Polyzwitterionic stabilized protein formulations

Abstract

Stabilized protein formulations and methods of using them are disclosed herein. In some embodiments, the formulations comprise a protein (e.g., insulin, glucagon, GLP-1 analogs, monoclonal antibodies) and a polyzwitterion, or a pharmaceutically acceptable salt thereof. The disclosed formulations promote delayed and reduced amounts of protein aggregation and subsequent protein fibrillation compared to formulations without polyzwitterions. These more stable formulations retain their potency for a longer period of time even at elevated temperatures.

Description

Attorney Docket No.: TAP.003 WOPOLYZWITTERIONIC STABILIZED PROTEIN FORMULATIONSCROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] The present application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 690,491, filed September 4, 2024, which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] The present invention relates to polyzwitterionic stabilized proteins, formulations comprising polyzwitterionic stabilized proteins, and methods of using polyzwitterionic stabilized protein formulations.BACKGROUND

[0003] Protein fibrillation is a process where proteins misfold and aggregate into insoluble, fibrous structures. These structures, often referred to as amyloid fibrils, are characterized by their P-sheet structure. As a result of this change in structure, the protein loses its biological activity. One notable example is insulin, which is essential for blood glucose regulation.

[0004] Under certain conditions, insulin aggregation can result in the formation of insulin fibrils, which are elongated, thread-like structures that can impair insulin's biological activity and contribute to amyloid formation. The particular conformation of insulin can be influenced by several factors, including pH, temperature, ionic strength, and the presence of specific excipients in insulin formulations. During its industrial production, purification, and transportation, insulin can form undesirable insulin fibrils. Fibrillation of insulin may occur during ex vivo storage of insulin, especially when stored for extended periods of time or at elevated temperatures. Insulin fibrils may also form at the site of insulin injections and the resulting amyloid deposit is associated with a need for increased insulin dosing. Insulin fibrils can interfere with insulin's ability to bind to its receptor and stimulate glucose uptake by cells. Fibril formation can promote insulin resistance, making it more difficult for the body to respond effectively to insulin.

[0005] Current strategies to enhance insulin stability include the use of formulation additives. For instance, the addition of excipients such as zinc, protamine, and surfactants canAttorney Docket No.: TAP.003 WO help to reduce fibrillation and improve insulin stability. Zinc is commonly used to form insulin hexamers, which are less prone to fibrillation than insulin monomers. Many commercial insulin formulations such as Humulin, Lantus, and NovoLog, utilize a combination of these stabilizers to achieve optimal stability.

[0006] Despite significant advancements in formulation and delivery, insulin can still aggregate over time into fibrils and stabilizing the protein remains a challenge. In addition to insulin, there are other proteins, such as glucagon, which are vulnerable to fibrillation and would benefit from a more stable and effective formulation.SUMMARY

[0007] The present disclosure provides a stabilized protein formulation comprising a protein, a polyzwitterion or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0008] The present disclosure provides a formulation comprising a protein and a polyzwitterion or a pharmaceutically acceptable salt thereof, wherein the formulation is a lyophilized formulation.

[0009] The present disclosure provides a method of regulating blood glucose in an individual in need thereof comprising administering a stabilized protein formulation disclosed herein.

[0010] The present disclosure provides a stabilized protein formulation for use in regulating blood glucose levels in an individual in need thereof.

[0011] The present disclosure provides a stabilized protein formulation for use in reducing the risk of hypoglycemia in an individual in need thereof.

[0012] The present disclosure provides a kit for the administration of insulin, comprising a stabilized protein formulation disclosed herein; a means for parenterally administering the stabilized protein formulation; and instructions for the administration of the stabilized protein formulation to an individual in need thereof.

[0013] Additional embodiments, features, and advantages of the invention will be apparent from the following detailed description and through practice of the invention.Attorney Docket No.: TAP.003 WOBRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. l is a representative plot of ThT fluorescence in the presence of insulin with P2 poly(SBMA), P2 poly(CBAA-2), or no polymer (control) over a period of 200 hours.

[0015] FIG. 2 is a representative plot of ThT fluorescence in the presence of insulin with P3 poly(SBMA), P3 poly(CBAA-2), or no polymer (control) over a period of 200 hours.

[0016] FIG. 3 is a representative plot of ThT fluorescence in the presence of insulin glulisine with P3 poly(CBAA-2), or no polymer (control) over a period of 145 hours.

[0017] FIG. 4 shows the in vitro functional activity of insulin in formulations containing no insulin (negative control), insulin with no polymer excipient (positive control), and insulin with Pl poly(CBAA-l), Pl poly(CBAA-2), P2 poly(CBAA-2), or P3 poly(CBAA- 2), as measured by a tyrosine auto-phosphorylation in-cell western assay.DETAILED DESCRIPTION

[0018] The present disclosure relates to stabilized protein formulations comprising a polyzwitterion or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients. In another aspect, the present disclosure relates to lyophilized formulations comprising a protein and a polyzwitterion or a pharmaceutically acceptable salt thereof. In another aspect, the present disclosure provides methods of using the formulations disclosed herein to control blood glucose levels in an individual in need thereof.I. FormulationsA. Polyzwitterion

[0019] The polyzwitterion is a class of polymers in which the monomers are zwitterions. The polyzwitterion is a biocompatible material. In one embodiment, the polyzwitterion is a polybetaine, wherein the cationic group is represented by quaternary ammonium groups and the anionic group is represented by a carboxylate group, sulfonate groups, or phosphate / phosphinate / phosphonate groups; poly(carboxybetaine), poly(sulfobetaine), and poly(phosphobetaine), respectively. In some embodiments, the cationic group is a quaternary ammonium and the anionic group is a carboxylate. In some embodiments, the cationic group is a dimethyl-substituted quaternary ammonium and the anionic group is a carboxylate.Attorney Docket No.: TAP.003 WO

[0020] The polyzwitterion may be a homopolymer or a copolymer. A homopolymer includes poly(carboxybetaine), poly(sulfobetaine), and poly(phosphobetaine). A copolymer is comprised of more than one type of monomer, wherein at least one of the monomer types is zwitterionic. Thus, a copolymer may encompass more than one kind of zwitterionic monomer, as well as at least one kind of zwitterionic monomer and a non-zwitterionic monomer. For instance, a polyzwitterion encompasses the copolymer poly(carboxybetaine-co-butyl methacrylate), which comprises zwitterionic carboxybetaine monomers and non-zwitterionic butyl methacrylate monomers.

[0021] "Poly(carboxybetaine)" refers to a polymer class comprising a monomer repeat unit having a carboxybetaine as a pendant group or alternatively known as a "side chain". The carboxybetaine has one carboxylate anion group and one cationic quaternary amine group, together resulting in a net neutral charge.

[0022] The poly(carboxybetaine) may be prepared, for example, from a polymerization reaction of carboxybetaine acrylates (e.g., carboxybetaine methacrylate) or carboxybetaine acrylamides, each of which may be substituted (e.g., C1-C6 alkyl groups).

[0023] Non-limiting examples of acrylate and acrylamide carboxybetaine monomers that may be used in the preparation of polyzwitterions are shown below in Table 1 :

[0024] Table 1.Attorney Docket No.: TAP.003 WO“CBAA” is short for carboxybetaine acrylamide and “CBMA” refers to carboxybetaine methacrylate. The number after each abbreviation refers to the number of methylenes between the ammonium and carboxylate groups. Exemplary structures are shown above. In some embodiments, the polycarboxybetaine is derived from any one of the monomers shown above.

[0025] In some embodiments, the number of methylenes between the ammonium and carboxylate groups is between 1 and 5. In some embodiments, the methylenes are optionally substituted with C1-C3 alkyl (e.g., methyl, ethyl, propyl, isopropyl).

[0026] As used herein, acrylate and acrylamide carboxybetaine monomers encompass derivatives and analogs such those with alkyl substituted (e.g., C1-C3 alkyl) vinyl groups. For example, carboxybetaine methacrylates and carboxybetaine methacrylamides. The resulting polymerized form of these monomer derivatives and analogs of acrylate and acrylamide carboxybetaine monomers, such as those having substituted vinyl groups (e.g., C1-C3 alkyl), are encompassed by polyzwitterions and poly(carboxybetaine) disclosed herein.

[0027] In some embodiments, the number of methylenes between the acrylamide or acryloyl and the ammonium group is between 1 and 5. In some embodiments, the methylenes are optionally substituted with C1-C3 alkyl.

[0028] In some embodiments, the quaternary amine group has two substituents, each one independently selected from C1-C4 alkyl (e.g., methyl, ethyl propyl, isopropyl, butyl, isobutyl).Attorney Docket No.: TAP.003 WO

[0029] In some embodiments, the polyzwitterionic group is a poly(carboxybetaine). In some embodiments, the polyzwitterion is poly(CBAA-2) or poly(CBAA-l). In some embodiments, the poly(carboxybetaine) is a poly(carboxybetaine methacrylate). In some embodiments, the polyzwitterion is a homopolymer. In some embodiments, the polyzwitterion is a copolymer. In some embodiments, the copolymer comprises poly(carboxybetaine-co-butyl methacrylate).

