in monomer content. Chemical degradation, i.e. glycation, is associated with the freezing process. With increasing protein concentration, the difference between the glass transition temperature and the collapse temperature gradually increases. Reconstitution times of freeze-dried HCPF have been observed to be very long, taking up to 30 minutes or more.

[0018] In summary, high concentration is often a requirement of clinicians who want a high dose of bioactive agent within a limited injection volume. This is not an ideal starting point for developing an efficient lyophilization cycle. High concentration and density of solids hinders water vapor transport and increases drying time. There is also a correlation between protein concentration in the lyophile and increased reconstitution time.

[0019] New constructs are needed to vitrify, store, transport, reconstitute, and / or deliver high concentrations of biological therapeutics to improve efficacy and address the needs of patient compliance and comfort.

[0020] The following summary is provided to facilitate understanding of some of the innovative features unique to the present disclosure and is not intended to be a complete description, although various aspects of the present disclosure can be fully understood by taking the specification, claims, drawings, and abstract as a whole.

[0021] Ultra-concentrated formulations of one or more bioactive agents in a dosing solvent are provided. The bioactive agent is present in a concentration greater than a solution of the bioactive agent in the dosing solvent formulated for subcutaneous administration to a subject. The dosing solvent may be aqueous and may include one or more monovalent or divalent salts. The bioactive agent may be present in a concentration equal to or greater than the concentration at which the bioactive agent aggregates in the solvent. The bioactive agent may be present in a concentration greater than 50 mg / mL, optionally greater than 90 mg / mL, optionally greater than 100 mg / mL, optionally greater than 150 mg / mL. The bioactive agent may be an antibody, a protein, a lipid particle, optionally a lipid nanoparticle, or other therapeutic agent. The bioactive agent may be an antibody, optionally a humanized antibody, including muromonab-CD3, infliximab, rituximab, solanezumab, bapinezumab, catumaxomab, trastuzumab, cetuximab, omalizumab, adalimumab, bevacizumab, BAN2401, tositumomab, or alemtuzumab. The bioactive agent may be a protein, including an interleukin or interferon, optionally including interferon beta-1b, pegylated interferon alpha-2b, roferon-A, or aldesleukin. The ultra-concentrated formulation may further include one or more pharmacologically acceptable excipients. The ultra-concentrated formulation may be up to 2.5 mL in volume. The ultra-concentrated formulation may be free of hyaluronidase. The ultra concentrated formulation may have a viscosity of 20 cP or less, optionally 12 cP or less, optionally 8 cP or less, optionally 2 cP or less, optionally 1 cP or less, where the viscosity is measured at 20° C. and 1 atm.

[0022] Also provided is a process for preparing an ultra-concentrated formulation of a bioactive agent in a dosing solvent, the process comprising: layering a vitrification mixture comprising a bioactive agent and a vitrification medium onto a membrane comprising a capillary network within a drying chamber; reducing air pressure within said drying chamber; providing sufficient thermal energy to the vitrification mixture to prevent the vitrification mixture from becoming cryogenic during vitrification; drying the vitrification mixture by capillary action until the vitrification mixture is in a glassy state; and reconstituting the bioactive agent in a dosing solvent such that the concentration of the bioactive agent in the dosing solvent is greater than the concentration of the bioactive agent in the vitrification medium. Includes.

[0023] Also provided is a method of treating or preventing a disease or condition, comprising administering to a subject in need thereof an ultra-concentrated formulation comprising a bioactive agent and an administration solvent. The bioactive agent is present at a concentration greater than a solution of the bioactive agent in the administration solvent formulated for subcutaneous administration to a subject. The administration solvent may be aqueous and may include one or more monovalent or divalent salts. The bioactive agent may be present at a concentration equal to or greater than the concentration at which the bioactive agent aggregates in the solvent. The bioactive agent may be present at a concentration greater than 50 mg / mL, optionally greater than 90 mg / mL, optionally greater than 100 mg / mL, optionally greater than 150 mg / mL. The bioactive agent may be an antibody, a protein, a lipid particle, optionally a lipid nanoparticle, or other therapeutic agent. The bioactive agent may be an antibody, optionally a humanized antibody, including muromonab-CD3, infliximab, rituximab, solanezumab, bapinezumab, catumaxomab, trastuzumab, cetuximab, omalizumab, adalimumab, bevacizumab, BAN2401, tositumomab, or alemtuzumab. The bioactive agent may be a protein, including an interleukin or interferon, optionally including interferon beta-1b, pegylated interferon alpha-2b, roferon-A, or aldesleukin. The ultra-concentrated formulation may further include one or more pharmacologically acceptable excipients. The ultra-concentrated formulation may be up to 2.5 mL in volume. The ultra-concentrated formulation may be free of hyaluronidase. The ultra concentrated formulation may have a viscosity of 20 cP or less, optionally 12 cP or less, optionally 8 cP or less, optionally 2 cP or less, optionally 1 cP or less, where the viscosity is measured at 20° C. and 1 atm.

[0024] These and additional features provided by the embodiments described herein will be more fully understood from the following detailed description taken in conjunction with the drawings. [Brief description of the drawings]

[0025] The drawings are not necessarily to scale, and some features may be exaggerated or minimized to show details of particular components. Therefore, specific structural and functional details disclosed herein should not be construed as limiting, but merely as a representative basis for teaching those skilled in the art various uses of the present disclosure. Example aspects will become more fully understood from the detailed description and the accompanying drawings.

[0026] FIG. 1 illustrates an exemplary chamber for capillary-assisted vitrification of a vitrification mixture supported by a substrate comprising a plurality of capillary channels, according to some embodiments provided herein.

[0027] 2A shows a hydrophilic bed 10 with a thin film of liquid 20 disposed on top where the capillary forces are significantly higher than the viscous forces, limiting the amount of liquid 21 that can dry.

[0028] FIG. 2B shows a contoured capillary bed in which drying occurs preferentially at the peak of the contour 30, and capillary action from the trough toward the peak during drying increases the overall vitrification rate, allowing for vitrification of larger volumes of sample compared to FIG. 2A.

[0029] FIG. 2C shows liquid filling the surface pattern when there is an excess of fluid within the contoured capillary 40, which results in bubbles nucleating under reduced pressure, boiling becoming dominant and sensitive molecules can be damaged.

[0030] FIG. 3 illustrates the accumulation of vitrified bioactive agents within capillaries of a capillary membrane upon multiple rounds of vitrification, according to some embodiments.

[0031] FIG. 4 illustrates the harvesting of vitrified material on the fibers of a membrane once the membrane has been formed according to some embodiments provided herein.

[0032] FIG. 5 illustrates an ELISA study demonstrating the structural stability and resistance to thermal degradation of vitrified, ultra-concentrated antibodies according to some embodiments provided herein.

[0033] FIG. 6 illustrates gel electrophoresis studies showing the structural stability and resistance to thermal degradation of vitrified, ultra-concentrated antibodies according to some embodiments provided herein.

[0034] 7 illustrates an XRD analysis of the sequentially vitrified material, showing that the amorphous structure was successfully maintained after vitrification within the channels of an exemplary membrane according to some embodiments provided herein.

[0035] The present disclosure provides methods and formulations that address the problems associated with administering highly concentrated biological agents that are typically not available for subcutaneous administration or that are desired to be administered more effectively or at lower doses than previously achievable. Thus, the present disclosure provides a method of vitrification at ambient temperature to stabilize highly concentrated protein and other therapeutic agent formulations. The absence of freezing, as occurs in conventional lyophilization methods, reduces or eliminates phase separation in the formulation, thereby forming a single-phase glass matrix. The resulting glass matrix is ​​unexpectedly able to withstand reconstitution in a suitable solvent for administration to a subject at a higher concentration than would otherwise be achievable, thereby creating a system that can be administered in a smaller volume for the same total therapeutic dose while maintaining the therapeutic function of the biological agent.

[0036] Thus, there is provided an ultra-concentrated formulation of a biological agent comprising a biological agent and an administration solvent, optionally the administration solvent being suitable for administration to an organism, optionally a human. In some embodiments, the biological agent is present in the formulation at a higher concentration than the biological agent is conventionally used for intravenous administration (if used), and when formulated and administered intravenously or by other conventional methods, exhibits a specific activity (activity per unit mass) that deviates by less than 10% from previously used solutions.

[0037] As used herein, the following terms or phrases have the exemplary meanings listed below, in the context of at least one embodiment.

[0038] As used herein, the term "ultra-concentrated" in some embodiments refers to a solution or suspension of a biological agent at a concentration that exceeds the solubility limit of the biological agent in a solvent, where the solution is substantially free of aggregates of the biological agent. For biological agents that are typically found as suspensions, such as lipid particles, "ultra-concentrated" is defined as a concentration higher than the concentration at which the material can be physically concentrated by evaporation or filtration without substantial aggregates of the biological agent being present. In some embodiments, "ultra-concentrated" is defined as a concentration higher than the concentration achievable in the same solution / suspension of the biological agent without an observable change in the colloidal stability of the solution, liquid-liquid phase separation, or tendency to form gels. In some embodiments, "ultra-concentrated" is defined as a concentration higher than the concentration at which the bioactive agent aggregates in a solution, such as the administration solvent. In some embodiments, an "ultra-concentrated" solution is one in which the thermodynamic stability of the biological agent is higher than would otherwise be observed in the same solvent.

[0039] As used herein, the term "cryogenic" or similarly "cryogenic" refers to a temperature at which a biological sample is cryopreserved such that biological activity therein is negligible or nonexistent. In some embodiments, it will be understood that cryogenic temperatures may include the freezing temperature of the biological sample and / or vitrification medium. Furthermore, it should be understood that cryogenic temperatures are not bound by a particular threshold or range of values ​​of temperature in either Fahrenheit or Celsius, but can be measured by the relationship between temperature, pressure, and molecular energy of the vitrification mixture of interest. Furthermore, it should be understood that as used herein, "cryogenic" and similar derivatives thereof are not limited to temperatures associated with liquid nitrogen at 1 atm or approximately -80°C, although this is certainly possible within the described definition.

