KR20220122993A - Excipient compounds for biopolymer formulations - Google Patents

Abstract

Disclosed herein are viscosity reducing liquid formulations comprising a protein and an excipient compound. Further disclosed are methods of reducing the viscosity of liquid formulations comprising proteins, methods of treatment, and methods of improving protein processing.

Description

Excipient Compounds for Biopolymer Formulations

Related applications

This application claims the benefit of U.S. Provisional Patent Application No. 62/940,683, filed on November 26, 2019. The entire contents of this application are incorporated herein by reference.

field of application

[0001] This application relates generally to formulations for delivering biopolymers.

[0002] Biopolymers can be used for therapeutic or non-therapeutic purposes. Biopolymer-based therapeutics, such as antibody or enzyme formulations, are widely used to treat disease. Non-therapeutic biopolymers such as enzymes, peptides, and structural proteins are useful for non-therapeutic applications such as household, nutritional, commercial and industrial uses.

[0003] Biopolymers used in therapeutic applications must be formulated so that they can be introduced into the body for the treatment of disease. Instead of administering these formulations, for example, by intravenous (IV) injection, under certain circumstances it is advantageous to deliver the antibody and protein/peptide biopolymer formulations by the subcutaneous (SC) or intramuscular (IM) route. However, in order to achieve better patient compliance and comfort for SC or IM injections, the liquid volume of the syringe is typically limited to 2-3 cc and the viscosity of the formulation is reduced using conventional medical devices and small caliber needles. typically less than about 20 centipoise (cP) to be delivered. These delivery parameters do not always fit well with the dosage requirements for the formulation to be delivered.

[0004] For example, antibodies may need to be delivered at high dose levels to exert their intended therapeutic effect. Using limited liquid volumes to deliver high dose levels of antibody formulations may require high concentrations of antibody in the delivery vehicle, sometimes exceeding levels of 150 mg/mL. At these dosage levels, the viscosity-versus-concentration plot of the formulations crosses their linear-nonlinear transition, so that the viscosity of the formulation rises dramatically with increasing concentration. However, the increased viscosity is not compatible with standard SC or IM delivery systems.

[0005] As a further concern, solutions of biopolymer-based therapeutics also have stability issues such as precipitation, fragmentation, oxidation, deamidation, hazing, opalescence, denaturation, liquid-liquid phase separation, and gel formation, reversible or irreversible aggregation. is likely to occur Stability issues limit the shelf life of the solution or require special handling.

[0006] As an example, one approach for producing an injectable protein formulation is to transform a therapeutic protein solution into a powder, which can be reconstituted to form a suspension suitable for SC or IM delivery. Lyophilization is a standard technique for producing protein powders. Freeze-drying, spray drying, and even precipitation followed by supercritical-fluid extraction have been used to produce protein powders for subsequent reconstitution. Powdered suspensions may be suitable for SC or IM injection if their viscosity prior to redissolving is low (compared to the same full dose of solution) and therefore the particles are small enough to fit through a needle. However, protein crystals present in the powder have an inherent risk of triggering an immune response. The uncertain protein stability/activity after redissolving raises additional issues. There is still a need in the art for techniques to produce low viscosity protein formulations for therapeutic purposes while circumventing the limitations introduced by protein powder suspensions.

[0007] In addition to the therapeutic applications of the proteins described above, biopolymers such as enzymes, peptides, and structural proteins can be used for non-therapeutic applications. Such non-therapeutic biopolymers can be produced from a number of different routes, for example, derived from plant sources, animal sources, or produced from cell culture.

[0008] Non-therapeutic proteins can be produced, transported, stored and handled as granular or powdered materials or solutions or dispersions, generally in water. Biopolymers for non-therapeutic applications may be spherical or fibrous proteins, and certain forms of these materials may have limited solubility in water or may exhibit high viscosity upon dissolution. Since these solution properties can present challenges for formulation, handling, storage, pumping, and performance of non-therapeutic substances, there is a need for methods to reduce viscosity and improve solubility and stability of non-therapeutic protein solutions. .

[0009] Proteins are complex biopolymers, each with a uniquely folded 3-D structure and a surface energy map (hydrophobic/hydrophilic region and charge). In concentrated protein solutions, these macromolecules can interact strongly and even engage in complex ways depending on their precise shape and surface energy distribution. "Hot-spots" for strong specific interactions cause protein clustering, increasing solution viscosity. To address this problem, a number of excipient compounds that aim to reduce solution viscosity by interfering with localized interactions and clustering are used in biotherapeutic formulations. These efforts are individually tuned, often empirically, and sometimes guided by in silico simulations. Combinations of excipient compounds may be provided, but re-optimizing such combinations should proceed empirically and on a case-by-case basis.

[0010] There is still a need in the art for a truly universal approach to reduce the viscosity of a protein formulation at a given concentration under non-linear conditions. There is a further need in the art to achieve this viscosity reduction while preserving the activity of the protein and avoiding stability issues. It would further be desirable to adapt the viscosity-reducing system for use with formulations having tunable and sustained release profiles and for use with formulations suitable for depot injection. It would also be desirable to improve processes for producing proteins and other biopolymers.

Summary of invention

[0011] In embodiments, proteins and hindered amines, aromatic or anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diones and sulfones, zwitterionic excipients, and crowding agents with hydrogen bonding elements Disclosed herein is a liquid formulation comprising an excipient compound selected from the group consisting of a crowding agent, wherein the excipient compound is added in a viscosity-reducing amount. In an embodiment, the protein is a PEGylated protein and the excipient is a low molecular weight aliphatic polyacid. In an embodiment, the excipient compound is a hindered amine compound.

[0012] In an embodiment, the hindered amine is ammonium chloride, ammonium bromide, ammonium fluoride, ammonium acetate, ammonium citrate, monoethanolamine hydrochloride, monoethanolamine hydrobromide, monoethanolamine hydrofluoride, monoethanolamine acetate, monoethanol amine citrate, diethanolamine hydrochloride, diethanolamine hydrobromide, diethanolamine hydrofluoride, diethanolamine acetate, diethanolamine citrate, triethanolamine hydrochloride, triethanolamine hydrobromide, triethanolamine hydrofluoride, triethanolamine acetate, triethanolamine citrate, urea hydrochloride, urea hydrobromide, urea hydrofluoride, urea acetate, and urea citrate. In an embodiment, the modified amine is a conjugate acid of ammonia or ammonium hydroxide. In an embodiment, the hindered amine compound is a conjugate acid of ammonia or ammonium hydroxide.

[0013] In an embodiment, the formulation is a pharmaceutical composition and the pharmaceutical composition comprises a therapeutic protein, wherein the excipient compound is a pharmaceutically acceptable excipient compound. In an embodiment, the formulation is a non-therapeutic formulation, and the non-therapeutic formulation comprises a non-therapeutic protein. In an embodiment, the therapeutic protein is bevacizumab, trastuzumab, adalimumab, infliximab, etanercept, darbepoetin alfa, epoetin alfa, cetuximab, pegfilgrastim, filgrastim, and is selected from the group consisting of rituximab. In an embodiment, the excipient compound is formulated as a concentrated excipient solution. In an embodiment, the viscosity-reducing amount reduces the viscosity of the formulation to a lower viscosity than that of the control formulation. In an embodiment, the viscosity of the formulation is at least about 10% less than the viscosity of the control formulation, at least about 30% less than the viscosity of the control formulation, at least about 50% less than the viscosity of the control formulation, or at least about the viscosity of the control formulation. about 70% lower, or at least about 90% lower than the viscosity of the control formulation. In an embodiment, the viscosity is less than about 100 cP, or less than about 50 cP, or less than about 20 cP, or less than about 10 cP. In an embodiment, the excipient compound has a molecular weight of <5000 Da, or <1500 Da, or <500 Da. In an embodiment, the formulation is at least about 1 mg/ml protein, or at least about 25 mg/mL protein, or at least about 50 mg/mL protein, or at least about 100 mg/mL protein, or at least about 200 mg protein. /mL of protein. In an embodiment, the formulation comprises from about 0.001 mg/mL to about 60 mg/mL of the excipient compound, from about 0.1 mg/mL to about 50 mg/mL of the excipient compound, or from about 1 mg/mL to about 40 mg/mL of the excipient compound, or about 5 mg/mL to about 30 mg/mL of the excipient compound. In an embodiment, the formulation has improved stability when compared to a control formulation. Improved stability may manifest as a reduction in the formation of visible particles, invisible particles, agglomerates, turbidity, opalescence, or gels. In an embodiment, the formulation has improved stability, wherein the improved stability is determined by comparison to a control formulation, wherein the control formulation contains no excipient compounds. In an embodiment, the improved stability prevents an increase in particle size as measured by light scattering. In an embodiment, improved stability is indicated by a higher percentage of monomer than the percentage of monomer in the control formulation, wherein the percentage of monomer is determined by size exclusion chromatography. In an embodiment, the excipient compound is a hindered amine, which may be caffeine. In an embodiment, the excipient compound is a hindered amine. In an embodiment, the hindered amine is from the group consisting of caffeine, theophylline, tyramine, imidazole, aspartame, saccharin, acesulfame potassium, pyrimidinone, 1,3-dimethyluracil, triaminopyrimidine, pyrimidine, and theacrine. is selected from In an embodiment, the hindered amine is caffeine, procaine, lidocaine, imidazole, aspartame, saccharin, and acesulfame potassium. In an embodiment, the hindered amine is caffeine. In an embodiment, the hindered amine is a local injectable anesthetic compound. The hindered amine may have independent pharmacological properties, and the hindered amine may be present in the formulation in an amount having an independent pharmacological effect. In embodiments, the hindered amine may be present in the formulation in an amount less than a therapeutically effective amount. The independent pharmacological activity may be local anesthetic activity. In an embodiment, a hindered amine having independent pharmacological activity is combined with a second excipient compound that further reduces the viscosity of the formulation. The second excipient compound may be selected from the group consisting of caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, aspartame, saccharin, and acesulfame potassium. In embodiments, the formulation may include additional agents selected from the group consisting of preservatives, surfactants, sugars, polysaccharides, arginine, proline, hyaluronidases, stabilizers, solubilizers, cosolvents, hydrotropes, and buffers. .

[0014] Further, a method of treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal a liquid therapeutic formulation, wherein the therapeutic formulation comprises a therapeutically effective amount of a therapeutic protein. wherein the dosage form comprises hindered amines, aromatics and anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diones and sulfones, zwitterionic excipients, and a hydrogen bonding element. further comprising a pharmaceutically acceptable acceptable excipient compound selected from the group consisting of Disclosed herein are methods wherein the therapeutic formulation is effective for the treatment of a disease or disorder. In an embodiment, the therapeutic protein is a pegylated protein and the excipient is a modified amine. In an embodiment, the modified amine is ammonium chloride, ammonium bromide, ammonium fluoride, ammonium acetate, ammonium citrate, monoethanolamine hydrochloride, monoethanolamine hydrobromide, monoethanolamine hydrofluoride, monoethanolamine acetate, monoethanol amine citrate, diethanolamine hydrochloride, diethanolamine hydrobromide, diethanolamine hydrofluoride, diethanolamine acetate, diethanolamine citrate, triethanolamine hydrochloride, triethanolamine hydrobromide, triethanolamine hydrofluoride, triethanolamine acetate, triethanolamine citrate, urea hydrochloride, urea hydrobromide, urea hydrofluoride, urea acetate, and urea citrate. In an embodiment, the modified amine is a conjugate acid of ammonia or ammonium hydroxide. In an embodiment, the protein is a pegylated protein and the excipient compound is a low molecular weight aliphatic polyacid. In an embodiment, the therapeutic protein is a pegylated protein, the excipient compound is a phenol or an aromatic compound, which may be a polyphenol, and the polyphenol may be a tannic acid. In an embodiment, the excipient is a hindered amine. In an embodiment, the formulation is administered by subcutaneous injection, or intramuscular injection, or intravenous injection. In an embodiment, the excipient compound is present in the therapeutic formulation in a viscosity-reducing amount, wherein the viscosity-reducing amount reduces the viscosity of the therapeutic formulation to a viscosity less than that of the control formulation. In an embodiment, the excipient compound is prepared as a concentrated excipient solution. In an embodiment, the therapeutic formulation has improved stability as compared to a control formulation. In an embodiment, the excipient compound is essentially pure.

[0015] In embodiments, there is provided a method of improving the stability of a liquid protein formulation comprising therapeutic proteins and hindered amines, aromatics and anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diones and and an excipient compound selected from the group consisting of a sulfone, a zwitterionic excipient, and a crowding agent having a hydrogen bonding component, wherein the liquid protein formulation exhibits improved stability compared to the control liquid protein formulation, and wherein the control liquid protein formulation comprises: Disclosed herein are methods that contain no excipient compounds and are otherwise substantially similar to liquid protein formulations. In an embodiment, the excipient compound is formulated as a concentrated excipient solution. The stability of the liquid formulation may be chemical stability exhibited by resistance to a chemical reaction selected from the group consisting of hydrolysis, photolysis, oxidation, reduction, deamidation, disulfide scrambling, fragmentation, and dissociation. The stability of the liquid formulation may be cold storage condition stability, room temperature stability or elevated temperature stability. The improved stability of the liquid protein formulation may be indicated by a higher percentage of monomer than the percentage of monomer in the control formulation, where the percentage of monomer is determined by size exclusion chromatography. The stability of the liquid formulation may be mechanical stability, which may be exhibited by improved resistance to stress conditions selected from the group consisting of agitation, pumping, filtration, filling, and gas bubble contact.

[0016] Also in embodiments, proteins and hindered amines, aromatic or anionic aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diones and sulfones, zwitterionic excipients, and hydrogen bonding elements A liquid formulation comprising an excipient compound selected from the group consisting of crowding agents, wherein the presence of the excipient compound in the formulation can be determined by a protein diffusion interaction parameter kD, or a second virial coefficient B22, for a more stable protein-protein interaction. or liquid formulations that result in improved protein-protein interaction properties. In an embodiment, the formulation is a therapeutic formulation and comprises a therapeutic protein. In an embodiment, the formulation is a non-therapeutic formulation, and the non-therapeutic formulation comprises a therapeutic protein.

[0017] The present invention also includes liquid formulations comprising a protein and an excipient compound, wherein the excipient compound is a pyrimidine and the excipient compound is added in a viscosity-reducing amount. In certain embodiments, the pyrimidine is a methyl-substituted pyrimidine. Non-limiting examples of pyrimidines include pyrimidine, pyrimidinone, triaminopyrimidine, 1,3-dimethyluracil, 1-methyluracil, 3-methyluracil, 1,3-diethyluracil, 5-methyluracil , 6-methyluracil, uracil, 1,3-dimethyl-tetrahydropyrimidinone, thymine, 1-methylthymine, O-4-methylthymine, 1,3-dimethylthymine, dimethylthymine dimer, theacrine, cyto sine, 5-methylcytosine, and 3-methylcytosine. In certain specific embodiments, the pyrimidine is 1,3-dimethyluracil. In a further aspect, the present invention provides a protein and an excipient compound selected from the group consisting of 3-aminopyridine, dicyclomine, 1-methyl-2-pyrrolidone, phenylserine, DL-3-phenylserine, and cycloserine. It relates to a liquid formulation comprising a liquid formulation, wherein an excipient compound is added in a viscosity-reducing amount. The liquid formulation may be a pharmaceutical composition, wherein the protein is a therapeutic protein and the excipient is a pharmaceutically acceptable excipient. Examples of therapeutic proteins include therapeutic antibodies (eg, bevacizumab, trastuzumab, adalimumab, infliximab, etanercept, cetuximab, rituximab, ipilimumab, and omalizumab) and pegylated proteins. Alternatively, the liquid formulation may be a non-therapeutic formulation, wherein the protein is a non-therapeutic protein. The viscosity-reducing amount of the excipient in the liquid formulations disclosed herein is about 250 mg/ml or less; about 10 mg/mL to about 200 mg/mL; about 10 mg/mL to about 120 mg/mL; about 20 mg/mL to about 120 mg/mL; about 1 mg/mL to about 100 mg/mL; about 2 mg/mL to about 80 mg/mL; about 5 mg/mL to about 50 mg/mL; about 10 mg/mL to about 40 mg/mL; about 1 mM to about 400 mM; about 2 mM to about 150 mM; about 5 mM to about 100 mM; or from about 15 mM to about 50 mM. In a further embodiment, the viscosity-reducing amount of the excipient compound reduces the viscosity of the formulation to a viscosity lower than that of the control formulation, wherein the control formulation contains no excipient but is otherwise identical on a dry weight basis to the therapeutic formulation; For example, the viscosity of the liquid formulation may be at least about 10%, at least about 30%, at least about 50%, at least about 70%, or at least about 90% lower than the viscosity of the control formulation. In a further aspect, the liquid formulation has a viscosity of less than about 100 cP, less than about 50 cP, less than about 20 cP, or less than about 10 cP. In yet a further aspect, the liquid formulation comprises an additional agent selected from the group consisting of preservatives, surfactants, sugars, polysaccharides, arginine, proline, hyaluronidase, stabilizers, solubilizers, cosolvents, hydrotropes, and buffers. do.

[0018] The present invention further provides a method for reducing the viscosity of a liquid formulation comprising a protein, e.g., a therapeutic protein or a non-therapeutic protein, comprising adding a viscosity-reducing amount of pyrimidine as described herein to the liquid formulation. further comprising a method comprising

[0019] In yet a further aspect, the present invention provides a method for reducing the viscosity of a liquid formulation comprising a protein, e.g., a therapeutic protein or a non-therapeutic protein, comprising: in the liquid formulation a viscosity-reducing amount of 3-aminopyridine; and adding an excipient selected from the group consisting of dicyclomine, 1-methyl-2-pyrrolidone, phenylserine, DL-3-phenylserine, and cycloserine.

[0020] The present invention further provides a method of treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal a liquid therapeutic formulation, wherein the liquid therapeutic formulation comprises a therapeutically effective amount a therapeutic protein, wherein the liquid therapeutic formulation further comprises a viscosity-reducing amount of a pharmaceutically acceptable excipient compound, wherein the excipient compound is pyrimidine; A therapeutic formulation includes a method effective for the treatment of a disease or disorder.

[0021] In a further aspect, the present invention provides a method of treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal a liquid therapeutic formulation, wherein the liquid therapeutic formulation is treated comprising an effective amount of a therapeutic protein, wherein the liquid therapeutic formulation comprises viscosity-reducing amounts of 3-aminopyridine, dicyclomine, 1-methyl-2-pyrrolidone, phenylserine, DL-3-phenylserine, and cyclo A method further comprising a pharmaceutically acceptable excipient compound selected from the group consisting of serine, wherein the therapeutic formulation is effective for the treatment of a disease or disorder.

[0022] Further, in an embodiment, disclosed herein is a method of improving a protein-related process comprising providing a liquid formulation as described herein and using the same in a method of processing. In an embodiment, the processing method comprises filtration, pumping, mixing, centrifugation, purification, membrane separation, lyophilization, or chromatography.

1 shows a graph of the particle size distribution for solutions of monoclonal antibodies under stress and non-stress conditions, as assessed by dynamic light scattering. The data curves of Figure 1 have baseline offsets that allow comparison: the curve for Sample 1-A is offset by 100 intensity units and the curve for Sample 1-FT is offset by 200 intensity units in the Y-axis.
2 shows a graph measuring the sample diameter versus multimodal size distribution for several molecular populations, as assessed by dynamic light scattering. The data curves of Figure 2 have baseline offsets that allow comparison: the curve for sample 2-A is offset by 100 intensity units and the curve for sample 2-FT is offset by 200 intensity units in the Y-axis.
3 shows a size exclusion chromatogram of a monoclonal antibody solution with a major monomer peak at 8-10 min retention time. The data curves of Figure 3 have baseline offsets to allow comparison: the curves for samples 2-C, 2-A, and 2-FT are offset in the Y-axis direction.

details

[0026] Formulations and methods for their production and use that allow for the delivery of concentrated protein solutions are disclosed herein. In embodiments, the approaches disclosed herein can produce lower viscosity liquid formulations or higher concentrations of therapeutic or non-therapeutic proteins in liquid formulations compared to typical protein solutions. In embodiments, the approaches disclosed herein can produce liquid formulations with improved stability when compared to typical protein solutions. A stable formulation is a formulation in which the protein contained therein substantially retains its physical and chemical stability and its therapeutic or non-therapeutic efficacy upon storage under storage conditions, regardless of cold storage conditions, room temperature conditions, or elevated temperature storage conditions. . Advantageously, stable formulations may also provide protection against aggregation or precipitation of proteins dissolved therein. For example, cold storage conditions may entail storage in a refrigerator or freezer. In some examples, cold storage conditions may involve storage at a temperature of 10° C. or less. In a further example, cold storage conditions involve storage at a temperature of about 2°C to about 10°C. In another example, cold storage conditions involve storage at a temperature of about 4°C. In a further example, cold storage conditions involve storage at a freezing temperature, such as about -20°C or less. In another example, cold storage conditions involve storage at a temperature of about -20°C to about 0°C. Room temperature storage conditions may involve storage at ambient temperature, for example, from about 10°C to about 30°C. Elevated storage conditions may entail storage at a temperature greater than about 30°C. For example, elevated temperature stability at temperatures of about 30° C. to about 50° C. can be used to predict long-term storage at typical ambient (10-30° C.) conditions as part of an accelerated aging study.