[0030] The molecular weight of the polyzwitterion may influence the rate and degree of fibrillation. The desirable molecular weight is a range that delays and minimizes protein fibrillation. In some embodiments, the molecular weight, Mn, of the polyzwitterion is 400 Da- 100 kDa, 400 Da-70 kDa, 400 Da-50 kDa, 400 Da-40 kDa, 400 Da-30kDa, 400 Da-20 kDa, 400 Da-10 kDa, 400 Da-5 kDa, 400 Da-3 kDa, 400 Da-2 kDa, 400 Da-1 kDa, 1 kDa-100 kDa, 1 kDa-70 kDa, 1 kDa-50 kDa, 1 kDa-40 kDa, 1 kDa-30 kDa, 1 kDa-20 kDa, 1 kDa-10 kDa, 1 kDa-5 kDa, lkDa-3 kDa, 1 kDa-2 kDa, 2 kDa-100 kDa, 2 kDa-70 kDa, 2 kDa-50 kDa, 2 kDa-40 kDa, 2 kDa-30 kDa, 2 kDa-20 kDa, 2 kDa-10 kDa, 2 kDa-5 kDa, or 2 kDa- 3 kDa. Mn is the number average molecular weight.

[0031] In some embodiments, the polyzwitterion comprises 5-50 monomers, 5-40 monomers, 5-30 monomers, 5-20 monomers, 5-10 monomers, 2-50 monomers, 2-40 monomers, 2-30 monomers, 2-20 monomers, 2-10 monomers, 2-5 monomers, or 7-15 monomers.

[0032] The concentration of the polyzwitterion may influence the rate and degree of fibrillation. The desirable concentration is one that delays and minimizes protein fibrillation. In some embodiments, the concentration of the polyzwitterion is 1-20 mg / mL, 1-15 mg / mL, 1-10 mg / mL, 1-7.5 mg / mL, 1-5 mg / mL, 1-3 mg / mL, 2-20 mg / mL, 2-15 mg / mL, 2-10 mg / mL, 2-7.5 mg / mL, 2-5 mg / mL, 2-3 mg / mL, 3-20 mg / mL, 3-15 mg / mL, 3-10 mg / mL, 3- 7.5 mg / mL, 3-5 mg / mL, 5-20 mg / mL, 5-15 mg / mL, 5-10 mg / mL, or 5-7.5 mg / mL.

[0033] In some embodiments, the concentration of the polyzwitterionic group is about 20 mg / mL, about 15 mg / mL, about 10 mg / mL, about 7.5 mg / mL, about 5 mg / mL, about 4 mg / mL, about 3 mg / mL, about 2 mg / mL, or about 1 mg / mL.

[0034] Under certain conditions (e.g., low pH), the polyzwitterion may exist as a pharmaceutically acceptable salt. In some embodiments, the stabilized protein formulation comprises a pharmaceutically acceptable salt of the polyzwitterion.Attorney Docket No.: TAP.003 WOB. Protein

[0035] The formulation described herein comprises a protein (e.g., insulin, glucagon, GLP-1 analogs, monoclonal antibodies), wherein the protein in the absence of the polyzwitterion is one that tends to misfold and form aggregates, leading to the formation of amyloid fibrils, and subsequently having reduced biological function. In some embodiments, the protein is a hormone (e.g., insulin, glucagon). In some embodiments, the protein is any protein whose therapeutic use benefits from the delay and reduction of protein aggregation.1. Insulin

[0036] Insulin is a peptide hormone produced by the beta cells of the pancreas. It plays a crucial role in regulating blood glucose levels. When blood glucose rises after a meal, insulin is released to facilitate the uptake of glucose from the bloodstream into the cells of the body, primarily in the liver, muscle, and fat tissues.

[0037] Insulin in its monomeric form is composed of two polypeptide chains, A and B, linked together by disulfide bonds. The monomeric form is the functional form of insulin but is unstable and prone to aggregation, which in turn leads to the formation of insulin fibrils. Depending on the environmental conditions, insulin can also exist as a hexamer, tetramer, or dimer. The predominant form of insulin in circulation and storage is the hexameric form where a cluster of six insulin molecules are arranged in a ring-like structure. The hexameric structure is more stable than the monomer and is essential for insulin's biological activity.

[0038] Insulin molecules may clump together to form aggregates, and can be without a specific structure, amorphous, or have a more defined structure such as insulin fibrils. Insulin that has undergone fibrillation is less effective in stimulating glucose uptake by cells. Aggregated insulin can trigger an immune response, leading to allergic reactions or decreased insulin sensitivity. Over time, insulin aggregation can result in a loss of potency, requiring increased doses to achieve the desired blood glucose control.

[0039] In some embodiments, insulin is human insulin. In some embodiments, insulin is animal-sourced insulin, and may be derived from, for example, from pigs (porcine insulin) or cattle (bovine insulin). In some embodiments, insulin is synthetic insulin, which is produced using recombinant DNA technology. Synthetic insulin includes human insulin and insulin analogs (including insulin glulisine, insulin aspart, and insulin lispro). In some embodiments, the protein is an insulin analog. In some embodiments, the protein is selected from the group consisting of insulin glulisine, insulin aspart, and insulin lispro.Attorney Docket No.: TAP.003 WO

[0040] In some embodiments, delayed release forms, including intermediate and long- acting insulin, may be used in the disclosed formulations and methods.

[0041] Monomeric insulin form means insulin in its monomer form. Dimeric insulin form means insulin in its dimeric form (e.g., two monomers associated or coupled together). Hexamer insulin form means insulin in its hexamer form (e.g. , three dimeric forms associated or coupled together).

[0042] In some embodiments, the concentration of the protein is 1-5 mg / mL, 5-10 mg / mL, 10-15 mg / mL, 15-20 mg / mL, 20-25 mg / mL, 3-5 mg / mL, 3-7 mg / mL, 15-18 mg / mL, or 16-18 mg / mL.2. Glucagon

[0043] Glucagon is a hormone produced by alpha cells of the pancreas and raises blood glucose levels by stimulating the breakdown of glycogen in the liver. Glucagon can also misfold and aggregate, forming amyloid fibrils. This can lead to glucagonomas, rare tumors that produce excessive amounts of glucagon, resulting in high blood glucose levels and other symptoms. In some embodiments, the protein is glucagon.3. GLP-l AnalogsGlucagon-like peptide 1 (GLP-l) is a 31-residue peptide hormone responsible for regulation of blood glucose levels and other physiological functions. Several GLP-l analogs including semaglutide, liraglutide, dulaglutide, tirzepatide and exenatide, have been developed for treatment of type 2 diabetes, obesity, and other conditions. GLP-l and GLP-l analogs can aggregate into amyloid fibrils in many conditions, hindering therapeutic effect or causing other undesired outcomes. In some embodiments, the protein is a GLP-l analog (also known as a GLP-l receptor agonist).4. Monoclonal antibody therapeutics (mAbs)

[0044] Monoclonal antibody (mAb) therapeutics are treatments that use native or recombinantly manufactured identical antibodies, designed to target specific physiological pathways to treat diseases such as cancer, autoimmune disorders, and infections. MAb therapeutics face formulation challenges including elevated viscosity, aggregation propensity, fibrillation potential, and narrow pH solubility windows. Current excipients have limitations including degradation susceptibility, biocompatibility or immunogenicity concerns, and incomplete efficacy in high concentration formulations (HCFs) required for subcutaneous (SC)Attorney Docket No.: TAP.003 WO delivery of the mAb therapeutics. In some embodiments, the protein is a monoclonal antibody therapeutic.5. Fibrillation

[0045] As mentioned previously, aggregation of proteins includes amorphous, disordered clumps of proteins as well as more ordered fibril formation. Fibrillation is a subset of aggregation and is irreversible. Protein fibrillation generally leads to more severe physiological consequences compared to aggregation. The formulations disclosed herein reduce the rate and the total amount of both protein fibrillation and protein aggregation. Reference to “fibrillation” and “aggregation” may be used interchangeably herein. By virtue of preventing or reducing protein fibrillation, such formulations therefore are also effective at preventing or reducing the broader category of protein aggregation.

[0046] " Stable proteins" refers to proteins which do not irreversibly aggregate and form protein fibrils, and do not have reduced activity as a result of fibrillation. The formulations contemplated herein comprise stable proteins and have reduced protein fibrillation (e.g., insulin, glucagon) compared to formulations without polyzwitterions or a pharmaceutically acceptable salt thereof, and thus preserve the physiological efficacy of the protein.