[0040] Thus, as used herein, "above cryogenic temperatures" refers to temperatures above the freezing point (Tf) of the vitrification mixture. "Above cryogenic temperatures" may further include temperature values ​​where cryogenic conditions do not exist in relation to the surrounding environment and molecular energy. As used herein, room temperature refers to a temperature of about 25°C.

[0041] As used herein, "boiling" may refer to the temperature at which a material transitions to a vapor, often characterized by the formation of vapor bubbles within the material that can escape to the surroundings and dissipate therein.

[0042] "Glass transition temperature" refers to the temperature above which a material behaves like a liquid and below which the material behaves like a solid phase and is in an amorphous / glassy state. This is not a fixed temperature, but rather varies depending on the properties of the vitrified mixture in question. In some embodiments, the glassy state can refer to the state that the vitrified mixture enters when it falls below its glass transition temperature.

[0043] "Amorphous" or "glass" refers to a non-crystalline material in which there is no long-range order of atomic positions, which refers to an order parameter of 0.3 or less. Solidification of a vitreous solid occurs at a glass transition temperature, Tg. In some embodiments, the vitrification medium may be an amorphous material.

[0044] "Crystal" means a three-dimensional atomic, ionic, or molecular structure that is repeated periodically and consists of one particular regular geometric arrangement called a lattice or unit cell.

[0045] "Crystalline" means a form of matter composed of constituents arranged in an atomically ordered structure, as opposed to glassy or amorphous. Solidification of a crystalline solid occurs at the crystallization temperature, Tc.

[0046] As used herein, "vitrification" is the process of converting a material into an amorphous solid, which may be devoid of crystalline structure.

[0047] As used herein, "vitrification mixture" means a heterogeneous mixture of a biological material and a vitrification medium that includes a vitrification agent and optionally other materials.

[0048] As used herein, "biological material" or "biological sample" refers to a material that can be isolated or derived from a living organism. Examples of biological materials include, but are not limited to, proteins, cells, tissues, organs, cell-based constructs, or combinations thereof. In some aspects, biological material may refer to mammalian cells. In other aspects, biological material may refer to human mesenchymal stem cells, mouse fibroblasts, platelets, bacteria, viruses, mammalian cell membranes, liposomes, enzymes, or combinations thereof. In other aspects, biological material may refer to reproductive cells, including sperm cells, spermatocytes, oocytes, eggs, blastocysts, embryos, germinal vesicles, or combinations thereof. In other aspects, biological material may refer to whole blood, red blood cells, white blood cells, platelets, viruses, bacteria, algae, fungi, or combinations thereof.

[0049] As used herein, a "vitrification agent" is a material that forms an amorphous structure or inhibits the formation of crystals in other materials when a mixture of the vitrification agent and other materials cools or dries. The vitrification agent may provide osmoprotection or allow cell survival during dehydration. In some aspects, the vitrification agent may be any aqueous solution that results in an amorphous structure suitable for storage of biomaterials. In other aspects, the vitrification agent may be soaked in a cell, tissue, or organ.

[0050] As used herein, "storable or storage" refers to the ability of a biomaterial to be stored and remain viable for later use.

[0051] As used herein, "hydrophilic" means attracting or preferentially binding with water molecules. A hydrophilic material, which has a particular affinity for water, maximizes contact with water and has a smaller contact angle with water than a hydrophobic material.

[0052] As used herein, "hydrophobic" means lacking an affinity for water. Hydrophobic materials naturally repel water, forming beads and having a large contact angle with water.

[0053] As used herein, a "capillary" is a tube having a diameter of about 2000 μm. 2 It relates to, is present within, or appears to be present within a tube of pores having a cross-sectional area of:

[0054] As used herein, a "subject" is an animal, optionally a human, non-human primate, horse, cow, mouse, sheep, pig, rabbit, or other mammal.

[0055] "Cryopreservation" usually refers to the rapid cooling of biological samples, often using liquid nitrogen due to its low temperature. This allows for rapid cooling of liquid materials, or small amounts of biological material by direct immersion. The rate of cooling reduces the mobility of the molecules of the material before they can solidify into a more thermodynamically favorable crystalline state. Over a longer period of time, the molecules can become crystallized, which can have detrimental consequences, especially for biological samples. Water is a major concern in biological samples, as it can crystallize quickly. It is also abundant in biological tissues, and can cause significant damage as it crystallizes. Protective additives that interfere with the ability of the main components to crystallize, often called cryoprotectants, can produce amorphous / vitrified material.

[0056] The ultra-concentrated formulations include one or more bioactive agents. Without limitation, the bioactive agents are optionally bio-derived or can be bio-derived. In other embodiments, the bioactive agents have functional activity by increasing, decreasing, or altering the concentration, activity, physiological modification, synthesis, or degradation of any other compound found in the subject.

[0057] Optionally, the bioactive agent is an antibody. As used herein, the term "antibody" includes monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, bispecific antibodies, simianized antibodies, and humanized antibodies, drug-antibody conjugates, and Fab fragments, including the products of a Fab immunoglobulin expression library. Intact antibodies, fragments thereof (e.g., Fab or F(ab')2), or engineered variants thereof (e.g., sFv) can also be used. Optionally, the antibody is humanized, as recognized in the art. Optionally, the antibody is not humanized. The antibody can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Specific examples of antibodies include bifunctional antibodies.

[0058] Non-limiting examples of antibodies include, but are not limited to, anti-CD3 mAbs (e.g., otelizumab, teplizumab, visilizumab), anti-staphylococcal therapeutic antibodies (e.g., tefibazumab, tosatoxumab, pagibaximab, subratoxumab, and other mAbs), antibodies against specific cell populations (F4 / 80-macrophages or c-kit-stem cells), antibodies against injury-associated antigens (VCAM or fibrinopeptide A), or antibodies targeting TGF-β, TNF-α, IL-6, IL-2, CD52, and IL-1β, or combinations of any two or more of the above, among others. Other specific examples of antibodies include, but are not limited to, muromonab-CD3, infliximab, rituximab, solanezumab, bapinezumab, catumaxomab, trastuzumab, cetuximab, omalizumab, adalimumab, infliximab, leticimab, bevacizumab, BAN2401, tositumomab, or alemtuzumab. Such antibodies are commercially available. Bifunctional antibodies may include, for example, an anti-CD3 antibody bound to one of the anti-staphylococcal therapeutic antibodies (e.g., tefibazumab, tosatoxumab, pagibaximab, subratoxumab, and other mAbs), an antibody fusion (either a hybridoma fusion or chemically generated) of a monoclonal antibody against a specific cell population (F4 / 80-macrophage or c-kit-stem cell) bound to a monoclonal antibody against an injury-associated antigen (VCAM or fibrinopeptide A). Methods for making bifunctional antibodies are known in the art and are provided, for example, in Nolan and O'Kennedy, Biochim Biophys Acta., 1990; 1040(1):1-11.

[0059] In other aspects, the biologic is a non-antibody therapeutic agent. Examples of such therapeutic agents include, but are not limited to, proteins, nucleotides, lipids, or other molecules that have biological activity in a subject. More specifically, specific examples of non-antibody therapeutic biological agents include, among others, aldesleukin (PROLEUKIN®), interferon alpha-2a, pegylated IFN-alpha-2b, interferon-alpha-2a (Roferon-A), interferon-alpha-2b, interferon-beta-1b (BETASERON®), insulin glargine, interleukin, abatacept, abobotrinumtoxin A, aflibercept, agalsidase beta, agalsidase beta, albiglitide, alglucosidase alfa, alteplase, catecholamine activating enzyme, anakinra, asfotase alfa, asparaginase, asparaginase blackleg bacteria, becaplermin, belatacept, collagenase, collagenase clostridium histolyticum, darbepoetin alfa, denileukin diftitox, dornase alfa, dulaglutide, ecallantide, elosulfase alfa, epoetin alfa, etanercept, etanercept-szzs, filgrastim, filgrastim-sndz, follitropin alfa, galsulfase, glucarpidase, idursulfase, incobotulinumtoxinA, interferon alfa-2b, interferon alfa-n3, interferon beta-1a, interferon beta-1b, interferon gamma-1b, laronidase, methoxypolyethyleneglycol-epoetin beta, metreleptin, ocriplasmin, onabotulinumtoxinA, opelvekin, palifermin, parathyroid hormone, pegaspargas, pegfilgrastim, pegloticase, rasuburicase, reteplase, rilonacept, rimabotulinumtoxinB, romiplostim, sargramostim, sebelipase alfa, tbo-filgrastim, tenecteplacen, vedolizumab, and dib-aflibercept.

[0060] In other embodiments, the biological product is suitable for use as a vaccine. Specific examples of vaccines include BNT162b2, mRNA-1273, JNJ-78436735, influenza virus vaccines (e.g., IIV or LAIV), measles, mumps, rubella (MMR) vaccines, DTaP vaccines, HepA vaccines, HebB vaccines, Hib vaccines, HPV9 vaccines, MenACWY, PCV13, PPSV23, polio vaccines, rabies vaccines, RV1, RV5, RZV, vaccinia, VAR, MMRV, and yellow fever vaccines, among others.

[0061] The formulation includes a bioactive agent present in an administration solvent. The administration solvent is optionally any fluid suitable for use in delivering a bioactive agent to a subject. Optionally, the administration solvent is an aqueous solvent, i.e., the solvent comprises water, and optionally is predominantly water. Optionally, the administration solvent is water and one or more of a buffer, a salt, an excipient, or other desired substance. The administration solvent is optionally or includes a sugar, optionally dextrose.