[0027] It is well known to those skilled in polymer science and engineering that proteins in solution have a tendency to form entanglements that can limit the translational mobility of the entangled chains and interfere with the therapeutic or non-therapeutic efficacy of the protein. In an embodiment, an excipient compound as disclosed herein is capable of inhibiting protein clustering due to a specific interaction between the excipient compound and a target protein in solution. Excipient compounds as disclosed herein may be natural or synthetic, and are preferably substances generally recognized as safe by the FDA (“GRAS”).

One. Justice

[0028] For the purposes of this disclosure, the term “protein” refers to a sequence of amino acids having a chain length long enough to create distinct tertiary structures, typically having a molecular weight of 1-3000 kDa. In some embodiments, the molecular weight of the protein is about 50-200 kDa; In other embodiments, the molecular weight of the protein is about 20-1000 kDa or about 20-2000 kDa. In contrast to the term “protein”, the term “peptide” refers to a sequence of amino acids that does not have a distinct tertiary structure. A wide variety of biopolymers are included within the scope of the term “protein”. For example, the term "protein" may refer to therapeutic or non-therapeutic proteins, including antibodies, aptamers, fusion proteins, pegylated proteins, synthetic polypeptides, protein fragments, lipoproteins, enzymes, structural peptides, and the like. .

a. Therapeutic Biopolymer Definition

[0029] Such biopolymers having a therapeutic effect may be referred to as “therapeutic biopolymers”. Such a protein having a therapeutic effect may be referred to as a “therapeutic protein”. A therapeutic protein contained in a therapeutic formulation may also be referred to as its “protein active ingredient”.

[0030] By way of non-limiting example, therapeutic proteins include mammalian proteins such as hormones and prohormones (eg, insulin and proinsulin, glucagon, calcitonin, thyroid hormones (T3 or T4 or thyroid-stimulating hormone), para thyroid hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone, growth hormone releasing factor, etc.); coagulation and anticoagulant factors (eg, tissue factor, von Willebrand factor, factor VIIIC, factor IX, protein C, plasminogen activator (urokinase, tissue type plasminogen activator), thrombin); cytokines, chemokines and inflammatory mediators; interferon; colony-stimulating factors; interleukins (eg, IL-1 to IL-10); growth factors (eg, vascular endothelial growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor, neurotrophic growth factor, insulin-like growth factor, etc.); albumin; collagen and elaskin; fibrin sealant; hematopoietic factors (eg, erythropoietin, thrombopoietin, etc.); osteoinductive factors (eg, osteogenic proteins); receptors (eg, integrins, cadherins, etc.); surface membrane proteins; transport protein; regulatory proteins; antigenic proteins (eg, viral components that act as antigens); and antibodies. The term "antibody", in its broadest sense, includes, by way of non-limiting examples, monoclonal antibodies (including, for example, full-length antibodies having an immunoglobulin Fc region), single chain molecules, bi-specific and multi-specific antibodies; Diabodies, antibody-drug conjugates, antibody compositions with polyepitopic specificity, polyclonal antibodies (e.g., polyclonal immunoglobulins used in therapy for immuno-compromised patients), and fragments of antibodies (e.g., Fc, Fab, Fv and F(ab')2)). Antibodies may also be referred to as “immunoglobulins”. Antibodies are understood to be directed against specific proteins or non-protein “antigens” that are biologically important substances; Administration of a therapeutically effective amount of an antibody to a patient may alter its biological properties such that the patient experiences a therapeutic effect by forming a complex with the antigen.

[0031] In an embodiment, the proteins are pegylated, meaning that they comprise poly(ethylene glycol) (“PEG”) and/or poly(propylene glycol) (“PPG”) units. PEGylated proteins, or PEG-protein conjugates, have found utility in therapeutic applications because of their beneficial properties such as solubility, pharmacokinetics, pharmacodynamics, immunogenicity, renal clearance, and stability. Non-limiting examples of pegylated proteins are cytokines, hormones, hormone receptors, cell signaling factors, coagulation factors, antibodies, antibody fragments, peptides, aptamers, and pegylated versions of enzymes. In an embodiment, the pegylated protein is interferon (PEG-IFN), pegylated anti-vascular endothelial growth factor (VEGF), pegylated human growth hormone (HGH), pegylated mutein antagonist, PEG protein conjugate drug, adagen , PEG-adenosine deaminase, PEG-uricase, pegaspargase, pegylated granulocyte colony-stimulating factor (GCSF), pegfilgrastim, pegloticase, pegvisomant, pegaptanib, peginesatide , pegylated erythropoiesis-stimulating agent, pegylated epoetin-α, pegylated epoetin-β, methoxy polyethylene glycol-epoetin beta, pegylated antihemophilic factor VIII, pegylated antihemophilic factor IX, and sertolizumab pegol (Certolizumab pegol).

[0032] PEGylated proteins can be synthesized by a variety of methods, such as reaction of a protein with a PEG reagent having one or more reactive functional groups. Reactive functional groups on PEG reagents can form linkages with proteins at targeted protein sites such as lysine, histidine, cysteine, and the N-terminus. Typical pegylation reagents have reactive functional groups such as aldehyde, maleimide, or succinimide groups that have specific reactivity with the targeted amino acid residue on the protein. The PEGylation reagent may have a PEG chain length of from about 1 to about 1000 PEG and/or PPG repeat units. Another method of pegylation involves glyco-pegylation, wherein the protein is first glycosylated and then the glycosylated residues are pegylated in a second step. Certain pegylation processes are assisted by enzymes such as sialyltransferase and transglutaminase.

[0033] While pegylated proteins may provide therapeutic advantages over native, non-pegylated proteins, such materials may have physical or chemical properties that make them difficult to purify, dissolve, filter, concentrate and administer. Pegylation of proteins can result in higher solution viscosities compared to native proteins, which generally require formulation of lower concentrations of pegylated protein solutions.

[0034] It is desirable to formulate protein therapeutics into stable, low-viscosity solutions so that they can be administered to patients in minimal injection volumes. For example, subcutaneous (SC) or intramuscular (IM) injection of a drug generally requires a small injection volume, preferably less than 2 mL. The SC and IM injection routes are well suited for self-administration treatment, which is a less costly and accessible form of treatment compared to intravenous (IV) injections performed only under direct medical supervision. Formulations for SC or IM injection require a low solution viscosity, generally less than 30 cP, and preferably less than 20 cP, to allow easy flow of the therapeutic solution through a narrow-gauge needle with minimal injection force. This combination of small injection volumes and low viscosity requirements poses challenges for the use of pegylated protein therapeutics in the SC or IM injection route.

[0035] "Treatment" is intended to treat, cure, alleviate, ameliorate, relieve or otherwise advantageously affect a disorder, including preventing or delaying the onset of the symptoms and/or alleviating or ameliorating the symptoms of the disorder. Including any action. Such patients in need of treatment include both those already with the particular disorder and those for whom prevention of the disorder is desired. A disorder is any condition that alters the homeostatic well-being of a mammal, including an acute or chronic disease, or a pathological condition that predisposes the mammal to an acute or chronic disease. Non-limiting examples of disorders include cancer, metabolic disorders (eg diabetes), allergic disorders (eg asthma), skin disorders, cardiovascular disorders, respiratory disorders, blood disorders, musculoskeletal disorders, inflammatory or rheumatic disorders, autoimmune disorders, gastrointestinal disorders, urological disorders, sexual and reproductive disorders, nervous system disorders, and the like. "Mammal" for therapeutic purposes may refer to any animal classified as a mammal, including humans, livestock, pet animals, farm animals, sports animals, working animals, and the like. Thus, “treatment” may include both veterinary and human treatments. For convenience, a mammal receiving such “treatment” may be referred to as a “patient”. In certain embodiments, the patient can be of any age, including in utero fetal animals.

[0036] A formulation containing a therapeutically effective amount of a therapeutic protein may be referred to as a "therapeutic formulation." In an embodiment, treating comprises providing a therapeutically effective amount of a therapeutic formulation to a mammal in need thereof. A "therapeutically effective amount" is at least the minimum concentration of a therapeutic protein administered to a mammal in need thereof to achieve treatment of an existing disorder or prevention of a contemplated disorder (such treatment or prophylaxis is a "therapeutic intervention"). ). Therapeutically effective amounts of the various therapeutic proteins that may be included as active ingredients in therapeutic formulations may be familiar to those skilled in the art; For therapeutic proteins found hereinafter or applied to therapeutic intervention, a therapeutically effective amount can be determined by standard techniques practiced by one of ordinary skill in the art using no more than routine experimentation. In an embodiment, a therapeutic formulation comprises a therapeutically effective amount of a protein active ingredient and an excipient, with or without other optional ingredients.

b. Non-therapeutic biopolymer definition

[0037] Such biopolymers used for non-therapeutic purposes (ie, non-therapeutic purposes) such as household, nutritional, commercial and industrial applications may be referred to as “non-therapeutic biopolymers”. Such proteins used for non-therapeutic purposes may be referred to as “non-therapeutic proteins”. A formulation containing a non-therapeutic protein may be referred to as a “non-therapeutic formulation”. Non-therapeutic proteins can be derived from plant sources, animal sources, or produced from cell culture; They may also be enzymes or structural proteins. Non-therapeutic proteins can be used in household, nutritional, commercial and industrial applications such as catalysts, human and animal nutrition, processing aids, detergents, and waste treatment.

[0038] An important category of non-therapeutic proteins is that of enzymes. Enzymes have numerous non-therapeutic uses, for example, as catalysts, human and animal nutritional ingredients, processing aids, detergents, and waste disposal agents. Enzyme catalysts are used to catalyze various chemical reactions. Examples of enzyme catalysts for non-therapeutic use include catalase, oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase. Human and animal nutritional uses of enzymes include nutraceuticals, nutritional sources of proteins, chelated or controlled delivery of micronutrients, digestive aids, and supplements; They may be derived from amylases, proteases, trypsin, lactases, and the like. Enzyme processing aids are used to improve the production of food and beverage products in operations such as baking, brewing, fermentation, juice processing, and winemaking. Examples of such food and beverage processing aids include amylase, cellulase, pectinase, glucanase, lipase, and lactase. Enzymes can also be used in the production of biofuels. For example, ethanol for biofuels can be assisted, for example, by enzymatic degradation of biomass feedstocks such as cellulosic and lignocellulosic materials. Treatment of these feedstock materials with cellulase and ligninase transforms the biomass into a substrate that can be fermented as fuel. In other commercial applications, enzymes are used as detergents, cleaners, and stain removal aids for laundry, dish washing, surface washing, and equipment washing applications. Typical enzymes for this purpose include proteases, cellulases, amylases, and lipases. In addition, non-therapeutic enzymes are used in various commercial and industrial processes such as textile softening by cellulase, leather processing, waste treatment, contaminated sediment treatment, water treatment, pulp bleaching, and pulp softening and debonding. Typical enzymes for this purpose are amylases, xylanases, cellulases, and ligninases.

[0039] Other examples of non-therapeutic biopolymers include fibrous or structural proteins such as keratin, collagen, gelatin, elastin, fibroin, actin, tubulin, or hydrolyzed, degraded, or derivatized forms thereof. These materials are used in the manufacture and formulation of food ingredients such as gelatin, ice cream, yogurt, and confectionery; They are also added to foods as thickeners, rheology modifiers, mouthfeel improvers, and sources of nutritional protein. In the cosmetic and personal care industries, collagen, elastin, keratin, and hydrolyzed keratin are widely used as ingredients in skin care and hair care formulations. Another example of a non-therapeutic biopolymer is whey proteins such as beta-lactoglobulin, alpha-lactalbumin, and serum albumin. This whey protein is mass produced as a by-product from dairy operations and has been used in a variety of non-therapeutic applications.

2. measurement

[0040] In an embodiment, the protein-containing formulations described herein are resistant to monomer loss as measured by size exclusion chromatography (SEC) analysis. In SEC analysis as used herein, a major analyte peak is generally associated with a target protein contained in the formulation, and this major peak of the protein is referred to as the monomeric peak. The monomer peak represents the amount of the target protein, eg, protein active ingredient, in the monomeric state as opposed to the aggregated (dimeric, trimer, oligomeric, etc.) or fragmented state. The monomer peak area can be compared to the total area of the monomer, aggregate, and fragment peaks associated with the target protein. Thus, the stability of the protein-containing formulation can be observed by the relative amount of monomer after the elapsed time; Thus, the improvement in stability of the protein-containing formulations of the present invention can be measured as a higher percentage of monomer after a certain elapsed time compared to the percentage of monomer in a control formulation containing no excipient.

[0041] In an embodiment, the ideal stability result is to have a monomer peak of 98-100% as determined by SEC analysis. In an embodiment, the improvement in stability of a protein-containing formulation of the invention is a higher percentage when the control formulation is exposed to the same stress condition as compared to the percentage of monomer in a control formulation that does not contain an excipient after exposure to stress conditions. can be measured as a monomer of In embodiments, the stress condition may be cold storage, high temperature storage, exposure to air, exposure to gas bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.

[0042] In an embodiment, the protein-containing formulation as described herein is resistant to an increase in protein particle size as measured by dynamic light scattering (DLS) analysis. In DLS analysis, as used herein, the particle size of a protein in a protein-containing formulation can be observed as the median particle size. Ideally, the median particle size of the target protein should be relatively unchanged when subjected to DLS analysis, which means that the particle diameter represents the active ingredient in the monomeric state as opposed to the aggregated (dimer, trimer, oligomer, etc.) or fragmented state. Because. An increase in the median particle size may indicate aggregated protein. Thus, the stability of the protein-containing formulation can be observed by the relative change in median particle size after elapsed time.

[0043] In an embodiment, the protein-containing formulation as described herein is resistant to forming a polydisperse particle size distribution as measured by dynamic light scattering (DLS) analysis. In an embodiment, the protein-containing formulation may contain a monodisperse particle size distribution of colloidal protein particles. In an embodiment, the ideal stability result is to have less than a 10% change in the median particle diameter compared to the initial median particle diameter of the formulation. In an embodiment, the improvement in stability of a protein-containing formulation of the present invention can be measured as a sub-percent change in median particle size after a predetermined elapsed time compared to median particle diameter in a control formulation containing no excipient. In an embodiment, the improvement in stability of a protein-containing formulation of the present invention occurs when the control formulation is exposed to the same stress condition as compared to the percent change in median particle size in a control formulation containing no excipient after exposure to the stress condition. It can be measured as a lower percentage change in the median particle size. In embodiments, the stress condition may be cold storage, high temperature storage, exposure to air, exposure to gas bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles. In an embodiment, the improvement in stability of a therapeutic formulation for a protein-containing formulation of the invention is measured by DLS compared to the polydispersity of particle size distribution in a control formulation containing no excipient when the control formulation is exposed to the same stress conditions. can be measured as a less polydisperse particle size distribution.

[0044] In an embodiment, a protein-containing formulation of the invention is resistant to sedimentation as measured by turbidity, light scattering, and/or particle counting analysis. In turbidity, light scattering, or particle count analysis, lower values generally indicate fewer suspended particles in the formulation. Turbidity, light scattering, or an increase in particle count may indicate that the solution of the target protein is not stable. Thus, the stability of a protein-containing formulation can be observed by the relative amount of turbidity, light scattering, or particle count after elapsed time. In embodiments, the ideal stability result is to have low and relatively constant haze, light scattering, or particle count values. In an embodiment, improved stability of a protein-containing formulation of the invention results in lower haze, lower light scattering, or fewer particles after a specified elapsed time as compared to the haze, light scattering, or particle number values of a control formulation that does not contain an excipient It can be measured as a number. In an embodiment, the improvement in stability of a protein-containing formulation as described herein is lower after exposure to stress conditions as compared to turbidity, light scattering, or particles of a control formulation that does not contain an excipient when the control formulation is exposed to the same stress conditions. It can be measured as haze, lower light scattering, or lower particle count. In embodiments, the stress condition may be cold storage, high temperature storage, exposure to air, exposure to gas bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.

3. therapeutic formulation

[0045] In one aspect, the formulations and methods disclosed herein provide a stable liquid formulation of improved or reduced viscosity comprising a therapeutically effective amount of a therapeutic protein and an excipient compound. In embodiments, formulations are capable of improving stability while providing acceptable concentrations of active ingredient and acceptable viscosities. In an embodiment, the formulation provides an improvement in stability when compared to a control formulation; For the purposes of the present disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar or identical by dry weight to a therapeutic formulation in all respects except for the absence of an excipient compound. In an embodiment, the formulation is stable under stress conditions of long-term storage, elevated temperature, such as 25-45° C., freeze/thaw conditions, shear or mixing, injection, dilution, gas bubble exposure, oxygen exposure, light exposure, and lyophilization. provide improvement of In an embodiment, the improved stability of the protein-containing formulation is in the form of a lower percentage of soluble aggregates, particulates, invisible particles, or gel formation as compared to a control formulation. In an embodiment, the improved stability of the protein-containing formulation is in the form of higher biological activity compared to the control formulation. In an embodiment, the improved stability of the protein-containing formulation is in the form of improved chemical stability, such as resistance to chemical reactions, such as hydrolysis, photolysis, oxidation, reduction, deamidation, disulfide scrambling, fragmentation, or dissociation. . In an embodiment, the improved stability of the protein-containing formulation is exhibited by a reduction in the formation of visible particles, invisible particles, aggregates, turbidity, opalescence, or gels.

[0046] The viscosity of a liquid protein formulation depends on the nature of the protein itself (eg, enzyme, antibody, receptor, fusion protein, etc.); its size, three-dimensional structure, chemical composition, and molecular weight; its concentration in the formulation; components of the formulation other than protein; desired pH range; storage conditions for the formulation; and how the formulation is administered to a patient. Therapeutic proteins most suitable for use with the excipient compounds described herein are preferably essentially pure, ie, free of contaminating proteins. In an embodiment, an "essentially pure" therapeutic protein comprises at least 90% by weight of the therapeutic protein, or preferably at least 95% by weight of the therapeutic protein, all based on the total weight of the therapeutic protein and the contaminating protein in the composition. protein, or more preferably a protein composition comprising at least 99% by weight of a therapeutic protein. For purposes of clarity, proteins added as excipients are not intended to be included in this definition. The therapeutic formulations described herein treat mammals in a form in which the desired therapeutic efficacy of the protein active ingredient can be achieved without containing ingredients that are toxic to the mammal to which the formulation is administered, i.e., a pharmaceutical-grade formulation. It is a formulation intended for use as a formulation intended for use in

[0047] In an embodiment, the therapeutic formulation contains at least 1 mg/mL of protein active ingredient. In another embodiment, the therapeutic formulation contains at least 10 mg/mL of protein active ingredient. In another embodiment, the therapeutic formulation contains at least 25 mg/mL of protein active ingredient. In another embodiment, the therapeutic formulation contains at least 25 mg/mL of protein active ingredient. In another embodiment, the therapeutic formulation contains at least 100 mg/mL of protein active ingredient. In another embodiment, the therapeutic formulation contains at least 200 mg/mL of protein active ingredient. In yet another embodiment, the therapeutic formulation solution contains at least 300 mg/mL of protein active ingredient. Generally, the excipient compounds disclosed herein are added to the therapeutic formulation in an amount from about 0.001 to about 60 mg/mL. In an embodiment, the excipient compound may be added in an amount from about 0.1 to about 50 mg/mL. In an embodiment, the excipient compound may be added in an amount from about 1 to about 40 mg/mL. In an embodiment, the excipient may be added in an amount from about 5 to about 30 mg/mL.

[0048] In certain embodiments, the therapeutic formulation solution contains at least 300 mg/mL of protein active ingredient. In certain embodiments, the excipient compounds disclosed herein are added to the therapeutic formulation in an amount from about 1 to about 300 mg/mL, or from about 5 to about 300 mg/mL. In an embodiment, the excipient compound may be added in an amount from about 10 to about 200 mg/mL. In embodiments, the excipient compound may be added in an amount from about 5 to about 100 mg/mL, or from about 20 to about 100 mg/mL. In an embodiment, the excipient compound may be added in an amount from about 10 to about 75 mg/mL, or from about 20 to about 75 mg/mL. In an embodiment, the excipient may be added in an amount of about 15 to about 50 mg/mL.

[0049] Excipient compounds of various molecular weights are selected for certain advantageous properties when combined with the protein active ingredient in a formulation. Examples of therapeutic formulations comprising an excipient compound are provided below. In an embodiment, the excipient compound has a molecular weight of <5000 Da. In an embodiment, the excipient compound has a molecular weight of <1000 Da. In an embodiment, the excipient compound has a molecular weight of <500 Da.

[0050] In an embodiment, an excipient compound disclosed herein is added to a therapeutic formulation in a viscosity-reducing amount. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 10% as compared to a control formulation; For the purposes of the present disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar or identical by dry weight to a therapeutic formulation in all respects except for the absence of an excipient compound. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 30% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 50% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 70% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 90% as compared to a control formulation.