[0047] Comparisons of protein fibrillation over a period of time (e.g., 24 hours, 3-10 days) of the disclosed formulations and analogous formulations without polyzwitterions may be made by any well-known method in the art (e.g., ThT fibrillation assay). The extent and rate of fibril formation can be measured at different pH and different temperatures. The rate and degree of aggregation measured is expected to increase as temperatures increase.6. Measuring Fibrillation

[0048] The thioflavin T (ThT) fluorescence assay is widely used to detect and monitor protein fibrillation. Mechanistically, ThT specifically binds to the side chain channels along the long axis of P-sheets in amyloid fibrils, with four consecutive P strands thought to be the minimum required for binding. When unbound, ThT can rotate freely around its central C-C bond connecting its benzothiazole and aniline rings, causing rapid decay of the excited state and preventing fluorescence. When bound to fibrils, ThT is rotationally immobilized and becomes fluorescent. ThT shows high specificity for amyloid fibrils and P-sheet-rich aggregates, and it does not bind significantly to monomeric proteins or oligomeric pre-fibril protein aggregates. Its increase in fluorescence intensity is proportional to the concentration of amyloid fibrils present, allowing quantitative measurement of fibril formation over time.Attorney Docket No.: TAP.003 WO

[0049] The rate and extent of fibrillation of the formulations disclosed herein may be determined by the use of the ThT fluorescence assay, or by other methods known to a person having ordinary skill in the art. A description of the particular conditions used to measure certain formulations disclosed herein is provided in Example 3.

[0050] In some embodiments, at least the amount of protein fibrillation or the rate of protein fibrillation in the formulation are reduced compared to an analogous formulation without a polyzwitterion.

[0051] In some embodiments, the total amount of protein fibrillation in the formulation is less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay. In some embodiments, the total amount of protein fibrillation in the formulation is less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 7 days at 45°C and pH 2, as measured by a ThT fluorescence assay.

[0052] In some embodiments, the amount of protein fibrillation in the formulation does not increase by more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 10 days at 45°C as measured by a ThT fluorescence assay. In some embodiments, the amount of protein fibrillation in the formulation does not increase by more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 7 days at 45°C and pH 2, as measured by a ThT fluorescence assay.

[0053] In some embodiments, the time to half maximum protein fibrillation is greater than 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, at 45°C and pH 2, as measured by a ThT fluorescence assay.

[0054] In some embodiments, the formulations described herein do not require refrigeration and are stable at room temperature.

[0055] In some embodiments, the stabilized protein formulation comprises insulin or glucagon; poly(CBAA-2) or a pharmaceutically acceptable salt thereof; and one or more excipients; wherein the stabilized protein formulation comprises at least 3 or more of the following features: a) the molecular weight, Mn, of poly(CBAA-2) is 1 kDa-10 kDa; b) the poly(CBAA-2) has 5-50 monomers; c) the concentration of poly(CBAA-2) is 5-15 mg / mL; d) the concentration of poly(CBAA-2) is 3-10 mg / mL; e) the total amount of protein fibrillation in the formulation is less than 15% over a period of 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay; e) the amount of protein fibrillation in the formulation does not increase by more than 15% over a period of 10 days at 45°C and pH 2, as measured by a ThTAttorney Docket No.: TAP.003 WO fluorescence assay; f) the time to half maximum protein fibrillation is greater 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay; and wherein the stabilized protein formulation is suitable for parenteral administration.

[0056] In some embodiments, the stabilized protein formulation comprises insulin or glucagon; poly(CBAA-2) or a pharmaceutically acceptable salt thereof; and one or more excipients; wherein at least 4 or more of the following features apply: a) the molecular weight, Mn, of poly(CBAA-2) is 1 kDa-10 kDa; b) the poly(CBAA-2) has 5-50 monomers; c) the concentration of poly(CBAA-2) is 5-15 mg / mL; d) the concentration of poly(CBAA-2) is 3- 10 mg / mL; e) the total amount of protein fibrillation in the formulation is less than 15% over a period of 10 days at 45 °C and pH 2, as measured by a ThT fluorescence assay; e) the amount of protein fibrillation in the formulation does not increase by more than 15% over a period of 10 days at 45 °C and pH 2, as measured by a ThT fluorescence assay; f) the time to half maximum protein fibrillation is greater 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay; and g) wherein the stabilized protein formulation is suitable for parenteral administration.

[0057] In some embodiments, the stabilized protein formulation comprises insulin or glucagon; poly(CBAA-2) or a pharmaceutically acceptable salt thereof; and one or more excipients; wherein at least 3 or more of the following features apply: a) the molecular weight, Mn, of poly(CBAA-2) is 1 kDa-10 kDa; b) the poly(CBAA-2) has 5-25 monomers; c) the concentration of poly(CBAA-2) is 4-10 mg / mL; d) the concentration of poly(CBAA-2) is 3-6 mg / mL; e) the total amount of protein fibrillation in the formulation is less than 10% over a period of 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay; e) the amount of protein fibrillation in the formulation does not increase by more than 10% over a period of 10 days at 45 °C and pH 2, as measured by a ThT fluorescence assay; f) the time to half maximum protein fibrillation is greater 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay; and g) wherein the stabilized protein formulation is suitable for parenteral administration.

[0058] In some embodiments, the stabilized protein formulation comprises insulin or glucagon; poly(CBAA-2) or a pharmaceutically acceptable salt thereof; and one or more excipients; wherein at least 4 or more of the following features apply: a) the molecular weight, Mn, of poly(CBAA-2) is 1 kDa-10 kDa; b) the poly(CBAA-2) has 5-25 monomers; c) the concentration of poly(CBAA-2) is 4-10 mg / mL; d) the concentration of poly(CBAA-2) is 3-6 mg / mL; e) the total amount of protein fibrillation in the formulation is less than 10% over aAttorney Docket No.: TAP.003 WO period of 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay; e) the amount of protein fibrillation in the formulation does not increase by more than 10% over a period of 10 days at 45 °C and pH 2, as measured by a ThT fluorescence assay; f) the time to half maximum protein fibrillation is greater 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay; and g) wherein the stabilized protein formulation is suitable for parenteral administration.C. Excipients

[0059] Certain formulations disclosed herein comprise a pharmaceutically acceptable excipient. Excipients are biocompatible, inactive ingredients added to the formulation to enhance stability, solubility, or delivery properties, for instance. Excipients include solvents, preservatives, buffers, isotonic agents, surfactants, or stabilizer (z.e., other stabilizing additives such as zinc or protamine.)

[0060] In some embodiments, the formulations disclosed herein are liquid formulations. In some embodiments, the formulations disclosed herein are lyophilized formulations comprising the polyzwitterion or a pharmaceutically acceptable salt thereof, and protein.II. Methods of Use

[0061] The formulations disclosed herein may be used for treating conditions that benefit from administration of the particular protein (e.g., insulin, glucagon, GLP-1 analogs, monoclonal antibodies) in the formulation.A. Conditions

[0062] In some embodiments, the individual has a condition that would benefit from blood glucose control. For instance, the individual in need thereof has diabetes (e.g., type 1, type 2, gestational). Alternatively, the individual is in need of treating or reducing the risk of hyperglycemia, or hypoglycemia.

[0063] Diabetes mellitus is a chronic condition characterized by the body's inability to produce enough or effectively use insulin. This leads to elevated blood glucose levels, which can cause a range of health problems. Insulin therapy is a cornerstone of diabetes management, providing a means to replace or supplement the body's natural insulin production with an external source of insulin. In some embodiments, the disclosure provides a method of treating diabetes in an individual in need thereof with a formulation disclosed herein.Attorney Docket No.: TAP.003 WO

[0064] Hypoglycemia, or low blood glucose, in an individual can be a serious condition if not treated promptly and can result in loss of consciousness, heart problems, brain damage, and impair judgment and physical coordination. In some embodiments, the disclosure provides a method of treating hypoglycemia with a formulation disclosed herein, wherein the formulation comprises the protein glucagon.

[0065] Hyperglycemia, or high blood sugar, can have long-term consequences if not managed properly. Hyperglycemia is associated with damage to blood vessels, which can lead to heart disease, stroke, kidney disease, retinopathy, and neuropathy. Uncontrolled hyperglycemia in people with type 1 diabetes can lead to diabetic ketoacidosis, which can be fatal. Chronic elevated glucose levels can constrict blood vessels, thus impairing wound healing. Hyperglycemia has also been associated with dysfunction of the immune response. In some embodiments, the disclosure provides a method of treating hyperglycemia with a formulation disclosed herein, wherein the formulation comprises the protein insulin.B. Administration

[0066] In some embodiments, liquid formulations disclosed herein are delivered to the individual in need thereof by parenteral administration. Examples of parenteral administration include subcutaneous, intramuscular, and intravenous injection. Solid, lyophilized formulations may be reconstituted by combining with a liquid solvent (e.g., saline, sterile water) and desired excipients.