[0062] The administration medium optionally contains one or more salts. The salts are optionally monovalent, divalent, or polyvalent salts. Monovalent salts are optionally Na, K, Mg, Ca, Zn, or other salts. Such salts optionally contain chloride, sulfate, phosphate, acetate, or other anions. Optionally, one, two, three, four, or more salts are present in the medium.

[0063] The administration medium optionally includes one or more buffers. Exemplary buffers may be, but are not limited to, phosphate, HEPES, TRIS, succinate, citrate, and acetate, among others.

[0064] The dosing solvent optionally includes an organic (e.g., non-aqueous) main component or components. Optionally, the organic is or includes, among others, dimethyl sulfoxide, N-methyl-2-pyrrolidone, glycofurol, solketal, glycerol formal, acetone, tetrahydrofurfuryl alcohol, diglyme, dimethyl isosorbide, and ethyl lactate. Optionally, the dosing solvent does not include an organic solvent. Optionally, the organic is present in the dosing solvent at a concentration that does not alter the coagulation properties of the bioactive agent in the remaining solution in the absence of the organic.

[0065] Illustratively, the administration medium is or includes sodium or phosphate buffered saline.

[0066] The administration medium may optionally contain one or more pharma- ceutically acceptable excipients. In this context, "pharma-ceutically acceptable" refers to an excipient that, at the dosage and concentration of the bioactive agent in the formulation, does not cause undesirable effects in the subject to which the excipient is administered. Such pharma-ceutically acceptable excipients are well known in the art, and may be, in particular, carriers, diluents, binders, disintegrants, flow improvers, pH adjusters, stabilizers, viscosity adjusters, preservatives, gelling or swelling agents, surfactants, emulsifiers, and suspending agents. As will be recognized by those skilled in the art, the specific selection of pharma-ceutically acceptable excipients depends on the particular form or formulation, e.g., dosage form. Those skilled in the art may find guidance on providing suitable pharma-ceutically acceptable excipients in various textbooks, for example, Remington's "Pharmaceutical Science and Practice."

[0067] The ultra-concentrated formulations provided herein contain a bioactive agent present at a concentration that maintains the functional activity of the bioactive agent, but also contains the bioactive agent at a higher concentration than is typically used or achievable by simple evaporation or filtration concentration methods. This provides for the first time a material that can be used to deliver an active agent by subcutaneous injection, which historically was not tolerated in the volumes required for an adequate dose of bioactive agent without the presence of substantial aggregates, viscosity that was too high for subcutaneous delivery devices (e.g., 20-30 gauge needles), or significant loss of activity.

[0068] In some embodiments, the ultra-concentrated formulations provided herein are free of or contain physiologically irrelevant aggregates of bioactive agents. Physiologically irrelevant aggregates are the absence of aggregates that cause undesirable immune responses to bioactive agents in subjects or reduce the specific activity (bioavailability) of the bioactive agent solution. The absence of aggregates is the absence of dimers, trimers, tetramers, or 10 or less monomers or other aggregates of bioactive agents, or the absence of aggregates with particle sizes up to 1 micrometer in diameter as measured by dynamic light scattering.

[0069] Thus, the formulations provided herein optionally contain a sufficient amount of bioactive agent so that the usual approved dose of bioactive agent can be administered in a single administration. The administration of the formulations provided herein is exemplified by subcutaneous administration, since it typically has the highest strictness for the tolerance of injection. As will be understood, the formulations may be administered by other routes, such as intravenous or intramuscular. Optionally, the formulations provided herein are formulated to be administered or are administered subcutaneously, optionally excluding intravenous and / or intramuscular administration.

[0070] Thus, the formulations provided herein optionally have a volume that contains a sufficient amount of bioactive agent for the recommended dose administered in a single administration.The formulations are optionally 5 milliliters (ml) or less, optionally 4 ml or less, and optionally 3 ml or less.In some embodiments, the formulations are administered in an amount that is tolerated painlessly in human subjects, optionally 2.5 ml or less, optionally 2.0 ml or less, and optionally 1.5 ml or less.

[0071] For example, when the bioactive agent is an antibody or other protein therapeutic, the bioactive agent is optionally present at a concentration of 50 milligrams (mg) / ml or greater. Optionally, the concentration of the antibody is 75 mg / ml or greater, optionally 90 mg / ml or greater, optionally 100 mg / ml or greater, optionally 125 mg / ml or greater, or optionally 150 mg / ml or greater.

[0072] The viscosity of the formulations provided herein is optionally greater than that of saline used for administration to humans, and optionally is 1 centipoise (cP) or greater. As used herein, viscosity is measured at 20° C. and 1 atmosphere (i.e., 760 mg / mm). Optionally, the viscosity of the formulation is 1.25 cP or greater, optionally 1.5 cP or greater, optionally 1.75 cP or greater, optionally 1.9 cP or greater, optionally 2 cP or greater, optionally 8 cP or greater, optionally 12 cP or greater, and optionally 20 cP or greater.

[0073] The formulation optionally maintains the activity of the bioactive agent at substantially the same level as the agent prior to forming the formulation. The activity of the bioactive agent depends on the nature of the bioactive agent. For example, the activity of an antibody is defined as its affinity for a target sequence. The activity of an enzyme is defined as its ability to react with a substrate. The activity of an immunogen is defined as its immunogenic potential. The activity of a nanoparticle, such as a nanoparticle vaccine, is its ability to enter a target cell, if desired. The activity of the bioactive agent in the formulation is optionally 75 percent (%) or greater than the activity of the bioactive agent prior to making the formulation. Optionally, the activity of the formulation relative to the activity of the bioactive agent prior to making the formulation is 80% or greater, optionally 85% or greater, optionally 90% or greater, optionally 95% or greater, optionally 99% or greater, and optionally 99.9% or greater.

[0074] The activity of the antibody can be tested and quantified by one of many techniques known in the art. For example, the affinity of the antibody can be obtained by enzyme-linked immunosorbent assay (ELISA) known in the art. Briefly, a sample of the formulation provided herein before preparation and a sample of the formulation provided herein can be tested against the target antigen, and the ability of each sample to bind to the target antigen that may be bound to the plate surface can be measured and directly compared. In another example, when the bioactive agent is a protein such as pegylated interferon-α-2a, it may be measured as described in Foser et al., Protein Expression and Purification, 2002; 30:78-87. Standard activity assays for other protein bioactive agents are also known in the art. The concentration of the bioactive agent may be measured by function and comparison to a standard curve, ELISA, or other known quantification methods.

[0075] Also provided is a process for preparing an ultra-concentrated formulation of a bioactive agent provided herein, the process comprising: layering a vitrification mixture comprising a bioactive agent and a vitrification medium onto a membrane comprising a capillary network within a drying chamber; reducing air pressure within said drying chamber; providing said biological sample with sufficient thermal energy to prevent the vitrification mixture from becoming cryogenic during vitrification; drying the vitrification mixture by capillary action until the vitrification mixture is in a glassy state; and reconstituting the bioactive agent in a dosing solvent such that the concentration of the bioactive agent in the dosing solvent is greater than the concentration of the bioactive agent in the vitrification medium. Includes. Method of Formulation of the Formulation

[0076] The formulations provided herein may be produced by vitrification of a vitrification mixture of a bioactive agent and a vitrification medium, while avoiding its cryogenic cooling, crystallization, and / or freezing. In certain embodiments, vitrification of a bioactive agent and a vitrification medium is performed, the vitrification medium comprising one or more glass formers. In the presence of a suitable glass former, it is possible to store the bioactive agent in a vitrification matrix at temperatures above cryogenic temperatures, and vitrification is achieved by dehydration. Some animals and many plants can survive complete dehydration. This ability to survive in dry conditions (anhydrobiosis) depends on several complex intracellular physicochemical and genetic mechanisms. Among these mechanisms is the intracellular accumulation of sugars (e.g., saccharides, disaccharides, oligosaccharides, etc.) that function as protective agents during desiccation. Trehalose is an example of a disaccharide that is naturally produced in desiccation-tolerant organisms.

[0077] Sugars such as trehalose may protect bioactive agents in several different ways. Due to the unique arrangement of hydroxyl groups on the trehalose molecule, trehalose molecules can effectively displace hydrogen-bonded water molecules from the surface of folded proteins without altering their conformation or folding. Sugar molecules may also prevent leakage from lipid nanoparticles or cells during rehydration by binding with the phospholipid head groups of the lipid bilayer. In addition, many sugars have high glass transition temperatures, allowing them to form glass at cryogenic temperatures or above room temperature even at low water contents. The highly viscous "glassy" state reduces molecular mobility, thereby preventing degradative biochemical reactions that lead to reduced cell function and death. Vitrification of biomaterials by dehydration in the presence of the glass-forming sugar trehalose has been disclosed in U.S. Pat. No. 10,433,540 and U.S. Patent Application Serial No. 63 / 115,936.

[0078] The formulations provided herein can be prepared by a vitrification process that combines low pressure and thermal energy to achieve uniform and rapid vitrification of biological samples in a vitrification mixture.In some embodiments, the supply of thermal energy to the vitrification mixture is carried out under reduced pressure.In some embodiments, the thermal energy is supplied to the vitrification mixture to prevent crystallization of the vitrification mixture.

[0079] The temperature of the vitrification mixture is controlled during drying and / or vitrification. For example, the vitrification mixture is placed in a drying chamber and thermal energy is provided to the vitrification mixture to limit or prevent exposure of the vitrification mixture to cryogenic temperatures. In some embodiments, thermal energy is transferred to the vitrification mixture to prevent crystallization of the vitrification mixture.