[0051] In an embodiment, the viscosity-reducing amount results in a therapeutic formulation having a viscosity of less than 100 cP. In another embodiment, the therapeutic formulation has a viscosity of less than 50 cP. In another embodiment, the therapeutic formulation has a viscosity of less than 20 cP. In another embodiment, the therapeutic formulation has a viscosity of less than 10 cP. As used herein, the term “viscosity” refers to the value of the kinematic viscosity as measured by the methods disclosed herein.

[0052] In an embodiment, the therapeutic formulation is administered to the patient at a high concentration of the therapeutic protein. High concentration solutions of therapeutic proteins formulated with the excipient compounds described herein can be administered to a patient using a syringe or pre-filled syringe. In embodiments, the therapeutic formulation is administered to the patient in a smaller injection volume and/or less discomfort than would be experienced with a similar formulation without the therapeutic excipient. In an embodiment, the therapeutic formulation is administered to the patient using a narrower gauge needle, or less syringe force that would be required with a similar formulation without the therapeutic excipient. In an embodiment, the therapeutic formulation is administered as a depot injection. In an embodiment, the therapeutic formulation prolongs the half-life of the therapeutic protein in the body. This characteristic of the therapeutic formulation as disclosed herein is such that the clinical situation, ie, the patient tolerance of intramuscular injection, is typical for IM/SC use and the use of acceptable (eg 2-3 cc) injection volumes. Circumstances involving the use of a needle, and those conditions that result in administration of an effective amount of the formulation in a single injection at a single injection site, will permit administration of such formulations by intramuscular or subcutaneous injection. In contrast, injection of comparable doses of therapeutic protein using conventional formulation techniques would be limited by the higher viscosity of conventional formulations and therefore SC/IM injections of conventional formulations would not be suitable for clinical situations. .

[0053] Therapeutic formulations according to the present disclosure may have certain advantageous properties consistent with improved stability. In embodiments, the therapeutic formulation is resistant to shear degradation, phase separation, clouding out, precipitation, oxidation, deamidation, aggregation, and/or denaturation. In embodiments, the therapeutic formulation is more effectively processed, purified, stored, injected, administered, filtered, and/or centrifuged as compared to a control formulation.

[0054] In embodiments, the therapeutic formulations disclosed herein are resistant to monomer loss as measured by size exclusion chromatography (SEC) analysis. In SEC analysis, a major analyte peak is usually associated with the active ingredient of a formulation, such as a therapeutic protein, and this major peak of the active ingredient is referred to as a monomeric peak. The monomer peak represents the amount of active ingredient in the monomeric state, as opposed to aggregated (dimer, trimer, oligomer, etc.) protein. Thus, the stability of the therapeutic formulation can be observed by the relative amount of the monomer after the elapsed time. In an embodiment, the improvement in stability of a therapeutic formulation as disclosed herein can be measured as the top monomer percent after a predetermined elapsed time compared to the monomer percent in a control formulation that does not contain an excipient. In an embodiment, the improvement in stability of a therapeutic formulation as disclosed herein can be measured as the top monomer percent after exposure to the stress condition compared to the monomer percent in a control formulation that does not contain the excipient after exposure to the stress condition. have. In an embodiment, a therapeutic formulation of the invention is resistant to an increase in protein particle size as measured by dynamic light scattering (DLS) analysis. In DLS analysis, the particle size of the therapeutic protein can be observed as the median particle size. Ideally, the median particle size of the therapeutic protein should be relatively unchanged. Therefore, an increase in the median particle size may indicate an aggregated protein. Therefore, the stability of the therapeutic formulation can be observed by the relative change in the median particle size after the elapsed time. In an embodiment, the therapeutic formulation as disclosed herein is resistant to forming a polydisperse particle size distribution as measured by dynamic light scattering (DLS) analysis. In an embodiment, the improvement in stability of a therapeutic formulation of the present invention can be measured as a sub-percent change in median particle size after a predetermined elapsed time compared to median particle diameter in a control formulation containing no excipient. In an embodiment, the improvement in stability of a therapeutic formulation as disclosed herein can be measured as a lower percent change in median particle size after exposure to stress conditions compared to the percent change in median particle diameter in a control formulation that does not contain an excipient. can In other words, in an embodiment, the improved stability prevents an increase in particle size as measured by light scattering. In embodiments, the stress condition may be cold storage, high temperature storage, exposure to air, exposure to gas bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles. In an embodiment, the improvement in stability of a therapeutic formulation as disclosed herein can be measured as a less polydisperse particle size distribution when measured by DLS compared to the polydispersity of the particle size distribution in a control formulation containing no excipient. have.

[0055] In an embodiment, a therapeutic formulation as disclosed herein is resistant to sedimentation as measured by turbidity, light scattering, or particle counting analysis. In an embodiment, improved stability of a therapeutic formulation as disclosed herein results in lower haze, lower light scattering, or less turbidity, light scattering, or less after a specified elapsed time as compared to the haze, light scattering, or particle number values of a control formulation that does not contain an excipient It can be measured as the number of particles. In an embodiment, improved stability of a therapeutic formulation as disclosed herein results in lower haze, lower light scattering, or lower haze after exposure to stress conditions, as compared to the haze, light scattering, or particle number values of a control formulation that does not contain an excipient It can be measured as a smaller number of particles. In embodiments, the stress condition may be cold storage, high temperature storage, exposure to air, exposure to gas bubbles, exposure to shear conditions, or exposure to freeze/thaw cycles.

[0056] In an embodiment, the therapeutic excipient has antioxidant properties that stabilize the therapeutic protein against oxidative damage. In an embodiment, the therapeutic formulation is stored at ambient temperature or in refrigerated conditions for an extended period of time without significant loss of potency to the therapeutic protein. In an embodiment, the therapeutic formulation is dried for storage until needed; It is then reconstituted with a suitable solvent, for example water. Advantageously, formulations prepared as described herein may be stable over long periods of time from months to years. If an exceptionally long shelf life is desired, the formulation can be stored in the freezer (and reactivated later) without concern for protein denaturation. In an embodiment, the formulation may be prepared for long-term storage that does not require refrigeration.

[0057] In an embodiment, an excipient compound disclosed herein is added to a therapeutic formulation in a stability-improving amount. In an embodiment, the stability-improving amount is an amount of an excipient compound that reduces denaturation of the formulation by at least 10% as compared to a control formulation; For the purposes of the present disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar on a dry weight basis to a therapeutic formulation except for the absence of an excipient compound. In an embodiment, the stability-improving amount is an amount of an excipient compound that reduces denaturation of the formulation by at least 30% as compared to a control formulation. In an embodiment, the stability-improving amount is an amount of an excipient compound that reduces denaturation of the formulation by at least 50% as compared to a control formulation. In an embodiment, the stability-improving amount is an amount of an excipient compound that reduces denaturation of the formulation by at least 70% when compared to a control formulation. In an embodiment, the stability-improving amount is an amount of an excipient compound that reduces denaturation of the formulation by at least 90% as compared to a control formulation.

[0058] Methods for preparing therapeutic formulations may be familiar to those skilled in the art. Therapeutic formulations of the present invention can be prepared, for example, by adding an excipient compound to the formulation before or after the therapeutic protein is added to the solution. A therapeutic formulation can be produced, for example, by combining a therapeutic protein and an excipient in a first (lower) concentration and then processing by filtration or centrifugation to produce a second (higher) concentration of the therapeutic protein. can Therapeutic formulations may be prepared with one or more excipient compounds having chaotropes, cosmotropes, hydrotropes, and salts. Therapeutic formulations may be prepared with one or more excipient compounds using techniques such as encapsulation, dispersion, liposome, vesicle formation, and the like. Methods for preparing a therapeutic formulation comprising an excipient compound disclosed herein may include a combination of excipient compounds. In embodiments, the combination of excipients may result in an advantage in lower viscosity, improved stability, or reduced injection site pain. Other additives may be incorporated into the therapeutic formulation during their manufacture, including preservatives, surfactants, sugars, sucrose, trehalose, polysaccharides, arginine, proline, hyaluronidases, stabilizers, buffers, and the like. As used herein, pharmaceutically acceptable excipient compounds are those that are non-toxic and suitable for animal and/or human administration.

3. Non-therapeutic formulations

[0059] In one aspect, the formulations and methods disclosed herein provide a stable liquid formulation of improved or reduced viscosity comprising an effective amount of a non-therapeutic protein and an excipient compound. In an embodiment, the formulation improves stability while providing an acceptable concentration of active ingredient and an acceptable viscosity. In an embodiment, the formulation provides an improvement in stability when compared to a control formulation; For the purposes of the present disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar or identical on a dry weight basis to a non-therapeutic formulation in all respects except for the absence of an excipient compound.

[0060] The viscosity of a liquid protein formulation depends on the nature of the protein itself (eg, enzymes, structural proteins, degree of hydrolysis, etc.); its size, three-dimensional structure, chemical composition, and molecular weight; its concentration in the formulation; components of the formulation other than protein; desired pH range; storage conditions for the formulation; and storage conditions for the formulation.

[0061] In an embodiment, the non-therapeutic formulation contains at least 25 mg/mL of protein active ingredient. In another embodiment, the non-therapeutic formulation contains at least 100 mg/mL of protein active ingredient. In another embodiment, the non-therapeutic formulation contains at least 200 mg/mL of protein active ingredient. In yet another embodiment, the non-therapeutic formulation solution contains at least 300 mg/mL of protein active ingredient. Generally, the excipient compounds disclosed herein are added to the non-therapeutic formulation in an amount from about 0.001 to about 60 mg/mL. In an embodiment, the excipient compound may be added in an amount from about 0.1 to about 50 mg/mL. In an embodiment, the excipient compound may be added in an amount from about 1 to about 40 mg/mL. In an embodiment, the excipient may be added in an amount from about 5 to about 30 mg/mL.

[0062] In certain embodiments, the non-therapeutic formulation solution contains at least 300 mg/mL of protein active ingredient. In certain embodiments, the excipient compounds disclosed herein are added to the therapeutic formulation in an amount from about 1 to about 300 mg/mL, or from about 5 to about 300 mg/mL. In an embodiment, the excipient compound may be added in an amount from about 10 to about 200 mg/mL. In embodiments, the excipient compound may be added in an amount from about 5 to about 100 mg/mL, or from about 20 to about 100 mg/mL. In an embodiment, the excipient compound may be added in an amount from about 10 to about 75 mg/mL, or from about 20 to about 75 mg/mL. In an embodiment, the excipient may be added in an amount from about 15 to about 50 mg/mL.

[0063] Excipient compounds of various molecular weights are selected for certain advantageous properties when combined with the protein active ingredient in a formulation. Examples of non-therapeutic formulations comprising an excipient compound are provided below. In an embodiment, the excipient compound has a molecular weight of <5000 Da. In an embodiment, the excipient compound has a molecular weight of <1000 Da. In an embodiment, the excipient compound has a molecular weight of <500 Da.

[0064] In an embodiment, an excipient compound disclosed herein is added to a non-therapeutic formulation in a viscosity-reducing amount. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 10% as compared to a control formulation; For the purposes of the present disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar or identical by dry weight to a therapeutic formulation in all respects except for the absence of an excipient compound. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 30% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 50% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 70% as compared to a control formulation. In an embodiment, the viscosity-reducing amount is an amount of an excipient compound that reduces the viscosity of the formulation by at least 90% as compared to a control formulation.

[0065] In an embodiment, the viscosity-reducing amount results in a non-therapeutic formulation having a viscosity of less than 100 cP. In another embodiment, the non-therapeutic formulation has a viscosity of less than 50 cP. In another embodiment, the non-therapeutic formulation has a viscosity of less than 20 cP. In another embodiment, the non-therapeutic formulation has a viscosity of less than 10 cP. As used herein, the term “viscosity” refers to a kinematic viscosity value.

[0066] Non-therapeutic formulations according to the present disclosure may have certain advantageous properties. In embodiments, the non-therapeutic formulation is resistant to shear degradation, phase separation, clouding out, oxidation, deamidation, aggregation, precipitation, and denaturation. In embodiments, therapeutic formulations can be processed, purified, stored, pumped, filtered, and centrifuged more effectively compared to control formulations.

[0067] In an embodiment, the non-therapeutic excipient has antioxidant properties that stabilize the non-therapeutic protein against oxidative damage. In an embodiment, the non-therapeutic formulation is stored for an extended period of time at ambient temperature or in refrigerated conditions without significant loss of potency to the non-therapeutic protein. In an embodiment, the non-therapeutic formulation is dried for storage until needed; It may then be reconstituted with a suitable solvent, for example water. Advantageously, formulations prepared as described herein are stable over long periods of time from months to years. If an exceptionally long shelf life is desired, the formulation is preserved in the freezer (and later reactivated) without concern for protein denaturation. In an embodiment, the formulation is prepared for long-term storage that does not require refrigeration.

[0068] In an embodiment, an excipient compound disclosed herein is added to a non-therapeutic formulation in a stability-improving amount. In an embodiment, the stability-improving amount is an amount of an excipient compound that reduces denaturation of the formulation by at least 10% when compared to a control formulation; For the purposes of the present disclosure, a control formulation is a formulation containing a protein active ingredient that is substantially similar or identical on a dry weight basis to a therapeutic formulation, except for the absence of an excipient compound. In an embodiment, the stability-improving amount is an amount of an excipient compound that reduces denaturation of the formulation by at least 30% when compared to a control formulation. In an embodiment, the stability-improving amount is an amount of an excipient compound that reduces denaturation of the formulation by at least 50% when compared to a control formulation. In an embodiment, the stability-improving amount is an amount of an excipient compound that reduces denaturation of the formulation by at least 70% when compared to a control formulation. In an embodiment, the stability-improving amount is an amount of an excipient compound that reduces denaturation of the formulation by at least 90% when compared to a control formulation.

[0069] Methods for preparing non-therapeutic formulations comprising the excipient compounds disclosed herein may be familiar to those skilled in the art. For example, the excipient compound may be added to the formulation before or after the non-therapeutic protein is added to the solution. The non-therapeutic formulation can be produced at a first (lower) concentration and then treated by filtration or centrifugation to produce a second (higher) concentration. Non-therapeutic formulations can be prepared with one or more excipient compounds having chaotropes, cosmotropes, hydrotropes, and salts. Non-therapeutic formulations may be prepared with one or more excipient compounds using techniques such as encapsulation, dispersion, liposome, vesicle formation, and the like. Other additives may be incorporated into non-therapeutic formulations during their manufacture, including preservatives, surfactants, stabilizers, and the like.

4. excipient compounds

[0070] Described herein are several excipient compounds, each suitable for use with one or more therapeutic or non-therapeutic proteins, and each allowing formulations to be configured to contain high concentrations of protein(s). Some categories of excipient compounds described below are: (1) hindered or modified amines; (2) aromatic and anionic aromatics; (3) functionalized amino acids; (4) oligopeptides; (5) short chain organic acids; (6) low molecular weight aliphatic polyacids; and (7) diones and sulfones.

[0071] In an embodiment, one or more viscosity-reducing excipient compounds may be added to the formulation simultaneously or sequentially. In an embodiment, at least one of the viscosity-reducing compounds is a hindered amine. In an embodiment, the at least one excipient compound is a pyrimidine, a methyl-substituted pyrimidine, or a phenethylamine. A pyrimidine is a compound comprising at least one pyrimidine ring; It is to be understood that the pyrimidine ring may be optionally substituted and/or may be part of a fused ring system, for example part of a fused bicyclic or tricyclic ring system. Exemplary pyrimidines include, for example, pyrimidine, pyrimidinone, triaminopyrimidine, purine, adenine, and guanine. A methyl-substituted pyrimidine is a compound comprising at least one methyl-substituted pyrimidine ring. Exemplary methyl-substituted pyrimidines are 1,3-dimethyluracil, 1-methyluracil, 3-methyluracil, 5-methyluracil, and 6-methyluracil. Additional exemplary pyrimidines are described in more detail below. In certain embodiments, the pyrimidine or methyl-substituted pyrimidine is not caffeine or xanthine. In yet a further aspect, the pyrimidine is a compound comprising at least one pyrimidine ring, wherein the pyrimidine ring is not part of a fused ring system. In an embodiment, the at least one excipient compound is from the group consisting of caffeine, saccharin, acesulfame potassium, aspartame, theophylline, taurine, 1-methyl-2-pyrrolidone, 2-pyrrolidinone, niacinamide, and imidazole is selected from In an embodiment, the at least one excipient compound is selected from the group consisting of caffeine, taurine, niacinamide, and imidazole. In an embodiment, the at least one excipient compound is uracil, 1-methyluracil, 6-methyluracil, 5-methyluracil, 1,3-dimethyluracil, cytosine, 5-methylcytosine, 3-methylcytosine, thymine , methylthymine, O-4-methylthymine, 1,3-dimethylthymine, and dimethylthymine dimer. In an embodiment, the at least one excipient compound is diphenhydramine, phenethylamine, N-methylphenethylamine, N,N-dimethylphenethylamine, beta-3-dihydroxyphenethylamine, beta-3-di hydroxy-N-methylphenethylamine, 3-hydroxyphenethylamine, 4-hydroxyphenethylamine, tyrosinol, tyramine, N-methyltyramine, and hordenine. In embodiments, the at least one excipient compound is an anionic aromatic excipient, and in some embodiments, the anionic aromatic excipient may be 4-hydroxybenzenesulfonic acid. In an embodiment, the viscosity-reducing amount is from about 1 mg/mL to about 100 mg/mL of at least one excipient compound, the viscosity-reducing amount is from about 1 mM to about 400 mM of at least one excipient compound, or the viscosity-reducing amount is is in an amount of about 2 mM to about 150 mM, or the viscosity-reducing amount is in an amount of about 2 mg/mL to about 80 mg/mL, or the viscosity-reducing amount is in an amount of about 5 mg/mL to about 50 mg/mL, or - The reduction amount is an amount from about 10 mg/mL to about 40 mg/mL. In an embodiment, the viscosity-reducing amount is from about 2 mM to about 150 mM, or from about 5 mM to about 100 mM, or from about 10 mM to about 75 mM, or from about 15 mM to about 50 mM. to be. In an embodiment, the carrier solution comprises an additional agent selected from the group consisting of preservatives, sugars, polyols, polysaccharides, arginine, proline, surfactants, stabilizers, and buffers.

[0072] Without wishing to be bound by theory, it is believed that the excipient compounds described herein associate with a particular fragment, sequence, structure, or section of a therapeutic protein that would otherwise be involved in an interparticle (ie, protein-protein) interaction. Association of such excipient compounds with therapeutic or non-therapeutic proteins can mask the protein-protein interactions so that the protein can be formulated at high concentrations without causing excessive solution viscosity. In an embodiment, an excipient compound can result in a more stable protein-protein interaction; Protein-protein interactions can be measured by the protein diffusion parameter kD, or the osmotic second virial coefficient B22, or by other techniques familiar to those skilled in the art.

[0073] The excipient compounds may advantageously be water-soluble and therefore suitable for use with aqueous vehicles. In an embodiment, the excipient compound has a solubility in water of >1 mg/mL. In an embodiment, the excipient compound has a water solubility of >10 mg/mL. In an embodiment, the excipient compound has a water solubility of >100 mg/mL. In an embodiment, the excipient compound has a water solubility of >500 mg/mL.

[0074] Advantageously, for therapeutic protein formulations, the excipient compounds may be derived from biologically acceptable and non-immunogenic substances and are therefore suitable for pharmaceutical use. In a therapeutic embodiment, the excipient compound can be metabolized in the body to produce a biologically compatible non-immunogenic by-product.

a. Excipient compound category 1: hindered or modified amines

[0075] High concentration solutions of therapeutic or non-therapeutic proteins can be formulated with small molecules of impaired or modified amines as excipient compounds. As used herein, the term “hindered amine” refers to a small molecule containing at least one bulky or sterically hindered group, consistent with the examples below. The term “modified amine,” as used herein, refers to a small molecule containing an amine functionality but without bulky or sterically hindered groups, as described in more detail below. The hindered or modified amines can be used in the free base form, in the protonated form, or a combination of the two. In the protonated form, the hindered or modified amine can be associated with an anionic counterion such as chloride, hydroxide, bromide, iodide, fluoride, formate, phosphate, sulfate, or carboxylate.

[0076] Hindered amine compounds useful as excipient compounds include secondary amines, tertiary amines, quaternary ammonium, pyridinium, pyrrolidone, pyrrolidine, piperidine, morpholine, or guanidinium groups. Hindered amine compounds also contain at least one bulky or sterically hindered group, such as a cyclic aromatic, cycloaliphatic, cyclohexyl, or alkyl group. In embodiments, the sterically hindered group may itself be an amine group, such as a dialkylamine, trialkylamine, guanidinium, pyridinium, or quaternary ammonium group. Without being bound by theory, it is believed that hindered amine compounds associate with aromatic sections of proteins such as phenylalanine, tryptophan, and tyrosine by cationic pi interactions. In an embodiment, the cationic group of a sterically hindered amine may have an affinity for the electron-rich pi structure of an aromatic amino acid residue in the protein, such that they may mask this section of the protein, whereby such masked protein associates and It can reduce the tendency to agglomerate.