[0067] The choice of delivery methods may depend on the particular therapy, release profile, and dosage requirements. For instance, insulin may be delivered in multiple daily injections (MDI), which involve injecting insulin several times a day, often before meals and at bedtime. Needles, syringes, and pen injectors (prefilled pens) may be used for injection. Alternatively, insulin may be delivered via continuous subcutaneous insulin infusion (CSII), wherein an insulin pump is used to deliver insulin continuously throughout the day.

[0068] Dosing regimens may depend on a variety of factors including age / weight / health of individual, type of insulin (rapid-acting, short-acting, intermediate acting, long acting), target blood glucose range, monitoring frequency, individual sensitivity to insulin, hypoglycemia risk, combination therapies, and delivery methods. Typical doses and dose ranges for the administration of insulin to control blood sugar levels can be determined by a medical care provider or any person having ordinary skill in the art. Furthermore, dosing mayAttorney Docket No.: TAP.003 WO be adjusted as needed depending on the physiological response to treatment. For instance, dosing of insulin may be adjusted based on results from blood glucose monitoring.

[0069] Individual” refers to a human or non-human mammal. Examples of non-human mammals include non-human primates, dog, cat, bovine, ovine, porcine, equine, mouse, rate, hamster, rabbit, or guinea pig.III. Miscellaneous

[0070] Where the context permits, singular or plural terms may also include the plural or singular term, respectively. As used herein, the terms “generally,” “substantially,” “about,” and similar terms are used as terms of approximation and not as terms of degree, and are intended to account for the inherent variations in measured or calculated values that would be recognized by those of ordinary skill in the art. As used herein, the term “about” is used synonymously with the term “approximately.” Illustratively, the use of the term “about” with regard to an amount indicates values slightly outside the cited values, e.g., plus or minus 10%, plus or minus 5%, plus or minus 2%, plus or minus 1%, plus or minus 0.5%, plus or minus 0.2%, or plus or minus 0.1%.

[0071] Moreover, unless the word “or” is expressly limited to mean only a single item exclusive from the other items in reference to a list of two or more items, then the use of “or” in such a list is to be interpreted as including (a) any single item in the list, (b) all of the items in the list, or (c) any combination of the items in the list.

[0072] Additionally, the term "comprising" is used throughout to mean including at least the recited feature(s) such that any greater number of the same feature and / or additional types of other features are not precluded.IV. Example Embodiments

[0073] Some of the embodiments of this disclosure relate to Embodiment I, as follows:

[0074] Embodiment 1-1. A stabilized protein formulation comprising: a protein; a polyzwitterion or a pharmaceutically acceptable salt thereof; and one or more pharmaceutically acceptable excipients.

[0075] Embodiment 1-2. The stabilized protein formulation of Embodiment 1-1, wherein the polyzwitterion is a poly(carboxybetaine).Attorney Docket No.: TAP.003 WO

[0076] Embodiment 1-3. The stabilized protein formulation of Embodiment 1-1 or 1-2, wherein the polyzwitterion is selected from the group consisting of poly(CBAA-2), poly(CBAA-l), poly(CBMA-l), and poly(CBMA-2).

[0077] Embodiment 1-4. The stabilized protein formulation of Embodiment 1-1 or 1-2, wherein the polyzwitterion is poly(CBAA-2).

[0078] Embodiment 1-5. The stabilized protein formulation of Embodiment 1-2, wherein the poly(carboxybetaine) is poly(carboxybetaine methacrylate).

[0079] Embodiment 1-6. The stabilized protein formulation of any one of Embodiments1-1 to 1-5, wherein the polyzwitterion is a homopolymer.

[0080] Embodiment 1-7. The stabilized protein formulation of any one of Embodiments1-1 to 1-5, wherein the polyzwitterion is a copolymer.

[0081] Embodiment 1-8. The stabilized protein formulation of Embodiment 1-7, wherein the copolymer comprises poly(carboxybetaine-co-butyl methacrylate).

[0082] Embodiment 1-9. The stabilized protein formulation of any one of Embodiments1-1 to 1-8, wherein the molecular weight, Mn, of the polyzwitterion is 400 Da-100 kDa, 400 Da-70 kDa, 400 Da-50 kDa, 400 Da-40 kDa, 400 Da-30kDa, 400 Da-20 kDa, 400 Da-10 kDa, 400 Da-5 kDa, 400 Da-3 kDa, 400 Da-2 kDa, or 400 Da-1 kDa 1 kDa-100 kDa, 1 kDa- 70 kDa, 1 kDa-50 kDa, 1 kDa-40 kDa, 1 kDa-30 kDa, 1 kDa-20 kDa, 1 kDa-10 kDa, 1 kDa- 5 kDa, lkDa-3 kDa, 1 kDa-2 kDa, 2 kDa-100 kDa, 2 kDa-70 kDa, 2 kDa-50 kDa, 2 kDa-40 kDa, 2 kDa-30 kDa, 2 kDa-20 kDa, 2 kDa-10 kDa, 2 kDa-5 kDa, or 2 kDa-3 kDa.

[0083] Embodiment 1-10. The stabilized protein formulation of any one of Embodiments 1-1 to 1-8, wherein the polyzwitterion comprises 5-50 monomers, 5-40 monomers, 5-30 monomers, 5-20 monomers, 5-10 monomers, 2-50 monomers, 2-40 monomers, 2-30 monomers, 2-20 monomers, 2-10 monomers, 2-5 monomers, or 7-15 monomers.

[0084] Embodiment 1-11. The stabilized protein formulation of any one of Embodiments 1-1 to 1-10, wherein the concentration of the polyzwitterion is 1-20 mg / mL, 1- 15 mg / mL, 1-10 mg / mL, 1-7.5 mg / mL, 1-5 mg / mL, 1-3 mg / mL, 2-20 mg / mL, 2-15 mg / mL,2-10 mg / mL, 2-7.5 mg / mL, 2-5 mg / mL, 2-3 mg / mL, 3-20 mg / mL, 3-15 mg / mL, 3-10 mg / mL, 3-7.5 mg / mL, 3-5 mg / mL, 5-20 mg / mL, 5-15 mg / mL, 5-10 mg / mL, or 5-7.5 mg / mL.Attorney Docket No.: TAP.003 WO

[0085] Embodiment 1-12. The stabilized protein formulation of any one of Embodiments 1-1 to I- 10, wherein the concentration of the polyzwitterion is about 20 mg / mL, about 15 mg / mL, about 10 mg / mL, about 7.5 mg / mL, about 5 mg / mL, about 4 mg / mL, about 3 mg / mL, about 2 mg / mL, or about 1 mg / mL.

[0086] Embodiment 1-13. The stabilized protein formulation of any one ofEmbodiments 1-1 to 1-12, wherein the protein is a hormone.

[0087] Embodiment 1-14. The stabilized protein formulation of any one ofEmbodiments 1-1 to 1-13, wherein the protein is selected from the group consisting of insulin, glucagon, a GLP-1 analog, and a monoclonal antibody.

[0088] Embodiment 1-15. The stabilized protein formulation of any one of Embodiments 1-1 to 1-13, wherein the protein is selected from the group consisting of insulin and glucagon.

[0089] Embodiment 1-16. The stabilized protein formulation of any one of Embodiments 1-1 to 1-15, wherein the protein is in an amount effective for the treatment of diabetes.

[0090] Embodiment 1-17. The stabilized protein formulation of any one of Embodiments 1-1 to 1-16, wherein the concentration of the protein is 1-5 mg / mL, 5-10 mg / mL, 10-15 mg / mL, 15-20 mg / mL, 20-25 mg / mL, 3-5 mg / mL, 3-7 mg / mL, 15-18 mg / mL, or 16- 18 mg / mL.

[0091] Embodiment 1-18. The stabilized protein formulation of any one of Embodiments 1-1 to 1-17, wherein the one or more pharmaceutically acceptable excipient is selected from the group consisting of solvent, preservative, buffer, surfactant, isotonic agent, and stabilizer.

[0092] Embodiment 1-19. The stabilized protein formulation of any one of Embodiments 1-1 to 1-18, wherein the amount or rate of protein fibrillation in the formulation is reduced compared to an analogous formulation without a polyzwitterion.

[0093] Embodiment 1-20. The stabilized protein formulation of any one of Embodiments 1-1 to 1-19, wherein the total amount of protein fibrillation in the formulation is less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay.Attorney Docket No.: TAP.003 WO

[0094] Embodiment 1-21. The stabilized protein formulation of any one of Embodiments 1-1 to 1-19, wherein the total amount of protein fibrillation in the formulation is less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 7 days at 45°C and pH 2, as measured by a ThT fluorescence assay.