[0080] Optionally, the temperature of the biological sample is controlled by applying a vacuum or reducing the atmospheric pressure around the vitrification mixture. Applying a reduced atmospheric pressure can significantly reduce the temperature of the vitrification mixture, potentially causing the vitrification mixture to crystallize. If crystallization occurs, irreparable damage can occur to the bioactive agent, which can adversely affect its desired activity and use upon reconstitution. Also, reducing the atmospheric pressure around the vitrification mixture can alter the molecular activity within the vitrification mixture, thereby reducing the boiling point. As with cryogenic cooling, boiling or overheating the biological sample and / or vitrification medium can be harmful. Boiling the vitrification mixture can cause loss of tertiary structure of the bioactive agent, crosslinking, and degradation of components including proteins, fatty acids, and nucleic acids, etc., resulting in impaired activity upon reconstitution. In certain aspects, the process of the present disclosure maintains the vitrification mixture at temperatures above cryogenic temperatures under reduced pressure, such as vacuum, partial vacuum, or generally reduced atmospheric pressure.

[0081] In certain embodiments, the vitrification mixture containing the bioactive agent and vitrification media may be heated directly to control the temperature during drying. In other embodiments, the temperature of the vitrification mixture containing the bioactive agent and vitrification media may be controlled by means of conduction, convection, and / or radiation. Optionally, the vitrification mixture containing the biological sample and vitrification media may be temperature controlled by controlling the temperature outside the drying chamber and relying on conduction through the drying chamber or a portion thereof to control the temperature of the vitrification mixture. In such cases, it will be understood that the physical properties of the walls of the drying chamber must be taken into consideration. For example, a less conductive material of the drying chamber may require application of a temperature different from that required by the vitrification mixture so that the vitrification mixture can receive the appropriate thermal energy. Such necessary adaptations will be readily understood by one of ordinary skill in the art. In some embodiments, heat may be provided through a heating pad, heating bath, flame, heating bed (glass beads, heating block, etc.). In some cases, the thermal energy may be from a heat generating source, and / or thermal energy released by combustion, and / or thermal energy generated by electrical resistance.

[0082] In some embodiments, thermal energy can be provided to the vitrification mixture through an underlying support substrate. A porous material with a continuous capillary network can also provide thermal energy to the vitrification mixture, but in some cases the porous material is a low-conductivity material such as glass or polymer. However, the underlying substrate can be made of metal or a similar efficient conductive material and can be easily connected to a heat source or power source outside the drying chamber and provide heat by an internally generated resistance. To aid in capillary evaporation, the supply of thermal energy from this solid support can also provide a temperature gradient.

[0083] In some aspects, the vitrification mixture, including the bioactive agent and vitrification media, is maintained above its cryogenic temperature during vitrification at low pressure. Optionally, the vitrification mixture is preheated prior to drying at low pressure. In other aspects, the vitrification mixture is heated during vitrification at low pressure. In other aspects, heat is provided at or around the beginning of vitrification. It will be appreciated that the amount of thermal energy provided to the vitrification mixture may be constant or may vary during vitrification at low pressure process. In some aspects, the introduction of low pressure in the drying chamber may cause the temperature of the vitrification mixture to drop rapidly. In such aspects, having the vitrification mixture ready to receive or having already received thermal energy may increase the speed of recovery from the temperature drop.

[0084] In certain embodiments, a constant temperature is applied to the vitrification mixture so that the mixture is maintained at a temperature between about its Tg (°C) and about 40°C, including about 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, and 39°C. In certain embodiments, a higher temperature may be applied to the drying chamber or porous material to provide the necessary thermal energy to the vitrification mixture. Such applied temperature may be between about 15°C and about 70°C, depending on the size of the drying chamber and the available means of transmission to effectively transport the biological sample and / or vitrification medium.

[0085] In some embodiments, the vitrification of the biological sample is performed at low pressure, defined as less than 1 atmosphere (760 mmHg). Optionally, the drying is performed in a drying chamber, which is placed in a drying chamber so that the vitrification mixture is exposed to low pressure. Such a drying chamber may be connected to a vacuum source to provide low pressure to the vitrification mixture. The vitrification mixture can be prepared with a vitrification medium or a cryopreservative such as trehalose and exposed to low pressure, such as through the application of a vacuum. Optionally, the low pressure is 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.255, 0.25, 0245, 0.24, 0.235, 0.23, 0.225, 0.22, 0.215, 0.21, 0.205, 0.2, 0.195, 0.19, 0.185, 0.18, 0.175, 0.17, 0.165, 0.16, 0.155, From about 0.9 atm to about 0.005 atm, including 0.15, 0.145, 0.14, 0.135, 0.13, 0.125, 0.12, 0.115, 0.11, 0.105, 0.1, 0.095, 0.09, 0.085, 0.08, 0.075, 0.07, 0.065, 0.06, 0.055, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, and 0.01 atm.

[0086] In some embodiments, the pressure in the drying chamber is reduced to a value above the triple point of the vitrification mixture. In other embodiments, the pressure is reduced to a value above the triple point of water, for example, above 0.006 atm. As described herein, the reduction in air pressure reduces the temperature of the vitrification mixture and simultaneously reduces its boiling point. In some embodiments, the pressure in the drying chamber is reduced to about 0.04 atm or about 29 mmHg.

[0087] The vitrification mixture may be placed in a vacuum or partial vacuum at elevated temperature, or the applied air pressure may maintain the vitrification mixture at a temperature above cryogenic temperatures, such that the vitrification mixture is not exposed to cryogenic temperatures if the air pressure is suddenly reduced. During vitrification, the temperature of the vitrification mixture is lower than the Tg of the vitrification media, allowing for vitrification of the biological specimen.

[0088] Maintaining the low atmospheric pressure may require placing the vitrification mixture in a sealed container such as a drying chamber. As will be appreciated by those skilled in the art, providing and / or maintaining the low atmospheric pressure around the vitrification mixture will typically require the drying chamber to be able to withstand the low pressure inside. Such may be of any suitable or desired shape and / or material, constrained by the requirement of maintaining the low atmospheric pressure inside, requiring sufficient sealing and sufficient wall strength. The drying chamber may be operably connected to a vacuum source to reduce the atmospheric pressure therein while simultaneously allowing air to return upon completion of vitrification. The drying chamber may be sufficiently sealed or sealed such that a vacuum can be applied to effectively reduce the atmospheric pressure within the drying chamber to the desired range.

[0089] In some embodiments, the vitrification mixture is left on or in a capillary network to promote vitrification of the vitrification mixture. The capillary network can prevent the vitrification mixture from boiling under reduced pressure. The principles of capillary-assisted evaporation are described in U.S. Pat. No. 10,568,318. Briefly, FIG. 1 illustrates the conditions favorable for capillary-assisted drying of the following vitrification mixtures: a plurality of capillary channels forming a capillary plate / membrane 53; These capillary channels have a first opening 54 and a second opening 56; leaving the vitrified mixture 52 over the first opening 54 and further exposing the second opening 56 and a surface 59 of the vitrified mixture to an ambient environment 58 that has a lower humidity than the vitrified mixture; Optionally, heat is applied to the vitrification mixture to dry the vitrification mixture by capillary action until the vitrification mixture is in a glassy state, thereby preserving the bioactive agent in a glassy state. The chemistry, humidity, pressure, and temperature within container 55 are controlled by control mechanism 57. In some embodiments, the amount of vitrification mixture is small enough that the entire vitrification mixture is retained within the pores of the capillary network, thereby improving the overall drying rate and preservation of the bioactive agent.

[0090] The control mechanism 57 is simplified for illustrative purposes only and can have multiple systems and mechanisms to achieve the most favorable conditions for drying and vitrification. Optionally, a second capillary plate / membrane similar to 53 is placed directly on top of the vitrification mixture 52 to benefit from the capillary-assisted drying method of the present disclosure on the upper surface of the vitrification mixture 52. However, gravity does not favor the capillary forces at the top. In some embodiments, a flow of low humidity gas (relative humidity less than 30%) is provided across the second opening 56 of the capillary plate / membrane to enhance the capillary effect. Inert or relatively inert gases such as nitrogen, argon, and xenon may be used as the low humidity gas. In some embodiments, a reduced pressure or vacuum is maintained within the container 55. In some embodiments, suction / pressure is provided across the second opening 56 to speed up the drying rate. It should be noted that maintaining low ambient humidity (optionally 5% relative humidity or less) is essential to prevent rehydration after drying.

[0091] JPEG2024527538000002.jpg182167

[0092] In some embodiments, the thermal energy is provided to the vitrification mixture as it undergoes vitrification on the capillary network. The underlying capillary network allows for uniform and complete vitrification of the vitrification mixture undergoing thermal energy while protecting the vitrification mixture from boiling. The capillary network can be a continuous network of capillaries. In some cases, the capillary network can be a continuous capillary channel that forms an uninterrupted structure from the first end to the second end. In some cases, the capillary network can be provided by an underlying porous material such as a membrane, or an underlying contoured or raised surface whose troughs and peaks provide a sufficient bed to expose the liquid vitrification mixture to capillary action during vitrification.

[0093] Referring to FIG. 2A, a continuous hydrophilic bed 10 is shown covered by the application of a thin liquid layer of vitrification mixture 20. In some embodiments, the protective fluid layer of vitrification mixture may protect the biological specimen from boiling during exposure to low pressure. Boiling under low pressure can be avoided and / or mitigated by an extremely thin liquid film on the hydrophilic surface as shown in FIG. 2A. However, while boiling can be prevented, the liquid thickness is limited, reducing the amount of liquid that can be vitrified due to the available surface area. The presence of a contoured surface as shown in FIG. 2B effectively provides a surface for the vitrification mixture to undergo capillary action due to preferential drying occurring at the peaks, thereby wicking up moisture during the vitrification process, as well as protecting the biological specimen (mainly located at the apex of the contour or ridge) from boiling, allowing very large volumes of sample to be vitrified. Furthermore, as the sample vitrifies at the peak of the contour, capillary action draws fluid from the underlying trough, thereby promoting superior vitrification and drying of the vitrification mixture. Similarly, if the porous material of the capillary membrane supports the biological sample within the capillary, capillary action will pull fluid out of the capillary channel during the vitrification process, resulting in a larger volume of biological sample being uniformly and completely vitrified and dried. However, if capillary action cannot successfully pull up the liquid, such as when the liquid load is too large, as shown in Figure 2C, the liquid will fill the surface pattern or be held in a trough, and bubble nucleation and boiling will become dominant under reduced pressure, which may lead to damage to sensitive molecules contained in the liquid.