[0077] In an embodiment, the hindered amine excipient compound is an imidazole, imidazoline, or imidazolidine group, or a salt thereof, such as imidazole, 1-methylimidazole, 4-methylimidazole, 1-hexyl- 3-methylimidazolium chloride, 1-ethylimidazole, 4-ethylimidazole, 1-hexyl-3-ethylimidazolium chloride, imidazoline, 2-imidazoline, imidazolidone, 2 -Imidazolidone, histamine, 4-methylhistamine, alpha-methylhistamine, betahistine, beta-alanine, 2-methyl-2-imidazoline, 1-butyl-3-methylimidazolium chloride, butylimi It has a chemical structure that includes dazole, uric acid, potassium urate, betaazole, carnosine, spermine, spermidine, aspartame, saccharin, acesulfame potassium, xanthine, theophylline, theobromine, caffeine, and anserine. In an embodiment, the hindered amine excipient compound is pyrimidine, pyrimidinone, triaminopyrimidine, 1,3-dimethyluracil, 1-methyluracil, 3-methyluracil, 1,3-diethyluracil, 6-methyluracil , uracil, 1,3-dimethyl-tetrahydropyrimidinone, thymine, 1-methylthymine, O-4-methylthymine, 1,3-dimethylthymine, dimethylthymine dimer, theacrine, cytosine, 5-methylcytosine , 3-methylcytosine, purine, guanine, and a pyrimidine selected from the group consisting of adenine. In another embodiment, the hindered amine excipient compound is selected from the group consisting of 1-methyl-2-pyrrolidone, phenylserine, DL-3-phenylserine, cycloserine, dicyclomine, and cysteamine. In an embodiment, the hindered amine excipient compound is guanidinoacetate, dimethylethanolamine, ethanolamine, dimethylaminoethanol, dimethylaminopropylamine, triethanolamine, 1,3-diaminopropane, 1,2-diaminopropane, poly Etheramine, Jeffamine® brand polyetheramine, polyether-monoamine, polyether-diamine, polyether-triamine, 1-(1-adamantyl)ethylamine, hordenine, benzylamine, dimethylbenzylamine, dimethyl Cyclohexylamine, diethylcyclohexylamine, dicyclohexylmethylamine, hexamethylene biguanide, poly(hexamethylene biguanide), imidazole, dimethylglycine, meglumine, agmatine, agmatine sulfate, diazabicyclo [2.2.2] Octane, tetramethylethylenediamine, N,N-dimethylethanolamine, ethanolamine phosphate, glucosamine, choline chloride, phosphocholine, niacinamide, isonicotinamide, N,N-diethyl nicotinamide, nicotinic acid , nicotinic acid sodium salt, isonicotinic acid, tyramine, N-methyltyramine, 3-aminopyridine, 4-aminopyridine, 2,4,6-trimethylpyridine, 3-pyridine methanol, dipyridamole, nicotinamide adenosine dinucleotide, biotin , folic acid, folinic acid, folinic acid calcium salt, morpholine, N-methylpyrrolidone, 2-pyrrolidinone, procaine, lidocaine, dicyandiamide-taurine adduct, 2-pyridylethylamine, dicyandiamide -benzyl amine adduct, dicyandiamide-alkylamine adduct, dicyandiamide-cycloalkylamine adduct, and dicyandiamide-aminomethanephosphonic acid adduct. In an embodiment, hindered amine compounds consistent with the present disclosure are formulated as protonated ammonium salts. In an embodiment, hindered amine compounds consistent with the present disclosure are formulated as salts with either an inorganic anion or an organic anion as a counterion. In an embodiment, a high concentration solution of a therapeutic or non-therapeutic protein is formulated with a combination of caffeine as an excipient compound and benzoic acid, hydroxybenzoic acid, or benzenesulfonic acid. In an embodiment, the hindered amine excipient compound is metabolized in the body to produce a biologically compatible byproduct. In some embodiments, the hindered amine excipient compound is present in the formulation at a concentration of about 250 mg/ml or less. In a further embodiment, the hindered amine excipient compound is present in the formulation at a concentration of about 10 mg/ml to about 200 mg/ml, or a concentration of about 10 mg/ml to about 120 mg/ml. In yet a further embodiment, the hindered amine excipient compound is present in the formulation at a concentration of about 20 mg/ml to about 120 mg/ml.

[0078] In embodiments, viscosity-reducing excipients of this hindered amine category may include methylxanthines such as caffeine and theophylline, although their use has typically been limited due to their low water solubility. In some applications, it may be advantageous to have higher concentrated solutions of these viscosity-reducing excipients despite their low water solubility. For example, in processing, it may be advantageous to have a concentrated excipient solution that can be added to the concentrated protein solution so that the addition of the excipient does not dilute the protein below the desired final concentration. In other cases, notwithstanding its low water solubility, additional viscosity-reducing excipients may be required to achieve the desired viscosity reduction, stability, isotonicity, etc. of the final protein formulation. In an embodiment, the highly concentrated solution of excipient is (i) as a viscosity-reducing excipient at a concentration of 1.5 to 50 times higher than the effective viscosity-reducing amount, or (ii) 1.5 to 1.5 to its literature-reported solubility in pure water at 298 K. Formulated as 50-fold higher concentration of viscosity-reducing excipient (eg, as reported in The Merck Index; Royal Society of Chemistry; Fifteenth Edition, (April 30, 2013)), or both can be

[0079] It has been found that certain co-solutes substantially increase the solubility limit of these low solubility viscosity-reducing excipients, allowing excipient solutions at concentrations several times higher than the solubility values reported in the literature. These co-solutes can be classified into the general category of hydrotropes. The co-solutes found to provide the greatest improvement in solubility for these applications are generally very soluble in water (>0.25 M) at ambient temperature and physiological pH and contain pyridine or benzene rings. Examples of compounds that may be useful as co-solutes include aniline HCl, isoniacinamide, niacinamide, n-methyltyramine HCl, phenol, procaine HCl, resorcinol, saccharin calcium salt, saccharin sodium salt, sodium aminobenzoic acid, Sodium benzoate, sodium parahydroxybenzoate, sodium metahydroxybenzoate, sodium 2,5-dihydroxybenzoate, sodium salicylate, sodium sulfanilate, sodium parahydroxybenzene sulfonate, synephrine, and tyramine HCl.

[0080] In embodiments, certain hindered amine excipient compounds may have other pharmacological properties. As an example, xanthine is a category of hindered amines that, when absorbed systemically, have independent pharmacological properties, including stimulant properties and bronchodilator properties. Representative xanthines include caffeine, aminophylline, 3-isobutyl-1-methylxanthine, paraxanthine, pentoxifylline, theobromine, theophylline, and the like. Methylated xanthine is understood to affect cardiac contractility, heart rate, and bronchodilation. In some embodiments, the xanthine excipient compound is present in the formulation at a concentration of about 30 mg/ml or less.

[0081] Another category of hindered amines with independent pharmacological properties are local injectable anesthetic compounds. Locally injectable anesthetic compounds are hindered amines having a three-component molecular structure of (a) a lipophilic aromatic ring, (b) an intermediate ester or amide linkage, and (c) a secondary or tertiary amine. It is understood that this category of hindered amines interferes with nerve conduction by inhibiting the influx of sodium ions to induce local anesthesia. The lipophilic aromatic ring for the local anesthetic compound may be formed of carbon atoms (eg, a benzene ring) or may contain heteroatoms (eg, a thiophene ring). Representative local injectable anesthetic compounds are amylocaine, articaine, bupivicaine, butacaine, butanilicaine, chlorprocaine, cocaine, cyclomethicaine, dimethocaine, editokaine, hexylcaine, isobucaine, levo bupivacaine, lidocaine, metabutetamine, metabutoxycaine, mepivacaine, mepricaine, propoxycaine, prilocaine, procaine, piperocaine, tetracaine, trimecaine, etc. not limited Locally injectable anesthetic compounds can have a number of advantages in formulations for protein therapy, such as reduced viscosity, improved stability, and reduced pain upon injection. In some embodiments, the local anesthetic compound is present in the formulation at a concentration of about 50 mg/ml or less.

[0082] In an embodiment, hindered amines with independent pharmacological properties are used as excipient compounds according to the formulations and methods described herein. In some embodiments, an excipient compound with independent pharmacological properties has no pharmacological effect and/or is present in an amount that is not therapeutically effective. In other embodiments, an excipient compound having independent pharmacological properties has a pharmacological effect and/or is present in a therapeutically effective amount. In certain embodiments, a hindered amine having independent pharmacological properties is used in combination with another excipient compound selected to reduce formulation viscosity, wherein the hindered amine having independent pharmacological properties benefits from its pharmacological activity. used to give For example, a locally injectable anesthetic compound can be used to reduce formulation viscosity and also to reduce pain upon injection of the formulation. The reduction in injection pain may be caused by the anesthetic properties; Also, a lower injection force may be required when the viscosity is reduced by the excipient. Alternatively, a locally injectable anesthetic compound may be used in combination with another excipient compound that reduces the viscosity of the formulation while conferring the desired pharmacological advantage of reduced local sensation during formulation injection.

[0083] In other embodiments, the viscosity-reducing excipient is selected based on its physiological impact on a potential patient or its deficiency. For example, certain substituted phenethylamines are understood to modulate a variety of neurotransmitters, such as the monoamine neurotransmitter system, which, due to their effect on the central nervous system, has a variety of psychoactive effects (e.g., stimulants, hallucinogens or hallucinogens), but do not produce a psychotropic effect, do not produce a clinically problematic psychotropic effect, or cause a psychotropic effect in a dose-related manner but have a psychotropic effect at dosages found in certain formulations. It may be desirable to use a viscosity-reducing amount of a viscosity-reducing phenethylamine excipient that can cause Similarly, it does not produce other physiological effects (eg, cardiovascular, respiratory, gastrointestinal, genitourinary, etc.), does not produce clinically problematic physiological effects, or does not produce physiological effects in a dose-related manner. It may be desirable to use other viscosity-reducing excipients that cause but do not have a physiological effect at the dosages found in certain formulations.

[0084] In an embodiment, the modified amine excipient may be a conjugate acid of a weak base. As used herein, the term “conjugate acid” refers to a protonated form of a base having some acidity; Thus, such molecules are known as conjugate acids of bases. Weak bases in which the conjugate acid is included as a modified amine excipient include, for example, ammonia (NH ₃ ), ammonium hydroxide (NH ₄ OH), alkanolamines, and amines. Modified amine excipients can be produced by reacting such weak bases with acids to form their conjugate acids. Exemplary modified amine excipients include conjugate acids of such weak bases, such as ammonium chloride, ammonium bromide, ammonium fluoride, ammonium acetate, ammonium citrate, monoethanolamine hydrochloride, monoethanolamine hydrobromide, monoethanolamine hydrofluoride , monoethanolamine acetate, monoethanolamine citrate, diethanolamine hydrochloride, diethanolamine hydrobromide, diethanolamine hydrofluoride, diethanolamine acetate, diethanolamine citrate, triethanolamine hydrochloride, triethanolamine hydro bromide, triethanolamine hydrofluoride, triethanolamine acetate, triethanolamine citrate, urea hydrochloride, urea hydrobromide, urea hydrofluoride, urea acetate, urea citrate. In an embodiment, the weak base is ammonia or ammonium hydroxide and the modified amine is its conjugate acid. In certain embodiments, the weak base is an ethoxylated amine, such as monoethanolamine, diethanolamine, and triethanolamine, and the modified amine is its conjugate acid.

[0085] In an embodiment, the monoethanolamine weak base may be generated by a synthetic route by reacting ammonia with ethylene oxide, or it may be produced by decarboxylation of serine or phosphatidylserine, optionally with the aid of a decarboxylase enzyme. can In another embodiment, the weak base is urea. The weak base may be modified with an acid (eg, HF, HCl, HBr, H ₃ PO ₄ , H ₂ SO ₄ , acetic acid, citric acid, etc.) to form a conjugate acid excipient prior to introduction into a solution containing the protein. In another embodiment, the weak base can be modified with an acid to form a modified amine excipient in the presence of the protein in solution.

[0086] Modified amines, such as those disclosed above, are particularly useful for reducing the viscosity of formulations comprising pegylated proteins. Without wishing to be bound by theory, the protonated amine group alters the hydrogen bonding and solvation of the PEG segment of the PEGylated protein, causing a conformational change in the backbone of the PEG chain and altering the PEG solution structure to decrease solution viscosity. It is considered to be fundamentally transformative.

b. Excipient Compound Category 2: Aromatic and Anionic Aromatics

[0087] High concentration solutions of therapeutic or non-therapeutic proteins may be formulated with aromatic small molecule compounds as excipient compounds. The aromatic excipient compound may contain aromatic functional groups such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, phenol, hydroxyaryl, heteroaromatic groups, or fused aromatic groups. Aromatic excipient compounds may also contain functional groups such as carboxylates, oxides, phenoxides, sulfonates, sulfates, phosphonates, phosphates, or sulfides. Aromatic excipients may be anionic, cationic, or uncharged.

[0088] Charged aromatic excipients may be described as acids, bases or salts (where applicable), which may exist in various salt forms. Without wishing to be bound by theory, it is believed that charged aromatic excipient compounds are bulky sterically hindered molecules capable of associating with oppositely charged segments of the protein, thereby masking these sections of the protein, thereby rendering the protein-containing formulation viscous. It can reduce the interactions between the protein molecules it makes. For example, anionic aromatic excipients can associate with the cationic segments of the protein, thereby masking these sections of the protein, reducing interactions between protein molecules that render the protein-containing formulation viscous.

[0089] In embodiments, examples of aromatic excipient compounds include anionic aromatic compounds such as salicylic acid, aminosalicylic acid, hydroxybenzoic acid, aminobenzoic acid, para-aminobenzoic acid, benzenesulfonic acid, hydroxybenzenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid Phonic acid, hydroquinone sulfonic acid, sulfanilic acid, vanillic acid, vanillin, vanillin-taurine adduct, aminophenol, anthranilic acid, cinnamic acid, coumaric acid, caffeic acid, isonicotinic acid, folic acid, folinic acid, folinic acid calcium salt, phenyl Serine, DL-3-phenylserine, adenosine monophosphate, deoxyadenosine, guanosine, deoxyguanosine, indole acetic acid, potassium urate, furan dicarboxylic acid, furan-2-acrylic acid, 2-furanpropionic acid, sodium phenylpyru baits, sodium hydroxyphenylpyruvate, dihydroxybenzoic acid, trihydroxybenzoic acid, pyrogallol, benzoic acid, and salts of these acids. In an embodiment, the anionic aromatic excipient compound is formulated in the form of an ionized salt. In an embodiment, the anionic aromatic compound is formulated as a salt of a hindered amine such as dimethylcyclohexylammonium hydroxybenzoate. In an embodiment, the anionic aromatic excipient compound is formulated with various counterions, such as organic cations. In an embodiment, a high concentration solution of a therapeutic or non-therapeutic protein is formulated with an anionic aromatic excipient compound and caffeine. In an embodiment, the anionic aromatic excipient compound is metabolized in the body to produce a biologically compatible by-product.

[0090] In an embodiment, examples of aromatic excipient compounds include phenols and polyphenols. As used herein, the term "phenol" refers to an organic molecule consisting of at least one aromatic group or fused aromatic group bonded to at least one hydroxyl group, and the term "polyphenol" refers to an organic molecule consisting of more than one phenol group do. Such excipients may be advantageous in certain circumstances, for example, used in formulations with high concentration solutions of therapeutic or non-therapeutic pegylated proteins to lower the solution viscosity. Non-limiting examples of phenols include benzenediol resorcinol (1,3-benzenediol), catechol (1,2-benzenediol) and hydroquinone (1,4-benzenediol), benzenetriol hydroxyquinol (1,2,4-benzenetriol), pyrogallol (1,2,3-benzenetriol), and phloroglucinol (1,3,5-benzenetriol), benzeneterol 1,2, 3,4-benzeneterol and 1,2,3,5-benzeneterol, and benzenepentol and benzenehexol. Non-limiting examples of polyphenols include tannic acid, ellagic acid, epigallocatechin gallate, catechins, tannins, ellagitannins, and gallotannins. More generally, phenolic and polyphenolic compounds include, but are not limited to, flavonoids, lignans, phenolic acids, and stilbenes. Flavonoid compounds include, but are not limited to, anthocyanins, chalcones, dihydrochalcones, dihydroflavanols, flavanols, flavanones, flavones, flavonols, and isoflavonoids. Phenolic acids include, but are not limited to, hydroxybenzoic acid, hydroxycinnamic acid, hydroxyphenylacetic acid, hydroxyphenylpropanoic acid, and hydroxyphenylpentanoic acid. Other polyphenol-based compounds include alkylmethoxyphenol, alkylphenol, curcuminoid, hydroxybenzaldehyde, hydroxybenzoketone, hydroxycinnamaldehyde, hydroxycoumarin, hydroxyphenylpropene, methoxyphenol, naphthoquinone. , hydroquinone, phenol terpenes, resveratrol, and tyrosol. In an embodiment, the polyphenol is tannic acid. In an embodiment, the phenol is gallic acid. In an embodiment, the phenol is pyrogallol. In an embodiment, the phenol is resorcinol. Without wishing to be bound by theory, the hydroxyl groups of phenolic compounds such as gallic acid, pyrogallol, and resorcinol form hydrogen bonds with ether oxygen atoms in the backbone of the PEG chain, thereby reducing the solution viscosity. A phenol/PEG complex is formed that radically alters the structure of the PEG solution.

Polyphenolic compounds such as tannic acid derive their viscosity-reducing properties from their respective phenolic group building blocks such as gallic acid, pyrogallol, and resorcinol. The specific organization of phenolic groups in polyphenolic compounds can lead to complex behavior in which the viscosity reduction achieved by the addition of phenol is enhanced by the addition of smaller amounts of each polyphenol.

c. Excipient Compound Category 3: Functionalized Amino Acids

[0091] High concentration solutions of therapeutic or non-therapeutic proteins may be formulated with one or more functionalized amino acids, wherein a single functionalized amino acid or an oligopeptide comprising one or more functionalized amino acids may be used as an excipient compound. . In an embodiment, a functionalized amino acid compound comprises a molecule capable of being hydrolyzed or metabolized to produce an amino acid (“amino acid precursor”). In embodiments, the functionalized amino acid may contain an aromatic functional group such as a phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl, hydroxyaryl, heteroaromatic group, or fused aromatic group. In an embodiment, the functionalized amino acid compound may contain an esterified amino acid, such as methyl, ethyl, propyl, butyl, benzyl, cycloalkyl, glyceryl, hydroxyethyl, hydroxypropyl, PEG, and PPG esters. have. In an embodiment, the functionalized amino acid compound is selected from the group consisting of arginine ethyl ester, arginine methyl ester, arginine hydroxyethyl ester, and arginine hydroxypropyl ester. In an embodiment, the functionalized amino acid compound is a charged ionic compound in an aqueous solution at neutral pH. For example, a single amino acid can be derivatized by forming an ester such as acetate or benzoate, and the hydrolysis product will be acetic acid or benzoic acid, both of which are natural, and an amino acid. In an embodiment, the functionalized amino acid excipient compound is metabolized in the body to produce a biologically compatible by-product.

d. excipient compound category 4: oligopeptides

[0092] High concentration solutions of therapeutic or non-therapeutic proteins can be formulated into oligopeptides as excipient compounds. In an embodiment, the oligopeptide is designed so that the structure has a charged section and a bulky section. In an embodiment, the oligopeptide consists of 2 to 10 peptide subunits. The oligopeptide may be a bifunctional, eg, a cationic amino acid coupled to a non-polar amino acid, or an anionic amino acid coupled to a non-polar amino acid. In an embodiment, the oligopeptide consists of 2 to 5 peptide subunits. In an embodiment, the oligopeptide is a homopeptide, such as polyglutamic acid, polyaspartic acid, poly-lysine, poly-arginine, and poly-histidine. In an embodiment, the oligopeptide has a net cationic charge. In another embodiment, the oligopeptide is a heteropeptide such as Trp2Lys3. In embodiments, the oligopeptide may have an alternating structure, such as an ABA repeat pattern. In an embodiment, the oligopeptide may contain both anionic and cationic amino acids, eg, Arg-Glu. Without wishing to be bound by theory, oligopeptides comprise structures capable of associating with proteins in a manner that reduces intermolecular interactions resulting in highly viscous solutions; For example, oligopeptide-protein association may be a charge-charge interaction, which may leave slightly non-polar amino acids, interfering with hydrogen bonding in the hydration layer around the protein, thereby lowering viscosity. In some embodiments, the oligopeptide excipient is present in the composition at a concentration of about 50 mg/ml or less.

e. Excipient Compound Category 5: Short Chain Organic Acids

[0093] As used herein, the term "short chain organic acid" refers to C2-C6 organic acid compounds and salts, esters, or lactones thereof. This category includes saturated and unsaturated carboxylic acids, hydroxyl functionalized carboxylic acids, and linear, branched, or cyclic carboxylic acids. In an embodiment, the acid group in the short chain organic acid is a carboxylic acid, a sulfonic acid, a phosphonic acid, or a salt thereof.