[0095] Embodiment 1-22. The stabilized protein formulation of any one of Embodiments 1-1 to 1-19, wherein the amount of protein fibrillation in the formulation does not increase by more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay.

[0096] Embodiment 1-23. The stabilized protein formulation of any one of Embodiments 1-1 to 1-19, wherein the amount of protein fibrillation in the formulation does not increase by more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 7 days at 45°C and pH 2, as measured by a ThT fluorescence assay.

[0097] Embodiment 1-24. The stabilized protein formulation of any one of Embodiments 1-1 to 1-23, wherein the time to half maximum protein fibrillation is greater than 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, at 45°C and pH 2, as measured by a ThT fluorescence assay.

[0098] Embodiment 1-25. The stabilized protein formulation of any one of Embodiments 1-1 to 1-24, wherein the stabilized protein formulation is suitable for parenteral administration.

[0099] Embodiment 1-26. The stabilized protein formulation of any one of Embodiments 1-1 to 1-25, wherein the stabilized protein formulation is suitable for subcutaneous, intradermal, intramuscular, or intravenous injection.

[0100] Embodiment 1-27. The stabilized protein formulation of any one of Embodiments 1-1 to 1-26, wherein the stabilized protein formulation is a liquid formulation.

[0101] Embodiment 1-28. A stabilized protein formulation comprising a protein and a polyzwitterion or a pharmaceutically acceptable salt thereof, wherein the stabilized protein formulation is a lyophilized formulation.

[0102] Embodiment 1-29. The stabilized protein formulation of Embodiment 1-28, wherein the polyzwitterion is a poly(carboxybetaine).Attorney Docket No.: TAP.003 WO

[0103] Embodiment 1-30. The stabilized protein formulation of Embodiment 1-28 or I-29, wherein the polyzwitterion is selected from the group consisting of poly(CBAA-2), poly(CBAA-l), poly(CBMA-l), and poly(CBMA-2).

[0104] Embodiment 1-31. The stabilized protein formulation of Embodiment 1-28 or I-29, wherein the polyzwitterion is poly(CBAA-2).

[0105] Embodiment 1-32. The stabilized protein formulation of any one ofEmbodiments 1-28 to 1-31, wherein the polyzwitterion is a homopolymer.

[0106] Embodiment 1-33. The stabilized protein formulation of any one ofEmbodiments 1-28 to 1-31, wherein the polyzwitterion is a copolymer.

[0107] Embodiment 1-34. The stabilized protein formulation of Embodiment 1-33, wherein the copolymer comprises poly(carboxybetaine-co-butyl methacrylate).

[0108] Embodiment 1-35. The stabilized protein formulation of any one of Embodiments 1-28 to 1-34, wherein the molecular weight (Mn) of the polyzwitterion is 400 Da-100 kDa, 400 Da-70 kDa, 400 Da-50 kDa, 400 Da-40 kDa, 400 Da-30kDa, 400 Da-20 kDa, 400 Da-10 kDa, 400 Da-5 kDa, 400 Da-3 kDa, 400 Da-2 kDa, or 400 Da-1 kDa, 1 kDa- 100 kDa, 1 kDa-70 kDa, 1 kDa-50 kDa, 1 kDa-40 kDa, 1 kDa-30 kDa, 1 kDa-20 kDa, 1 kDa-10 kDa, 1 kDa-5 kDa, lkDa-3 kDa, 1 kDa-2 kDa, 2 kDa-100 kDa, 2 kDa-70 kDa, 2 kDa-50 kDa, 2 kDa-40 kDa, 2 kDa-30 kDa, 2 kDa-20 kDa, 2 kDa-10 kDa, 2 kDa-5 kDa, or 2 kDa-3 kDa.

[0109] Embodiment 1-36. The stabilized protein formulation of any one of Embodiments 1-28 to 1-34, wherein the polyzwitterion comprises 5-50 monomers, 5-40 monomers, 5-30 monomers, 5-20 monomers, 5-10 monomers, 2-50 monomers, 2-40 monomers, 2-30 monomers, 2-20 monomers, 2-10 monomers, 2-5 monomers, or 7-15 monomers.

[0110] Embodiment 1-37. The stabilized protein formulation of any one ofEmbodiments 1-28 to 1-36, wherein the protein is a hormone.

[0111] Embodiment 1-38. The stabilized protein formulation of any one ofEmbodiments 1-28 to 1-37, wherein the protein is selected from the group consisting of insulin, glucagon, a GLP-1 analog, and a monoclonal antibody.Attorney Docket No.: TAP.003 WO

[0112] Embodiment 1-39. The stabilized protein formulation of any one of Embodiments 1-28 to 1-37, wherein the protein is selected from the group consisting of insulin and glucagon.

[0113] Embodiment 1-40. The stabilized protein formulation of any one of Embodiments 1-28 to 1-39, wherein the protein is in an amount effective for the treatment of diabetes.

[0114] Embodiment 1-41. A method of regulating blood glucose in an individual in need thereof comprising: administering a stabilized protein formulation of any one of Embodiments 1-1 to 1-40 to an individual in need thereof.

[0115] Embodiment 1-42. The method of Embodiment 1-41, wherein the stabilized protein formulation is parenterally administered.

[0116] Embodiment 1-43. The method of Embodiment 1-41 or 1-42, wherein the method comprises injecting the stabilized protein formulation into the individual in need thereof, and the injection is subcutaneous, intradermal, intramuscular, or intravenous.

[0117] Embodiment 1-44. The method of any one of Embodiments 1-41 to 1-43, wherein insulin fibrillation in the individual in need thereof is reduced compared to administration of an insulin formulation without a polyzwitterion.

[0118] Embodiment 1-45. The method of any one of Embodiments 1-41 to 1-44, wherein the individual is at risk of hypoglycemia.

[0119] Embodiment 1-46. The method of any one of Embodiments 1-41 to 1-45, wherein the individual is at risk of hyperglycemia.

[0120] Embodiment 1-47. The method of any one of Embodiments 1-41 to 1-46, wherein the individual in need thereof is diabetic.

[0121] Embodiment 1-48. The method of any one of Embodiments 1-41 to 1-47, wherein the protein is insulin.

[0122] Embodiment 1-49. The method of any one of Embodiments 1-41 to 1-48, wherein the protein is glucagon.

[0123] Embodiment 1-50. The stabilized protein formulation of any one of Embodiments 1-1 to 1-40 for use in regulating blood glucose levels in an individual in need thereof.Attorney Docket No.: TAP.003 WO

[0124] Embodiment 1-51. The stabilized protein formulation of any one of Embodiments 1-1 to 1-13, 1-15 to 1-38, and 1-40 for use in reducing the risk of hypoglycemia in an individual in need thereof.

[0125] Embodiment 1-52. The stabilized protein formulation of any one of Embodiments 1-1 to 1-14 and 1-16 to 1-40 for use in reducing the risk of hyperglycemia in an individual in need thereof.

[0126] Embodiment 1-53. A kit for the administration of insulin, comprising: the stabilized protein formulation of any one of Embodiments 1-1 to 1-40; a means for parenterally administering the stabilized protein formulation; and instructions for the administration of the stabilized protein formulation to an individual in need thereof.V. Examples

[0127] The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.A. Example 1 : Monomer Synthesis

[0128] This example describes the preparation of two carboxybetaine monomers: CBAA-1 and CBAA-2.1. CBAA-2 Monomer Synthesis

[0129] 3-((3-acrylamidopropyl)dimethylammonio)propanoate (CBAA-2) monomer was synthesized by reacting N-(3-dimethylaminopropyl) acrylamide with P-propiolactone. [3- Propiolactone (5.76 g, 80 mmol) was dissolved in 40 mL of anhydrous acetone and added dropwise to a solution of N-(3 -dimethylaminopropyl) acrylamide (7.80 g, 50 mmol) dissolved in 100 mL of anhydrous acetone. The reaction mixture was stirred under argon at 15 °C for 5 h and formed a white precipitate. The precipitate was washed with 200 mL anhydrous acetone followed by 150 mL anhydrous ether. The product was dried under vacuum to obtain the final CBAA-2 monomer product (white powder, 98% yield). The chemical structure was confirmed by H NMR spectroscopy. The monomer was stored at 2-8°C before polymerization.Attorney Docket No.: TAP.003 WO2. CBAA-1 Monomer Synthesis

[0130] 2-((3-acrylamidopropyl)dimethylammonio)acetate (CBAA-1) monomer was synthesized via a two-step reaction. First, N,N-dimethylaminopropyl acrylamide (10 g) was reacted with ethyl bromoacetate (14 g) in acetonitrile (50 mL) at 60°C for 12 h. Upon reaction completion, the acetonitrile was removed via rotary evaporator, and the product, 3-acrylamido- N-(2-ethoxy-2-oxoethyl)-N,N-dimethylpropan-l-aminium bromide, was precipitated with diethyl ether (100 mL) at quantitative yield. The product was washed twice with acetone, and then hydrolyzed in aqueous solution using Amberlite anion exchange resin (OH- form) to get CBAA-1 monomer (white powder, 97% yield). The chemical structure was confirmed by 'H NMR spectroscopy.B. Example 2: Zwitterionic Polymer Synthesis

[0131] This example describes the synthesis of various carboxybetaine and sulfobetaine zwitterionic homopolymers having target molecular weights of 10 kDa and 20 kDa. In addition to the SBMA, CBAA-1, and CBAA-2 homopolymers, copolymers with butyl methacrylate (BuMA) are also described.