[0094] Thus, in some aspects of the present disclosure, the volume of fluid present in the vitrification mixture can be configured to allow the fluid to fill the capillary network without spilling or pooling on surfaces.

[0095] In certain embodiments, the drying chamber or the membrane contained therein may be appropriately patterned such that when left therein, the chamber walls or membrane provide a capillary network for the vitrification mixture. For example, contours or ridges as shown in FIG. 2B may cover the chamber walls to provide an underlying capillary network. In other embodiments, a continuous capillary network porous material containing the vitrification mixture therein is provided within a sealable drying chamber. In certain embodiments, the porous material may include a membrane of multiple continuous capillary channels.

[0096] The capillary membrane can be placed in a support that can itself be heated to avoid exposing the vitrification mixture to temperatures below Tf. In some embodiments, a support scaffold can be included between the membrane and the heating block or the side of the chamber in which the membrane is placed to separate the membrane from the surface where heat is generated. In some embodiments, this prevents direct heating of the sample from below, allows for substantially two-sided drying, or avoids exposing a larger amount of heat to the trough of membrane material.

[0097] Alternatively, or additionally, heating elements may be introduced into the chamber to provide heat directly to the surface of the vitrification membrane, which promotes effective vitrification at the desired location in / on the membrane and promotes good capillary action and prevents boiling of the vitrification medium during vitrification.

[0098] Optionally, a support substrate may be utilized to provide and / or transfer thermal energy to the vitrification mixture. As will be appreciated by those skilled in the art, in some embodiments, the support substrate may be a well-conductive material, such as a metal, to effectively provide thermal energy to the vitrification mixture. In other embodiments, a porous material may be disposed between the vitrification mixture and the underlying solid support substrate to provide a continuous capillary network.

[0099] As shown in FIG. 2, the presence of contours and / or ridges provides capillary ridges and promotes vitrification. When the vitrification mixture is present on the surface, capillary action draws the vitrification mixture towards the peaks, allowing rapid evaporation from the peaks. The presence of a continuous capillary network allows the vitrification mixture fluids to evaporate evenly, preventing boiling while also preventing the accumulation of excess fluids that can cause harmful boiling. Similarly, a porous material such as a membrane with continuous capillary channels can provide an underlying capillary network. In such an embodiment, the porous material such as a membrane contains the vitrification mixture and capillary action therein promotes vitrification. Thus, in some embodiments of the present disclosure, the vitrification mixture is disposed on a continuous capillary network. In further embodiments, the vitrification mixture is disposed on any porous material that is patterned and / or raised and / or contoured. In further embodiments, the continuous capillary network is formed by patterns and / or ridges and / or contours in the walls of the drying chamber. In other aspects, the capillary network is provided by a porous material that includes a plurality of interconnected capillary channels.

[0100] The drying chamber must further be capable of or arranged to accommodate the vitrification mixture therein. In some embodiments, the drying chamber must be capable of accommodating the vitrification mixture on a porous material and / or a supporting substrate. In some embodiments, the vitrification mixture is prepared for vitrification by disposing it on a substrate. In some embodiments, the substrate may be a porous material such as a membrane and / or a bed of arranged capillaries. In further embodiments, the walls of the drying chamber act as a supporting substrate and are raised and / or patterned and / or contoured to provide a capillary network therein.

[0101] The capillaries in the capillary network membrane can provide an interface for rapid evaporation. The capillary network formed from the underlying patterned raised support or porous material such as the membrane may be made of a material that is non-toxic, non-reactive to the biological material or biological sample, and does not react chemically or physically with the vitrification medium. The material may be a suitable polymer, metal, ceramic, glass, or combinations thereof. In some embodiments, the continuous capillary network is formed from polydimethylsiloxane (PDMS), polycarbonate, polyurethane, polyethersulfone (PES), polyester (e.g., polyethylene terephthalate) materials, among others. Examples of capillary channels including membranes suitable as surfaces for the devices and processes provided herein include hydrophilic filtration membranes such as those sold by EMD Millipore (Bellerica, Massachusetts). In certain embodiments, the porous material does not substantially bind, change, or otherwise chemically or physically bind with the components of the biological sample and / or the vitrification medium. The porous material is not optionally derivatized. Optionally, capillary channels can be formed in a substrate (e.g., drying chamber walls) of desired material and thickness by PDMS forming techniques, laser drilling, or other hole forming techniques known in the art.

[0102] The capillary network underlying or containing the vitrification mixture may aid in the evaporation process during drying. As described herein, the capillaries may be provided by patterning or contouring the walls of the drying chamber to effectively provide an underlying capillary bed, or by providing a porous material with a continuous capillary network, such as with a membrane. In some embodiments, the capillary network provided by the porous material and / or the patterned and / or contoured surface is characterized by pores of about 20 μm or less, which pores provide underlying capillaries that aid in vitrification. In some embodiments, the average opening of the pores may be about 20 μm to about 0.1 μm, including about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 μm. The capillary channels may have a length optionally defined by the thickness of the substrate forming the channel or by the individual channel or channels themselves. The length of the capillary channels is optionally about 1 millimeter or less, but should not be construed as being limited to such dimensions. Optionally, the length of the capillary channels is about 0.1 to about 1000 microns, or any value or range therebetween. Optionally, the length of the capillary channels is about 5 to about 100 microns, optionally about 1 to about 200 microns, and / or optionally about 1 to about 100 microns. The length of the capillary channels is optionally about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 microns. In some embodiments, the length of the capillary channels is optionally non-uniformly varied throughout the plurality of capillary channels.

[0103] The cross-sectional area of ​​the capillary channel is about 2000 μm 2 Optionally, the cross-sectional area is less than about 0.01 μm 2 ~about 2000μm 2 , optionally about 100 μm 2 ~about 2000μm 2Optionally, the cross-sectional area of ​​the capillary channel is about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 μm 2 The following is the result.

[0104] The rate of capillary-assisted evaporation can be influenced by both the demands of the surrounding environment (humidity, temperature, air / gas velocity at the evaporative surface) and (i) the properties of the capillary channel that generate the driving capillary force, (ii) the depth of the liquid meniscus, and (iii) the viscous resistance to flow through the capillary. As a result, a wide range of evaporation behaviors arise due to the complex and highly dynamic interplay between capillary properties, transport processes, and boundary conditions. Parameters important for rapid drying can include (1) conditions that favor the formation and maintain a liquid network at the evaporative surface, and (2) properties that promote the formation of capillary pressure that induces sufficient flow to supply water to the evaporative surface.

[0105] In some embodiments, the porous material may be raised and / or contoured, or disposed on a raised and / or contoured underlying support substrate, so that it will assume a similar shape when placed or pressed onto it. As shown in Figure 2B, the contours and / or ridges of the patterned material may increase the surface area and thus the exposure for evaporation.

[0106] In some aspects, the capillary network is comprised of a hydrophilic material, while in other aspects, the capillary network is comprised of a hydrophobic material and may be further treated to become inherently hydrophilic or more hydrophilic, such as by exposure to a plasma.

[0107] In some embodiments, the bioactive agent is left in, coated with, or mixed with the vitrification media to form a vitrification mixture. The presence of an appropriate vitrification agent in the vitrification media may be essential for the vitrification media to dry under ambient conditions as described herein. A fast drying method in itself does not necessarily guarantee successful survival of the bioactive agent after drying. A vitrification media that forms glass and / or inhibits the formation of crystals in other materials may be required. The vitrification media may provide osmoprotection and allow cell survival during dehydration of the biological specimen. Specific examples of agents that may be included in the vitrification media include one or more of the following: dimethyl sulfoxide, glycerol, sugars, polyalcohols, methylamines, betin, antifreeze proteins, synthetic antinucleating agents, polyvinyl alcohol, cyclohexanetriol, cyclohexanediol, inorganic salts, organic salts, ionic liquids, or combinations thereof. In some embodiments, the vitrification media optionally includes one, two, three, four, or more vitrification agents.

[0108] The vitrification media may include a vitrification agent at a concentration depending on the type of vitrification agent. Optionally, the concentration of the vitrification agent is less than that which would be toxic to the biological specimen being vitrified if it were so toxic that functional or biological viability would not be achieved during subsequent use of the sample. The concentration of the vitrification agent is optionally about 500 μM to about 6 M, or any value or range therebetween, including about 1, 2, 3, 4, or 5 M. In the case of the vitrification agent trehalose, its concentration is about 1 M to about 6 M, optionally including 2, 3, 4, or 5 M. Optionally, when combined, the total concentration of all vitrification agents is about 1 M to about 6 M, optionally including 2, 3, 4, or 5 M.

[0109] Trehalose, a glass-forming sugar, is utilized in anhydrous vitrification and may provide desiccation resistance in some ways. However, the glass transition temperature of 1.8 moles per liter (M) trehalose vitrified in water is −15 to 43° C. To achieve vitrification above 0° C., higher concentrations (6 to 8M) are required that may be damaging to biomaterials. Alternatively, the vitrification medium may include a buffer and / or salt to increase the Tg value of the VM. In some embodiments, the vitrification medium may optionally include water or a solvent, and / or a buffer, and / or one or more salts, and / or other components. A buffer may be any reagent with a pKa at 25° C. of about 6 to about 8.5. Specific examples of buffers include HEPES, TRIS, PIPES, MOPS, among others. A buffer may be provided at a concentration suitable to stabilize the pH of the vitrification medium at a desired level.