[0094] In addition to the above four excipient categories, high concentration solutions of therapeutic or non-therapeutic proteins can be used as excipient compounds of short-chain organic acids, such as acids of sorbic acid, valeric acid, propionic acid, glucuronic acid, caproic acid, and ascorbic acid, or It can be formulated with salt form. Examples of excipient compounds in this category include potassium sorbate, taurine, sodium propionate, calcium propionate, magnesium propionate, and sodium ascorbate.

f. Excipient compound category 6: low molecular weight aliphatic polyacids

[0095] High concentration solutions of therapeutic or non-therapeutic pegylated proteins can be formulated with certain excipient compounds that allow for lower solution viscosities, wherein such excipient compounds are low molecular weight aliphatic polyacids. As used herein, the term “low molecular weight aliphatic polyacid” refers to an organic aliphatic polyacid having a molecular weight of less than about 1500 and having at least two acidic groups, wherein the acidic group is understood to be a proton-donating moiety. The acidic group can be in the protonated acid form, in the salt form, or a combination thereof. Non-limiting examples of acidic groups include carboxylate, phosphonate, phosphate, sulfonate, sulfate, nitrate, and nitrite groups. The acidic groups on the low molecular weight aliphatic polyacid can be in the form of anionic salts such as carboxylates, phosphonates, phosphates, sulfonates, sulfates, nitrates, and nitrites; Their counterions may be sodium, potassium, lithium, and ammonium. Specific examples of low molecular weight aliphatic polyacids useful for interacting with pegylated proteins as described herein include maleic acid, tartaric acid, glutaric acid, malonic acid, itaconic acid, citric acid, ethylenediaminetetraacetic acid (EDTA), aspartic acid , glutamic acid, alendronic acid, etidronic acid and salts thereof. Further examples of low molecular weight aliphatic polyacids in their anionic salt form are phosphate (PO ₄ ^3- ), hydrogen phosphate (HPO ₄ ^3- ), dihydrogen phosphate (H ₂ PO ₄ ^- ), sulfate (SO ₄ ^2- ), bisulfate (HSO ₄ ^- ), pyrophosphate (P ₂ O ₇ ^4- ), hexametaphosphate, carbonate (CO ₃ ^2- ), and bicarbonate (HCO ₃ ^- ). The counterion for the anionic salt may be a Na, Li, K, or ammonium ion. Such excipients may also be used in combination with excipients. As used herein, a low molecular weight aliphatic polyacid can also be an alpha hydroxy acid, wherein there are hydroxyl groups adjacent to a first acidic group, such as glycolic acid, lactic acid, and gluconic acid and salts thereof. In an embodiment, low molecular weight aliphatic polyacids are oligomeric forms having more than two acidic groups, eg, polyacrylic acid, polyphosphates, polypeptides and salts thereof. In some embodiments, the low molecular weight aliphatic polyacid excipient is present in the composition at a concentration of about 50 mg/ml or less.

g. Excipient compound category 7: dione and sulfone

[0096] An effective viscosity-reducing excipient can be a molecule containing a sulfone, sulfonamide, or dione functional group that is soluble in pure water at 298K to at least 1 g/L and has a net neutral charge at pH 7. Preferably, the molecule has a molecular weight of less than 1000 g/mol and more preferably less than 500 g/mol. Diones and sulfones effective in reducing viscosity have multiple double bonds, are water soluble, have no net charge at pH 7, and are not strong hydrogen bond donors. Without wishing to be bound by theory, the double bond nature may enable weak pie-stacking interactions with proteins. In an embodiment, in high protein concentrations and in proteins that produce high viscosity only at high concentrations, charged excipients are not effective because the electrostatic interactions are longer range interactions. Solvated protein surfaces are primarily hydrophilic, making them water-soluble. While hydrophobic regions of proteins are usually masked within their three-dimensional structure, their structures are constantly evolving, unfolding, and refolding (sometimes referred to as “respiration”), and hydrophobic regions of adjacent proteins come into contact with each other and interact with each other in hydrophobic interactions. may lead to aggregation. The pie-stacking nature of dione and sulfone excipients can mask hydrophobic patches that may be exposed during this "breathing". Another important role of excipients is to disrupt hydrophobic interactions and hydrogen bonds between proteins in close proximity, which will effectively reduce solution viscosity. Dione and sulfone compounds suitable for the present description include dimethylsulfone, ethyl methyl sulfone, ethyl methyl sulfonyl, ethyl isopropyl sulfone, bis(methylsulfonyl)methane, methane sulfonamide, methionine sulfone, sodium bisulfite, menadione sodium bi sulfite, 1,2-cyclopentanedione, 1,3-cyclopentanedione, 1,4-cyclopentanedione, and butane-2,3-dione.

h. Excipient Compound Category 8: Zwitterionic Excipients

[0097] Solutions of therapeutic or non-therapeutic proteins may be formulated with certain zwitterionic compounds as excipients to improve stability or reduce viscosity. As used herein, the term “zwitterionic” refers to a compound having a cationic charged section and an anionic charged section. In an embodiment, the zwitterionic excipient compound is an amine oxide. In an embodiment, opposite charges are separated from each other by 2 to 8 chemical bonds. In an embodiment, the zwitterionic excipient compound may be a small molecule, such as having a molecular weight of from about 50 to about 500 g/mol, or may be a medium molecular weight molecule, such as having a molecular weight of from about 500 to about 2000 g/mol, or It may be a high molecular weight molecule, such as a polymer having a molecular weight of about 2000 to about 100,000 g/mol.

Examples of zwitterionic excipient compounds include (3-carboxypropyl) trimethylammonium chloride, 1-aminocyclohexane carboxylic acid, homocycloleucine, 1-methyl-4-imidazoleacetic acid, 3-(1-pyridinio)-1 -Propanesulfonate, 4-aminobenzoic acid, alendronate, aminoethyl sulfonic acid, aminohipuric acid, aspartame, aminotris (methylenephosphonic acid) (ATMP), calcobutrol, calteridol, cocamidopropyl butane, coca Midopropyl hydroxysultaine, creatine, cytidine, cytidine monophosphate, diaminopimelic acid, diethylenetriaminepentaacetic acid, dimethyl phenylalanine, methylglycine, sarcosine, dimethylglycine, zwitterionic dipeptides (e.g. , Arg-Glu, Lys-Glu, His-Glu, Arg-Asp, Lys-Asp, His-Asp, Glu-Arg, Glu-Lys, Glu-His, Asp-Arg, Asp-Lys, Asp-His), Diethylenetriamine penta(methylene phosphonic acid) (DTPMP), dipalmitoyl phosphatidylcholine, ectoin, ethylenediamine tetra(methylenephosphonic acid) (EDTMP), folate benzoate mixture, folate niacinamide mixture, gelatin, hydroxy Proline, iminodiacetic acid, isogubacin, lecithin, myristamine oxide, nicotinamide adenine dinucleotide (NAD), aspartic acid, N-methyl aspartic acid, N-methylproline, lysine, N-trimethyl lysine, orni Tin, oxolinic acid, lysendronate, allyl cysteine, S-allyl-L-cysteine, somafacitan, taurine, theanine, trigonelline, vivatrin, ectoin, 4-(2-hydroxyethyl)-1 -Piperazineethanesulfonate, o-octylphosphorylcholine, nicotinamide mononucleotide, triglycine, tetraglycine, β-guanidinopropionic acid, 5-aminolevulinic acid hydrochloride, picolinic acid, lidophenin, phospho Choline, 1- (5-carboxypentyl) -4-methylpyridin-1-ium bromide, L-anserine nitrate, L-glutathione reduced form, N-ethyl-L-glutamine, N-methyl proline, (Z) -1-[N-(2-aminoethyl)-N-(2-aminoethyl)amino]diazen-1-ium- 1,2-diolate (DETA-NONOate), (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2- diolate (DPTA-NONate), and zoledronic acid.

[0099] Without wishing to be bound by theory, the zwitterionic excipient compound may interact with the protein by interaction with the protein, for example, by charge interactions, hydrophobic interactions, and steric interactions, such that the protein may inhibit aggregation. reducing viscosity or stabilizing effect by making it more resistant, or by affecting the bulk properties of water in the protein formulation, such as electrolyte contribution, reduced surface tension, change in the amount of unbound water available, or change in dielectric constant can apply

i. Excipient Compound Category 9: Crowding Agents with Hydrogen Bonding Elements

[00100] Solutions of therapeutic or non-therapeutic proteins may be formulated with crowding agents having hydrogen bonding elements as excipients to improve stability or reduce viscosity. As used herein, the term “crowding agent” refers to a formulation additive that increases the effective protein concentration by reducing the amount of water available to dissolve the protein in solution. In an embodiment, a crowding agent can reduce protein particle size or reduce the amount of protein unfolding in solution. In embodiments, the crowding agent may act as a solvent modifier causing the structuring of water by hydrogen bonding and hydration effects. In an embodiment, a crowding agent can reduce the amount of intermolecular interactions between proteins in solution. In an embodiment, the crowding agent has a structure containing at least one hydrogen bond donor element, such as an oxygen, sulfur, or hydrogen attached to a nitrogen atom. In an embodiment, the crowding agent has a structure containing at least one weakly acidic hydrogen bond donor element having a pKa of from about 6 to about 11. In an embodiment, the crowding agent has a structure containing from about 2 to about 50 hydrogen bond donor elements. In an embodiment, the crowding agent has a structure containing at least one hydrogen bond acceptor element, such as a Lewis base. In an embodiment, the crowding agent has a structure containing from about 2 to about 50 hydrogen bond acceptor elements. In an embodiment, the crowding agent has a molecular weight of about 50 to 500 g/mol. In an embodiment, the crowding agent has a molecular weight of about 100 to 350 g/mol. In other embodiments, the crowding agent may have a molecular weight greater than 500 g/mol, for example, raffinose, inulin, pullulan, or synistrin.

Examples of crowding agent excipients having a hydrogen bonding element are 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidione, 15-crown-5, 18-crown-6, 2 -Butanol, 2-butanone, 2-phenoxyethanol, acetaminophen, allatoin, arabinose, meglumine, arabitol, benzyl acetone acetate, benzyl alcohol, chlorobutanol, cholestanol tetraacetyl-b-gluco Cid, cinnamaldehyde, cyclohexanone, deoxyribose, diethyl carbonate, dimethyl carbonate, dimethyl isosorbide, dimethylacetamide, dimethylformamide, dimethylol ethylene urea, dimethyluracil, epilactose, erythritol, erythrose , ethyl lactate, ethyl maltol, ethylene carbonate, formamide, fucose, galactose, genistein, gentisic acid ethanolamide, gluconolactone, glyceraldehyde, glycerol, glycerol carbonate, glycerol formal, glycerol urethane, glycyrrhizic acid , gosipine, harpagoside, hederacoside C, icodextrin, iditol, imidazolidine, inositol, inulin, isomaltitol, kojic acid, lactitol, lactobionic acid, lactose, lactulose, lyxos, made Casoside, maltotriose, mamaniferin, mannose, melgitose, methyl lactate, methylpyrrolidone, mogroside V, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid, N- Methyl acetamide, N-methyl formamide, N-methyl propionamide, pentaerythritol, pinoresinol diglucoside, glucuronic acid, piracetam, propyl gallate, propylene carbonate, psicose, pullulan, pyrogallol , quinic acid, raffinose, rebaudioside A, rhamnose, ribitol, ribose, ribulose, saccharin, sedoheptulose, sinistrin, solketal, stachiose, sucralose, tagatose, t-butanol, tetraglycol, Triacetin, N-acetyl-d-mannosamine, nystose, kestose, turanose, acarbose, D-saccharic acid 1,4-lactone, thiodigalactoside, fucoidan, hydroxysaflor yellow A, shikimic acid, diosmin, pravastatin sodium salt, D-altrose, L-gulonic acid gamma-lactone, neomycin, ruburocy De-dihydroartemisinin, phloroglucinol, naringin, baicalin, hesperidin, apigenin, pyrogallol, morin, salsalate, camphorol, myricetin, 3',4',7-trihydroxyiso Flavones, (±)-taxifolin, silybin, perceitol diformal, 4-hydroxyphenylpyruvic acid, sulfacetamide, isopropyl β-D-1-thiogalactopyranoside, ethyl 2,5- Dihydrobenzoate, spectinomycin, leveratrol, quercetin, kanamycin sulfate, 1-(2-pyrimidinyl)piperazine, 2-(2-pyridyl)ethylamine, 2-imidazolidone, DL- 1,2-isopropylideneglycerol, metformin, m-xylylenediamine, x-xylylenediamine, demeclocycline, tripropylene glycol, tubeimoside I, verbenaloside, xylitol, and xylose .

6. Protein/Excipient Solutions: Characteristics and Process

[00102] In certain embodiments, a solution of therapeutic or non-therapeutic protein is administered by a protein diffusion interaction parameter, kD, by biolayer interferometry, by surface plasmon resonance, or by a second virial coefficient, B22, or similar method. The above-identified excipient compounds, or combinations thereof (“excipient additives”), such as hindered or modified amines, aromatics, to cause improved protein-protein interaction characteristics or protein self-interactions when measured by It is formulated with functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diones and sulfones, zwitterionic excipients, and crowding agents. As used herein, an “improvement” in one or more protein-protein interaction parameters achieved by a test formulation using the excipient compounds identified above or a combination thereof means that the test formulation does not contain an excipient compound or excipient additive. It may refer to a decrease in attractive protein-protein interactions when compared under conditions with similar formulations that do not. Measurements of KD and B22 can be made using standard techniques in the art and can be indicative of improved solution properties or stability of the protein in solution. For example, high negative kD values may indicate that the protein has strong attractive interactions, which may lead to aggregation, instability, and rheological problems. When formulated in the presence of certain excipient compounds or combinations thereof identified above, the same protein may have an improved proxy parameter with a lower negative kD value, or a kD value close to or greater than zero, such improved proxy The parameter relates to the improvement of the formulation-related parameter.

[00103] In an embodiment, any of the above-described excipient compounds or combinations thereof, such as hindered or modified amines, aromatics, functionalized amino acids, oligopeptides, short chain organic acids, low molecular weight aliphatic polyacids, diones and sulfones, zwitterionic excipients, and/or the crowding agent is filtration, injection, transfer, pumping, mixing, heating or cooling by heat transfer, gas transfer, centrifugation, chromatography, membrane separation, centrifugal concentration, tangential flow filtration, radial flow filtration, axial flow filtration It is used to improve protein-related processes such as preparation, processing, sterile filling, purification, and analysis of protein-containing solutions using processing methods such as , lyophilization, and gel electrophoresis. Such processes and processing methods can have improved efficiency, improved solubility, or improved stability due to the lower viscosity of the protein in solution during the manufacturing, processing, purification, and analytical steps. In addition, equipment-related processes such as cleaning, sterilization, and maintenance of protein processing equipment can be facilitated by the use of the excipients identified above because of reduced contamination, reduced denaturation, reduced viscosity, and improved solubility of proteins, such processes The parameters related to the improvement of , are similarly improved.

Example

[00104] ingredient:

● Bovine Gamma Globulin (BGG), >99% Purity, Sigma Aldrich

● Histidine, Sigma Aldrich

● Other materials described in the examples below were from Sigma Aldrich unless otherwise specified.

Example 1 Preparation of Formulations Containing Excipient Compounds and Test Protein

[00105] Formulations were prepared using an excipient compound and a test protein, wherein the test protein was to simulate a therapeutic protein to be used in a therapeutic formulation, or a non-therapeutic protein to be used in a non-therapeutic formulation. These formulations were prepared in 50 mM histidine with different excipient compounds for viscosity measurement in the following manner. Histidine hydrochloride was first dissolved in 1.94 g of histidine (Sigma-Aldrich, St. Louis, MO) in distilled water and the pH was adjusted to about 6.0 using 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, MO). , prepared by diluting with distilled water to a final volume of 250 mL in a volumetric flask. The excipient compound was then dissolved in 50 mM histidine HCl. A list of excipients is provided in Examples 4, 5, 6, and 7 below. In some cases, the excipient compound was adjusted to pH 6 prior to dissolution in 50 mM histidine HCl. In this case, the excipient compound was first dissolved in deionized water to about 5 wt %, and the pH was adjusted to about 6.0 with hydrochloric acid or sodium hydroxide. The prepared salt solution was then placed in a convection laboratory oven at about 150 degrees Fahrenheit (about 65 degrees C.) to evaporate water and separate the solid excipients. Once the excipient solution in 50 mM histidine HCl was prepared, the test protein (bovine gamma globulin (“BGG”) (Sigma-Aldrich, St. Louis, Mo.)) was dissolved at a rate of about 0.336 g BGG per 1 mL excipient solution. This resulted in a final protein concentration of about 280 mg/mL. A solution of BGG in 50 mM histidine HCl with excipients was formulated in a 20 mL vial and shaken overnight at 100 rpm on an orbital shaker table. The solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 min in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

Example 2: Viscosity Measurement

[00106] Viscosity measurements of formulations prepared as described in Example 1 were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as the viscosity for the sample.

Example 3: Protein concentration measurement

[00107] The concentration of protein in the experimental solution was determined by measuring the absorbance of the protein solution at a wavelength of 280 nm in a UV/VIS spectrometer (Perkin Elmer Lambda 35). First, the instrument was calibrated to zero absorbance with 50 mM histidine buffer at pH 6. Next, the protein solution was diluted 300-fold with the same histidine buffer and the absorbance at 280 nm was recorded. The final concentration of protein in solution was calculated by using an extinction coefficient value of 1.264 mL/(mg x cm).

Example 4: Formulations with hindered amine excipient compounds

[00108] Formulations containing 280 mg/mL of BGG were prepared as described in Example 1, and some samples contained added excipient compounds. In this test, the hydrochloride salts of dimethylcyclohexylamine (DMCHA), dicyclohexylmethylamine (DMACHMA), dimethylpropylamine (TEA), dimethylethanolamine (DMEA), and niacinamide were used as hindered amine excipient compounds as examples. tested. In addition, the hydroxybenzoic acid salt of DMCHA and the taurine-dicyandiamide adduct were tested as examples of hindered amine excipient compounds. The viscosity of each protein solution was measured as described in Example 2 and the results are presented in Table 1 below, showing the advantage of the added excipient compound in reducing the viscosity.

Table 1

Example 5: Formulations with Anionic Aromatic Excipient Compounds

[00109] A formulation of BGG at 280 mg/mL was prepared as described in Example 1, and some samples contained added excipient compounds. The viscosity of each solution was measured as described in Example 2 and the results are presented in Table 2 below, which shows the advantage of the added excipient compound in reducing the viscosity.

Table 2

Example 6: Formulations with Oligopeptide Excipient Compounds

[00110] Oligopeptides (n=5) were synthesized by NeoBioLab Inc. (Woburn, Mass.) with >95% purity with N-terminus free amine and C-terminus free acid. The dipeptide (n=2) was synthesized by LifeTein LLC in 95% purity. A formulation of BGG at 280 mg/mL was prepared as described in Example 1, and some samples contained synthetic oligopeptides as added excipient compounds. The viscosity of each solution was measured as described in Example 2 and the results are presented in Table 3 below, showing the advantage of the added excipient compound in reducing the viscosity.

Table 3

Example 8: Synthesis of Guanyl Taurine Excipient

[00111] Guanyl taurine was prepared according to the following method described in US Pat. No. 2,230,965. 3.53 parts of taurine (Sigma-Aldrich, St. Louis, MO) were mixed with 1.42 parts of dicyandiamide (Sigma-Aldrich, St. Louis, MO) and ground in a mortar and pestle until a homogeneous mixture was obtained. Next, the mixture was placed in a flask and heated at 200° C. for 4 hours. The product was used without further purification.

Example 9: Protein Formulations Containing Excipient Compounds

[00112] Formulations were prepared using an excipient compound and a test protein, wherein the test protein was to simulate a therapeutic protein to be used in a therapeutic formulation, or a non-therapeutic protein to be used in a non-therapeutic formulation. These formulations were prepared in 50 mM aqueous histidine buffer solution with different excipient compounds for viscosity measurement in the following manner. A histidine hydrochloride buffer solution was first dissolved in 1.94 g of histidine (Sigma-Aldrich, St. Louis, MO) in distilled water and the pH was adjusted to about 6.0 using 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, MO). Then, it was prepared by diluting with distilled water to a final volume of 250 mL in a volumetric flask. The excipient compound was then dissolved in 50 mM histidine HCl buffer solution. A list of excipient compounds is provided in Table 4. In some cases, the excipient compound was dissolved in 50 mM histidine HCl and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein. In some cases, the excipient compound was adjusted to pH 6 prior to dissolution in 50 mM histidine HCl. In this case, the excipient compound was first dissolved in deionized water to about 5 wt %, and the pH was adjusted to about 6.0 with hydrochloric acid or sodium hydroxide. The prepared salt solution was then placed in a convection laboratory oven at about 150 degrees Fahrenheit (65° C.) to evaporate water and separate the solid excipients. Once the excipient solution in 50 mM histidine HCl was prepared, the test protein, bovine gamma globulin (Sigma-Aldrich, St. Louis, Mo.) was dissolved at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in 50 mM histidine HCl with excipients was formulated in a 20 mL vial and shaken overnight at 100 rpm on an orbital shaker table. The solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 min in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

[00113] Viscosity measurements of formulations prepared as described above were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as the viscosity for the sample. The viscosity of the solution with excipients was normalized to the viscosity of the model protein solution without excipients. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein solution without excipients.