[0132] Sulfobetaine methacrylate (SBMA) and butyl methacrylate (BuMA) monomers were purchased from MilliporeSigma and used as received. CBAA-1 and CBAA-2 monomers were synthesized as described in Example 1. A total of 15 polymers and copolymers were prepared as listed in the following five polymer groups P1-P5, shown in Table 2.

[0133] Table 2.Pl: 10 kDa Homopolymers: poly(SBMA), poly(CBAA-l), poly(CBAA-2)20 kDa Homopolymers: poly(SBMA), poly(CBAA-l), poly(CBAA-2)10 kDa Copolymers with 10% BuMA: poly(SBMA-co-BuMA), poly(CBAA-l-co-BuMA), poly(CBAA-2-co-BuMA)10 kDa Copolymers with 30% BuMA: poly(SBMA-co-BuMA), poly(CBAA-l-co-BuMA), poly(CBAA-2-co-BuMA)20k Da Copolymers with 30% BuMA: poly(SBMA-co-BuMA), poly(CBAA-l-co-BuMA), poly(CBAA-2-co-BuMA)Attorney Docket No.: TAP.003 WO1. Synthesis of homopolymers CBAA-L CBAA-2, SBMA (Groups Pl and P2)[0134J Reversible addition fragmentation transfer (RAFT) homopolymerizations of each CBAA-1, CBAA-2 and SBMA monomers were conducted in the presence of chain transfer agent (CTA) 4-((((2-Carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid. 4.6 mmol of monomer (SBMA, CBAA-1, or CBAA-2) was dissolved in 10 mL acetic buffer, with initiator V-501 (4,4-azobis(4-cyanopentanoic acid)). The final [monomer]: [initiator]: [CTA] molar ratios in the reactions were set as shown in Table 3.

[0135] Table 3.

[0136] All reactions were conducted under argon after degassing and stirred at 70°C for 12 h. Polymerization was stopped via rapid cooling and exposure to air. Polymers were purified by dialysis against deionized water for 72 h in 3500 MW cutoff dialysis tubing (Repligen SpectraPor 3). Purified polymers were then lyophilized (Labconco FreeZone).2. Synthesis of copolymers CBAA-l / BuMA, CBAA-2 / BuMA, SBMA / BuMA (Groups P3, P4, and P5)

[0137] Copolymers comprising a zwitterionic polymer (e.g., CBAA-1, CBAA-2 or SBMA) and 10 or 30% of a non-zwitterionic, hydrophobic polymer (e.g., poly(butyl methacrylate) (BuMA)) were prepared. Reversible addition fragmentation transfer (RAFT) copolymerizations of the zwitterionic monomers with butyl methacrylate were conducted in the presence of chain transfer agent (CTA) 4-((((2-Carboxyethyl)thio)carbonothioyl)thio)-4- cyanopentanoic acid. A total of 4.6 mmol of monomer (SBMA, CBAA-1, or CBAA-2 with BuMA) was dissolved in 10 mL mixed solvent (1: 1 ratio of acetic buffer and methanol) and combined with initiator V-501 (4,4-azobis(4-cyanopentanoic acid)). The final [monomer]: [initiator]: [CTA] molar ratios in the reactions were set as shown in Table 4.Attorney Docket No.: TAP.003 WO

[0138] Table 4.

[0139] All reactions were conducted under argon after degassing and stirred at 70°C for 12 h. Polymerization was stopped via rapid cooling and exposure to air. Polymers were purified by dialysis against deionized water for 72 h in 3500 MW cutoff dialysis tubing (Repligen SpectraPor 3). Purified polymers were then lyophilized (Labconco FreeZone).3. Molecular Weight Analysis of Synthesized Polymers

[0140] The molecular weight of the lyophilized polymers was characterized via aqueous SEC-MALS (Agilent 1260 Infinity, Wyatt dRI and MALS detectors) with a Waters UltraHydrogel 2000 column. Table 5 summarizes the molecular weight (Mn) and poly dispersity (Mw / Mn) of the prepared homo- and copolymers.

[0141] Table 5.C. Example 3: Fibrillation Measurements with ThT Fluorescence Assay

[0142] This example describes the use of a ThT Fluorescence Assay to measure protein fibrillation over an 8- to 10-day period at 45°C.Attorney Docket No.: TAP.003 WO1. Procedure for Measuring Fibrillation

[0143] A 2 mg / mL solution of recombinant human insulin (Roche Cat. No. 11376497001) was prepared in 0.01 HC1, 140 mM NaCl buffer to pH 2.0. Insulin exists as a monomeric form in these conditions. Polymer stock solutions (50 mg / mL) and thioflavin T (ThT, Sigma Aldrich T3516) were prepared separately in the same buffer. Volumes of stock solutions were calculated to arrive at final concentrations of 100 mM (0.58 mg / mL) insulin, 7.2 pM ThT, and polymer concentrations between 0.5% and 1.5% w / v (5 mg / mL, 7.5 mg / mL, 10 mg / mL, and 15 mg / mL) in each well of a 96-well microplate (Coming, black walls with transparent bottom), to a total volume of 200 pL per well. The plates were sealed with transparent film and placed in a BioTek Synergy HIM monochromator-based plate reader preset to 45°C incubation temperature. Each plate contained SBMA-based, CBAA-1 based, and CBAA-2 based formulations of all polymer designs (P1-P5) in addition to polymer-free controls. Fibrillation experiments were conducted through 200-228 h (8-10 days) continuously, in situ in the plate reader at a constant 45°C temperature, with readings of the entire plate taken every 20 minutes. The fluorescence gain, read height, excitation / emission wavelengths (Ex = 450 nm, Em = 484 nm), and all other measurement parameters were fixed for all wells in all experiments to allow direct comparison. All samples were prepared at the same time just prior to each experiment using identical methods. Two polymer concentrations per sample were tested in each plate, with n > 3 replicates of each formulation / concentration pair.

[0144] Kinetics data was fit to the following function in DataGraph 4 (Visual Data Tools):

[0146] Wherein, F is the detected fluorescence, yo is the fluorescence at time 0, yf is the final fluorescence, K is the apparent rate constant for the growth of the fibrils corresponding to 1 / t, and xo is the time at which fluorescence reaches half its maximum value.

[0147] Insulin with no polymer additive (control) reached xo (time to half maximum fluorescence) at 20 h ± 2 h, which was repeatable over multiple runs and set to 1.0 in the normalized xo calculations. The maximum fluorescence yf reached by the same control samples was set to 100% in the normalized maximum fluorescence calculations.Attorney Docket No.: TAP.003 WO2. Performance Measured by Time to Half Maximum Fluorescence (xo) and Maximum Final Fluorescence

[0148] The key metrics used to compare the stabilizing effect of various polymers were (1) time to half maximum fluorescence, xo, normalized to control, and (2) maximum final fluorescence, yr, reached by a formulation over the entire fibrillation kinetics run, normalized to control. Those two values x and yr indicate the delay and suppression of protein fibrillation, respectively. The n > replicates of each condition were modeled separately, and the x and yr values calculated for each replicate were averaged to give the reported values.

[0149] In interpreting the results, for example, a reported xp of 3.0 indicates that the time to half maximum fluorescence was delayed by a factor of 3 (occurring at 60 h) relative to the control (occurring at 20 h). If no fibrillation was observed for a sample over the full experiment, x could not be quantified and was reported as ‘>10’. Note that while half-max values of ‘>10’ indicate no fibril accumulation observed over the experiment, the relative performance between these ‘>10’ polymers in delaying fibrillation cannot necessarily be ranked until longer-term studies are conducted (e.g., any relative xp >10 in the currently reported data may be found to be xp=12, xp=40, etc. in a sufficiently long future study). In general, a higher x is desirable because it indicates a longer delay for half the protein to undergo fibrillation compared to a protein formulation without a polymer.