[0110] Vitrification media containing 1.8 M trehalose, 20 mM HEPES, 120 mM ChCl, and 60 mM betin provides a glass transition temperature of +9° C. Exemplary vitrification media for capillary assisted vitrification may include one or more buffers containing large organic ions (>120 kDa) such as trehalose, choline, betin, or HEPES, and buffers containing small molecular ions such as K, Na, or Cl.

[0111] A bioactive agent may be coated or soaked into the vitrification medium and left on a support substrate to hold the vitrification medium during the vitrification steps described herein. In certain aspects, the capillary network absorbs a portion of the vitrification mixture while allowing a thin layer of fluid to remain on the surface.

[0112] In some cases, the biological sample may be coated and / or mixed with the vitrification medium while in the drying chamber, while in other embodiments, the biological sample may be prepared with the vitrification medium before being placed in the drying chamber.

[0113] In some embodiments, the method of the present disclosure may be carried out over a drying time period. The drying time period is sufficient to promote adequate drying to vitrify the vitrification medium. The drying time period is from about 1 second to about 1 hour, including but not exceeding about 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, and 55 minutes. Optionally, the drying time period is from about 1 second to about 30 minutes, optionally from about 5 seconds to about 10 minutes.

[0114] The bioactive agent remains viable in the vitrified state during storage at temperatures above cryogenic temperatures and may vary over time for different sample materials. Optionally, the bioactive agent may remain viable during storage at temperatures above cryogenic temperatures for 2-20 days. In other embodiments, the bioactive agent may remain viable during storage at temperatures above cryogenic temperatures for 10 weeks. In other embodiments, the bioactive agent may remain viable during storage at temperatures above cryogenic temperatures for up to 1 year. In other embodiments, the bioactive agent may remain viable during storage at temperatures above cryogenic temperatures for up to 10 years.

[0115] Alternatively, the vitrified bioactive agent can be stored for very long periods of time in cryogenic and / or liquid nitrogen after vitrification at temperatures above cryogenic temperatures using the teachings and devices of the present disclosure. This is the preferred approach for many bioactive agents to avoid the freeze damage that typically occurs during direct vitrification at cryogenic temperatures. In one aspect, the preferred approach is to vitrify the bioactive agent at room temperature using a low concentration of vitrification agent (e.g., <2M trehalose) and then immediately store at cryogenic temperatures. Thus, the device is optionally made from a material that can be stored at temperatures between -196°C and 60°C after vitrification according to the teachings of the present disclosure.

[0116] Optionally, the bioactive agent is vitrified in successive layers on or within the vitrified membrane. It has been found that successive vitrification not only increases the amount of bioactive agent stored within the membrane prior to reconstitution, but also promotes successful concentration of viable and active bioactive agent after reconstitution in the dosing solvent. As shown in FIG. 3, vitrification of the bioactive agent within the channels of the membrane can be achieved by adding a vitrification mixture containing the bioactive agent and vitrification media to the channels of the membrane and subjecting the membrane to drying as provided herein until glass formation occurs on the walls of the channel. A second vitrification mixture can then be added to the same membrane, optionally to a level below that which fills the remaining channels, and a second drying process can be performed to add additional or different bioactive agents, cofactors, coenzymes, or other desired molecules to the vitrified channels. Additional vitrification mixtures containing more of the same or different bioactive agents can be added one, two, three, four, five, six, or more times. In some aspects, the number of times additional vitrification media can be added is limited by the requirement that the membrane with the vitrified material therein still have a pore structure or shape sufficient to allow the additional vitrification mixture to be absorbed into or onto the channels of the membrane.

[0117] The formulations provided herein are formed by reconstituting a vitrified bioactive agent in a dosing solvent at a volume such that rehydration of the bioactive agent produces a fluid, producing a supersaturated fluid of the bioactive agent in the dosing solvent. The amount of dosing solvent is optionally less than the amount of vitrification medium used to prepare the vitrified sample, such that the reconstituted bioactive agent is present in the dosing solvent at a higher concentration than in the vitrification mixture.

[0118] The amount of administration solvent used may be adjusted according to the desired number of administrations of the bioactive agent.For example, for subcutaneous administration, administration solvent may be added in a volume of 5ml or less, optionally 4ml or less, optionally 3ml or less.In some embodiments, the formulation is administered in an amount that is tolerated painlessly in human subjects, optionally 2.5ml or less, optionally 2.0ml or less, optionally 1.5ml or less.In other embodiments, administration solvent is added in a volume greater than 5ml depending on the desired downstream administration, such as intravenous or intramuscular injection.

[0119] The administration vehicle is referred to as an administration vehicle for purposes of example only. The composition of the vehicle need not be, but may be, made up solely of materials acceptable for administration to a subject, but may also include other materials, including in addition to or as an alternative, depending on the intended downstream use of the bioactive agent. The administration vehicle may be as described herein. Methods for preventing or treating a disease or condition

[0120] The formulations provided herein may be used to prevent or treat a disease or condition, or a symptom of a disease or condition. The identity of the disease or condition that can be prevented, treated, etc. will depend on the type of biological activity performed by the bioactive agent upon administration to a subject having the disease or condition, or a subject at risk of suffering from or developing the disease or condition.

[0121] Optionally, diseases or conditions for which the formulations provided herein may be administered include, but are not limited to, rheumatoid arthritis; cancer (especially breast cancer, lung cancer, colorectal cancer, pancreatic cancer, leukemia, etc.); autoimmune diseases; acute organ rejection; ankylosing spondylitis; inflammatory diseases; hypercholesterolemia; Crohn's disease; ulcerative colitis; psoriasis; diabetes; multiple sclerosis; heart attack; systemic lupus erythematosus; age-related macular degeneration; diabetic retinopathy, pneumonia; anemia; chronic migraine headaches; infectious diseases; hepatitis B, hemophilia, respiratory syncytial virus infection; HPV infection; chickenpox virus infection; growth hormone deficiency; osteoporosis; asthma; allergic asthma; chronic idiopathic urticaria; infertility; HIV infection; meningococcal disease; cystic fibrosis; paroxysmal nocturnal hemoglobinuria; procoagulant states; myocardial ischemia; myocardial infarction; and Alzheimer's disease, among others.

[0122] The formulations provided herein are optionally administered by any desired route suitable for administering the bioactive agent. Administration is optionally subcutaneous, intravenous, intramuscular, etc. In certain embodiments, the formulations are administered subcutaneously to a subject. Increasing the concentration of the bioactive agent in the administration medium allows for administration of smaller volumes while still providing a sufficient and adequate dose of the bioactive agent. When administered subcutaneously, the formulations are optionally administered without the need for the presence or co-administration of hyaluronidase. However, in some embodiments, hyaluronidase is present when the bioactive agent is administered.

[0123] The formulations are optionally administered one or more times daily, weekly, monthly, or yearly. Optionally, the formulations are administered once a day. The frequency of administration is as defined for the bioactive agent of interest.

[0124] Optionally, the bioactive agent is an antibody at a concentration of 50 mg / ml or more in a dosing solvent and the formulation is administered subcutaneously. Optionally, the antibody is at a concentration of 90 mg / ml or more in a dosing solvent and the formulation is administered subcutaneously. Optionally, the antibody is at a concentration of 150 mg / ml or more in a dosing solvent and the formulation is administered subcutaneously. Optionally, the dosing solvent is buffered saline or normal saline.

[0125] Optionally, the bioactive agent is an anti-proprotein convertase subtilisin / kexin type 9 (PCSK9) antibody at a concentration of 50 mg / ml or more in a dosing vehicle and the formulation is administered subcutaneously. Optionally, the antibody is at a concentration of 90 mg / ml or more in a dosing vehicle and the formulation is administered subcutaneously. Optionally, the antibody is at a concentration of 150 mg / ml or more in a dosing vehicle and the formulation is administered subcutaneously. Optionally, the dosing vehicle is buffered saline or normal saline.

[0126] Optionally, the bioactive agent is an antibody-drug (toxin) conjugate at a concentration of 50 mg / ml or more in a dosing vehicle and the formulation is administered subcutaneously. Optionally, the antibody is at a concentration of 90 mg / ml or more in a dosing vehicle and the formulation is administered subcutaneously. Optionally, the antibody is at a concentration of 150 mg / ml or more in a dosing vehicle and the formulation is administered subcutaneously. Optionally, the dosing vehicle is buffered saline or normal saline.

[0127] The following non-limiting examples illustrate various aspects of the present disclosure. These examples are for illustrative purposes and are not intended to limit the practice of the present invention. It will be understood that variations and modifications can be made without departing from the spirit and scope of the present invention. EXAMPLES

[0128] Example 1: To vitrify the exemplary antibody IgG, a polyethylene terephthalate membrane was prepared by immersion in 0.05 wt% phosphate buffered saline containing Tween 20 with continuous agitation for 20 minutes at 25° C. The membrane was then dried in an oven at 55° C. for 1 hour.

[0129] A vitrification mixture was prepared by mixing 20 mg / mL powdered IgG (IHUIGGGGLYIGM-34158, Innovative Research, Inc.), 5 wt. % trehalose dihydrate, and 0.25 wt. % glycerol in phosphate buffered saline. 150 microliters of the vitrification mixture was added to the treated membrane. The membrane was left on a metal screen in a vitrification chamber and dried for 30 minutes at a bed temperature of 37°C. In each case, the chamber was evacuated to 29.5 mmHg. An additional 150 microliters of vitrification medium was then added to the membrane and the vitrification process was repeated. This was repeated for a total of five more vitrification cycles. The process produced a total of three separate samples.