Table 4

Example 10: Preparation of Formulations Containing Excipient Combinations and Test Proteins

[00114] A formulation was prepared using a primary excipient compound, a secondary excipient compound and a test protein, wherein the test protein was to simulate a therapeutic protein to be used in a therapeutic formulation, or a non-therapeutic protein to be used in a non-therapeutic formulation. The primary excipient compounds were selected from compounds having both anionic and aromatic functionality as listed in Table 5 below. Secondary excipient compounds were selected from compounds having a nonionic or cationic charge and an imidazoline or benzene ring at pH 6 as listed in Table 5 below. Formulations of these excipients were prepared in 50 mM histidine with excipient solutions for viscosity measurement in the following manner. Histidine hydrochloride was first dissolved in 1.94 g of histidine (Sigma-Aldrich, St. Louis, MO) in distilled water and the pH was adjusted to about 6.0 using 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, MO). , prepared by diluting with distilled water to a final volume of 250 mL in a volumetric flask. The individual primary or secondary excipient compounds were then dissolved in 50 mM histidine HCl. The combination of primary and secondary excipients was dissolved in 50 mM histidine HCl and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve pH 6 prior to dissolution of the model protein. Once the excipient solution was prepared as described above, test protein (bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis, Mo.)) was added to each test at a rate to achieve a final protein concentration of about 280 mg/mL. dissolved in the solution. A solution of BGG in 50 mM histidine HCl with excipients was formulated in a 20 mL vial and shaken overnight at 100 rpm on an orbital shaker table. The solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 min in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

[00115] Viscosity measurements of formulations prepared as described above were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as the viscosity for the sample. The viscosity of the solution with excipient was normalized to the viscosity of the model protein solution without excipient and summarized in Table 5 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein solution without excipients. The examples show that the combination of primary and secondary excipients can provide better results than single excipients.

Table 5

Example 11: Preparation of Formulations Containing Excipient Combination and Test Protein

[00116] A formulation was prepared using a primary excipient compound, a secondary excipient compound and a test protein, wherein the test protein was to simulate a therapeutic protein to be used in a therapeutic formulation, or a non-therapeutic protein to be used in a non-therapeutic formulation. The primary excipient compounds were selected from compounds having both anionic and aromatic functionality as listed in Table 6 below. Secondary excipient compounds were selected from compounds having a nonionic or cationic charge and an imidazoline or benzene ring at pH 6 as listed in Table 6 below. Formulations of these excipients were prepared in distilled water for viscosity measurement in the following manner. The combination of primary and secondary excipients was dissolved in distilled water and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve pH 6 prior to dissolution of the model protein. Once the excipient solution in distilled water was prepared, the test protein (bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis, Mo.)) was dissolved at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in distilled water with excipients was formulated in 20 mL vials and shaken overnight at 100 rpm on an orbital shaker table. The solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 min in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

[00117] Viscosity measurements of formulations prepared as described above were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as the viscosity for the sample. The viscosity of the solution with excipient was normalized to the viscosity of the model protein solution without excipient and summarized in Table 6 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein solution without excipients. The examples show that the combination of primary and secondary excipients can provide better results than single excipients.

Table 6

Example 12: Preparation of Formulations Containing Excipient Compounds and PEG

[00118] Materials: All materials were purchased from Sigma-Aldrich (St. Louis, MO). Formulations were prepared using excipient compounds and PEG, where the PEG was intended to simulate a therapeutic pegylated protein to be used in a therapeutic formulation. These formulations were prepared by mixing an equal volume of the PEG solution with the solution of the excipient. Both solutions were prepared in Tris buffer consisting of 10 mM Tris, 135 mM NaCl, and 1 mM trans-cinnamic acid at a pH of 7.3.

[00119] A PEG solution was prepared by mixing 3 g of poly(ethylene oxide) with an average Mw of ~1,000,000 (Aldrich catalog #372781) with 97 g of Tris buffer solution. The mixture was stirred overnight for complete dissolution.

[00120] An example of excipient solution preparation is as follows: Prepare a solution of approximately 80 mg/mL citric acid in Tris buffer by dissolving 0.4 g of citric acid (Aldrich cat. # 251275) in 5 mL of Tris buffer and with a minimum amount of 10 M NaOH The pH was adjusted to 7.3 with the solution.

[00121] A PEG excipient solution was prepared by mixing 0.5 mL of PEG solution with 0.5 mL of excipient solution, and mixing using a vortex for a few seconds. Control samples were prepared by mixing 0.5 mL of PEG solution with 0.5 mL of Tris buffer solution.

Example 13: Measurement of Viscosity of Formulations Containing Excipient Compounds and PEG

[00122] Viscosity measurements of the prepared formulations were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25°C. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as the viscosity for the sample.

[00123] The results presented in Table 7 show the effect of the added excipient compound on reducing the viscosity.

Table 7

Example 14: Preparation of PEGylated BSA with one PEG chain per BSA molecule

[00124] To a beaker was added 200 mL of phosphate buffered saline (Aldrich Cat. # P4417) and 4 g of BSA (Aldrich Cat. # A7906) and mixed with a magnetic bar. Then 400 mg of methoxy polyethylene glycol maleimide, MW = 5,000 (Aldrich Cat. #63187) was added. The reaction mixture was reacted overnight at room temperature. The next day, the reaction was stopped by adding 20 drops of 0.1 M HCl. The reaction product was characterized by SDS-Page and SEC, which clearly indicated pegylated BSA. The reaction mixture was placed in an Amicon centrifuge tube with a molecular weight cutoff (MWCO) of 30,000 and concentrated to a few milliliters. Next, the sample was diluted 20-fold with 50 mM histidine buffer at a pH of approximately 6, and then concentrated until a highly viscous fluid was obtained. The absorbance was measured at 280 nm and the extinction coefficient for BSA of 0.6678 was used to obtain the final concentration of the protein solution. The results indicated that the final concentration of BSA in the solution was 342 mg/mL.

Example 15: Preparation of pegylated BSA with multiple PEG chains per BSA molecule

[00125] A 5 mg/mL solution of BSA (Aldrich A7906) in 25 mM phosphate buffer at a pH of 7.2 was prepared by mixing 0.5 g of BSA with 100 mL of buffer. Next, 1 g of methoxy PEG propionaldehyde Mw=20,000 (JenKem Technology, Plano, TX 75024) and then 0.12 g of sodium cyanoborohydride (Aldrich 156159) were added. The reaction was allowed to proceed overnight at room temperature. The next day, the reaction mixture was diluted 13 times with Tris buffer (10 mM Tris, 135 mM NaCl at pH=7.3) and concentrated using an Amicon centrifuge tube MWCO at 30,000 until a concentration of approximately 150 mg/mL was reached. did it

Example 16: Preparation of pegylated lysozyme with multiple PEG chains per lysozyme molecule

[00126] A 5 mg/mL solution of lysozyme (Aldrich L6876) in 25 mM phosphate buffer at a pH of 7.2 was prepared by mixing 0.5 g of lysozyme with 100 mL of buffer. Next, 1 g of methoxy PEG propionaldehyde Mw=5,000 (JenKem Technology, Plano, TX 75024) and then 0.12 g of sodium cyanoborohydride (Aldrich 156159) were added. The reaction was allowed to proceed overnight at room temperature. The next day, the reaction mixture was diluted 49-fold with phosphate buffer, 25 mM, pH 7.2 and concentrated using a 30,000 Amicon centrifuge tube MWCO. The absorbance was measured at 280 nm and the extinction coefficient for lysozyme of 2.63 was used to obtain the final concentration of the protein solution. The final concentration of lysozyme in the solution was 140 mg/mL.

Example 17: Effect of excipients on the viscosity of PEGylated BSA with one PEG chain per BSA molecule

[00127] Formulations of pegylated BSA with excipients (from Example 14 above) were prepared by adding 6 or 12 mg of excipient salt to 0.3 mL of pegylated BSA solution. The solutions were mixed by gentle shaking and the viscosity was measured by a RheoSense microVisc equipped with an A10 channel (100 microns deep) at a shear rate of 500 sec ⁻¹ . Viscometer measurements were completed at ambient temperature.

[00128] The results presented in Table 8 show the effect of the added excipient compound on reducing the viscosity.

Table 8

Example 18: Effect of excipients on viscosity of PEGylated BSA with multiple PEG chains per BSA molecule

[00129] A formulation of pegylated BSA (from Example 15 above) with Na citrate salt as excipient was prepared by adding 8 milligrams of excipient salt to 0.2 mL of pegylated BSA solution. The solutions were mixed by gentle shaking and the viscosity was measured by a RheoSense microVisc equipped with an A10 channel (100 microns deep) at a shear rate of 500 sec ⁻¹ . Viscometer measurements were completed at ambient temperature. The results presented in Table 9 show the effect of the added excipient compound on reducing the viscosity.

Table 9

Example 19: Effect of excipients on the viscosity of pegylated lysozyme with multiple PEG chains per lysozyme molecule

[00130] A formulation of pegylated lysozyme (from Example 16 above) with potassium acetate as excipient was prepared by adding 6 milligrams of excipient salt to 0.3 mL of pegylated lysozyme solution. The solutions were mixed by gentle shaking and the viscosity was measured by a RheoSense microVisc equipped with an A10 channel (100 microns deep) at a shear rate of 500 sec ⁻¹ . Viscometer measurements were completed at ambient temperature. The results presented in the following table show the advantage of the added excipient compound in reducing viscosity.

Table 10

Example 20: Protein Formulations Containing Excipient Compounds

[00131] Formulations were prepared using an excipient compound or a combination of two excipient compounds and a test protein, wherein the test protein was intended to simulate a therapeutic protein to be used in the therapeutic formulation. These formulations were prepared in 20 mM histidine buffer with different excipient compounds for viscosity measurement in the following manner. The excipient combination was dissolved in 20 mM histidine (Sigma-Aldrich, St. Louis, MO) and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve a pH of 6 prior to dissolution of the model protein. The excipient compounds for this example are listed in Table 11 below. Once the excipient solution was prepared, the test protein (bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis, MO)) was dissolved at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in excipient solution was formulated in a 5 mL sterile polypropylene tube and shaken overnight at 80-100 rpm on an orbital shaker table. The solution was then transferred to a 2 mL microcentrifuge tube and centrifuged in an IEC MicroMax microcentrifuge at 2300 rpm for about 10 minutes to remove entrained air prior to viscosity measurement.

[00132] Viscosity measurements of formulations prepared as described above were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 degrees Celsius. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as the viscosity for the sample. The viscosity of the solution with excipient was normalized to the viscosity of the model protein solution without excipient, and the results are shown in Table 11 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein solution without excipients.

Table 11

Example 21: Protein Formulations Containing Excipients to Reduce Viscosity and Injection Pain

[00133] A formulation was prepared using a combination of an excipient compound, a second excipient compound, and a test protein, wherein the test protein was to simulate a therapeutic protein to be used in the therapeutic formulation. The first excipient compound, Excipient A, was selected from the group of compounds having local anesthetic properties. The first excipient, Excipient A, and the second excipient, Excipient B, are listed in Table 12. These formulations were prepared in 20 mM histidine buffer using excipients A and B in the following manner so that their viscosities could be measured. The amounts of excipients disclosed in Table 12 were dissolved in 20 mM histidine (Sigma-Aldrich, St. Louis, Mo.) and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to pH prior to dissolution of the model protein. 6 was achieved. Once the excipient solution was prepared, the test protein (bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis, Mo.)) was dissolved in the excipient solution at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in excipient solution was formulated in a 5 mL sterile polypropylene tube and shaken overnight at 80-100 rpm on an orbital shaker table. The BGG-excipient solution was then transferred to a 2 mL microcentrifuge tube and centrifuged in an IEC MicroMax microcentrifuge at 2300 rpm for about 10 minutes to remove entrained air prior to viscosity measurement.

[00134] Viscosity measurements of formulations prepared as described above were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 degrees Celsius. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as the viscosity for the sample. The viscosity of the solution with excipients was normalized to the viscosity of the model protein solution without excipients, and the results are shown in Table 12 below. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein solution without excipients.

Table 12

Example 22: Formulations Containing Excipient Compounds and PEG

[00135] Formulations were prepared using an excipient compound and PEG, where the PEG was to simulate a therapeutic PEGylated protein to be used in a therapeutic formulation, providing the excipient compound in amounts as listed in Table 13. These formulations were prepared by mixing an equal volume of the PEG solution with the solution of the excipient. Both solutions were prepared in DI-water.

[00136] A PEG solution was prepared by mixing 16.5 g of poly(ethylene oxide) with an average Mw of ~100,000 (Aldrich catalog # 181986) with 83.5 g of DI water. The mixture was stirred overnight for complete dissolution.

[00137] Excipient solutions were prepared by this general method and as detailed in Table 13 below: an approximately 20 mg/mL solution of tribasic potassium phosphate (Aldrich catalog # P5629) in DI-water with 0.05 g of potassium phosphate 5 prepared by dissolving in mL. A PEG excipient solution was prepared by mixing 0.5 mL of PEG solution with 0.5 mL of excipient solution, and mixing using a vortex for a few seconds. A control sample was prepared by mixing 0.5 mL of PEG solution with 0.5 mL of DI-water. The viscosity was measured and the results are reported in Table 13 below.

Table 13

Example 23: Improved Processing of Protein Solutions with Excipients

[00138] Two BGG solutions were prepared by mixing 0.25 g of solid BGG (Aldrich catalog number G5009) with 4 ml of buffer solution. For Sample A: The buffer solution was 20 mM histidine buffer (pH=6.0). For sample B: The buffer solution was 20 mM histidine buffer (pH=6) containing 15 mg/ml caffeine. Dissolution of solid BGG was performed by placing the sample on an orbital shaker set at 100 rpm. Buffer samples containing caffeine excipients were observed to dissolve proteins faster. For the sample with caffeine excipient (Sample B), complete dissolution of BGG was achieved within 15 minutes. For the caffeine-free sample (Sample A), 35 min was required for dissolution.

[00139] Next, the samples were placed in two separate Amicon Ultra 4 centrifugal filter units with a 30,000 molecular weight cutoff, and the samples were centrifuged at 2,500 rpm at 10 minute intervals. The filtrate volume recovered after each 10 min centrifugation run was recorded. The results in Table 14 show a faster recovery of the filtrate for Sample B. Also, while sample B remained concentrated with all further runs, sample A reached its maximum concentration point and further centrifugation resulted in no additional sample concentration.

Table 14

Example 24: Protein Formulations Containing Multiple Excipients

[00140] This example shows how the combination of caffeine and arginine as excipients has a beneficial effect on reducing the viscosity of the BGG solution. Four BGG solutions were prepared by mixing 0.18 g of solid BGG (Aldrich catalog number G5009) with 0.5 mL of 20 mM histidine buffer at pH 6. Each buffer solution contained a different excipient or combination of excipients as described in the table below. The viscosity of the solution was measured as described in the previous examples. The results show that caffeine, a hindered amine excipient, can be combined with known excipients such as arginine, and that the combination has superior viscosity reducing properties than the individual excipients themselves.

Table 15

[00141] Arginine was added to a 280 mg/mL solution of BGG in histidine buffer at pH 6. At levels above 50 mg/mL, adding more arginine did not further decrease the viscosity as shown in Table 16.

Table 16

[00142] Caffeine was added to a 280 mg/mL solution of BGG in histidine buffer, pH 6. At levels above 10 mg/mL, adding more caffeine did not further decrease the viscosity as shown in Table 17.

Table 17

Example 25: Preparation of a solution of co-solute in deionized water

[00143] Compounds used as co-solutes to increase caffeine solubility in water were obtained from Sigma-Aldrich (St. Louis, Mo.) and contain niacinamide, proline, procaine HCl, ascorbic acid, 2,5-dihydroxybenzoic acid, lidocaine, saccharin, acesulfame K, tyramine, and aminobenzoic acid. of each co-solute by dissolving the dry solid in deionized water and, in some cases, adjusting the pH to a pH value of about 6 to about 8 with 5 M hydrochloric acid or 5 M sodium hydroxide as needed. A solution was prepared. The solution was then diluted to a final volume of 25 mL or 50 mL using a class A volumetric flask, and the concentration was recorded based on the mass of the dissolved compound and the final volume of the solution. The prepared solution was used purely or diluted with deionized water.

Example 26: Caffeine solubility test

[00144] The effect of different co-solutes on the solubility of caffeine at ambient temperature (about 23° C.) was evaluated in the following manner. Dry caffeine powder (Sigma-Aldrich, St. Louis, MO) was added to a 20 mL glass scintillation vial and the mass of caffeine was recorded. 10 mL of the co-solute solution prepared according to Example 25 was added to the caffeine powder in certain cases (reported in Table 18); In other cases (reported in Table 18), a blend of the co-solute solution and deionized water was added to the caffeine powder while maintaining a final addition volume of 10 mL. The volume contribution of dry caffeine powder was assumed to be negligible in any such mixture. A small magnetic stir bar was added to the vial and the solution was vigorously mixed on a stir plate for about 10 minutes. After about 10 minutes, the vial was observed for dissolution of the dry caffeine powder and the results are provided in Table 18 below. This observation shows that niacinamide, procaine HCl, 2,5-dihydroxybenzoic acid sodium salt, saccharin sodium salt, and tyramine chloride salt have all reported caffeine solubility limits (about 16 mg/mL at room temperature according to Sigma-Aldrich). indicated that it enabled the dissolution of caffeine up to at least about 4 times of

Table 18

Example 27: Effect of Higher Caffeine Concentrations on Protein Formulations

[00145] A formulation was prepared using a primary excipient compound, a secondary excipient compound and a test protein, wherein the test protein was intended to simulate a therapeutic protein to be used in the therapeutic formulation. The primary excipient compounds were both selected from compounds with low solubility and demonstrated reduced viscosity. Secondary excipient compounds were selected from compounds with higher solubility and pyridine or benzene rings. These formulations were prepared in the following manner in 20 mM histidine buffer with different excipient compounds for viscosity measurement: The excipient combination was dissolved in 20 mM histidine buffer solution and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or pH 6 was achieved prior to dissolution of the model protein by adjustment with hydrochloric acid. In certain experiments, the primary excipient was added in an amount that greatly exceeded its room temperature solubility limit as reported in the literature. Once the excipient solution was prepared, the test protein (bovine gamma globulin (BGG, Sigma-Aldrich, St. Louis, Mo.)) was dissolved at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in excipient solution was formulated in 20 mL vials and shaken overnight at 100 rpm on an orbital shaker table. The solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 min in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

[00146] Viscosity measurements of formulations prepared as described above were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 degrees Celsius. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as viscosity for the sample as shown in Table 19 below. The viscosity of the solution with excipients was normalized to the viscosity of the model protein solution without excipients. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein solution without excipients.

Table 19

Example 28: Improved stability of adalimumab solution with caffeine as excipient

[00147] The stability of adalimumab solutions in the presence and absence of caffeine excipients was evaluated after exposing the samples to two different stress conditions: agitation and freeze-thaw. An adalimumab drug formulation HUMIRA® with the properties described in more detail in Example 32 was used. HUMIRA® samples were concentrated to a concentration of 200 mg/ml adalimumab in original buffer solution as described in Example 32; This concentrated sample was designated "Sample 1". A second sample was prepared as described in Example 33 with -200 mg/mL of adalimumab and 15 mg/mL of added caffeine; This concentrated sample with added caffeine was designated "Sample 2". Both samples were diluted to a final concentration of 1 mg/ml adalimumab using diluents as follows: sample 1 diluent was the original buffer solution, sample 2 diluent was 20 mM histidine, 15 mg/ml caffeine, pH = 5. Both HUMIRA® dilutions were filtered through a 0.22 μ syringe filter. For all diluted samples, three batches of 300 μl each were prepared in 2 ml Eppendorf tubes in a laminar flow hood. The samples were subjected to the following stress conditions: for agitation, the samples were placed on an orbital shaker at 300 rpm for 91 hours; For freeze-thaw, samples were cycled 7 times from -17°C to 30°C for an average of 6 hours per condition. Table 20 describes the samples prepared.