[0150] The normalized maximum final fluorescence (yf) results are critical data, as they indicate the maximum amount of protein fibrillation compared to a formulation without a zwitterionic polymer. A relative yr reported as <0.1% indicates no significant fibrillation occurred over the complete study and is considered the highest performance tier in the current data. In other words, the presence of the zwitterionic polymer results in substantially complete suppression of protein fibrillation over the experimental period. Thus, a lower yf value is desirable and indicates reduced protein fibrillation correlating to increased suppression of protein fibrillation as a result of the polymer in the formulation. In general, total suppression of yf is seen as the most desirable effect of the polymer additive regardless of xo. In comparison, a sample showing a nominal delay to fibril accumulation (e.g., xo = 2) but high eventual total fibrillation (e.g., 80% of the control) is much less desirable in real-world applications of this technology, as fibrillation is primarily delayed rather than suppressed.Attorney Docket No.: TAP.003 WO3. Discussion of Experimental Results

[0151] Table 6 summarizes the ThT fluorescence kinetics of insulin fibrillation, specifically (1) the time to half maximum fluorescence, xo, normalized to control, and (2) maximum final fluorescence, normalized to control. Each of the SBMA based, CBAA-1 based, and CBAA-2 based polymers of each of the five polymer groups, P1-P5, were evaluated at various concentrations (5 mg / mL, 7.5 mg / mL, 10 mg / mL, and 15 mg / mL).

[0152] Table 6. Summary of ThT fluorescence kinetics of fibrillation.

[0153] Shaded cells indicate no significant fibrillation above baseline observed during full experiment duration (200 h).

[0154] [*] indicates that fibrillation had started in all replicates and was in early exponential growth at the end of the experiment.Attorney Docket No.: TAP.003 WO

[0155] “N / A” indicates insufficient replicates to make fair comparisons to other conditions.

[0156] Overall, for sulfobetaine-based polymers, the greatest stabilization was seen for SB-P1, SB-P4, and SB-P5 samples at the highest concentration tested. At 15 mg / mL, these polymers delayed the exponential growth phase of fibrillation until just prior to the end of the experiment (200 h), so maximum fibrillation (yf) could not be calculated. For all other SBbased samples, yr was between 14% and 85% of the control, and exponential fibril growth had finished.

[0157] Surprisingly, analogous CBAA-2-based polymers worked significantly better in both delaying and suppressing fibrillation than the other zwitterionic polymers at the conditions and compositions tested. All CBAA-2 -based polymers in groups P1-P5 delayed xo beyond 10X of control at 7.5 mg / mL or greater (i.e., xo could not be calculated because fibrillation was delayed past the full duration of the experiment). In addition, of the 20 total CBAA-2 conditions tested, 16 conditions kept the maximum fluorescence yr to 0.4% or less compared to control over the full experiment, showing no significant increase from baseline yo. A representative fluorescence plot of 15 mg / mL P2 poly(CBAA-2) and P2 poly(SBMA) compared to control is provided in Fig. 1.

[0158] Notably, we observed that CBAA-2-based P3 (10 kDa, 10% BuMA) completely suppressed fibrillation at 5 mg / mL. A representative fluorescence plot of 5 mg / mL of P3 poly(CBAA2) and P3 poly(SBMA) compared to control is provided in Fig. 2.

[0159] The insulin analog insulin glulisine was also evaluated for fibrillation with and without polymer additives using an equivalent ThT fluorescence protocol. We observed that CBAA-2-based P3 (10 kDa, 10% BuMA) completely suppressed fibrillation at 5 mg / mL. A representative fluorescence plot of 5 mg / mL of P3 poly(CBAA2) compared to control is provided in Fig. 3.D. Example 4: Assessing the Efficacy of the Formulation

[0160] This example describes the use of an FDA-validated in vitro insulin potency assay to evaluate the biological activity of insulin-excipient formulations.

[0161] Several bioassays have been described to accurately assess the biological activity of insulin formulations in vitro. For instance, a validated cell-based insulin potency assay, modified from a similar test method described in USP <121> has been described in theAttorney Docket No.: TAP.003 WO literature. (Methods Protoc. 2023, 6, 33) This in-cell western assay enables quantification of tyrosine auto-phosphorylation in insulin receptors, which is induced by insulin binding. In this example, this assay was used to evaluate the biological activity of selected insulin-excipient formulations.

[0162] The molecular basis of this assay is the biological effect of insulin when it binds to the insulin receptor. When insulin or an insulin analogue in a test formulation binds to insulin receptors in the cultured cells, it triggers the auto-phosphorylation of tyrosine residues of these receptors. The phospho-tyrosine residues are then recognized by an anti -phospho-tyrosine primary antibody (e.g., Upstate clone 4G10, Cat. No. 05-321), followed by a secondary antibody tagged with a fluorescent dye (e.g., Invitrogen #A28175, Goat anti-Mouse IgG-Alexa Fluor 488). DNA intercalating stain (Hoechst) is used to simultaneously normalize the results to the cell population. A commercially available CHO-K1 cell line stably over-expressing human insulin receptor is recommended (CHO INSR 1284, ATCC® CRL-3307™), though other cell lines expressing human insulin receptors can be used.

[0163] Procedure: In this study, cells (CHO INSR 1284, ATCC® CRL-3307) were cultured in complete medium with 10% FBS in a 37°C, 5% CO2 incubator. Several representative insulin-excipient formulations of interest were diluted to 0.34 U / mL insulin with 5 mg / mL excipient concentrations. When cells reached confluency in a 96-well assay plate, insulin test dilutions and standards were added to the cells and incubated for 20 min. Cells were then fixed, washed, and treated with primary and secondary antibodies (1 : 1000 dilutions) with and overnight wash between antibody treatments and Hoechst solution applied with the secondary antibody for normalization. After a final wash, detection was performed with a fluorescence plate reader (Ex=493nm, Em=519nm for Alexa Fluor 488, Ex=361nm, Em=486nm for Hoechst 33342). Relative potency of the test formulations was compared to USP reference standards used as controls, with insulin-free cultures used as negative controls.

[0164] Results: The results are summarized in Fig 4. No statistical difference between the activity of excipient-free and excipient-containing insulin was observed for any of the formulations tested, including Pl-pCBAAl, Pl-pCBAA2, P2-pCBAA2, and P3-pCBAA2, all at 5 mg / mL, indicating stabilization with tested excipients did not harm the biological activity of insulin.Attorney Docket No.: TAP.003 WOVI. Conclusion

[0165] While embodiments of the present invention have been shown and described herein, those skilled in the art will understand that such embodiments are provided by way of example only. The descriptions of embodiments of the technology are not intended to be exhaustive or to limit the technology to the precise form disclosed above. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. For example, while steps are presented in a given order, alternative embodiments may perform steps in a different order. The various embodiments described herein may also be combined to provide further embodiments. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0166] It will also be appreciated that specific embodiments have been described herein for purposes of illustration, but that various modifications may be made without deviating from the technology. Further, while advantages associated with certain embodiments of the technology have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the technology. Accordingly, the present disclosure and associated technology can encompass other embodiments not expressly shown or described herein.

Claims

Attorney Docket No.: TAP.003 WOCLAIMSI / We claim:

1. A stabilized protein formulation comprising: a protein; a polyzwitterion or a pharmaceutically acceptable salt thereof; and one or more pharmaceutically acceptable excipients.

2. The stabilized protein formulation of claim 1, wherein the polyzwitterion is a poly(carboxybetaine).

3. The stabilized protein formulation of claim 1 or 2, wherein the polyzwitterion is selected from the group consisting of poly(CBAA-2), poly(CBAA-l), poly(CBMA-l), and poly(CBMA-2).

4. The stabilized protein formulation of claim 1 or 2, wherein the polyzwitterion is poly(CBAA-2).

5. The stabilized protein formulation of claim 2, wherein the poly(carboxybetaine) is poly(carboxybetaine methacrylate).

6. The stabilized protein formulation of any one of claims 1-5, wherein the polyzwitterion is a homopolymer.

7. The stabilized protein formulation of any one of claims 1-5, wherein the polyzwitterion is a copolymer.

8. The stabilized protein formulation of claim 7, wherein the copolymer comprises poly(carboxybetaine-co-butyl methacrylate).

9. The stabilized protein formulation of any one of claims 1-8, wherein the molecular weight, Mn, of the polyzwitterion is 400 Da-100 kDa, 400 Da-70 kDa, 400Attorney Docket No.: TAP.003 WODa-50 kDa, 400 Da-40 kDa, 400 Da-30kDa, 400 Da-20 kDa, 400 Da-10 kDa, 400 Da-5 kDa, 400 Da-3 kDa, 400 Da-2 kDa, or 400 Da-1 kDa 1 kDa-100 kDa, 1 kDa-70 kDa, 1 kDa-50 kDa, 1 kDa-40 kDa, 1 kDa-30 kDa, 1 kDa-20 kDa, 1 kDa-10 kDa, 1 kDa-5 kDa, lkDa-3 kDa, 1 kDa-2 kDa, 2 kDa-100 kDa, 2 kDa-70 kDa, 2 kDa-50 kDa, 2 kDa- 40 kDa, 2 kDa-30 kDa, 2 kDa-20 kDa, 2 kDa-10 kDa, 2 kDa-5 kDa, or 2 kDa-3 kDa.