[0130] The resulting membrane was subjected to scanning electron microscopy (SEM) to analyze the collection of vitrified antibodies within the channels of the membrane. The results are shown in FIG. 4A, which shows the structure of the membrane before vitrification. FIG. 4B shows the membrane after a first round of vitrification of the antibodies, showing the initial deposition / coating of the antibodies on the fibers of the membrane. FIG. 4C shows the membrane after a second round of vitrification showing further collection of vitrified material. FIG. 4D shows the membrane after a third round of vitrification, shown at 100x magnification in FIG. 4E, both of which show further filling of the pores within the channels of the membrane and greater continuity of the coating of vitrified antibodies.

[0131] The membrane containing the vitrified antibody was then contacted with 150 microliters of water and incubated for 5 minutes, after which the membrane was rolled into a tube shape with the mesh support and centrifuged at 10×g for 10 minutes to collect all the reconstituted antibody.

[0132] The concentration of the reconstituted antibody was then assayed by BCA protein assay using standard techniques. The starting concentration of antibody in the vitrification mixture was 20 mg / ml. After vitrification and reconstitution as described, the antibody concentration was 91.59 mg / ml with a standard error of 1.82 mg / ml. This represents a greater than 4.5-fold increase in antibody concentration, demonstrating the availability of ultra-concentrated antibodies beyond those typically available in antibody biopharmaceuticals for intravenous delivery. Example 2:

[0133] The vitrified material of Example 1 was analyzed for the functional stability of its antibodies. The vitrified sample of Example 1 was subjected to heat treatment at 75° C. for 1 hour. The sample was then concentrated as in Example 1 and subjected to analysis by ELISA. Briefly, polystyrene plates were coated with anti-IgG antibodies. The reconstituted samples were serially diluted and incubated with the coated plates. After washing, detection of the presence or absence of the reconstituted antibodies was performed with a secondary anti-IgG antibody conjugated to horseradish peroxidase. Antibody sandwich detection was performed using TMB substrate. As a control, an identical antibody solution not subjected to vitrification at 2 mg / ml was prepared, diluted to the same concentration, and subjected to the same ELISA analysis. As a second control, an antibody solution at 2 mg / ml was heat treated at 75° C. for 1 hour and subjected to the same ELISA analysis. As shown in FIG. 5, the vitrified antibody reconstituted at 91.59 mg / ml maintained the same structure and solvation as the non-vitrified and non-heat treated control, as shown by the substantially overlapping detection levels at each identical concentration. In contrast, the heat-treated solution samples that were not vitrified were essentially destroyed by the heat treatment.

[0134] In another series of tests, the test samples in 2 mg / ml solution and the heat-treated solutions were subjected to SDS-PAGE and detected with Coomassie Brilliant Blue. 1 μg or 5 μg of sample antibody was loaded per lane. As shown in FIG. 6, the ultra-concentrated, vitrified, and heat-treated samples maintain intact heavy and light chain structures, while the non-vitrified and heat-treated antibody is almost completely degraded. In summary, as revealed by these tests, vitrification maintains the functional conformation of the antibody despite the generation of an ultra-concentrated reconstituted solution of the antibody, and also maintains the apparent thermal stability of the antibody in the vitrified state when prepared as provided herein. Example 3:

[0135] The antibody of Example 1 was vitrified under the conditions of Example 1, except that 200 μl of sample was added to each membrane. After each successive vitrification, the membranes containing the vitrified material were subjected to analysis by X-ray diffraction (XRD). As shown in FIG. 7, only the dried trehalose showed the typical crystalline structure. The filter membrane itself showed the expected characteristic amorphous structure before vitrification of any material. After the first round of vitrification with the vitrified material, as shown in FIG. 4B, this amorphous structure was maintained and no crystalline structure was formed during vitrification, indicating the presence of fully vitrified material. A similar amorphous structure was observed after a second round of vitrification on the same membrane. In contrast, the third round of vitrification showed the presence of some crystalline structure formation, indicating that the amount of solution was too large to be accommodated in the channels of the membrane because the previous vitrified material was occupying the space. Under the conditions of the third round, the formation of crystals would occur during the drying of the material on the surface.

[0136] These data show that the number of successive vitrification rounds and the total amount of material that can be accommodated within the membrane and retain the vitrified biologic effectively stored is limited by the presence of sufficient pore volume to accommodate the vitrification mixture during vitrification and before overloading the membrane, maintaining an amorphous structure and indicating the presence of an adequately stabilized biologic.

[0137] The following list of exemplary aspects is also relevant to this disclosure:

[0138] Aspect 1: An ultra-concentrated formulation of a bioactive agent comprising a bioactive agent and an administration solvent, wherein the bioactive agent is present at a concentration greater than the concentration of the bioactive agent in the administration solvent formulated for subcutaneous administration to a subject, and optionally greater than the concentration at which physiologically relevant aggregation of the bioactive agent would occur in the administration solvent if not prepared by a method provided herein, and optionally prepared by a preparation method of any one of Aspects 14-27, or wherein the concentration of the bioactive agent in the administration solvent has similar or the same colloidal stability, liquid-liquid phase separation, or tendency to form a gel as compared to the aggregate-free concentration of the bioactive agent in the formulation in the absence of the administration solvent as provided herein or as provided by any one of Aspects 14-27.

[0139] Aspect 2. The ultra concentrated formulation of aspect 1, wherein the solvent is aqueous.

[0140] Aspect 3. The ultra-concentrated formulation of any one of aspects 1 and 2, wherein the solvent comprises water and one or more monovalent or divalent salts.

[0141] Aspect 4. The ultra-concentrated formulation of any one of aspects 1-3, wherein the bioactive agent is present at a concentration in the solvent that is equal to or greater than the concentration at which the bioactive agent aggregates.

[0142] Aspect 5. The ultra-concentrated formulation of any one of aspects 1-4, wherein the bioactive agent is present in a concentration greater than 50 mg / mL, optionally greater than 90 mg / mL, optionally greater than 100 mg / mL, optionally greater than 150 mg / mL.

[0143] Aspect 6. The ultra-concentrated formulation of any one of aspects 1 to 5, further comprising one or more pharmacologically acceptable excipients.

[0144] Aspect 7. The ultra concentrated formulation of any one of aspects 1-6, wherein the volume of the formulation is up to 2.5 milliliters.

[0145] Aspect 8. The ultra-concentrated formulation of any one of aspects 1 to 7, which does not contain hyaluronidase.

[0146]

[0023] Aspect 9. The ultra-concentrated formulation of any one of aspects 1 to 8, wherein the bioactive agent is an antibody, a protein, a lipid particle, optionally a lipid nanoparticle, or another therapeutic agent.

[0147] Aspect 10. The ultra-concentrated formulation of aspects 1-9, wherein the bioactive agent is an antibody, optionally a humanized antibody.

[0148]

[0023] Aspect 11. The ultra-concentrated formulation of aspect 9 or 10, wherein said antibody comprises muromonab-CD3, infliximab, rituximab, solanezumab, bapinezumab, catumaxomab, trastuzumab, cetuximab, omalizumab, adalimumab, bevacizumab, BAN2401, tositumomab, or alemtuzumab.

[0149]

[0036] Aspect 12. The ultra-concentrated formulation of any one of aspects 1-9, wherein the bioactive agent is a protein, and the protein comprises an interleukin or an interferon, optionally interferon beta-1b, pegylated interferon alpha-2b, roferon-A, or aldesleukin.

[0150] Aspect 13. The ultra concentrated formulation of any one of aspects 1-12, wherein the formulation has a viscosity of 20 cP or less, optionally 12 cP or less, optionally 8 cP or less, optionally 2 cP or less, optionally 1 cP or less, wherein the viscosity is measured at 20° C. and 1 atm.

[0151] A process for preparing the ultra-concentrated formulation of any one of aspects 1 to 13, comprising the steps of: a) layering a vitrification mixture comprising said bioactive agent and a vitrification medium onto a membrane comprising a capillary network in a drying chamber; b) reducing the air pressure within the drying chamber; c) providing sufficient thermal energy to the vitrification mixture to prevent the vitrification mixture from becoming cryogenic; d) drying the vitrification mixture by capillary action until it reaches a glassy state; and e) reconstituting the bioactive agent in the administration solvent such that the concentration of the bioactive agent in the administration solvent is equal to or greater than the concentration of the bioactive agent in the administration solvent when formulated for subcutaneous administration to a subject, and optionally equal to or greater than the concentration of the bioactive agent in the vitrification mixture. The process includes:

[0152] Embodiment 15. The process of embodiment 14, further comprising repeating steps a)-d) prior to step e).

[0153] Aspect 16. The process of aspect 15, wherein the repetition is 1 to 3 times.

[0154]

[0031] Example 17. The process of any one of Examples 14 to 16, wherein the capillary network is provided by a contour along a surface of the membrane.

[0155]

[0041] Embodiment 18. The process of any one of embodiments 14 to 17, wherein the capillary network in the drying chamber is supported by an underlying solid support substrate.

[0156]

[0041] Aspect 19. The process of any one of aspects 14-18, wherein vitrification of the vitrification mixture occurs in less than 30 minutes, optionally less than 10 minutes.

[0157]

[0031] Aspect 20. The process of any one of aspects 14-19, wherein the thermal energy is provided by heating the vitrification mixture.

[0158]

[0041] Aspect 21. The process of any one of aspects 14 to 20, wherein the pressure is reduced to a value of about 0.9 atm to about 0.005 atm.

[0159]

[0046] Aspect 22. The process of aspect 21, wherein the atmospheric pressure is reduced to about 0.004 atm.

[0160]

[0041] Aspect 23. The process of any one of aspects 14-22, wherein the thermal energy provided is sufficient to prevent crystallization of the bioactive agent within the vitrification mixture during vitrification.

[0161]

[0031] Aspect 24. The process of any one of aspects 14-23, wherein the thermal energy provided is sufficient to maintain the bioactive agent at a temperature of about 0° C. to about 40° C. during the vitrification.