Table 20

[00148] Stability evaluation by dynamic light scattering (DLS)

[00149] A Brookhaven Zeta Plus dynamic light scattering instrument was used to measure the hydrodynamic radius of the adalimumab molecules in the samples and look for evidence of the formation of aggregate populations. Table 21 shows the DLS results for the 6 samples listed in Table 20; Some of these (1-A, 1-FT, 2-A, and 2-FT) were exposed to stress conditions (stressed samples) and others (1-C and 2-C) were not stressed. In the absence of caffeine as an excipient, stressed samples 1-A and 1-FT exhibited higher effective diameters than unstressed samples 1-C, and they also produced a second population of significantly larger diameter particles. showed; This new grouping of particles with larger diameters was evidence of agglomeration into invisible particles. Stressed samples containing caffeine (Samples 2-A and 2-FT) exhibited only one population with a particle size similar to that of unstressed Sample 2-C. These results demonstrate the effect of adding caffeine to reduce or minimize the formation of aggregates or invisible particles. Table 21 and Figures 1, 2, and 3 present the DLS data, where the multimodal particle size distribution of the monoclonal antibody is evident in the stressed sample containing no caffeine.

Table 21

[00150] Table 22A and Table 22B show the DLS raw data of adalimumab samples showing particle size distribution. G(d) is the intensity-weighted differential size distribution. C(d) is the cumulative intensity-weighted differential size distribution.

Table 22A

Table 22B

Example 29: Stability evaluation by size-exclusion chromatography (SEC)

[00151] SEC was used to detect any invisible particulates less than about 0.1 microns in size from the stressed and unstressed adalimumab samples described above. A TSKgel SuperSW3000 column with a guard column (Tosoh Biosciences, Montgomeryville, PA) was used and elution was monitored at 280 nm. A total of 10 μl of each stressed and unstressed sample was eluted isocratically with pH 6.2 buffer (100 mM phosphate, 325 mM NaCl) at a flow rate of 0.35 ml/min. The residence time of the adalimumab monomer was approximately 9 minutes. The data showed that the samples containing caffeine as an excipient did not show any detectable aggregates. In addition, the amount of monomer was kept constant in all three samples.

Example 30: HERCEPTIN ^® decrease in viscosity of

[00152] The monoclonal antibody trastuzumab (HERCEPTIN ^® from Genentech) was obtained as a lyophilized powder and reconstituted to 21 mg/mL in DI water. The resulting solution was concentrated as-is in an Amicon Ultra 4 centrifuge concentrator tube (molecular weight cut-off, 30 KDa) by centrifugation at 3500 rpm for 1.5 h. Concentrations were determined by diluting the samples 200-fold in the appropriate buffer and measuring the absorbance at 280 nm using an extinction coefficient of 1.48 mL/mg. Viscosity was measured using a RheoSense microVisc viscometer.

[00153] An excipient buffer containing salicylic acid and caffeine alone or in combination was prepared by dissolving histidine and excipients in distilled water and then adjusting the pH to an appropriate level. The conditions for buffer systems 1 and 2 are summarized in Table 23.

Table 23

[00154] The HERCEPTIN ^® solution was diluted in excipient buffer in a ratio of ˜1:10 and concentrated in an Amicon Ultra 15 (MWCO 30 KDa) concentrator tube. Concentrations were determined using the Bradford assay and compared to a standard calibration curve made from stock HERCEPTIN ^® samples. Viscosity was measured using a RheoSense microVisc. Concentration and viscosity measurements of various HERCEPTIN ^® solutions are shown in Table 24 below, where Buffer Systems 1 and 2 refer to the buffers identified in Table 23.

Table 24

[00155] Buffer System 1 containing both salicylic acid and caffeine had a maximum viscosity reduction of 76% at 215 mg/mL compared to the control sample. Buffer System 2 containing caffeine only had a viscosity reduction of up to 59% at 200 mg/mL.

Example 31: AVASTIN ^® decrease in viscosity of

[00156] ^AVASTIN® (bevacizumab formulation marketed by Genentech) was received as a 25 mg/ml solution in histidine buffer. Samples were concentrated at 3500 rpm in Amicon Ultra 4 centrifuge concentrator tubes (MWCO 30 KDa). Viscosity was measured by RheoSense microVisc and concentration was determined by absorbance at 280 nm (extinction coefficient, 1.605 mL/mg). Excipient buffer was prepared by adding 10 mg/mL caffeine with 25 mM histidine HCl. AVASTIN ^® stock solution was diluted with excipient buffer and then concentrated in an Amicon Ultra 15 centrifugal concentrator tube (MWCO 30 KDa). The concentration of the excipient sample was determined by Bradford assay and the viscosity was measured using a RheoSense microVisc. The results are presented in Table 25 below.

Table 25

[00157] AVASTIN ^® exhibited a maximum viscosity reduction of 73% when 10 mg/mL of caffeine was concentrated to 213 mg/ml when compared to the control AVASTIN sample.

Example 32: HUMIRA ^® profile of

[00158] HUMIRA ^® (AbbVie Inc., Chicago, IL) reduces the inflammatory response in autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate to severe chronic psoriasis and juvenile idiopathic arthritis is a commercially available formulation of the therapeutic monoclonal antibody adalimumab, a TNF-alpha blocker typically prescribed for HUMIRA ^® contains 40 mg of adalimumab, 4.93 mg of sodium chloride, 0.69 mg of sodium phosphate monobasic dihydrate, 1.22 mg of sodium phosphate dibasic dihydrate, 0.24 mg of sodium citrate, 1.04 mg of citric acid monohydrate, It is sold as a 0.8 mL single use dose containing 9.6 mg of mannitol and 0.8 mg of polysorbate-80. The viscosity versus concentration profile of this formulation was generated in the following manner. An Amicon Ultra 15 centrifugal concentrator (EMD-Millipore, Billerica, MA) with a 30 kDa molecular weight cut-off was charged with approximately 15 mL of deionized water and a Sorvall Legend RT (Kendro Laboratory Products, Newtown, CT) at 4000 rpm for 10 min. ) to wash the membrane by centrifugation. After that, residual water was removed and 2.4 mL of HUMIRA ^® liquid formulation was added to the concentrator tube and centrifuged at 4000 rpm at 25° C. for 60 minutes. 10 microliters of the residue was diluted with 1990 microliters of deionized water, the absorbance of the diluted sample was measured at 280 nm, and the concentration of the residue was calculated using the dilution factor and extinction coefficient of 1.39 mL/mg-cm. The concentration was determined. The viscosity of the concentrated samples was measured by a microVisc viscometer equipped with an A05 chip (RheoSense, San Ramon, CA) at 23° C. and a shear rate of 250 sec ⁻¹ . After the viscosity measurement, the sample was diluted with a small amount of filtrate and the concentration and viscosity measurements were repeated. This process was used to generate viscosity values at various adalimumab concentrations as described in Table 26 below.

Table 26

Example 33: HUMIRA Using Viscosity-Reducing Excipients ^® reformulation of

[00159] The following examples describe the general process for reformulation of HUMIRA ^® in a buffer with viscosity-reducing excipients. A solution of viscosity-reducing excipient was prepared in 20 mM histidine by dissolving about 0.15 g of histidine (Sigma-Aldrich, St. Louis, Mo.) and 0.75 g of caffeine (Sigma-Aldrich, St. Louis, Mo.) in deionized water. did. The pH of the resulting solution was adjusted to about 5 with 5 M hydrochloric acid. The solution was then diluted with deionized water to a final volume of 50 mL in a volumetric flask. Thereafter, HUMIRA ^® was reformulated to high mAb concentrations using the resulting buffered viscosity-reducing excipient solution. Next, about 0.8 mL of HUMIRA ^® was added to a cleaned Amicon Ultra 15 centrifugal concentrator tube with a 30 kDa molecular weight cutoff and centrifuged at 4000 rpm and 25° C. for 8-10 minutes in a Sorvall Legend RT. Then, about 14 mL of the buffered viscosity-reducing excipient solution prepared as described above was added to the concentrated HUMIRA ^® in a centrifugal concentrator. After gentle mixing, the samples were centrifuged at 4000 rpm and 25° C. for about 40 to 60 minutes. The residue was a concentrated sample of HUMIRA ^® reformulated in buffer with viscosity-reducing excipients. The viscosity and concentration of the samples were measured and, in some cases, diluted with a small amount of filtrate to determine the viscosity at lower concentrations. Viscosity measurement was completed with a microVisc viscometer in the same manner as the concentrated HUMIRA ^® formulation in the previous example. Concentrations were determined by the Bradford assay using a standard curve generated from a HUMIRA ^® stock solution diluted in deionized water. Reformulation of HUMIRA ^® with viscosity-reducing excipients provided a viscosity reduction of 30% to 60% compared to the viscosity values of HUMIRA ^® concentrated in commercial buffer without reformulation, as shown in Table 27 below.

Table 27

Example 34: Preparation of Formulations Containing Caffeine, Secondary Excipients and Test Protein

[00160] Formulations were prepared using caffeine or a combination of caffeine and a second excipient compound as the excipient compound, and a test protein, wherein the test protein was to simulate a therapeutic protein to be used in the therapeutic formulation. These formulations were prepared in 20 mM histidine buffer with different excipient compounds for viscosity measurement in the following manner. The excipient combination (Excipients A and B as described in Table 28 below) was dissolved in 20 mM histidine (Sigma-Aldrich, St. Louis, Mo.) and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid. Adjustments were made to achieve pH 6 prior to dissolution of the model protein. Once the excipient solution was prepared, the test protein (bovine gamma globulin (BGG) (Sigma-Aldrich, St. Louis, MO)) was dissolved at a rate to achieve a final protein concentration of about 280 mg/mL. A solution of BGG in excipient solution was formulated in 20 mL glass scintillation vials and shaken overnight at 80-100 rpm on an orbital shaker table. The solution was then transferred to a 2 mL microcentrifuge tube and centrifuged in an IEC MicroMax microcentrifuge at 2300 rpm for about 10 minutes to remove entrained air prior to viscosity measurement.

[00161] Viscosity measurements of formulations prepared as described above were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 degrees Celsius. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as the viscosity for the sample. The viscosity of the solution with excipients was normalized to the viscosity of the model protein solution without excipients. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein solution without excipients.

Table 28

Example 35: Preparation of Formulations Containing Dimethyl Sulfone and Test Protein

[00162] Formulations were prepared using dimethyl sulfone (Jarrow Formulas, Los Angeles, CA) as excipient compounds and a test protein, where the test protein was intended to simulate a therapeutic protein to be used in the therapeutic formulation. This formulation was prepared in 20 mM histidine buffer for viscosity measurement in the following manner. Dimethyl sulfone was dissolved in 20 mM histidine (Sigma-Aldrich, St. Louis, MO) and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to achieve pH 6 prior to dissolution of the model protein. , filtered through a 0.22 micron filter. Once the excipient solution was prepared, the test protein (bovine gamma globulin (BGG) from Sigma-Aldrich, St. Louis, MO)) was dissolved at a concentration of about 280 mg/mL. A solution of BGG in excipient solution was formulated in 20 mL glass scintillation vials and shaken overnight at 80-100 rpm on an orbital shaker table. The solution was then transferred to a 2 mL microcentrifuge tube and centrifuged in an IEC MicroMax microcentrifuge at 2300 rpm for about 10 minutes to remove entrained air prior to viscosity measurement.

[00163] Viscosity measurements of formulations prepared as described above were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 degrees Celsius. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as the viscosity for the sample. The viscosity of the solution with excipients was normalized to the viscosity of the model protein solution without excipients. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein solution without excipients.

Table 29

Example 36: Preparation of Formulations Containing Tannic Acid

[00164] In this example, a high molecular weight poly(ethylene oxide) (PEG) molecule was used as a model compound to mimic the viscosity behavior of pegylated proteins. By mixing PEG (Sigma-Aldrich, Saint Louis, MO) having a viscosity average molecular weight (MV) of 1,000,000 with deionized water (DI) in a sterile 5 mL polypropylene centrifuge tube to prepare an approximately 1.8 wt % PEG solution A control sample (#36.1 in Table 30) was prepared. Two samples #36.2 and 36.3 with tannic acid (TA, Spectrum Chemicals, New Brunswick, NJ) were mixed approximately 1:100 and 1:1000 respectively by mixing 1 mM KCl prepared in appropriate amounts of PEG, TA, and DI water. TA: PEG mass ratio was prepared. All samples were placed on a Daigger Scientific (Vernon Hills, IL) Labgenius orbital shaker overnight at 200 rpm. Viscosity measurements were performed on a microVISC rheometer (RheoSense, San Ramon, CA) at 20° C. and a wall shear rate of 250 s ⁻¹ . After measurement, the viscosity was normalized to each PEG concentration to account for the concentration dependence of the viscosity. The PEG concentration, TA concentration, viscosity, normalized viscosity, and viscosity reduction for samples 36.1, 36.2, and 36.3 are listed in Table 30 below. The addition of TA to the concentrated PEG solution substantially reduces the solution viscosity by approximately 40%.

Table 30

Example 37: Formulations with Excipient Dosage Profiles

[00165] Formulations were prepared using different molar concentrations of excipient and test protein, where the test protein was intended to simulate a therapeutic protein to be used in the therapeutic formulation. This formulation was prepared in 20 mM histidine buffer for viscosity measurement in the following manner. Stock solutions of 0 and 80 mM caffeine excipients were prepared in 20 mM histidine (Sigma-Aldrich, St. Louis, MO) and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to dissolve the model protein. pH 6 was achieved before. Additional solutions of varying caffeine concentrations were prepared by blending the two stock solutions at various volume ratios. Once the excipient solution was prepared, a final protein of about 280 mg/mL was obtained by adding a 0.7 mL solution of the test protein (bovine gamma globulin (BGG, Sigma-Aldrich, St. Louis, Mo.)) to 0.25 g lyophilized protein powder. Dissolve in proportions to achieve the concentration. A solution of BGG in excipient solution was formulated in a 5 mL sterile polypropylene tube and dissolved by shaking at 100 rpm overnight on an orbital shaker table. The solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2400 rpm for 5 min in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

[00166] Viscosity measurements of formulations prepared as described above were performed with a microVisc viscometer (RheoSense, San Ramon, CA). The viscometer was equipped with an A-10 chip with a channel depth of 100 microns, operated at a shear rate of 250 s ^-1 and at 25 degrees Celsius. To measure the viscosity, the formulation was loaded into the viscometer taking care to remove any air bubbles from the pipette. A pipette with loaded sample formulation was placed in the instrument and allowed to incubate at the measurement temperature for 5 minutes. The instrument was then run until the channels were fully equilibrated with the test fluid indicated by a stable viscosity reading, and then the viscosity was recorded in centipoise. The results of these measurements are listed in Table 31.

Table 31

Example 38: Preparation of Formulations Containing Phenolic Compounds

[00167] In this example, a high molecular weight poly(ethylene oxide) (PEG) molecule was used as a model compound to mimic the viscosity behavior of pegylated proteins. Control samples were prepared by mixing PEG (Sigma-Aldrich, Saint Louis, MO) having a viscosity average molecular weight (MV) of 1,000,000 with deionized water (DI) at approximately 1.8 wt % PEG in a sterile 5 mL polypropylene centrifuge tube. prepared. The phenolic compounds gallic acid (GA, Sigma-Aldrich, Saint Louis, MO), pyrogallol (PG, Sigma-Aldrich, Saint Louis, MO) and resorcinol (R, Sigma-Aldrich, Saint Louis, MO) were followed by It was also tested as an excipient. Three PEG-containing samples were prepared with GA, PG and R added in excipient:PEG mass ratios of approximately 3:50, 1:2, and 1:2, respectively, by mixing appropriate amounts of PEG, excipient, and DI water. Samples were placed on a Daigger Scientific (Vernon Hills, IL) Labgenius orbital shaker overnight at 200 rpm. Viscosity measurements were performed on a microVISC rheometer (RheoSense, San Ramon, CA) at 20° C. and a wall shear rate of 250 s ⁻¹ . After measurement, the viscosity was normalized to each PEG concentration to account for the concentration dependence of the viscosity. PEG concentrations, excipient concentrations, viscosities, normalized viscosities, and viscosity reductions for control and excipient-containing samples are listed in Table 32 below.

Table 32

Example 39: Preparation of Formulations Containing Test Protein and Excipient with Pyrimidine Ring

[00168] Formulations were prepared using an excipient compound having a structure comprising one pyrimidine ring and a test protein, wherein the test protein was to simulate a therapeutic protein to be used in the therapeutic formulation. These formulations were prepared in 20 mM histidine buffer with different excipient compounds in the following manner. Excipient compounds as listed in Table 33 were dissolved in an aqueous solution of 20 mM histidine (Sigma-Aldrich, St. Louis, Mo.), and the pH of the resulting solution was adjusted with a small amount of concentrated sodium hydroxide or hydrochloric acid to prepare the model protein. A pH of 6 was achieved prior to dissolution. Once the excipient solution was prepared, the test protein (bovine gamma globulin (BGG, Sigma-Aldrich, St. Louis, Mo.)) was dissolved at a rate to achieve a final protein concentration of 280 mg/mL each. A solution of BGG in excipient solution was formulated in a 20 mL glass scintillation vial and stirred overnight at 100 rpm on an orbital shaker table. The solution was then transferred to a 2 mL microcentrifuge tube and centrifuged at 2300 rpm for 10 min in an IEC MicroMax microcentrifuge to remove entrained air prior to viscosity measurement.

[00169] Viscosity measurements of formulations prepared as described above were performed with a DV-IIT LV cone and plate viscometer (Brookfield Engineering, Middleboro, Mass.). The viscometer was equipped with a CP-40 cone and operated at 3 rpm and 25 degrees Celsius. The formulation was loaded into the viscometer in a volume of 0.5 mL and incubated for 3 minutes at the given shear rate and temperature followed by a measurement collection period of 20 seconds. Two additional steps were then performed consisting of a 1 min shear incubation followed by a 20 sec measurement collection period. The three data points collected were then averaged and reported as the viscosity for the sample. The viscosity of the solution with excipients was normalized to the viscosity of the model protein solution without excipients. The normalized viscosity is the ratio of the viscosity of the model protein solution with excipients to the viscosity of the model protein solution without excipients. The results of these measurements are shown in Table 33.

Table 33

Example 40: Preparation of Formulations Containing Excipients and PEG Model Compounds

[00170] The material for this example comprised a linear PEG molecule with a molecular weight of 20,000, abbreviated as PEG-20K. Formulations were prepared using an excipient compound having a structure comprising a conjugate acid of a weak base, and a PEG-20K model compound, wherein the PEG-20K model compound was to simulate a pegylated protein.

[00171] First, the viscosity profile of PEG-20K at different concentrations in water and 20 mM citrate buffer was determined as baseline conditions as shown in Table 34 below with n=3 replicate measurements per condition.

Table 34

[00172] Next, excipient stock solutions were prepared at a concentration of 1 M in 20 mM citrate buffer. The pH of the stock solution was adjusted to about 5. A PEG-20K solution was prepared by mixing 140 mL of 300 mg/mL PEG-20K stock with 20-60 μL of excipient stock. Depending on the amount of excipient spiked, the final sample volume was adjusted to 200 μL by adding 40-0 μL of citrate buffer. Since the blended solutions containing PEG-20K and excipients were all prepared with citrate buffer adjusted to pH 4.9-5.0, any weak base excipients were converted to their conjugate acids in the buffer solution. The viscosity of each PEG-20K sample containing different excipients and 210 mg/mL of PEG-20K was measured on a MicroVISC at least three times, and the results are summarized in Table 35 below.

Table 35

Example 41: Improvement of Tangential Flow Filtration Using Caffeine

400 mL of human gamma globulin (Octagam, Octapharma, USA) at a concentration of 35 mg/mL was prepared by diluting a stock of 100 mg/mL in phosphate buffered saline (PBS). Buffer was prepared by dissolving 1.8 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 137 mM NaCl, 2.7 mM KCl in 1 L of Milli-Q water. Caffeine PBS solution was prepared by dissolving 50 mM caffeine, 1.8 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 137 mM NaCl, 2.7 mM KCl in 1 L of Milli-Q water. Tap water was purified with a Direct-Q 3 UV purification system from EMD Millipore (Billerica, MA) to produce DI water. The human gamma globulin (HGG) solution was transferred to the reservoir of a Labscale Tangential Flow Filtration (TFF) system (Millipore, Billerica, MA) equipped with a 30 KDa MWCO Pellicon XL TFF cassette (Millipore, Billerica, MA). Prior to use, the cassettes were flushed with Milli-Q water followed by PBS and a water permeability test was performed to ensure membrane integrity and efficiency. The HGG solution was pumped through the cassette using a Quattroflow pump (Cole-Parmer, IL), the retentate line was returned to the sample reservoir and the permeate was collected in a graduated measuring cylinder. The stirrer bar ensured proper mixing of feed and residue. The feed pump was set to deliver a 120 mL/min feed to the cassette. A retentate restrictor was used to achieve a transmembrane pressure (TMP) in the range of approximately 20-30 psi, and the feed pump and retentate restrictor were adjusted to ensure that the TMP remained constant throughout the run. Data logging of pressure and flow was performed and samples were taken every 30 minutes. To calculate the feed concentration, samples were analyzed by SE-HPLC, where 50 mg was loaded onto a TSKgel SuperSW3000 column (30 cm × 4.6 mm ID, Tosoh Bioscience, King of Prussia, PA) and an Agilent G1351B Diode array detector. loaded onto an Agilent 1100 HPLC system. PBS was used as the mobile phase at a flow rate of 0.35 mL/min. The area under the peak was integrated to calculate the protein concentration. TFF efficiencies in the presence of caffeine were compared to TFF using a control system using feed concentrations plotted as a function of time. As shown in Table 36 below, a higher percentage change in concentration from the initial feed concentration was observed in a shorter time for caffeine compared to the control, demonstrating increased TFF efficiency.