10. The stabilized protein formulation of any one of claims 1-8, wherein the polyzwitterion comprises 5-50 monomers, 5-40 monomers, 5-30 monomers, 5-20 monomers, 5-10 monomers, 2-50 monomers, 2-40 monomers, 2-30 monomers, 2-20 monomers, 2-10 monomers, 2-5 monomers, or 7-15 monomers.

11. The stabilized protein formulation of any one of claims 1-10, wherein the concentration of the polyzwitterion is 1-20 mg / mL, 1-15 mg / mL, 1-10 mg / mL, 1-7.5 mg / mL, 1-5 mg / mL, 1-3 mg / mL, 2-20 mg / mL, 2-15 mg / mL, 2-10 mg / mL, 2-7.5 mg / mL, 2-5 mg / mL, 2-3 mg / mL, 3-20 mg / mL, 3-15 mg / mL, 3-10 mg / mL, 3-7.5 mg / mL, 3-5 mg / mL, 5-20 mg / mL, 5-15 mg / mL, 5-10 mg / mL, or 5-7.5 mg / mL.

12. The stabilized protein formulation of any one of claims 1-10, wherein the concentration of the polyzwitterion is about 20 mg / mL, about 15 mg / mL, about 10 mg / mL, about 7.5 mg / mL, about 5 mg / mL, about 4 mg / mL, about 3 mg / mL, about 2 mg / mL, or about 1 mg / mL.

13. The stabilized protein formulation of any one of claims 1-12, wherein the protein is a hormone.

14. The stabilized protein formulation of any one of claims 1-13, wherein the protein is selected from the group consisting of insulin, glucagon, a GLP-1 analog, and a monoclonal antibody.

15. The stabilized protein formulation of any one of claims 1-13, wherein the protein is selected from the group consisting of insulin and glucagon.Attorney Docket No.: TAP.003 WO16. The stabilized protein formulation of any one of claims 1-15, wherein the protein is in an amount effective for the treatment of diabetes.

17. The stabilized protein formulation of any one of claims 1-16, wherein the concentration of the protein is 1-5 mg / mL, 5-10 mg / mL, 10-15 mg / mL, 15-20 mg / mL, 20-25 mg / mL, 3-5 mg / mL, 3-7 mg / mL, 15-18 mg / mL, or 16-18 mg / mL.

18. The stabilized protein formulation of any one of claims 1-17, wherein the one or more pharmaceutically acceptable excipient is selected from the group consisting of solvent, preservative, buffer, surfactant, isotonic agent, and stabilizer.

19. The stabilized protein formulation of any one of claims 1-18, wherein the amount or rate of protein fibrillation in the formulation is reduced compared to an analogous formulation without a polyzwitterion.

20. The stabilized protein formulation of any one of claims 1-19, wherein the total amount of protein fibrillation in the formulation is less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 10 days at 45°C and pH 2, as measured by a ThT fluorescence assay.

21. The stabilized protein formulation of any one of claims 1-19, wherein the total amount of protein fibrillation in the formulation is less than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 7 days at 45°C and pH 2, as measured by a ThT fluorescence assay.

22. The stabilized protein formulation of any one of claims 1-19, wherein the amount of protein fibrillation in the formulation does not increase by more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 10 days at 45 °C and pH 2, as measured by a ThT fluorescence assay.

23. The stabilized protein formulation of any one of claims 1-19, wherein the amount of protein fibrillation in the formulation does not increase by more than 50%,Attorney Docket No.: TAP.003 WO40%, 30%, 20%, 15%, 10%, 5%, or 2% over a period of 7 days at 45 °C and pH 2, as measured by a ThT fluorescence assay.

24. The stabilized protein formulation of any one of claims 1-23, wherein the time to half maximum protein fibrillation is greater than 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, at 45 °C and pH 2, as measured by a ThT fluorescence assay.

25. The stabilized protein formulation of any one of claims 1-24, wherein the stabilized protein formulation is suitable for parenteral administration.

26. The stabilized protein formulation of any one of claims 1-25, wherein the stabilized protein formulation is suitable for subcutaneous, intradermal, intramuscular, or intravenous injection.

27. The stabilized protein formulation of any one of claims 1-26, wherein the stabilized protein formulation is a liquid formulation.

28. A stabilized protein formulation comprising: a protein; and a polyzwitterion or a pharmaceutically acceptable salt thereof; wherein the stabilized protein formulation is a lyophilized formulation.

29. The stabilized protein formulation of claim 28, wherein the polyzwitterion is a poly(carboxybetaine).

30. The stabilized protein formulation of claim 28 or 29, wherein the polyzwitterion is selected from the group consisting of poly(CBAA-2), poly(CBAA-l), poly(CBMA-l), and poly(CBMA-2).

31. The stabilized protein formulation of claim 28 or 29, wherein the polyzwitterion is poly(CBAA-2).Attorney Docket No.: TAP.003 WO32. The stabilized protein formulation of any one of claims 28-31, wherein the polyzwitterion is a homopolymer.

33. The stabilized protein formulation of any one of claims 28-31, wherein the polyzwitterion is a copolymer.

34. The stabilized protein formulation of claim 33, wherein the copolymer comprises poly(carboxybetaine-co-butyl methacrylate).

35. The stabilized protein formulation of any one of claims 28-34, wherein the molecular weight (Mn) of the polyzwitterion is 400 Da-100 kDa, 400 Da-70 kDa, 400 Da-50 kDa, 400 Da-40 kDa, 400 Da-30kDa, 400 Da-20 kDa, 400 Da-10 kDa, 400 Da-5 kDa, 400 Da-3 kDa, 400 Da-2 kDa, or 400 Da-1 kDa, 1 kDa-100 kDa, 1 kDa-70 kDa, 1 kDa-50 kDa, 1 kDa-40 kDa, 1 kDa-30 kDa, 1 kDa-20 kDa, 1 kDa-10 kDa, 1 kDa-5 kDa, lkDa-3 kDa, 1 kDa-2 kDa, 2 kDa-100 kDa, 2 kDa-70 kDa, 2 kDa-50 kDa, 2 kDa- 40 kDa, 2 kDa-30 kDa, 2 kDa-20 kDa, 2 kDa-10 kDa, 2 kDa-5 kDa, or 2 kDa-3 kDa.

36. The stabilized protein formulation of any one of claims 28-34, wherein the polyzwitterion comprises 5-50 monomers, 5-40 monomers, 5-30 monomers, 5-20 monomers, 5-10 monomers, 2-50 monomers, 2-40 monomers, 2-30 monomers, 2-20 monomers, 2-10 monomers, 2-5 monomers, or 7-15 monomers.

37. The stabilized protein formulation of any one of claims 28-36, wherein the protein is a hormone.

38. The stabilized protein formulation of any one of claims 28-37, wherein the protein is selected from the group consisting of insulin, glucagon, a GLP-1 analog, and a monoclonal antibody.

39. The stabilized protein formulation of any one of claims 28-37, wherein the protein is selected from the group consisting of insulin and glucagon.Attorney Docket No.: TAP.003 WO40. The stabilized protein formulation of any one of claims 28-39, wherein the protein is in an amount effective for the treatment of diabetes.

41. A method of regulating blood glucose in an individual in need thereof comprising: administering a stabilized protein formulation of any one of claims 1-40 to an individual in need thereof.

42. The method of claim 41, wherein the stabilized protein formulation is parenterally administered.

43. The method of claim 41 or 42, wherein the method comprises injecting the stabilized protein formulation into the individual in need thereof, and the injection is subcutaneous, intradermal, intramuscular, or intravenous.

44. The method of any one of claims 41-43, wherein insulin fibrillation in the individual in need thereof is reduced compared to administration of an insulin formulation without a polyzwitterion.

45. The method of any one of claims 41-44, wherein the individual is at risk of hypoglycemia.

46. The method of any one of claims 41-45, wherein the individual is at risk of hyperglycemia.

47. The method of any one of claims 41-46, wherein the individual in need thereof is diabetic.

48. The method of any one of claims 41-47, wherein the protein is insulin.

49. The method of any one of claims 41-48, wherein the protein is glucagon.Attorney Docket No.: TAP.003 WO50. The stabilized protein formulation of any one of claims 1-40 for use in regulating blood glucose levels in an individual in need thereof.

51. The stabilized protein formulation of any one of claims 1-40 for use in reducing the risk of hypoglycemia in an individual in need thereof.

52. The stabilized protein formulation of any one of claims 1-40 for use in reducing the risk of hyperglycemia in an individual in need thereof.

53. A kit for the administration of insulin, comprising: the stabilized protein formulation of any one of claims 1-40; a means for parenterally administering the stabilized protein formulation; and instructions for the administration of the stabilized protein formulation to an individual in need thereof.

Images