[0162] Aspect 25. The process according to any one of aspects 14 to 24, wherein the vitrification medium comprises trehalose, glycerol, betaine, and / or choline.

[0163]

[0041] Example 26. The process of any one of examples 14 to 25, wherein the capillary network is hydrophilic.

[0164]

[0041] Example 27. The process of any one of examples 14 to 26, wherein the capillary network comprises a continuous capillary channel.

[0165] Embodiment 28. A process for treating or preventing a disease or condition comprising administering to a subject in need thereof a composition according to any one of embodiments 1 to 13.

[0166] Aspect 29. The process of aspect 28, wherein the administering is subcutaneous administration.

[0167] Aspect 30. The process according to aspect 28 or 29, wherein said disease or condition is an autoimmune disease, cancer, asthma, an inflammatory disease, an infectious disease, hypercholesterolemia, acute organ rejection, osteoporosis, or Alzheimer's disease.

[0168] Aspect 31. The use according to any one of aspects 1 to 13 for the treatment of a disease.

[0169] Embodiment 32. The use of embodiment 31, wherein said disease or condition is an autoimmune disease, cancer, asthma, an inflammatory disease, an infectious disease, hypercholesterolemia, acute organ rejection, osteoporosis, or Alzheimer's disease.

[0170] While embodiments of the present disclosure have been illustrated and described, it is not intended that these embodiments illustrate and describe all possible forms of the present disclosure. Rather, the terms used herein are terms of description rather than limitation. It is also understood that various modifications and substitutions can be made without departing from the spirit and scope of the present disclosure.

[0171] Reference patent documents

[0172] 6,808,651 B1 10 / 2004 Katagiri, et al.

[0173] 7,883,664 B2 2 / 2011 G. Elliott; N. Chakraborty.

[0174] 8,349,252 B2 1 / 2013 G. Elliott; N. Chakraborty.

[0175] 10,568,318 B2 2 / 2020 PS Mohanty, N. Chakraborty.

[0176] US 2013 / 0157250 A1 6 / 2013 Gutierrez et al.

[0177] US 2013 / 0260452 A1 10 / 2013 Toner et al.

[0178] Reference non-patent literature

[0179] Chakraborty N, Menze MA, Malsam J, Aksan A, Hand SC, et al. (2011) Cryopreservation of spin-dried mammalian cells. PLoS ONE 6(9): e24916.

[0180] Chakraborty N, Biswas D, Elliott GD (2010) A simple mechanical method to improve mammalian cell survival during dry storage, biopreservation, and biobanking processes, Biopreservation and Biobanking, 8(2), 107-114.

[0181] Various modifications of the present disclosure in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description, and such modifications are intended to fall within the scope of the appended claims.

[0182] Unless otherwise stated, it is understood that all reagents are available from sources known in the art.

[0183] It should also be understood that the disclosure is not limited to the specific embodiments and methods described herein, as the specific components and / or conditions may of course vary. Moreover, the terms used herein are used only for the purpose of describing the specific embodiments of the disclosure, and are not intended to be limiting in any way. It should also be understood that, although the terms "first", "second", and "third", etc., may be used herein to describe various elements, components, regions, layers, and / or sections, these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are used only to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, a "first element", "component", "region", "layer", or "section" discussed below can be referred to as a second (or other) element, component, region, layer, or section without departing from the teachings of the present specification. Similarly, the singular forms "a" and "this" as used herein are intended to include the plural forms, including "at least one", unless the content clearly dictates otherwise. "Or" means "and / or." As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. It should be further understood that, as used herein, the terms "comprise" and / or "including," or "comprises" and / or "comprising" specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof. The term "or combinations thereof" refers to combinations including at least one of the preceding elements.

[0184] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. It should be further understood that terms as defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and this disclosure, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0185] Reference will now be made in detail to exemplary compositions, embodiments and methods of the present disclosure, which constitute the best modes of carrying out the present disclosure currently known to the inventors. The drawings are not necessarily to scale. However, it should be understood that the disclosed embodiments are merely exemplary of the present disclosure, which may be embodied in various alternative forms. Thus, the specific details disclosed herein should not be construed as limiting, but merely as a representative basis for any embodiment of the present disclosure and / or as a representative basis for teaching those skilled in the art how to utilize the present disclosure in various ways.

[0186] The patents, publications, and applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains, and are herein incorporated by reference to the same extent as if each individual patent, publication, or application was specifically and individually indicated to be incorporated by reference.

[0187] The above description illustrates certain embodiments of the present disclosure, but is not intended to limit its practice. The following claims, including all equivalents thereof, are intended to define the scope of the present disclosure.

Claims

1. 1. An ultra-concentrated formulation of a bioactive agent comprising a bioactive agent and an administration solvent, wherein the bioactive agent is present at a concentration greater than the concentration of the bioactive agent in the administration solvent formulated for subcutaneous administration to a subject.

2. 10. The ultra-concentrated formulation of claim 1, wherein the solvent is aqueous.

3. 10. The ultra-concentrated formulation of claim 1, wherein the solvent comprises water and one or more monovalent or divalent salts.

4. 10. The ultra-concentrated formulation of claim 1, wherein the bioactive agent is present at a concentration in the solvent at or above the concentration at which the bioactive agent aggregates.

5. 10. The ultra-concentrated formulation of claim 1, wherein the bioactive agent is present at a concentration greater than 50 mg / mL.

6. 6. The ultra-concentrated formulation of any one of claims 1 to 5, further comprising one or more pharmacologically acceptable excipients.

7. 6. The ultra-concentrated formulation of any one of claims 1 to 5, wherein the volume of the formulation is up to 2.5 milliliters.

8. The ultra-concentrated formulation of any one of claims 1 to 5, which is free of hyaluronidase.

9. 6. The ultra-concentrated formulation of any one of claims 1 to 5, wherein the bioactive agent is an antibody, a protein, a lipid particle, or another therapeutic agent, or the bioactive agent comprises a lipid nanoparticle.

10. 10. The ultra-concentrated formulation of claim 9, wherein the bioactive agent is an antibody or a humanized antibody.

11. 10. The ultra-concentrated formulation of claim 9, wherein the antibody comprises muromonab-CD3, infliximab, rituximab, solanezumab, bapineumab, catumaxomab, trastuzumab, cetuximab, omalizumab, adalimumab, bevacizumab, BAN2401, tositumomab, or alemtuzumab.

12. 10. The ultra-concentrated formulation of claim 9, wherein the bioactive agent is a protein, and the protein comprises an interleukin or an interferon.

13. The ultra-concentrated formulation of claim 12, wherein the protein comprises interferon beta-1b, pegylated interferon alpha-2b, roferon-A, or aldesleukin.

14. 6. The ultra concentrated formulation of any one of claims 1 to 5, wherein the formulation has a viscosity of 20 cP or less, the viscosity being measured at 20°C and 1 atm.

15. 10. A process for preparing the ultra-concentrated formulation of claim 1, comprising: a) layering a vitrification mixture comprising the bioactive agent and a vitrification medium onto a membrane comprising a capillary network within a drying chamber; b) reducing the air pressure within the drying chamber; c) providing sufficient thermal energy to the vitrification mixture to prevent the vitrification mixture from becoming cryogenic; d) drying the vitrification mixture by capillary action until it reaches a glassy state; and e) reconstituting the bioactive agent in the administration solvent such that the concentration of the bioactive agent in the administration solvent is equal to or greater than the concentration of the bioactive agent in the administration solvent when formulated for subcutaneous administration to a subject, and optionally equal to or greater than the concentration of the bioactive agent in the vitrification mixture. The process includes:

16. 16. The process of claim 15, further comprising repeating steps a) through d) before step e).

17. 17. The process of claim 16, wherein the repetition is 1 to 3 times.

18. 16. The process of claim 15, wherein the capillary network is provided by a contour along the surface of the membrane.

19. 16. The process of claim 15, wherein the capillary network within the drying chamber is supported by an underlying solid support substrate.

20. 20. The process of any one of claims 15 to 19, wherein vitrification of the vitrification mixture occurs in less than 30 minutes, optionally less than 10 minutes.

21. The process of any one of claims 15 to 19, wherein the thermal energy is provided by heating the vitrification mixture.

22. 20. The process of any one of claims 15 to 19, wherein the atmospheric pressure is reduced to a value of from about 0.9 atm to about 0.005 atm.

23. 23. The process of claim 22, wherein the pressure is reduced to 0.004 atm.

24. 20. The process of any one of claims 15 to 19, wherein the provided thermal energy is sufficient to prevent crystallization of the bioactive agent within the vitrification mixture during vitrification.

25. 20. The process of any one of claims 15 to 19, wherein the provided thermal energy is sufficient to maintain the bioactive agent at a temperature of between 0°C and 40°C during the vitrification.

26. 20. The process of any one of claims 15 to 19, wherein the vitrification medium comprises trehalose, glycerol, betaine, and / or choline.

27. The process of any one of claims 15 to 19, wherein the capillary network is hydrophilic.

28. 20. The process of any one of claims 15 to 19, wherein the capillary network comprises continuous capillary channels that form an uninterrupted structure from the first end to the second end.

29. An ultra-concentrated formulation according to any one of claims 1 to 5 for treating or preventing a disease or condition, comprising administering the ultra-concentrated formulation according to any one of claims 1 to 5 to a subject in need thereof.

30. 30. The ultra-concentrated formulation of claim 29, wherein the ultra-concentrated formulation is for subcutaneous administration.

31. 30. The ultra-concentrated formulation of claim 29, wherein the disease or condition is an autoimmune disease, cancer, asthma, an inflammatory disease, an infectious disease, hypercholesterolemia, acute organ rejection, osteoporosis, or Alzheimer's disease.

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