Table 36

Example 42: Improving Purification Yield from Protein A Resin Using Caffeine

[00174] Study-grade omalizumab, purchased from Bioceros (Utrecht, The Netherlands) at 15 mg/mL in 20 mM sodium phosphate, pH 7 buffer, was used as test sample. This protein solution was filtered through a 0.2 μm polyethersulfone (PES) filter. The filtered material was mixed with binding buffer consisting of 20 mM sodium phosphate at pH 7 in DI water in a 1:1 ratio. Tap water was purified with a Direct-Q 3 UV purification system from EMD Millipore (Billerica, MA) to produce DI water. Protein-A purification was performed using a HiTrap Protein-A HP 1 mL column from GE Healthcare (Chicago, IL). After 10 mL of binding buffer was used for column equilibration, 30 mg of protein was loaded. The column was then washed with 5 mL of binding buffer to remove unbound protein. Bound omalizumab was eluted from the column in 1 mL fractions using either 0.1 M glycine buffer, pH 3.5, or 0.1 M glycine, 50 mM caffeine buffer, pH 3.5 as control buffer. Control buffer was prepared by dissolving 7.5 g of glycine in DI water, adjusting the pH to 3.5 with 6M HCl and adjusting the volume to 1 L. Caffeine buffer was prepared by dissolving 7.5 g of glycine and 10 g of caffeine in DI water, adjusting the pH to 3.5 with 6 M HCl and adjusting the volume to 1 L. Collect five 1 mL fractions; These eluted fractions were labeled E1, E2, E3, E4, and E5. Finally, Protein-A was regenerated by washing the column with 5 mL of 0.1 M glycine pH 3.0 buffer. The flow rate for each step was 1 mL/min, which was maintained by a Fusion 100 infusion pump (Chemyx, Stafford, TX). A 10 mL NormJect Luer Lok syringe was used (Henke Sass Wolf, Tuttlingen, Germany, reference number 4100-000V0).

[00175] Five eluted fractions, E1, E2, E3, E4, and E5 were assayed for total protein content by high performance size-exclusion chromatography (SEC) analysis. SEC analysis was performed using a TSKgel SuperSW3000 column (30 cm × 4.6 mm ID, Tosoh Bioscience, King of Prussia, PA) connected to an Agilent HP 1100 HPLC system. PBS was used as mobile phase at room temperature at a flow rate of 0.35 mL/min. Protein concentration was monitored by absorbance at 280 nm using a G1315B diode array detector. The total amount of protein eluted from the Protein-A resin was determined by integrating the chromatogram, and a yield increase of about 8% was observed in the presence of caffeine as shown in Table 37 below.

Table 37

Example 43: Excipients for stabilization during low pH maintenance

[00176] Study-grade ripilimumab, purchased from Bioceros (Utrecht, The Netherlands) at 15 mg/mL in 20 mM sodium phosphate, pH 7 buffer, was used as test sample. The protein solution was filtered through a 0.2 μm polyethersulfone (PES) filter. Raffinose pentahydrate was obtained from Sigma (St Louis, Mo.). An excipient stock was prepared by dissolving raffinose pentahydrate at a concentration of 1 M in 0.15 M glycine buffer, pH 2.75. A buffer was prepared by dissolving 7.5 g of glycine in 0.9 L Milli-Q water, adjusting the pH to 2.75 with 1 M HCl, and making the volume to 0.1 L. One control formulation and three excipient-containing formulations were prepared by adding excipients to ipilimumab solutions at final excipient concentrations of 0 mM, 100 mM, 200 mM and 400 mM and final ipilimumab concentrations of 2 mg/mL. . Samples were incubated overnight at acidic pH (2.75) for 24 h and samples were analyzed by SE-HPLC, where 50 mg was administered on a TSKgel SuperSW3000 column (30 cm × 4.6 mm ID, Tosoh Bioscience, King of Prussia, PA) and Agilent Loaded into an Agilent 1100 HPLC system equipped with a G1351B Diode array detector. PBS was used as the mobile phase at a flow rate of 0.35 mL/min. The monomer protein (ipilimumab) concentration was calculated by integrating the area under the monomer peak. The monomer fraction from untreated samples not exposed to low pH was normalized to 100%, and the monomer fraction of treated samples was expressed as the percentage change in this untreated sample. The results in Table 38 below show that the presence of raffinose in the samples resulted in a higher percentage of ipilimumab in the monomeric form after maintaining low pH.

Table 38

Example 44: Preparation of Buffers and Excipients

[00177] A stock 20 mM histidine hydrochloride (His HCl) buffer was prepared for use in excipient formulation and protein buffer exchange. Two liters of His HCl were prepared by dissolving 6.206 grams of histidine (Sigma-Aldrich, St. Louis, MO) in Type 1 ultrapure water. A solution of dissolved histidine was titrated to pH 6.0 using concentrated hydrochloric acid. The His HCl solution was then made up to 2 liters using a volumetric flask and filtered through a 0.2 μm membrane bottle-top filter apparatus (Sigma Aldrich, St. Louis, MO). The excipients to be tested in Example 46 (listed in Table 39) were prepared as excipient solutions for subsequent testing as follows. Each excipient was prepared at 10X (1 M) by dissolving each excipient in His HCl buffer as described above and adjusting the pH with concentrated sodium hydroxide or concentrated hydrochloric acid. Then, each excipient solution was filtered using a 0.2 μm membrane filter.

Example 45: Protein Solution Preparation

[00178] The two test proteins, purified omalizumab purchased from Bioceros (The Netherlands) and polyclonal IgG from human serum (Octagam 10%) were mixed with His HCl (conducted) using a 20 kDa molecular weight cut-off dialysis cassette (Fisher Scientific). as prepared in Example 44). Each protein solution was transferred to a dialysis cassette attached to a buoy and placed in a flask for buffer exchange. A total of 3 buffer exchanges were performed with >50x starting protein volume. In the final buffer exchange step, the protein solution was removed from the dialysis cassette, filtered through a 0.2 μm membrane filter, and the protein concentration was determined via A280 by diluting 100-fold with His HCl buffer. Then, 100 μL was transferred to a UV transparent 96 half-well microplate (Greiner Bio-One, Austria) and absorbance was measured at a wavelength of 280 nm with a Synergy HT plate reader (BioTek, Winooski, VT). The blanked pathlength corrected A280 measurements were then divided by each extinction coefficient and multiplied by a dilution factor to determine protein concentration. A subsequent concentration step was required to concentrate the protein in the preparation for dynamic light scattering (DLS) viscosity measurement in Example 46. Concentration was performed using an Amicon-15 centrifuge unit (EMD Millipore, Billerica, MA) with a 30 kDa molecular weight cut-off, followed by centrifugation at 4000 x g in a bench-top centrifuge (Sorvall Legend RT). It was concentrated to 175 mg/mL based on the mass of the residue.

Example 46: DLS Measurement of Diffusion Interaction Parameters

[00179] In this example, the diffusion interaction parameters (kD) of diluted protein solutions were determined by DLS in the presence of 0.1 M excipient solution. The excipients tested are listed in Table 39. For each excipient, a 0.2 M excipient solution was prepared separately from the previously prepared 1 M excipient stock. KD was determined by DLS using 5 different concentrations of omalizumab (prepared as described in Example 45) ranging from 10 mg/mL to 0.6 mg/mL in the presence of 0.1 M excipient. The same set of control samples were prepared containing the same concentration of omalizumab in the absence of any excipient. For each test sample, 20 μL of protein solution was combined with 20 μL of 0.2 M excipient solution (1:1 mixture) on a 384-well plate (Aurora Microplates, Whitefish, MT). After loading the samples, the well plates were shaken on a plate shaker to mix the contents for 5 minutes. Upon mixing, the well plates were centrifuged at 400×g for 1 min in a Sorvall Legend RT to forcefully remove any air pockets. The well plates were then loaded into a DynaPro II DLS plate reader (Wyatt Technologies Corp., Goleta, CA) and the diffusion coefficient of each sample was measured at 25°C. For each excipient, the measured diffusion coefficient was plotted as a function of protein concentration and the slope of the linear fit of the data was reported as kD. In this example, each measurement was normalized to the control mean and reported as a percentage of the control. These results are shown in Table 39 below.

Table 39

Example 47: Viscosity Measurement by DLS

[00180] Purified omalizumab purchased from Bioceros (The Netherlands) and human serum-derived polyclonal IgG (Octagam 10%, Pfizer) were used as model protein systems to investigate the viscosity effect of excipients. Concentrated excipient stock solutions (listed in Tables 40 and 41) were prepared at 10X (1M) in His HCl buffer according to the protocol described in Example 44. Omalizumab was buffer exchanged into His HCl buffer using an Amicon-15 centrifuge (30 kDa MWCO) apparatus and concentrated to 175 mg/mL based on residue mass in a centrifuge apparatus. Excipients and concentrated protein were combined in a 200 μL PCR tube, and 1 part 10X excipient and 9 parts protein were added. An additional 2 µL of a 5-fold diluted solution of polyethylene glycol surface-modified gold nanoparticles (nanoComposix, San Diego, CA) was added to each PCR tube and mixed thoroughly by inversion. A control sample was prepared identically except that no excipient was added. Each sample (test sample and control) was transferred in duplicate (25 μL per well) into 384-well microplates (Aurora Microplates, Whitefish, MT) and centrifuged at 400 x g for 1 min prior to analysis. The apparent particle size of gold nanoparticles was measured at 25° C. using a DynaPro II DLS plate reader (Wyatt Technology Corp., Goleta, CA). The viscosity of the protein formulation was determined according to the Stokes-Einstein equation using the ratio of the apparent particle size of the gold nanoparticles to the known particle size of the gold nanoparticles in water. In this example, each measurement was normalized to the control mean and reported as a percentage reduction compared to the control, standard deviations are shown, and the results are shown in Tables 40 and 41 below.

Table 40

Table 41

Example 48: Viscosity Measurement by Viscometer

[00181] Excipient solutions for the excipients listed in Tables 42 and 43 were prepared at 0.1 M or 0.075 M in His HCl buffer, and the pH was adjusted using concentrated sodium hydroxide or concentrated hydrochloric acid. Omalizumab and human IgG were buffer exchanged into their respective excipient formulations using an Amicon-15 centrifuge apparatus (30 kDa MWCO). After buffer exchange, the protein solution was concentrated in a centrifuge apparatus to 150 mg/mL for omalizumab and 250 mg/mL for human IgG based on residue mass. A control formulation was prepared in the same manner except for the absence of excipients. Viscosity measurements were performed on a RheoSense micro-viscometer using an A05 chip enclosed in a temperature-controlled enclosure set at 25°C. The shear rate was set to 250 s ⁻¹ . After measuring the viscosity of each formulation three times, it was diluted by adding 20 μL of each buffer, and the viscosity was measured again. This was repeated 5-6 times each to generate viscosity data for 5-6 different protein concentrations. Protein concentration was determined by absorbance at 280 nm using an Agilent 1100 series high pressure liquid chromatography instrument paired with a size exclusion column (TOSOH TSKgel SuperSW3000). Scatter plots were generated by plotting the viscosity as a function of concentration for each excipient formulation. An exponential trend line was fitted to each formulation, and the viscosity at concentration was calculated based on exponential fitting with the equation y = a*e ^(b*x) , where y is the viscosity in cP, and x is mg/mL is the protein concentration of , a and b are the fitting parameters to the equation, and R ² is the statistical coefficient of determination. For this example, viscosity is reported as a function of fixed concentration and the results are provided in Tables 42 and 43 below.

Table 42

Table 43

Example 49: BLI measurement of self-interactions

[00182] In this example, biolayer interferometry (BLI) testing was performed with a ForteBio Octet Red-96 instrument. An amine responsive second generation (AR2G) biosensor (Molecular Devices, CA) was conjugated with omalizumab to detect protein self-interactions in the presence of excipients. Excipient solutions for the excipients listed in Table 44 were prepared at 0.1 M in His HCl buffer. 20 mM sodium phosphate by dissolving 1.679 g of dibasic sodium phosphate, heptahydrate (Sigma, St. Louis) and 1.895 g of monobasic sodium phosphate, monohydrate (Sigma, St. Louis) in DI water and adjusting the volume; A pH 6.4 buffer was prepared. Study-grade omalizumab, purchased from Bioceros (Utrecht, The Netherlands), was buffer exchanged into phosphate buffer at pH 6.4 using an Amicon-15 centrifuge (30 kDa MWCO) apparatus. This omalizumab stock solution at 15 mg/mL in 20 mM sodium phosphate, pH 6.4 buffer was further buffer exchanged using a Sephadex G-25 PD-10 desalting column (GE Healthcare Life Sciences) and excipients prepared at 0.1 M 20 mM sodium phosphate, pH 6.4 buffer containing A control was prepared similarly using a Sephadex G-25 PD-10 desalting column (GE Healthcare Life Sciences) and eluted with 20 mM sodium phosphate, pH 6.4 buffer. Protein concentration was measured using a UV transparent 96 half-well microplate (Greiner Bio-One, Austria), and absorbance was measured at a wavelength of 280 nm with a Synergy HT plate reader (BioTek, Winooski, VT). The protein concentration was adjusted to 5 mg/mL by dilution in the prepared excipient buffer. In a black bottom 96-well microplate (Greiner Bio-One, Austria), transfer 250 μL of a solution of each excipient 0.1 M in 20 mM sodium phosphate, pH 6.4 buffer to column B, 5 mg/ml containing 0.1 M excipient 250 μL of omalizumab solution was transferred to column C. 96-well plates were set up such that columns represent individual formulations and rows differentiate protein-containing formulations. The trays were then transferred to a ForteBio Octet Red-96 for analysis. A baseline was created by immersing the omalizumab conjugated biosensor in the protein-free formulation for 120 seconds. The biosensor was then removed and immersed in the formulation containing the protein for 300 seconds. In this example, we reported the delta as a percentage of the binding signal at 300 seconds compared to the binding signal of the control, and the results are summarized in Table 44 below.

Table 44

equality

[00183] While specific embodiments of the invention have been disclosed herein, the foregoing specification is illustrative and not restrictive. While the present invention has been shown and described with reference to particularly preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention encompassed by the appended claims. Many modifications of the present invention will become apparent to those skilled in the art upon review of this specification. Unless otherwise indicated, all numbers expressing reaction conditions, amounts of ingredients, etc. used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth herein are approximations which may vary depending upon the desired properties to be obtained by the present invention.

Claims (36)

A liquid formulation comprising a protein and an excipient compound, wherein the excipient compound is a pyrimidine and the excipient compound is added in a viscosity-reducing amount. The liquid formulation of claim 1 , wherein the pyrimidine is a methyl-substituted pyrimidine. The method of claim 1, wherein the pyrimidine is pyrimidine, pyrimidinone, triaminopyrimidine, 1,3-dimethyluracil, 1-methyluracil, 3-methyluracil, 1,3-diethyluracil, 5-methyluracil , 6-methyluracil, uracil, 1,3-dimethyl-tetrahydro pyrimidinone, thymine, 1-methylthymine, O-4-methylthymine, 1,3-dimethylthymine, dimethylthymine dimer, theacrine, cytosine , 5-methylcytosine, and 3-methylcytosine. 4. The liquid formulation of claim 3, wherein the pyrimidine is 1,3-dimethyluracil. The liquid formulation of claim 1 , wherein the formulation is a pharmaceutical composition, the protein is a therapeutic protein, and the excipient compound is a pharmaceutically acceptable excipient compound. The liquid formulation of claim 1 , wherein the formulation is a non-therapeutic formulation and the protein is a non-therapeutic protein. The liquid formulation according to claim 5, wherein the therapeutic protein is a PEGylated protein. 6. The liquid formulation of claim 5, wherein the therapeutic protein is a therapeutic antibody. 9. The method of claim 8, wherein the therapeutic antibody is selected from the group consisting of bevacizumab, trastuzumab, adalimumab, infliximab, etanercept, cetuximab, rituximab, ipilimumab, and omalizumab , liquid formulations. The liquid formulation of claim 1 , wherein the viscosity-reducing amount of the excipient compound is about 250 mg/mL or less. The liquid formulation of claim 1 , wherein the viscosity-reducing amount of the excipient compound is from about 10 mg/mL to about 200 mg/mL. The liquid formulation of claim 11 , wherein the viscosity-reducing amount of the excipient compound is from about 10 mg/mL to about 120 mg/mL. 13. The liquid formulation of claim 12, wherein the viscosity-reducing amount of the excipient compound is from about 20 mg/mL to about 120 mg/mL. The liquid formulation of claim 1 , wherein the viscosity-reducing amount of the excipient compound is from about 1 mg/mL to about 100 mg/mL. 15. The liquid formulation of claim 14, wherein the viscosity-reducing amount of the excipient compound is from about 2 mg/mL to about 80 mg/mL. 16. The liquid formulation of claim 15, wherein the viscosity-reducing amount of the excipient compound is from about 5 mg/mL to about 50 mg/mL. The liquid formulation of claim 16 , wherein the viscosity-reducing amount of the excipient compound is from about 10 mg/mL to about 40 mg/mL. The liquid formulation of claim 1 , wherein the viscosity-reducing amount of the excipient compound is from about 1 mM to about 400 mM. 19. The liquid formulation of claim 18, wherein the viscosity-reducing amount of the excipient compound is from about 2 mM to about 150 mM. 20. The liquid formulation of claim 19, wherein the viscosity-reducing amount of the excipient compound is from about 5 mM to about 100 mM. 21. The liquid formulation of claim 20, wherein the viscosity-reducing amount of the excipient compound is from about 10 mM to about 75 mM. 22. The liquid formulation of claim 21, wherein the viscosity-reducing amount of the excipient compound is from about 15 mM to about 50 mM. The liquid formulation of claim 1 , wherein the viscosity-reducing amount reduces the viscosity of the formulation to a viscosity lower than that of the control formulation, wherein the control formulation contains no excipient compound but is otherwise identical on a dry weight basis to the therapeutic formulation. 24. The liquid formulation of claim 23, wherein the viscosity of the liquid formulation is at least about 10% less than the viscosity of the control formulation. 25. The liquid formulation of claim 24, wherein the viscosity of the liquid formulation is at least about 30% less than the viscosity of the control formulation. 26. The liquid formulation of claim 25, wherein the viscosity of the liquid formulation is at least about 50% less than the viscosity of the control formulation. 27. The liquid formulation of claim 26, wherein the viscosity of the liquid formulation is at least about 70% less than the viscosity of the control formulation. 28. The liquid formulation of claim 27, wherein the viscosity of the liquid formulation is at least about 90% less than the viscosity of the control formulation. 24. The liquid formulation of claim 23, wherein the viscosity of the liquid formulation is less than about 100 cP. 30. The liquid formulation of claim 29, wherein the viscosity of the liquid formulation is less than about 50 cP. 31. The liquid formulation of claim 30, wherein the viscosity of the liquid formulation is less than about 20 cP. 32. The liquid formulation of claim 31, wherein the viscosity of the liquid formulation is less than about 10 cP. The method of claim 1, further comprising an additional agent selected from the group consisting of preservatives, surfactants, sugars, polysaccharides, arginine, proline, hyaluronidases, stabilizers, solubilizers, cosolvents, hydrotropes, and buffers. , form. A liquid formulation comprising a protein and an excipient compound selected from the group consisting of 3-aminopyridine, dicyclomine, 1-methyl-2-pyrrolidone, phenylserine, DL-3-phenylserine, and cycloserine, said A liquid formulation wherein the excipient compound is added in a viscosity-reducing amount. A method of treating a disease or disorder in a mammal in need thereof, comprising:
administering to said mammal a liquid therapeutic formulation, wherein said liquid therapeutic formulation comprises a therapeutically effective amount of a therapeutic protein, and wherein said liquid therapeutic formulation comprises a viscosity-reducing amount of a pharmaceutically acceptable excipient compound. further comprising, wherein the pharmaceutically acceptable excipient compound is pyrimidine; wherein said therapeutic formulation is effective for the treatment of said disease or disorder. A method of treating a disease or disorder in a mammal in need thereof, comprising:
administering to said mammal a liquid therapeutic formulation, said liquid therapeutic formulation comprising a therapeutically effective amount of a therapeutic protein, said liquid therapeutic formulation comprising a viscosity-reducing amount of 3-aminopyridine, dicyclomine , 1-methyl-2-pyrrolidone, phenylserine, DL-3-phenylserine, and further comprising a pharmaceutically acceptable excipient compound selected from the group consisting of cycloserine, wherein the therapeutic formulation is the disease or An effective method for the treatment of a disorder.

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